Methods and Protocols

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Methods in Molecular Biology 1187

Hugo J. Bellen Shinya Yamamoto Editors

Notch Signaling Methods and Protocols

METHODS

IN

M O L E C U L A R B I O LO G Y

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Notch Signaling Methods and Protocols

Edited by

Hugo J. Bellen Department of Molecular and Human Genetics, Program in Developmental Biology, Department of Neuroscience, Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, USA

Shinya Yamamoto Department of Molecular and Human Genetics, Program in Developmental Biology, Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA

Editors Hugo J. Bellen Department of Molecular and Human Genetics Program in Developmental Biology Department of Neuroscience Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital Howard Hughes Medical Institute Baylor College of Medicine Houston, TX, USA

Shinya Yamamoto Department of Molecular and Human Genetics Program in Developmental Biology Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital Baylor College of Medicine Houston, TX, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1138-7 ISBN 978-1-4939-1139-4 (eBook) DOI 10.1007/978-1-4939-1139-4 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014942725 © Springer Science+Business Media New York 2014 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com)

Preface Notch signaling has a long history that originated 100 ago. However, 98 % or more of the knowledge related to Notch signaling has been gathered in the past 30 years, and the period between 1990 and 2013 has been exciting, both because of the extent of basic knowledge accumulated and potential implications for therapy in diseases associated with Notch. Most of the methods discussed in this book have been developed in the past 10–15 years and they cover a wide array of approaches related or based on mouse and human cell lines, flies, and mice. The first set of chapters focus on genetic methods in flies and mice, methods to image Notch signaling in live organisms or cells, techniques to monitor Notch activity in cells, and procedures to visualize oscillation associated with Notch signaling in cells and tissues. The next set of chapters focus on molecular, biochemical, and bioinformatics aspects of Notch signaling and include analyzing the Notch interactome, posttranslational modifications of Notch, ligand binding assays, and methods to assess proteolytic cleavage and transcriptional targets. Finally, strategies to diminish Notch signaling using small molecules, anti-Notch antibodies, and anti-ligand antibodies are discussed. It is impossible to cover all methods using all organisms related to Notch signaling, but we believe that these 25 chapters will be a valuable contribution to hundreds of labs and thousands of scientists who pursue this research area. We are especially grateful to Karen L. Schulze who provided advice and performed skillful editing. We thank all authors for their expert contributions and their diligence. Houston, TX, USA

Shinya Yamamoto Hugo J. Bellen

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Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 Introduction to Notch Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shinya Yamamoto, Karen L. Schulze, and Hugo J. Bellen 2 Genetic Screens to Identify New Notch Pathway Mutants in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nikolaos Giagtzoglou 3 Structure-Function Analysis of Drosophila Notch Using Genomic Rescue Transgenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jessica Leonardi and Hamed Jafar-Nejad 4 Overview of Genetic Tools and Techniques to Study Notch Signaling in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thomas Gridley and Andrew K. Groves 5 Immunohistochemical Tools and Techniques to Visualize Notch in Drosophila melanogaster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emiliana Tognon and Thomas Vaccari 6 Antibody Uptake Assay and In Vivo Imaging to Study Intracellular Trafficking of Notch and Delta in Drosophila . . . . . . . . . . . . . . . . Lydie Couturier and François Schweisguth 7 Tracking Trafficking of Notch and Its Ligands in Mammalian Cells . . . . . . . . . Patricia Chastagner and Christel Brou 8 Visualizing Notch Signaling In Vivo in Drosophila Tissues . . . . . . . . . . . . . . . . Benjamin E. Housden, Jinghua Li, and Sarah J. Bray 9 Monitoring Notch Activity in the Mouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Swananda Marathe and Lavinia Alberi 10 Notch Signaling Assays in Drosophila Cultured Cell Lines . . . . . . . . . . . . . . . . Jinghua Li, Benjamin E. Housden, and Sarah J. Bray 11 Monitoring Notch Activation in Cultured Mammalian Cells: Transcriptional Reporter Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ma. Xenia G. Ilagan and Raphael Kopan 12 Monitoring Notch Activation in Cultured Mammalian Cells: Luciferase Complementation Imaging Assays . . . . . . . . . . . . . . . . . . . . . . . . . Ma. Xenia G. Ilagan and Raphael Kopan 13 Visualization of Notch Signaling Oscillation in Cells and Tissues . . . . . . . . . . . Hiromi Shimojo, Yukiko Harima, and Ryoichiro Kageyama 14 Proteomic Analysis of the Notch Interactome . . . . . . . . . . . . . . . . . . . . . . . . . K.G. Guruharsha, Kazuya Hori, Robert A. Obar, and Spyros Artavanis-Tsakonas

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15 Bacterial Expression and In Vitro Refolding of Limited Fragments of the Notch Receptor and Its Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pat Whiteman, Christina Redfield, and Penny A. Handford 16 Analyzing the Posttranslational Modification Status of Notch Using Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shinako Kakuda and Robert S. Haltiwanger 17 Assay to Probe Proteolytic Processing of Notch by γ-Secretase . . . . . . . . . . . . Lutgarde Serneels, Ina Tesseur, and Bart De Strooper 18 Analyzing the Nuclear Complexes of Notch Signaling by Electrophoretic Mobility Shift Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kelly L. Arnett and Stephen C. Blacklow 19 Identifying Direct Notch Transcriptional Targets Using the GSI-Washout Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Will Bailis, Yumi Yashiro-Ohtani, and Warren S. Pear 20 Probing the Epigenetic Status at Notch Target Genes . . . . . . . . . . . . . . . . . . . Robert Liefke and Tilman Borggrefe 21 Notch-Ligand Binding Assays in Drosophila Cells . . . . . . . . . . . . . . . . . . . . . . Aiguo Xu and Kenneth D. Irvine 22 Modeling Notch Signaling: A Practical Tutorial. . . . . . . . . . . . . . . . . . . . . . . . Pau Formosa-Jordan and David Sprinzak 23 Small Molecules That Inhibit Notch Signaling. . . . . . . . . . . . . . . . . . . . . . . . . Gerdien E. De Kloe and Bart De Strooper 24 Application and Evaluation of Anti-Notch Antibodies to Modulate Notch Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wendy R. Gordon and Jon C. Aster 25 Application of Anti-ligand Antibodies to Inhibit Notch Signaling . . . . . . . . . . Jun-ichiro Koga and Masanori Aikawa Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors MASANORI AIKAWA • The Center for Excellence in Vascular Biology and Center for Interdisciplinary Cardiovascular Sciences, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA LAVINIA ALBERI • Unit of Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland KELLY L. ARNETT • Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA SPYROS ARTAVANIS-TSAKONAS • Department of Cell Biology, Harvard Medical School, Boston, MA, USA JON C. ASTER • Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA WILL BAILIS • Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, and Institute for Immunology, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA HUGO J. BELLEN • Department of Molecular and Human Genetics, Program in Developmental Biology, Department of Neuroscience, Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, USA STEPHEN C. BLACKLOW • Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA TILMAN BORGGREFE • Institute of Biochemistry, Faculty of Medicine, University of Giessen, Giessen, Germany SARAH J. BRAY • Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK CHRISTEL BROU • Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur and CNRS, Paris, France PATRICIA CHASTAGNER • Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur and CNRS, Paris, France LYDIE COUTURIER • Département de Biologie du Développement, Unité de Génétique du Développement de la Drosophile, Institut Pasteur and CNRS, Paris, France PAU FORMOSA-JORDAN • Department of Structure and Constituents of Matter, Physics, University of Barcelona, Barcelona, Spain; Sainsbury Laboratory, Cambridge University, Cambridge, UK NIKOLAOS GIAGTZOGLOU • Department of Neurology, Jan and Dan Duncan Neurological Institute, Baylor College of Medicine, Houston, TX, USA WENDY, R. GORDON • Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA THOMAS GRIDLEY • Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME, USA ANDREW K. GROVES • Department of Neuroscience, Program in Developmental Biology, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA K.G. GURUHARSHA • Department of Cell Biology, Harvard Medical School, Boston, MA, USA

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ROBERT S. HALTIWANGER • Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA PENNY A. HANDFORD • Department of Biochemistry, University of Oxford, Oxford, UK YUKIKO HARIMA • Institute for Virus Research, Kyoto University, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kyoto, Japan KAZUYA HORI • Department of Cell Biology, Harvard Medical School, Boston, MA, USA BENJAMIN E. HOUSDEN • Department of Genetics, Harvard Medical School, Boston, MA, USA; Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK MA. XENIA G. ILAGAN • Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA KENNETH D. IRVINE • Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA HAMED JAFAR-NEJAD • Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA RYOICHIRO KAGEYAMA • Institute for Virus Research and World Premier International Research Initiative–Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kyoto, Japan SHINAKO KAKUDA • Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA GERDIEN E. DE KLOE • VIB Center for the Biology of Disease, Leuven, Belgium JUN-ICHIRO KOGA • The Center for Excellence in Vascular Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA RAPHAEL KOPAN • Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA JESSICA LEONARDI • Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA JINGHUA LI • Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK ROBERT LIEFKE • Cell Biology Department, Harvard Medical School and Division of Newborn Medicine, Boston Children’s Hospital, Boston, MA, USA SWANANDA MARATHE • Unit of Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland ROBERT A. OBAR • Department of Cell Biology, Harvard Medical School, Boston, MA, USA WARREN S. PEAR • Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute and Institute for Immunology, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA CHRISTINA REDFIELD • Department of Biochemistry, University of Oxford, Oxford, UK KAREN L. SCHULZE • Department of Molecular and Human Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, USA FRANÇOIS SCHWEISGUTH • Département de Biologie du Développement, Unité de Génétique du Développement de la Drosophile, Institut Pasteur and CNRS, Paris, France LUTGARDE SERNEELS • VIB Center for the Biology of Disease- VIB11 and Center for Human Genetics, VIB and KU Leuven, Leuven, Belgium HIROMI SHIMOJO • Institute for Virus Research and World Premier International Research Initiative–Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan

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DAVID SPRINZAK • Department of Biochemistry and Molecular Biology, George S Wise Faculty of Life Science, Tel Aviv University, Tel Aviv, Israel BART DE STROOPER • VIB Center for the Biology of Disease and Center for Human Genetics and Institute of Neuroscience & Disease (LIND), KU Leuven and universitaire ziekenhuizen, Leuven, Belgium INA TESSEUR • VIB Center for the Biology of Disease - VIB11 and Center for Human Genetics, VIB and KU Leuven, Leuven, Belgium EMILIANA TOGNON • Istituto FIRC di Oncologia Molecolare (IFOM), Milano, Italy THOMAS VACCARI • Istituto FIRC di Oncologia Molecolare (IFOM), Milano, Italy PAT WHITEMAN • Department of Biochemistry, University of Oxford, Oxford, UK AIGUO XU • Primera Analytical Solutions Corp., Princeton, NJ, USA SHINYA YAMAMOTO • Department of Molecular and Human Genetics, Program in Developmental Biology, Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA YUMI YASHIRO-OHTANI • Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, and Institute for Immunology, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

Chapter 1 Introduction to Notch Signaling Shinya Yamamoto, Karen L. Schulze, and Hugo J. Bellen Abstract Notch signaling is probably the most widely used intercellular communication pathway. The Notch mutant in the fruit fly Drosophila melanogaster was isolated about 100 years ago at the dawn of genetics. Since then, research on Notch and its related genes in flies, worms, mice, and human has led to the establishment of an evolutionarily conserved signaling pathway, the Notch signaling pathway. In the past few decades, molecular cloning of the Notch signaling components as well as genetic, cell biological, biochemical, structural, and bioinformatic approaches have uncovered the basic molecular logic of the pathway. In addition, genetic screens and systems approaches have led to the expansion of the list of genes that interact and finetune the pathway in a context specific manner. Furthermore, recent human genetic and genomic studies have led to the discovery that Notch plays a role in numerous diseases such as congenital disorders, stroke, and especially cancer. Pharmacological studies are actively pursuing key components of the pathway as drug targets for potential therapy. In this chapter, we will provide a brief historical overview of Notch signaling research and discuss the basic principles of Notch signaling, focusing on the unique features of this pathway when compared to other signaling pathways. Further studies to understand and manipulate Notch signaling in vivo in model organisms and in clinical settings will require a combination of a number of different approaches that are discussed throughout this book. Key words Review, Notch signaling, History, Development and disease, Experimental approaches

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Historical Overview Notch was “discovered” in the laboratory of Thomas Hunt Morgan in March of 1913 [1, 2]. The oldest publication record of description of a “notch” defect in Drosophila, a loss of wing margin tissue from the distal tip of the wing, goes back to 1914 by John S. Dexter [3]. The first allele of Notch was established in 1917 [4]. In addition to notched wings, heterozygous Notch mutant flies were reported to exhibit additional wing vein and bristle abnormalities [5], providing a glimpse of its pleiotropic nature. The first link between Notch and development was established by the pioneering work on hemizygous Notch mutant embryos by Donald F. Poulson in the 1930s [6, 7]. Notch mutant embryos lacked mesodermal and endodermal tissue while most of the remaining

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_1, © Springer Science+Business Media New York 2014

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ectodermal tissue produced nervous system at the expense of hypodermal cells. This unique phenotype, later called the “neurogenic” phenotype [8], was one of the first indications that Notch functions during cell–cell signaling. Furthermore, genetic screens looking for mutants with similar neurogenic phenotypes lead to the identification of core Notch signaling components such as Delta, mastermind, and Enhancer of Split (E(spl)) [8, 9]. In addition, analysis and genetic screens focusing on wing notching and bristle defects lead to the identification of genes such as Suppressor of Hairless (Su(H), also known as CSL), Serrate, and other factors of the pathway [10, 11]. An important breakthrough was the cloning [12, 13] and sequencing of the Notch gene [14, 15]. Notch was shown to be a very large transmembrane domain protein with large extracellular and intracellular domains. Molecular characterization of the Notch gene in Drosophila lead to the identification of direct homologs in other species including C. elegans (lin-12 and glp-1) [16–18] and vertebrates (Xotch and TAN-1) [19, 20], expanding the Notch field from Drosophila to other model organisms and human biology. Evidence that Notch functions as a receptor of a novel intercellular signaling pathway accumulated during the late 1980s and the early 1990s [21–23]. Pioneering work in C. elegans made the link between Notch activation and γ-secretase activity [24–27], which later led to the model that the intracellular portion of the Notch receptor translocates into the nucleus and directly regulates transcription [28–31]. Identification of Presenilin, a key protein in Alzheimer’s disease pathogenesis, as the core catalytic subunit of the γ-secretase complex responsible for the proteolytic cleavage of Notch broadened the significance of Notch studies in biomedical research [32]. Biochemical and structural approaches have also greatly contributed to the understanding of chemical, physical, and mechanistic properties of Notch signal regulations [33]. In parallel to the efforts to reveal the genes and mechanisms that coordinate the Notch signaling pathway using model organisms and cultured cell lines, research also uncovered a strong link between Notch and a diverse set of human diseases [34]. Mutations in the receptors and the ligands have been shown to be causative for the pathogenesis of multiple developmental disorders, and misregulation of the pathway has been linked to an array of tumors in various tissues [35]. The core components of the Notch signaling pathway have emerged as major drug targets for anticancer therapy, although less important or context specific components of the pathway may be much better targets as the core components are almost certainly extremely pleiotropic and are required almost continuously in gut, hematopoiesis, skin, bone, etc. [36]. More recently, an active debate is ongoing whether Notch signaling pathway dysregulation may participate in neurological and psychiatric disorders as well [37, 38].

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In summary, Notch research that started out by noticing a mild phenotype at the tip of the Drosophila wing has grown into an interdisciplinary field involving hundreds if not thousands of geneticists, developmental, cell, molecular, structural biologists, bioinformaticians, chemists, and clinicians. 1.1 Unique Properties of Notch Signaling

Compared to other intercellular signaling pathways such as Wnt, Hedgehog, and TGF-β/BMP, Notch signaling pathway is unique in multiple aspects. First, canonical Notch signaling occurs in a “juxtacrine” manner meaning that the signaling takes place between juxtaposed neighboring cells and requires direct cell–cell contact, while most other signaling pathways rely on paracrine signaling mediated by ligands that are secreted and reach distant cells through diffusion and/or active transport mechanisms. This is because both ligands and receptors of the canonical Notch pathway are transmembrane proteins that are embedded into the membrane of the cells [39]. However, some noncanonical Notch signaling events can be mediated by a secreted ligand in a paracrine manner, which has been documented in C. elegans [40]. Second, Notch signaling is extremely dose sensitive due to the lack of a signal amplification step or utilization of secondary messengers to transmit the signal from the cell surface to the nucleus. Notch signaling is mediated through the release and translocation of the intracellular domain of Notch (NICD) into the nucleus and NICD directly functions as a transcriptional coactivator. Notch in Drosophila is one of the very few examples where the locus is haploinsufficient, and a duplication also causes visible phenotypes. Furthermore, Delta, which encodes one of the two Notch signaling ligands in Drosophila, also exhibits haploinsufficient phenotypes in flies [41]. Strict dosage dependence of Notch signaling during development is also observed in mammals, including human [42, 43]. In addition, both hyper- and hypo-activation of the pathway are associated with different types of cancer in mice [44–46] and in humans [47–51]. These studies point out that a tight regulation of signal activity in both the signal sending and receiving cells is crucial for optimal signal output in physiological settings. Third, Notch is a highly pleiotropic signaling pathway whose output depends on developmental and cellular contexts. For example, Notch signaling can be used for lateral inhibition, cell fate decisions, and in an inductive or a permissive manner to select out certain cell types, induce specific cell fates, and define boundaries during morphogenesis in Drosophila [23, 52]. In addition, activation or inhibition of the pathway can lead to a wide range of cellular responses such as proliferation, differentiation, or cell death depending on the context and cell type. Finally, activation of the pathway is now being associated with synaptic plasticity and learning

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and memory in both fly [53–57] and mammalian nervous systems [58–60], suggesting a post-developmental role of Notch in differentiated cells. In sum, given these characteristic properties, Notch signaling can be thought of as a “double-edged sword” that needs to be carefully controlled and constantly monitored for proper activity.

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Core Components of Notch Signaling The core components of the Notch pathway are depicted in Fig. 1. The ligand binds to the receptor at the interface between the two signaling cells. This leads to the release of the intracellular portion of the receptor that translocates into the nucleus, and interacts with a transcription factor and coactivators to activate transcription. However, due to the unique properties of Notch signaling and the extreme dosage sensitivity, numerous factors seem to have evolved to fine-tune the activity of the Notch signaling pathway in different tissues, cell types, and contexts [61, 62]. Some of these factors are general regulators and affect the core components of the canonical Notch signaling pathway in all contexts, whereas others are context and tissue specific regulators. In addition, although many of the phenotypes caused by dysregulation of the pathway can be explained by the canonical signaling pathway, several phenomena exist where we need to consider a noncanonical branch of Notch signaling that is mediated without the involvement of certain core canonical pathway members [63]. Here, we mainly focus on the receptors, ligands, and nuclear factors (NICD, CSL, and Mastermind) that comprise the core of the Notch signaling pathway.

2.1 Receptors and Ligands

Notch receptors are large multidomain type I transmembrane proteins (Fig. 2) [33, 39]. Four Notch paralogs (Notch1-4) are present in mammals including human, while only one Notch homolog exists in Drosophila. Notch receptors are translated in the endoplasmic reticulum (ER) and travel to the plasma membrane through the exocytic pathway while undergoing numerous posttranslational modification events. In the ER, the signal sequence at the N-terminus is cleaved off and the extracellular domain of Notch, most of which consists of EGF repeats (EGFrs), undergoes sugar modifications mediated by protein glycosyltransferases [64]. For example, O-fucosylation by O-fut1 (Pofut1 in mammals) and subsequent elongation of this sugar chain with O-GlcNAc by Fringe (Lunatic, Manic, and Radical Fringe in mammals) in the Golgi complex modulates the ligand selectivity of the Notch receptor [65–67]. In addition, O-glucosylation by Rumi (Poglut in mammals) in the ER is necessary for receptor activation [68], while further elongation of this chain with O-xylose negatively regulates

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Fig. 1 A simplified diagram of the canonical Notch signaling pathway. For simplicity, the nomenclature of proteins depicted is based on Drosophila. Notch signaling occurs between juxtaposed signal sending cell (top) and signaling receiving cell (bottom). (1) Notch is translated in the rough ER and becomes glycosylated by glycosyltransferases such as O-fut1 and Rumi. (2) Notch traffics to the Golgi complex and undergoes S1 cleavage by Furin-like proteases. In addition, elongation of O-fucose chains on EGFrs occurs in cells that express Fringe. (3) Notch traffics to the cell surface. (4) Notch ligands, Delta and Serrate, are translated in the ER and traffic to the cell surface through the Golgi complex. Endocytosis and recycling of the ligands towards the site of ligand–receptor interaction is critical in certain contexts. (5) Ligands and Notch receptor physically interact. (6) The ligand–receptor complex is endocytosed into the signal sending cell. E3 ubiquitin ligases Neur or Mib and endocytic proteins such as Dynamin are essential for this trans-endocytosis. This physically “pulls” the Notch receptor so that conformational changes to reveal the S2 cleavage site can occur. (7) Notch undergoes S2 cleavage by ADAM proteases to generate NEXT. (8) NEXT undergoes S3 cleavage by the γ-secretase complex to release NICD. (9) γ-secretase complex cleavage of Notch/NEXT can also occur on the endosomal membrane. (10) NICD translocates into the nucleus. (11) NICD interacts with CSL and Mam on the target DNA. In the absence of NICD, CSL recruits corepressors to turn off gene expression. When NICD binds CSL and Mam, corepressors become replaced by coactivators to turn on gene expression. See main text for abbreviations

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Fig. 2 Schematic diagram of the structure of the Notch receptor. For simplicity, Drosophila Notch receptor is depicted. Notch receptors are type I transmembrane proteins that have a signal sequence (SS) at the N terminal. The SS becomes cleaved off after translation. The majority of the extracellular domain consists of EGFrs. 36 EGFrs are present in Drosophila Notch. EGFrs-11 and -12 are essential for ligand binding, and EGFr-8 is involved in ligand selectivity. EGFrs-24 ~ 29 (Abruptex domain) are involved in negative regulation of the signaling. The NRR consists of three LNR domains and a HD motif. S1 and S2 cleavage sites are present here. The S3 cleavage occurs in the transmembrane domain (TMD). The NICD consists of a RAM domain, seven ANK repeats, several NLS, the TAD, and the PEST domain. The RAM domain and ANK repeats interact with CSL and Mam. NLS are required for nuclear translocation of NICD. TAD is involved in the recruitment of additional coactivators. PEST domain is necessary for proteasome mediated degradation of NICD for signal termination. See text for abbreviations

Notch signaling [69]. The Notch extracellular domain undergoes its first proteolytic cleavage (S1) in the Golgi complex by furin-like proteases [70]. While the S1 cleavage is not absolutely required for signal activation in flies as well as in mammals, this processing is thought to contribute to the net signaling activity by facilitating exocytosis of Notch [71, 72]. The cleaved fragments are held together through non-covalent interactions at the heterodimerization (HD) domain, and the receptor subsequently traffics to the cell surface to interact with its ligands. Both ligands, Delta and Serrate (grouped together as DSL family ligands), are type I transmembrane proteins with a large extracellular domain and a relatively short intracellular domain [39, 40]. In mammals, three Delta-family ligands (Dll1, Dll3, and Dll4) and two Serrate-family ligands (Jagged1 and Jagged2) are present. Dll2 was initially identified in Xenopus [73], but its ortholog was later found to be absent in mammalian species, hence Dll2 is not present in mammalian nomenclature. The ligands are also synthesized in the ER, trafficked through the Golgi complex, and exocytosed. Since the ligands need to meet their receptors at the interface of the two signaling cells, vesicular trafficking to the membrane and endocytosis and recycling of proteins play important roles in fine-tuning the signaling strength [74–76]. Furthermore, upon ligand–receptor interactions, the ligand–receptor complex becomes endocytosed into the signal sending cell (trans-endocytosis) creating a “pulling force” that leads to a conformational change that promotes receptor activation. Monoubiquitination of the intracellular domains of ligands by the E3 ligase Neuralized (Neur) or Mindbomb (Mib) is critical for this

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ligand endocytosis. In the absence of ligand ubiquitination, the ligand–receptors can interact but fail to activate the signal. Interestingly, the intracellular domain of Dll3 lacks ubiquitination sites and is thought to act as a decoy ligand in vivo. Upon ligand binding and endocytosis of the ligand–receptor complex, the Notch receptor undergoes a conformational change that reveals a proteolytic cleavage site that permits cleavage by ADAM (a disintegrin and metalloproteinase) proteases. This leads to the second (S2) cleavage of the Notch receptor and the release of the extracellular domain [77]. This S2 cleaved Notch is still embedded in the membrane and is often referred to as the Notch extracellular truncated form (NEXT). NEXT is the substrate of the γ-secretase complex, an intramembrane protease [78]. Hence, Notch, via NEXT, undergoes a third (S3) cleavage that releases the NICD from the membrane. Where the cleavage is occurring within the cell is still a matter of debate [76]. The NICD that is freed from the membrane can translocate into the nucleus by the nuclear import machinery to engage in transcription regulation [79]. 2.2

Nuclear Complex

NICD consists of a single RAM (RBP-jκ Associated Molecule) domain, seven ankyrin repeats (ANK), a transactivation domain (TAD), and a PEST (proline (P)/glutamic acid (E)/serine (S)/ threonine (T)-rich motif) sequence at the carboxy-terminus (Fig. 2). In the fly NICD, a poly-glutamine (Q)-rich domain is present within the TAD, which is referred to as the OPA domain [80]. In addition, NICD carries multiple nuclear localization sequences (NLS) [29] as well as target sites for multiple posttranslational modifications such as phosphorylation [81] and ubiquitination [82]. In the nucleus, NICD interacts with a DNA binding transcription factor CSL (also known as Su(H) in Drosophila, RBP-jκ in mammals) and a coactivator Mastermind (Mam in Drosophila, MAML1, MAML2, MAML3 in mammals) through its RAM and ANK domains [83, 84]. In the absence of NICD, CSL binds to its consensus sequence on the DNA and recruits transcription corepressors such as Hairless, CtBP (C-terminal Binding Protein), and Groucho to further recruit histone deacetylases (HDACs) and other repressive cofactors to negatively regulate the expression of Notch target genes [85–90]. Upon Notch signaling activation and NICD binding to CSL and Mam, the corepressor complex is disassembled, leading to derepression of the gene targets [91–93] and recruitment of the transcription activation complex including histone acetyltransferases (HATs) and chromatin remodeling complexes [94–97]. To prevent continuous signal activation, Notch signaling is shut off through phosphorylation of NICD by kinases such as cyclin-dependent kinase-8 (CDK8) [82] followed by polyubiquitination via E3 ubiquitin ligases such as SEL10/FBXW7 [98]. Poly-ubiquitination of NICD leads to proteasome mediated degradation and termination of the signal [99–101].

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Fine-Tuning Notch Signaling in Development and in Disease As a molecule of Notch is consumed upon a single round of signal activation through proteolytic processing at the plasma membrane and eventual degradation in the nucleus, proteins and factors that regulate any of the above mentioned events during signal activation and termination have the potential to regulate and finetune the output of the canonical pathway [52, 61, 62]. For example, the requirement of trans-endocytosis of the ligand–receptor complex for signal activation allows an additional layer of finetuning the signal strength at the cell surface. In addition to the ability of the ligands and receptors to interact (in trans) with one another at the interface of the signaling cells, the two can interact (in cis) within the same cell when co-expressed. Since the cisinteraction does not permit the conformational change of the Notch receptor required for S2 cleavage, and the ligands expressed in cis and trans are thought to compete for the ligand–binding domain of the Notch receptor, cis-interactions lead to negative regulation of signal activation (cis-inhibition) [102]. Furthermore, Notch receptors that bind to the ligand in cis reduce the concentration of the ligand that can trans-activate the neighboring cells [103, 104]. Thus, cis-inhibition not only inhibits the signal receptive ability of the signal receiving cell, but also the signal transmission ability of the signal sending cell (mutual inactivation). By altering the amount of ligands and receptors expressed in a cell, and by modulating the affinity of the ligands and receptors through posttranslational modifications such as glycosylation, numerous scenarios of Notch activation patterns can be generated in a tissue of interest [105]. While ligand-mediated activation of Notch signaling is thought to be responsible for most physiologic Notch signaling events, ligand-independent activation of Notch receptors can occur in vitro and in vivo and has been linked to pathogenic events such as cancer. In cultured cells, Notch can be activated in a ligandindependent fashion by chelating extracellular Ca2+ [106, 107]. This has been used as a convenient experimental manipulation to monitor and follow the time course of Notch activation. Ligandindependent activation of Notch can also be seen in vivo in a number of mutants in which endocytic trafficking towards the lysosome is disrupted [108, 109]. If Notch is not degraded properly in the lysosome, a conformational change in the extracellular domain of Notch and/or dissociation of the heterodimer is thought to happen in the endolysosomal pathway [76]. This leads to ectopic production of NEXT, which in turn undergoes S3 cleavage at the endosome/lysosome membrane to generate a functional NICD. Furthermore, ligand-independent activation of Notch can occur when a specific domain of the Notch extracellular domain is mutated.

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In human patients who suffer from T cell acute lymphoblastic leukemia (T-ALL), mutations in the negative regulatory region (NRR), which includes three LNR repeats and the HD domain (Fig. 2), in Notch1 can be found in more than 50 % of the cases [47]. These mutations are proposed to lead to ectopic exposure of the S2 cleavage site that allows the ADAM protease and γ-secretase mediated cleavages that release the NICD in the absence of ligand binding [110, 111]. Additionally, late Notch1 truncation mutations in T-ALL patients lead to the loss of the PEST domain [47]. This prevents the ubiquitin-proteasome system dependent degradation of NICD, leading to ectopic and/or prolonged activation of Notch signaling. Thus, in addition to studying and manipulating the canonical ligand-dependent Notch signaling events, understanding the factors and mechanisms that regulate ligand-independent Notch signaling is important. Continuous efforts to uncover novel genes and proteins that regulate and fine-tune Notch signaling are important as they may provide specific targets for drugs. Model organisms continue to play critical roles in this process. For example, forward genetic screens in Drosophila and C. elegans are revealing novel components of the Notch signaling pathway, many of which act in context specific manners (Chapter 2). Further studies to understand the function of such genes in vivo can be performed using a number of tools and methods that have been developed in flies (Chapters 5, 6, 8, and 10) and in mice (Chapters 4, 9, and 13) over the years. In addition, experiments in cultured cells provide us with systems that allow more temporally precise manipulations and provide us with more quantitative data sets (Chapters 7, 11, 12, 17, and 21). Recently, a large scale protein–protein interaction study has revealed a large network of diverse molecular complexes that potentially regulate and/or interact with Notch signaling (Chapter 14). Also, several efforts are being made to uncover downstream targets of Notch by monitoring gene expression and chromatin status at a genome scale (Chapters 19 and 20). Finally, bioinformatics is starting to be used to model the signaling pathway in silico to provide a mathematical view of the properties and dynamics of Notch signaling (Chapter 22). By combining these diverse approaches and by integrating and analyzing these large data sets, the field is making progress to draw out a more complete map of the Notch signaling world. Understanding the physical, chemical, and mechanistic properties of the core components of the Notch pathway is as important as the discovery of novel factors in the pathway. Notch receptors, ligands, proteases (e.g., γ-secretase complex), and nuclear complexes (e.g., CSL, Mastermind) are now being considered as key drug targets for a number of clinical applications. By understanding the function of different domains of these proteins through structural biological methodologies (Chapters 15 and 18)

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and in vivo structure function studies (Chapter 3), we will more likely be able to design specific drugs that modulate specific aspects of the pathway. Uncovering posttranslational modifications of the relevant proteins will also reveal novel mechanisms by which these proteins are regulated (Chapter 16). Drugs that are being developed based on these structural studies will not only be useful in the clinics but also in labs to study different biological and pathological events that are regulated by Notch signaling. Small molecules against the γ-secretase complex (Chapter 23) and monoclonal antibodies against Notch receptors (Chapter 24) and ligands (Chapter 25) are currently available and are useful to pharmacologically manipulate Notch activity in vitro and in vivo.

4

Conclusion Starting out with the discovery of a fly with a tiny notch on the tip of its wings, Notch signaling has sparked the interest of numerous scientists and clinicians with diverse backgrounds and specialties over the last century. Research in the past two decades has revealed that Notch is a unique signaling pathway that is involved in diverse biological processes and numerous human diseases. Since the pathway is utilized in so many different biological settings, batteries of proteins seem to form regulatory networks that operate in different contexts. By expanding the list of Notch signaling regulatory factors and downstream target gene networks, and through understanding of the molecular logic of Notch signaling mechanisms in vivo, important questions will be answered. Furthermore, we foresee that novel Notch related human diseases will continue to be discovered through recent advances in genomic methodologies such as copy number variation (CNV) detection [112] and whole-exome sequencing (WES) techniques [113]. By combining numerous experimental methodologies and integrating the multidisciplinary knowledge that is derived from such studies, Notch signaling research will continue to thrive. In summary, the better we understand Notch signaling in development, physiology, and pathology, the better our knowledge becomes about the mechanisms that fine-tune Notch signaling. These include factors that are required for the production and maturation of the ligands and receptor, enzymes involved in posttranslational modification of these proteins, exocytic and endocytic vesicular trafficking pathway machineries, lysosomal and ubiquitinproteasome mediated protein degradation complexes, nuclear import and export factors, transcriptional coactivators and corepressors, and protein complexes that regulate chromatin status in the nucleus. It is therefore no surprise that the Notch field flourishes, and that the translational shift that has been initiated in the past 10 years will have one of the richest substrates.

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Acknowledgements We apologize to our colleagues for not being able to cite their work given the length restrictions. S.Y. is a fellow of the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital. H.J.B. is a Howard Hughes Medical Institute Investigator. References 1. Morgan TH, Bridges CB (1916) Sex-linked inheritance in Drosophila. Carnegie Inst Wash Publ 237:1–88 2. Morgan TH (1917) The theory of the gene. Am Nat 51:513–544 3. Dexter JS (1914) The analysis of a case of continuous variation in Drosophila by a study of its linkage relations. Am Nat 48:712–758 4. Mohr OL (1919) Character changes caused by mutation of an entire region of a chromosome in Drosophila. Genetics 4:275–282 5. Lindsley DL, Zimm GG (1992) The genome of Drosophila melanogaster. Academic, Waltham 6. Poulson DF (1937) Chromosomal Deficiencies and the Embryonic Development of Drosophila Melanogaster. Proc Natl Acad Sci U S A. 23(3):133–7 7. Poulson DF (1936) Chromosome deficiencies and embryonic development. Ph.D. thesis, Caltech, CA 8. Lehmann R, Jimenez F, Dietrich U, CamposOrtega JA (1983) On the phenotype and development of mutants of early neurogenesis in Drosophila melanogaster. Rouxs Arch Dev Biol 192:62–74 9. Lehmann R, Dietrich U, Jiménez F, CamposOrtega JA (1981) Mutations of early neurogenesis in Drosophila. Rouxs Arch Dev Biol 190:226–229 10. Fortini ME, Artavanis-Tsakonas S (1994) The suppressor of hairless protein participates in notch receptor signaling. Cell 79:273–282 11. Fleming RJ, Scottgale TN, Diederich RJ et al (1990) The gene Serrate encodes a putative EGF-like transmembrane protein essential for proper ectodermal development in Drosophila melanogaster. Genes Dev 4:2188–2201 12. Artavanis-Tsakonas S, Muskavitch MA, Yedvobnick B (1983) Molecular cloning of Notch, a locus affecting neurogenesis in Drosophila melanogaster. Proc Natl Acad Sci U S A 80:1977–1981 13. Kidd S, Lockett TJ, Young MW (1983) The Notch locus of Drosophila melanogaster. Cell 34:421–433 14. Wharton KA, Johansen KM, Xu T et al (1985) Nucleotide sequence from the neurogenic locus notch implies a gene product that shares

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44. Demehri S, Turkoz A, Kopan RE (2009) Epidermal Notch1 loss promotes skin tumorigenesis by impacting the stromal microenvironment. Cancer Cell 16:55–66 45. Nicolas M, Wolfer A, Raj K et al (2003) Notch1 functions as a tumor suppressor in mouse skin. Nat Genet 33:416–421 46. Pear WS, Aster JC, Scott ML et al (1996) Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles. J Exp Med 183: 2283–2291 47. Weng AP, Ferrando AA, Lee W et al (2004) Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306:269–271 48. Rosati E, Sabatini R, Rampino G et al (2009) Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells. Blood 113:856–865 49. Agrawal N, Frederick MJ, Pickering CR et al (2011) Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science 333: 1154–1157 50. Wang NJ, Sanborn Z, Arnett KL et al (2011) Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma. Proc Natl Acad Sci U S A 108: 17761–17766 51. Stransky N, Egloff AM, Tward AD et al (2011) The mutational landscape of head and neck squamous cell carcinoma. Science 333:1157–1160 52. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689 53. de Bivort BL, Guo HF, Zhong Y (2009) Notch signaling is required for activitydependent synaptic plasticity at the Drosophila neuromuscular junction. J Neurogenet 23: 395–404 54. Song Q, Sun K, Shuai Y et al (2009) Suppressor of hairless is required for longterm memory formation in Drosophila. J Neurogenet 23:405–411 55. Ge X, Hannan F, Xie Z et al (2004) Notch signaling in Drosophila long-term memory formation. Proc Natl Acad Sci U S A 101:10172–10176 56. Presente A, Boyles RS, Serway CN et al (2004) Notch is required for long-term memory in Drosophila. Proc Natl Acad Sci U S A 101:1764–1768 57. Lieber T, Kidd S, Struhl G (2011) DSLNotch signaling in the Drosophila brain in response to olfactory stimulation. Neuron 69:468–481 58. Costa RM, Honjo T, Silva AJ (2003) Learning and memory deficits in Notch mutant mice. Curr Biol 13:1348–1354

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prenyltransferase α subunit, regulates notch signaling via Rab1 and Rab11. PLoS Biol 12(1):e1001777 Giagtzoglou N, Yamamoto S, Zitserman D et al (2012) dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions. J Cell Biol 196:65–83 Yamamoto S, Charng WL, Bellen HJ (2010) Endocytosis and intracellular trafficking of Notch and its ligands. Curr Top Dev Biol 92:165–200 Weinmaster G, Fischer JA (2011) Notch ligand ubiquitylation: what is it good for? Dev Cell 21:134–144 Jorissen E, De Strooper B (2010) Gammasecretase and the intramembrane proteolysis of Notch. Curr Top Dev Biol 92:201–230 Huenniger K, Kramer A, Soom M et al (2010) Notch1 signaling is mediated by importins alpha 3, 4, and 7. Cell Mol Life Sci 67:3187–3196 Wharton KA, Yedvobnick B, Finnerty VG et al (1985) opa: a novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster. Cell 40:55–62 Ramain P, Khechumian K, Seugnet L et al (2001) Novel Notch alleles reveal a Deltexdependent pathway repressing neural fate. Curr Biol 11:1729–1738 Fryer CJ, White JB, Jones KA (2004) Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol Cell 16:509–520 Borggrefe T, Liefke R (2012) Fine-tuning of the intracellular canonical Notch signaling pathway. Cell Cycle 11:264–276 Tanigaki K, Honjo T (2010) Two opposing roles of RBP-J in Notch signaling. Curr Top Dev Biol 92:231–252 Furriols M, Bray S (2001) A model Notch response element detects suppressor of hairless-dependent molecular switch. Curr Biol 11:60–64 Nagel AC, Krejci A, Tenin G et al (2005) Hairless-mediated repression of notch target genes requires the combined activity of Groucho and CtBP corepressors. Mol Cell Biol 25:10433–10441 Morel V, Lecourtois M, Massiani O et al (2001) Transcriptional repression by suppressor of hairless involves the binding of a hairless-dCtBP complex in Drosophila. Curr Biol 11:789–792 Kao HY, Ordentlich P, Koyano-Nakagawa N et al (1998) A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev 12:2269–2277

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89. Oswald F, Winkler M, Cao Y et al (2005) RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes. Mol Cell Biol 25:10379–10390 90. Dou S, Zeng X, Cortes P et al (1994) The recombination signal sequence-binding protein RBP-2 N functions as a transcriptional repressor. Mol Cell Biol 14:3310–3319 91. Morel V, Schweisguth F (2000) Repression by suppressor of hairless and activation by Notch are required to define a single row of single-minded expressing cells in the Drosophila embryo. Genes Dev 14:377–388 92. Barolo S, Stone T, Bang AG et al (2002) Default repression and Notch signaling: hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to suppressor of hairless. Genes Dev 16:1964–1976 93. Bray S, Furriols M (2001) Notch pathway: making sense of suppressor of hairless. Curr Biol 11:R217–R221 94. Kurooka H, Honjo T (2000) Functional interaction between the mouse notch1 intracellular region and histone acetyltransferases PCAF and GCN5. J Biol Chem 275:17211–17220 95. Wallberg AE, Pedersen K, Lendahl U et al (2002) p300 and PCAF act cooperatively to mediate transcriptional activation from chromatin templates by notch intracellular domains in vitro. Mol Cell Biol 22:7812–7819 96. Oswald F, Tauber B, Dobner T et al (2001) p300 acts as a transcriptional coactivator for mammalian Notch-1. Mol Cell Biol 21: 7761–7774 97. Fryer CJ, Lamar E, Turbachova I et al (2002) Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes Dev 16:1397–1411 98. Hubbard EJ, Wu G, Kitajewski J et al (1997) sel-10, a negative regulator of lin-12 activity in Caenorhabditis elegans, encodes a member of the CDC4 family of proteins. Genes Dev 11:3182–3193 99. Oberg C, Li J, Pauley A et al (2001) The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian Sel-10 homolog. J Biol Chem 276:35847–35853 100. Wu G, Lyapina S, Das I et al (2001) SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation. Mol Cell Biol 21:7403–7415

101. Gupta-Rossi N, Le Bail O, Gonen H et al (2001) Functional interaction between SEL10, an F-box protein, and the nuclear form of activated Notch1 receptor. J Biol Chem 276:34371–34378 102. del Alamo D, Rouault H, Schweisguth F (2011) Mechanism and significance of cisinhibition in Notch signalling. Curr Biol 21:R40–R47 103. Becam I, Fiuza UM, Arias AM et al (2010) A role of receptor Notch in ligand cis-inhibition in Drosophila. Curr Biol 20:554–560 104. Sprinzak D, Lakhanpal A, Lebon L et al (2011) Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature 465:86–90 105. Yamamoto S, Charng WL, Rana NA et al (2012) A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands. Science 338: 1229–1232 106. Aster JC, Simms WB, Zavala-Ruiz Z et al (1999) The folding and structural integrity of the first LIN-12 module of human Notch1 are calcium-dependent. Biochemistry 38:4736–4742 107. Rand MD, Grimm LM, Artavanis-Tsakonas S et al (2000) Calcium depletion dissociates and activates heterodimeric notch receptors. Mol Cell Biol 20:1825–1835 108. Hori K, Sen A, Kirchhausen T et al (2012) Regulation of ligand-independent Notch signal through intracellular trafficking. Commun Integr Biol 5:374–376 109. Fortini ME, Bilder D (2009) Endocytic regulation of Notch signaling. Curr Opin Genet Dev 19:323–328 110. Gordon WR, Vardar-Ulu D, Histen G et al (2007) Structural basis for autoinhibition of Notch. Nat Struct Mol Biol 14:295–300 111. Gordon WR, Roy M, Vardar-Ulu D et al (2009) Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL. Blood 113:4381–4390 112. Stankiewicz P, Lupski JR (2010) Structural variation in the human genome and its role in disease. Annu Rev Med 61:437–455 113. Lupski JR, Belmont JW, Boerwinkle E et al (2013) Clan genomics and the complex architecture of human disease. Cell 147:32–43

Chapter 2 Genetic Screens to Identify New Notch Pathway Mutants in Drosophila Nikolaos Giagtzoglou Abstract Notch signaling controls a wide range of developmental processes, including proliferation, apoptosis, and cell fate specification during both development and adult tissue homeostasis. The functional versatility of the Notch signaling pathway is tightly linked with the complexity of its regulation in different cellular contexts. To unravel the complexity of Notch signaling, it is important to identify the different components of the Notch signaling pathway. A powerful strategy to accomplish this task is based on genetic screens. Given that the developmental context of signaling is important, these screens should be customized to specific cell populations or tissues. Here, I describe how to perform F1 clonal forward genetic screens in Drosophila to identify novel components of the Notch signaling pathway. These screens combine a classical EMS (ethyl methanesulfonate) chemical mutagenesis protocol along with clonal analysis via FRT-mediated mitotic recombination. These F1 clonal screens allow rapid phenotypic screening within clones of mutant cells induced at specific developmental stages and in tissues of interest, bypassing the pleiotropic effects of isolated mutations. More importantly, since EMS mutations have been notoriously difficult to map to specific genes in the past, I briefly discuss mapping methods that allow rapid identification of the causative mutations. Key words Drosophila, Notch, Forward genetic screen, EMS mutagenesis, Transposable element, RNA interference

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Introduction Drosophila melanogaster is a model organism used to elucidate the molecular and cellular mechanisms of intercellular signaling cascades that coordinate the development and physiology of multicellular organisms [1]. There are several key features that underlie the contributions of Drosophila genetics to biomedical research [1–4]. First, most Drosophila genes and proteins involved in signaling pathways are evolutionarily conserved. Second, the complexity of signaling pathways is often easier to unravel in flies than in vertebrates because of the paucity of functional redundancy conferred by paralogs. Third, the wealth of genetic tools and methods available in Drosophila facilitates experimental design

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_2, © Springer Science+Business Media New York 2014

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and permits high cellular resolution [5], allowing assembly of pathways. Finally, Drosophila permits the application of large scale, unbiased, forward genetic screens either via spatiotemporally targeted protein overexpression, RNA interference (RNAi), or by generating new mutations that impair intercellular signaling in vivo. Genetic screens in Drosophila can be categorized into three main types depending on whether one relies on (1) gain-of-function, (2) interactions with preexisting mutations, or (3) loss-of-function. Each type of screen has its advantages and disadvantages. Gain-of-function screens can lead to the identification of molecular components whose function may have been masked by functional redundancy [6, 7] using the GAL4/UAS binary expression system [8]. In the past, these screens relied on the availability of transposable element insertions carrying UAS enhancer sequences that are inserted in the proper orientation and location to drive expression of neighboring genes. To enhance the coverage, different transposable elements were engineered [9, 10]. More recently, UAStransgenic libraries were created, to allow more efficient misexpression screens [11–13]. Modifier screens allow the identification of genetic interactors in a given signaling pathway, and have been productive in the Notch pathway [14–20]. However, the success of genetic modifier screens is very labor intensive and mapping of the mutations can be difficult, unless collections of preexisting mutations are available and mapped. Loss-of-function screens aim to systematically reduce the function and/or expression of endogenous genes while screening for phenotypes of interest. One approach relies on overexpression of a library of RNAi transgenes that cover the fly genome in a spatiotemporally controlled manner and are expressed in the whole animal or in clones of cells [21]. This strategy was used to identify novel components in the Notch signaling pathway [22, 23]. However, inefficient knockdown of gene and/or off-target effects reduce the accuracy and efficiency of such screens. A similar strategy is to overexpress a set of UAS-micro-RNA transgenes to knockdown their target genes [24–26]. Notch signaling related phenotypes have been used to validate this approach, revealing a complex role of microRNAs in Notch signaling [24]. However, the identification of the miRNA targets remains difficult. Ethyl methanesulfonate (EMS) is the most widely used mutagen in Drosophila because it can be easily administered to flies and causes a high frequency of mutations. Different parameters of EMS mutagenesis have been extensively covered previously [27–29]. In summary, EMS induces mainly point mutations, the vast majority of which are transitions from pyrimidines (G/C) to purines (A/T) and vice versa. However, small deletions, frameshifts, and transversions can also be recovered at low frequency, depending on the conditions of the screen, such as age of flies and dosage of EMS.

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EMS mutagenesis is unbiased, covers the whole genome, and is the most efficient method to induce mutations. In standard EMS mutagenesis protocols, the dosage of EMS is 25 mM. This typically induces an average of 1 mutation per 1,000 genes [27, 28]. Importantly, the effective dosage of EMS also varies with respect to the size of the genes, the feeding conditions, the temperature, etc. Hence, one or more pilot runs to define the optimal EMS concentration is advised. A powerful technique in Drosophila genetics is the analysis of loss-of-function mutations in mitotic clones, based on the FLP/ FRT system, initially adapted from yeast [30, 31]. When FLP recombinase is expressed under the control of an inducible or tissue specific promoter, it can drive recombination between FRT (FLP Recombination Target) sites (Fig. 1). When FRT sites are located at the same chromosomal position of homologous chromosomes, chromatid exchange can be induced quite efficiently. Upon chromatid segregation during mitosis, some of the progeny cells are homozygous for the induced mutation, while most cells

A: wild type allele, a: recessive loss of function allele (A>a) Mitotic Recombination FRT42D

FRT42D

A A a a

A a A a

A a Heterozygous

Recombinant

Parental

Resolution of chromatids A a

Heterozygous

A A

Homozygous wild type

a A

Heterozygous

a a

Homozygous mutant

Fig. 1 Schematic diagram indicating FLP/FRT-mediated recombination gives rise to homozygous mutant clones, otherwise not possible to obtain if the mutation is in an essential gene. In the absence of recombination, chromatids from homologous chromosomes bearing different alleles of a given gene segregate to progeny cells. If “A” is the wild-type allele and “a” is the loss-of-function mutant recessive allele, then the progeny cells are all heterozygous without any phenotype. However, upon mitotic recombination, non-sister chromatids from homologous chromosomes exchange material. After their segregation into progeny cells, homozygous mutant and homozygous wild-type cells arise along with heterozygous ones. The phenotype of the homozygous mutant cells can be examined in comparison to surrounding wild-type tissue. Visible markers such as w (red or white eyes) and yellow (brown or yellow bristles) can be used to distinguish between the homozygous mutant cells and wild-type cells

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are heterozygous. Hence, the loss-of-function phenotype of a mutation can be examined only in subset of cells, bypassing potentially detrimental effects in other tissues or earlier developmental stages. In addition, there are ways to enhance the size of the mutant clones by favoring the growth of mutant tissue over the wild-type neighboring cells [2, 32, 33]. The aforementioned advantages of clonal analysis, namely the efficiency and speed of phenotypic analysis, in combination with the efficacy of EMS mutagenesis have created the unique opportunity to screen rapidly and reliably for novel components of Notch signaling pathway [34–46]. Evidently, the phenotypes of mutants for Notch signaling may range from pattern formation, cell fate acquisition and differentiation to proliferation and apoptosis [47]. For such an endeavor, a collection of males bearing FRT chromosomes is mutagenized and subsequently crossed to females carrying the same FRT chromosome. These females also express FLP under the control of tissue specific promoters, like the regulatory elements of Ultrabithorax (Ubx), which is active in most imaginal discs [36]. Thus, FLP-mediated recombination between FRT chromosomes can create clones of homozygous cells for a randomly induced mutation in the thorax, wings, and eyes. The effect of the mutations can be scored in the F1 generation by visually inspecting the number and morphology of the mechanosensory bristles in these areas, where they develop under the control of Notch signaling pathway [48]. One of the main disadvantages of EMS mutagenesis is the mosaicism in the progeny of mutagenized males, which may negatively affect the yield of recovery of mutations, especially when one performs F1 clonal screens. EMS mutagenizes the post-meiotic sperm DNA that can be repaired in the embryo, upon the first cleavages of division [27, 28]. Inevitably, F1 progeny are therefore often mosaic, i.e., not all somatic cells and, most importantly, not all germ cells carry the induced mutation. Therefore, even if F1 progeny have the desirable phenotype, these animals may not transmit the mutation. Consequently, mosaicism imposes a constraint on the yield of the recovery of new mutations. To avoid the consequences of mosaicism, one needs to expand the chromosome bearing the mutations via backcross and confirm the transmission of the visible phenotype into the subsequent generations.

2

Materials

2.1 Drosophila Stocks

The list below is specific for an example of screening the right arm of the second chromosome, using FRT42D chromosomes. 1. Isogenized y w; FRT42D. or, FRT42D iso—where iso: isogenic, see Note 1. Stock for males for mutagenesis.

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2. y w UbxFLP;FRT42D y+ for collecting FRT bearing virgins expressing FLP in imaginal discs under the promoter of Ubx. The FRT chromosome also carries the body color marker yellow (y+). 3. y w UbxFLP;FTR42D l(2R)cl y+w+/CyO for collecting FRT bearing virgins expressing FLP in imaginal discs and favoring the growth of mutant tissue over neighboring cells, which are dying because of homozygosity of a cell lethal (cl) mutation. This chromosome also contains the body color marker yellow (y+) and the eye color marker white (w+). 4. w;nocSco/CyO: Stock for balancing and amplification of mutations during backcrossing. The above and/or similar stocks can be ordered from Bloomington Drosophila Stock Center (BDSC, http://flystocks. bio.indiana.edu). 2.2

Equipment

1. Basic equipment to perform Drosophila work (e.g., stereomicroscope, light source, CO2 anesthetizer, fly food media, etc.). 2. 18 and 25 °C incubators for Drosophila culture You will be performing the EMS mutagenesis in the fume hood. Keep your supplies at the ready. 3. Empty fly stock culture bottles (e.g., Applied Scientific AS-359) and closures. An old-fashioned glass half-pint milk bottle is ideal. 4. Whatman No. 1 filter paper. 5. One 1 l beaker—for denaturing solution. 6. One 250 ml Erlenmeyer flask—for EMS-sucrose solution. 7. Pipets 5 and/or 10 ml. 8. 10 ml and 1 ml syringes. 9. Needles (18 G). 10. A container for EMS waste. 11. Handy extra gloves, Kimwipes, diapers, and paper towels.

2.3

Chemicals

1. Ethyl methanesulfonate (EMS) (Sigma #M0880). 2. Sucrose. 3. NaOH. 4. Thioglycolic acid (Sigma #T6750). 5. Inactivating-Denaturing solution: Dissolve 20 g of NaOH in 500 ml of ddH2O in the 1 l beaker. Add 2.5 ml thioglycolic acid. Thioglycolic acid should be kept at −20 °C to protect from oxidization. The solution should be prepared and used in the fume hood as it can decompose towards hydrogen sulfide.

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Methods The following mutagenesis protocol applies to screening of 1,000 genomes after mutagenizing 100 isogenized males (10 genomes per 1 starter mutagenized male, see Note 1). The number of males to be mutagenized can be scaled accordingly. To identify 1–2 novel complementation groups/genes from an F1 clonal screen for visible phenotypes of loss-of-function of Notch signaling, approximately 60,000 flies need to be screened, based on previous experience. In case that larger numbers of genomes need to be screened, it would be better if one follows a “rolling” scheme of mating, which tackles the labor intensive task of genetic screening. Thus, small batches of males (e.g., 100 males) are mutagenized weekly and outcrossed to virgin females of appropriate genotypes, while at the same time previous batches are phenotypically scored and balanced.

3.1 EMS Mutagenesis Protocol

1. Days 1–5: Collect newly eclosed males from the isogenic strain to be mutagenized. Age them for 3–5 days (see Note 2). In parallel, collect approximately three female virgins for every mutagenized male to be crossed. Maintain all flies in vials with yeast and food at 18 °C. 2. Day 6: Place two pieces of Whatman No. 1 filter paper cut to fit on the bottom of clean empty stock bottles. Lightly moisten with approximately 300 μl of water. Place 100 3–5 day old male flies to be mutagenized into each bottle. Incubate overnight at 22–25 °C to starve the males without dehydrating them. Starved flies will efficiently uptake EMS through feeding the next day. 3. Day 7: Prepare the chemical hood for EMS mutagenesis according to safety instructions described in Note 3. Briefly, do not forget to use appropriate protective clothing and double layers of gloves. There must be chemical waste disposal beakers with inactivating solution previously prepared (see Note 3), where all equipment that has been exposed to EMS, such as syringes, fly bottles, and Whatman filter paper, may be discarded and decontaminated. Make sure you decontaminate any spills using Kimwipes previously dipped into inactivating solution. Handle EMS with care in a fume hood, as it evaporates readily. 4. Prepare new clean stock bottles with two pieces of Whatman No. 1 filter paper cut to fit on the bottom. 5. Prepare 100 ml of 1 % sucrose solution in ddH2O in an Erlenmeyer flask. 6. Use a disposable 1 ml syringe with 18 G needle to measure and dispense the EMS into the sucrose solution. Add 0.26 ml

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EMS per 100 ml of sucrose solution to achieve a 25 mM final concentration of EMS. Dosage concentration can vary according to pilot tests (see Note 4). 7. Fill the used syringe with inactivating solution and place it in the beaker containing inactivating solution. 8. EMS is oily and forms droplets that sink in the sucrose solution. Disperse the EMS bubbles by repeated cycles of uptake and release through a 10 ml syringe with 18 G needle, applying moderate pressure and taking care not to expel the air in the syringe, so that the solution will not splash. Be sure to keep the needle below the surface of the sucrose solution. 9. Using the same 10 ml syringe, gently uptake and dispense 1.1 ml of EMS solution onto the center of the filter papers at the bottom of each empty stock bottle. Ensure that the EMS solution is applied evenly so that no puddles are formed. Once all bottles have been treated, discard used syringe by filling with inactivating solution then placing within the beaker. 10. Pour an equal volume of inactivating solution into the remaining EMS-sucrose solution. Keep equipment that have been exposed to EMS overnight (~15 h) in the inactivating solution and discard the waste according to the regulations of your facility, after which the glassware is safe to be washed and re-used. 11. Transfer starved males into the EMS treated bottles and leave in the fume hood during the treatment. Males are usually exposed to EMS for an approximate period of 10–12 h. However, one might expose the male flies for 6–15 h. The optimal duration of exposure must be determined empirically in a pilot screen. 12. Day 8: Prepare another 500 ml of inactivating solution. Using a funnel, transfer males onto fresh media to allow them to clean from excess EMS. Pour inactivating solution into the stock bottles used for mutagenesis (leaving the filter paper inside) and leave in the fume hood overnight. Discard appropriately the next day. Let males recover for several hours. 13. During the recovery period, distribute virgin females of the desired genotype into vials with fly food and extra yeast for performing the first cross (F0) (Fig. 2). 14. Anesthetize males. Cross each male with three females in a series of single crosses. Incubate crosses in 25 °C incubator. 15. Day 12: After 4 days remove all males from each cross to prevent the appearance of clusters of identical mutants derived from mutagenized germline stem cells (see Note 5). Transfer the females into fresh food vials with yeast (optional).

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Fig. 2 Schematic diagram of crosses designed in the context of an F1 clonal forward genetic screen performed on the right arm of the second chromosome. In the first generation (F0) crosses are set between mutagenized males and appropriate females. In the next generation (F1), progeny that are scored positive phenotypically are backcrossed for testing for genetic transmission of the mutations and for confirming the phenotypes. F2 progeny are then balanced. To establish a stock, F3 siblings are crossed. All crosses are performed with single males. Asterisk represents the EMS-induced mutation. cl = l(2) cl (cell lethal mutation)

16. Day 18: At a minimum of 10 days after setting the crosses, start collecting F1 males for phenotypic scoring. Isolate flies of interest and backcross them to ensure genetic transmission as well as amplification of the mutation into a stock (F1) (Fig. 2). 17. Day 28: At a minimum of 10 days after setting the F1 crosses, start collecting F2 males for confirmation of genetic transmission of the pre-observed phenotype. Isolate the flies with the genotype of interest and backcross them to balancer stocks (F2) for amplification of the chromosome and generation of a stock after crossing sibling progeny of the next generation (F3) (Fig. 2). 3.2 Formation of Complementation Groups and Mapping of EMS Mutations

After screening, the retrieved EMS alleles must be placed in complementation groups, which most likely correspond to single genes. Overall, the average number of alleles in each complementation group can be used to calculate the saturation of the genetic screen, which corresponds to what degree the specific region of the genome has been probed to retrieve all relevant EMS-induced mutations. The calculation is based on Poisson distribution and is equal to S = 1 − P(0) = 1 − (m* (e−m)/0!), where S stands for the degree of saturation, P(0) is probability of no-hits, i.e., the probability of the genes that might have not been mutated, m is the average number of mutations per gene (average number of alleles per complementation group), e is the natural logarithm, and 0! is the factorial of 0, which is equal to 1. Calculations of saturation

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of a genetic screen are only an estimate, since they can be confounded by multiple factors. For example, the rare cases of extragenic (nonallelic) non-complementation may increase the number of alleles in a given complementation group, while intragenic complementation may also undermine the formation of potential complementation groups, splitting them into single allele groups. However, both cases of extragenic non-complementation and intragenic complementation can be particularly informative with respect to pathway structure and protein function respectively. Extragenic non-complementation may reveal novel protein interactors of a given gene, which when mutated together lead to the manifestation of a visible phenotype. Intragenic complementation may become apparent in multi-allelic complementation groups that correspond to large genes with a complex domain structure, such as Notch. In such cases, they may prove to be extremely informative with regard to the interplay among different protein domains [49]. Complementation groups are formed based on a number of genetic schemes which may rely on different characteristics of the alleles. In an F1 clonal screen for Notch pathway mutants, it is expected that the isolated mutations may function pleiotropically in early developmental stages, given the requirement for the Notch signaling pathway in multiple aspects of the development of the nervous system and other organ systems. Thus, the gene of interest is likely to be essential genes, and majority of the alleles may be homozygous lethal, unless they are weak hypomorphs. Consequently, the complementation groups may be formed on the basis of lethality and subsequently, checked individually for the phenotype of interest or even for additional phenotypes in other aspects of Notch signaling. Such analysis can lead to the conclusion whether a given gene affects all or only a subset of Notch signalingmediated developmental decisions, i.e. whether the affected genes are obligatory or context dependent members of Notch signaling. On that note, one can perform a secondary screen using not visible markers, but in vivo reporters of Notch signaling activity, as described by Housden et al. in Chapter 8. The most obvious, but also the most tedious, method for establishing complementation groups is to set up crosses among all possible combinations of all isolated alleles in the screen [50]. Alternatively, one can form an initial complementation map based on the non-complementation of different alleles with a set of molecularly defined set of deficiencies that cover the entire fly genome (as performed in [51]; these tools are readily available from the BDSC, http://flystocks.bio.indiana.edu, [52–55]). A variation on this method is to rescue the alleles by a set of molecularly defined chromosomal duplications [56]. Alleles that fail to complement the same deficiency or rescued by the same chromosomal duplication can be further grouped

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with respect to the complementation profile amongst themselves as well as against molecularly defined deficiencies in the region of interest [57–59]. Another, more important aspect of EMS screens is the difficulty of mapping of EMS-induced mutations to causative genes. However, recent technical advances have increased the resolution and accuracy of complementation tests and molecular mapping by means of P element based meiotic mapping [60]. P element meiotic mapping is best performed when two alleles of the same complementation group are used, to avoid genetic background effects that will lead to perturbations in the generated genetic map. P element mapping is fast and quite accurate, substituting alternative methods based on SNP maps [61–65] and complementing the power and resolution provided by the engineering and utilization of molecularly defined deletions and duplications [52–54, 56, 66]. Nevertheless, whole genome sequencing has facilitated the identification of molecular lesions in an increasingly effective and affordable fashion [67–69]. Since EMS can cause multiple mutations in the genome in an unbiased fashion, it is advisable to work with multi-allelic complementation groups, consisting of at least two alleles, which provide the opportunity to work with heteroallelic combinations precluding the possibility of analyzing unrelated genes. Accordingly, it is of crucial importance to perform rescue experiments by expressing a wild-type copy of the mutated gene (either as cDNA or in the context of a genomic rescue construct) in the mutant genetic background. Such rescue experiments will confirm that the observed phenotypes are indeed due to the loss-of-function of the gene where the mutations of interest map.

4

Notes 1. It is important to isogenize the parental chromosome(s), and therefore the genetic background where the new mutations will be induced, prior to any type of mutagenesis and forward genetic screening experiment. Isogenization ensures that existing polymorphisms or mutations do not interfere with the recovery of newly induced mutations. Isogenization is carried out by the use of balancer chromosomes, which suppress recombination between homologous chromosomes. Multiple isogenized stocks must be established from single balanced males, so that they can be subsequently tested for viability, fertility, and especially the lack of particular phenotypes. Proceed with the healthiest stock. In addition, it is recommended to preserve flies or genomic DNA from the isogenized line immediately after isogenization if one considers gene identification through whole genome sequencing.

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2. Forward genetic screens are usually designed so that males are treated with EMS and subsequently crossed to female virgins of the appropriate phenotype. To compensate for loss in fertility and viability of males upon EMS exposure, more males should be treated with EMS. Furthermore, males should be approximately 3–5 days old. 3. EMS is mutagenic, carcinogenic, and teratogenic. Furthermore, EMS evaporates readily, and has a half-life of more than 2 days in water at 25 °C. Contamination by EMS cannot be measured. Thus, EMS should be handled with extreme caution. Any experiments with EMS must be conducted in a closed chemical hood wearing a lab coat, safety glasses, a mask, and double layers of gloves, to avoid accidental tearing. The chemical hood should be lined with a diaper to absorb EMS spills. The highest risk of contamination occurs mainly when the EMS solution is prepared and distributed. In the case of a contaminating spill, one should wipe the spot with Kimwipes, previously dipped in inactivating solution, available in a beaker in the hood. It may seem counterintuitive in terms of accuracy to use a syringe and needle rather than a pipettor for measuring, but you might contaminate your pipettor whereas the syringe is disposable. 4. To avoid inducing multiple lethal hits per chromosome, which may confound subsequent steps of analysis such as for a screen on the X chromosome, a 10 mM EMS concentration should be used [39, 57, 58]. 5. Progeny originating from sperm that was in pre-meiotic stages during EMS treatment may lead to the isolation of identical mutations (clonal events). Since spermatogenesis occurs over a period of 5 days at 25 °C, parents must be discarded on the fourth day of treatment. References 1. Bellen HJ, Tong C, Tsuda H (2010) 100 years of Drosophila research and its impact on vertebrate neuroscience: a history lesson for the future. Nat Rev Neurosci 11:514–522 2. St Johnston D (2002) The art and design of genetic screens: Drosophila melanogaster. Nat Rev Genet 3:176–188 3. Adams MD, Sekelsky JJ (2002) From sequence to phenotype: reverse genetics in Drosophila melanogaster. Nat Rev Genet 3:189–198 4. Bier E (2005) Drosophila, the golden bug, emerges as a tool for human genetics. Nat Rev Genet 6:9–23 5. Venken KJ, Simpson JH, Bellen HJ (2011) Genetic manipulation of genes and cells in the

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Screening Drosophila for Notch Pathway Genes 37. Rajan A, Tien AC, Haueter CM et al (2009) The Arp2/3 complex and WASp are required for apical trafficking of Delta into microvilli during cell fate specification of sensory organ precursors. Nat Cell Biol 11:815–824 38. Tien AC, Rajan A, Schulze KL et al (2008) Ero1L, a thiol oxidase, is required for Notch signaling through cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster. J Cell Biol 182:1113–1125 39. Yamamoto S, Charng WL, Rana NA et al (2012) A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands. Science 338:1229–1232 40. Charng WL, Yamamoto S, Jaiswal M et al (2013) Drosophila Tempura, a novel protein prenyltransferase α subunit, regulates Notch signaling via Rab1 and Rab11. PLoS Biol 12(1):e1001777 41. Berdnik D, Török T, González-Gaitán M et al (2002) The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila. Dev Cell 3:221–231 42. Herz HM, Chen Z, Scherr H et al (2006) vps25 mosaics display non-autonomous cell survival and overgrowth, and autonomous apoptosis. Development 133:1871–1880 43. Hutterer A, Knoblich JA (2005) Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs. EMBO Rep 6:836–842 44. Vaccari T, Bilder D (2005) The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating notch trafficking. Dev Cell 9:687–698 45. Yan Y, Denef N, Schupbach T (2009) The vacuolar proton pump, V-ATPase, is required for notch signaling and endosomal trafficking in Drosophila. Dev Cell 17:387–402 46. Gallagher CM, Knoblich JA (2006) The conserved c2 domain protein lethal (2) giant discs regulates protein trafficking in Drosophila. Dev Cell 11:641–653 47. Yamamoto S, Charng WL, Bellen HJ (2010) Endocytosis and intracellular trafficking of Notch and its ligands. Curr Top Dev Biol 92:165–200 48. Kandachar V, Roegiers F (2012) Endocytosis and control of Notch signaling. Curr Opin Cell Biol 24:534–540 49. Brennan K, Tateson R, Lewis K et al (1997) A functional analysis of Notch mutations in Drosophila. Genetics 147:177–188 50. Andrews HK, Giagtzoglou N, Yamamoto S et al (2009) Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila. EMBO Rep 10:636–641

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51. Hiesinger PR, Fayyazuddin A, Mehta SQ et al (2005) The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila. Cell 121:607–620 52. Cook KR, Parks AL, Jacobus LM et al (2010) New research resources at the Bloomington Drosophila Stock Center. Fly (Austin) 4: 88–91 53. Cook RK, Deal ME, Deal JA et al (2010) A new resource for characterizing X-linked genes in Drosophila melanogaster: systematic coverage and subdivision of the X chromosome with nested, Y-linked duplications. Genetics 186:1095–1109 54. Parks AL, Cook KR, Belvin M et al (2004) Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat Genet 36:288–292 55. Ryder E, Blows F, Ashburner M et al (2004) The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster. Genetics 167:797–813 56. Venken KJ, Popodi E, Holtzman SL et al (2010) A molecularly defined duplication set for the X chromosome of Drosophila melanogaster. Genetics 186:1111–1125 57. Xiong B, Bayat V, Jaiswal M et al (2012) Crag is a GEF for Rab11 required for rhodopsin trafficking and maintenance of adult photoreceptor cells. PLoS Biol 10:e1001438 58. Zhang K, Li Z, Jaiswal M et al (2013) The C8ORF38 homologue Sicily is a cytosolic chaperone for a mitochondrial complex I subunit. J Cell Biol 200:807–820 59. Yamamoto S, Bayat V, Bellen HJ et al (2013) Protein phosphatase 1β limits ring canal constriction during Drosophila germline cyst formation. PLoS One 8:e70502 60. Zhai RG, Hiesinger PR, Koh TW et al (2003) Mapping Drosophila mutations with molecularly defined P element insertions. Proc Natl Acad Sci U S A 100:10860–10865 61. Berger J, Suzuki T, Senti KA et al (2001) Genetic mapping with SNP markers in Drosophila. Nat Genet 29:475–481 62. Hoskins RA, Phan AC, Naeemuddin M et al (2001) Single nucleotide polymorphism markers for genetic mapping in Drosophila melanogaster. Genome Res 11:1100–1113 63. Schnorrer F, Ahlford A, Chen D et al (2008) Positional cloning by fast-track SNP-mapping in Drosophila melanogaster. Nat Protoc 3:1751–1765 64. Martin SG, Dobi KC, St Johnston D (2001) A rapid method to map mutations in Drosophila. Genome Biol 2(9): RESEARCH0036

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in Drosophila melanogaster by whole-genome sequencing. Genetics 182:25–32 68. Hobert O (2010) The impact of whole genome sequencing on model system genetics: get ready for the ride. Genetics 184:317–319 69. Wang H, Chattopadhyay A, Li Z et al (2010) Rapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing. Genome Res 20:981–988

Chapter 3 Structure-Function Analysis of Drosophila Notch Using Genomic Rescue Transgenes Jessica Leonardi and Hamed Jafar-Nejad Abstract One of the evolutionarily conserved posttranslational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a specific consensus sequence by the protein O-glucosyltransferase Rumi (POGLUT1 in human). Loss of rumi in flies results in a temperature-sensitive loss of Notch signaling. To demonstrate that the Notch receptor itself is the biologically relevant target of Rumi in flies, and to determine the role of the 18 Rumi target sites on Notch in regulating Notch signaling, we have performed an in vivo structure-function analysis of Drosophila Notch. In this chapter, we provide a detailed protocol for this analysis. To avoid the potential artifacts associated with overexpression of Notch and random insertion of transgenes, we have used recombineering and site-specific integration technologies, which have been adapted for usage in Drosophila in recent years. Using gene synthesis and site-directed mutagenesis, we generated a series of Notch genomic transgenes which harbor mutations in all or specific subsets of Notch O-glucose sites. Gene dosage and rescue experiments in animals raised at various temperatures allowed us to dissect the contribution of O-glucosylation sites to the regulation of the Notch signaling strength. The reagents and methods presented here can be used to address similar questions about other posttranslational modifications of Notch or other Drosophila proteins. Key words Notch, Drosophila, Recombineering, Site-specific integration, Glycosylation, Genomic transgene

1

Introduction Notch receptors undergo a number of evolutionarily conserved posttranslational modifications which serve to regulate Notch pathway activity and the strength of signaling [1, 2]. Genetic experiments using loss- and gain-of-function mutations in Notch modifiers have provided a wealth of knowledge on the role of the corresponding modifications in Notch pathway regulation. However, to demonstrate that the Notch receptor itself is the biologically relevant target of a specific modification and to determine the bona fide sites involved in signaling, it is preferable—and often imperative—to ablate or alter these sites in a given Notch

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_3, © Springer Science+Business Media New York 2014

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receptor in vivo. In this way, one can assess the effects of loss (or gain) of the corresponding modification on the activity of the Notch pathway in a living organism. Drosophila melanogaster has long been used as a model organism of choice for such studies on Notch and many other proteins. Drosophila has a single ortholog of Notch whereas four Notch receptors exist in mouse and human (Notch 1–4), and this simplifies in vivo analysis of pathway activity. Moreover, identification of various morphological and molecular readouts for Notch in vivo activity provides the means to perform a detailed comparison between wild-type and mutant versions in flies [3]. Last but certainly not the least, establishment of technologies for efficient generation of transgenic flies and tissue-specific over- or misexpression of a cDNA of interest has provided a very powerful toolkit for in vivo structure-function analysis [4–6]. Although the abovementioned strategies have provided insight into the role of certain posttranslational modifications in flies [7], several limitations exist which have restricted their usage. Notch is a dosage-sensitive gene in Drosophila: both loss and gain of one genomic copy of Notch result in morphological abnormalities [8, 9]. Therefore, studies based on overexpression can generate misleading results, especially if the modification under study has a modulating role as opposed to an all-or-none role in Notch signaling. The obvious remedy to this problem is to perform the in vivo structure-function analysis in the context of a genomic transgene, in which the expression of Notch is driven by endogenous enhancers and promoters. Indeed, more than 20 years ago, cosmid transgenesis was used to show that a ~40 kb long genomic fragment contains the sequences necessary for the expression and function of Notch, as it was able to fully rescue Notch loss-of-function phenotypes [10]. However, to our knowledge, this transgene was not used in structure-function studies of Notch in future studies. We can think of at least three reasons: (1) manipulation of a construct of this size (~40 kb plus vector) to introduce point mutations or generate small deletions is quite difficult by using standard molecular biology techniques; (2) the efficiency of obtaining transgenic animals significantly decreases for constructs larger than 20 kb, and dramatically so for constructs larger than 40 kb (which is the case for Notch genomic transgenes); (3) even if both of the previous issues were solved, the random nature of transgene insertion using traditional P element transgenesis requires that multiple independent insertions be evaluated and compared for the wildtype and each mutant transgenes. If multiple sites of the posttranslational modification under study exist on the Notch receptor, a prohibitively large amount of work will be necessary to generate independent insertions for each mutant and compare them to wild-type Notch. In the last 10 years, significant advances have been made in technologies used for the generation of large Drosophila transgenes and for the integration of transgenes into specific sites in the

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genome [11–13]. Specifically, the recombineering technology, which has been used to manipulate large constructs for functional genomic studies in mice [14], and ΦC31 site-specific genomic integration, which was previously used in other systems [15], were adapted for use in Drosophila and combined with each other to enable the researchers to readily generate transgenic Drosophila harboring large (>70 kb) transgenes integrated in specific genomic sites [ 11 , 16 ]. We took advantage of these technologies and performed a systematic analysis of the role of a specific posttranslational modification, namely “protein O-glucosylation” [17–19], in the regulation of Drosophila Notch signaling [20, 21]. Protein O-glucosylation is the addition of an O-linked glucose residue to a serine (S) residue on epidermal growth factor-like (EGF) repeats harboring a C1XSX(P/A)C2 consensus sequence, where C1 and C2 are the first two cysteine residues of the EGF repeat, and S is the modified serine [22, 23]. The O-glucose residue can be extended by the addition of one or two xylose residues to form the longer forms of this type of glycosylation, i.e., O-linked xylose–glucose disaccharides and xylose–xylose–glucose trisaccharides [24–26]. A number of years ago, the enzyme responsible for protein O-glucosylation, Rumi, was discovered in a genetic screen in Drosophila [19]. Both protein-null alleles and a missense mutation which abolishes the enzymatic activity of Rumi but does not affect its protein levels show a temperature-sensitive loss of Notch signaling [19], strongly suggesting that addition of O-glucose by Rumi to one or more of its target proteins is required for Notch signaling. A number of genetic experiments suggested that the Notch receptor itself is the biologically relevant target of Rumi in the context of Notch signaling [19], in agreement with the observation that the Notch receptors in flies and other species have by far the largest number of Rumi target sites among animal proteins (Fig. 1) [18, 19]. To demonstrate that this is indeed the case, and more importantly, to determine the relative contribution of the 18 Rumi target sites found in Drosophila Notch in tuning the signaling, we generated a platform for in vivo structure-function analysis of Notch using recombineering and site-specific integration [11], combined with additional steps that allowed us to generate 11 mutant Notch genomic transgenes harboring serine-to-alanine mutations in all 18 Rumi target sites in Notch, or in various subsets of these sites [20]. Genetic rescue experiments with these transgenes showed that while all Rumi target sites contribute to Notch signaling, those at EGF10–15 promote Notch signaling more than others [20]. More recently, analysis of the enzyme responsible for the addition of xylose to O-glucose residues on Notch has indicated that although O-glucose promotes Notch signaling, extending the O-glucose by adding xylose residues negatively regulates Notch signaling [21]. Again, our in vivo mutational analysis allowed us to

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Fig. 1 Structure of the Notch extracellular domain and the Notch genomic locus. (a) Schematic of the Notch extracellular domain (NotchECD) with 36 EGF repeats, three LNR domains, heterodimerization (HD), and transmembrane (TM) regions. EGF repeats with an O-glucosylation motif are marked blue. Corresponding exons encoding the NotchECD are labeled with Roman numbers on the horizontal line below the schematic. (b) The Notch genomic region (introns in white, noncoding regions of the exons in gray, and coding region of the exons in black) and the 40 kb Notch genomic transgene (N gt-wt ) (reproduced from ref. 20)

identify the subset of Notch EGF repeats that mediate the negative function of xylose in vivo [21]. Here, we provide a protocol for the generation of the wild-type Notch genomic construct, for introducing mutations in this construct, for site-specific integration of all constructs in a specific docking site in the Drosophila genome, and for the genetic analysis of the function of the wild-type and mutant Notch transgenes. Although our studies focused on a specific modification of Notch, the reagents and techniques discussed here can be used to ask similar questions about other posttranslational modifications of Notch or other Drosophila proteins.

2

Materials

2.1 Molecular Biology

1. Bacterial strains: SW102 [27], EPI300 (Epicentre, Inc.). 2. attB-P[acman]-ApR [11] (Drosophila Genomics Resource Center, Indiana University; stock #1245). 3. BAC RP98-1A14 (BACPAC Resources Center, Children’s Hospital Oakland Research Institute) [28]. 4. Copy Control solution (Epicentre, Inc.). 5. pCR-Blunt II-TOPO-KmR (Invitrogen). 6. CAT/SacB-pCR-Blunt II-TOPO-KmR. 7. 30 °C and 37 °C incubators. 8. 30 °C and 42 °C water baths. 9. LB medium and plates with ampicillin (100 μg/ml), chloramphenicol (12.5 μg/ml), or kanamycin (30 μg/ml).

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10. Electroporator and cuvettes. 11. Additional reagents and equipment for PCR, agarose gel electrophoresis of DNA, DNA purification from bacteria and from agarose gels, restriction digestion, and ligation. 2.2 Drosophila Genetics

Detailed information about various options available for fly food and supplies used for maintaining fly cultures is available at the Bloomington Drosophila Stock Center’s website: http://flystocks. bio.indiana.edu/Fly_Work/media-recipes/media-recipes.htm. The following Drosophila strains were used in this chapter: 1. y w (Bloomington Drosophila Stock Center, Indiana University; BDSC stock #6598). 2. N55e11/FM7c, Kr-GAL4 UAS-GFP sn+ [20]. 3. Df(1)N-54l9, y1/C(1)DX, y1 w1 f1; Dp(1;2)51b/+ (BDSC stock #6894). 4. y w; PBac{attP}VK22 (BDSC stock #9740) [11]. 5. y M{vas-int.Dm}ZH-2A w; PBac{attP}VK22 (BDSC stock #24868). 6. y w; PBac{Ngt-wt}VK22 [20]. 7. y w; PBac{Ngt-mut}VK22 [20] (mut indicates any of the lines harboring one or more serine-to-alanine mutations in the Rumi target sites of Notch).

3

Methods

3.1 Generation of the Wild-Type Notch Genomic Construct

In this section we will describe the protocol used to clone the wildtype Notch locus into the conditionally amplifiable attB-P[acman]ApR vector [11]. This vector can be used to integrate a DNA fragment of interest into the fly genome by ΦC31-mediated transgenesis or traditional P-element-mediated transgenesis, and contains the following elements: ampicillin resistance gene (ApR), conditionally amplifiable origin of replication (oriV), white (w+) minigene marker, multiple cloning site (MCS), attB integration site, and 5′ and 3′ P element transposase recognition sites (Fig. 2a). 1. Prepare the targeting construct (Fig. 2b), which contains 500 bp left and right homology arms (LA and RA, respectively) corresponding to the 5′ and 3′ ends of the Notch genomic locus to be cloned in the attB-P[acman]-ApR vector. Generate the LA flanked by restriction sites (AscI and BamHI) and the RA flanked by restriction sites (BamHI and PacI) by PCR using the following primers (restriction sites underlined): LA-F AGGCGCGCCGGATATACACACACATCTTTCTA. LA-R GTGGGATCCTGATATACGATTTTTTACTCGATA AGTA.

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Fig. 2 Generation of the Notch genomic transgenes and gap-repair mutagenesis. (a–d) Generation of the wildtype Notch genomic transgene. (a) Schematic of the attB-P[acman]-Ap R vector. (b) Cloning of the left and right homology arms (LA, RA respectively) into the attB-P[acman]-Ap R vector. (c) Recombination between the linearized targeting vector and the RP98-1A14 BAC in recombineering-induced SW102 cells. The Notch locus is not drawn to scale. (d) ΦC31-mediated integration of the Notch-attB-P[acman]-Ap R construct into the VK22 docking site in the fly genome to generate the wild-type Notch genomic transgene (N gt-wt ). (e–h) Gap-repair mutagenesis to generate mutant Notch genomic transgenes. (e, f) Replacement of the region harboring the EGF repeats which will be mutagenized by a CAT/SacB (CS) cassette. (g) Recombination between the linearized N EGF/CS-attB-P[acman]-ApR and the LA-EGF-RA-pCR-Blunt II-TOPO-KmR construct in SW102 cells to obtain the Notch genomic transgene with mutations in EGF repeats of interest. (H) ΦC31-mediated integration of the NEGF-attB-P[acman]-Ap R construct into the VK22 docking site in the fly genome to generate the mutant Notch genomic transgene (N gt-mut ). MCS multiple cloning site, CAT Chloramphenicol acetyl transferase (reproduced from ref. 20)

RA-F CGC GGATCC AGACAGTAACCAGCCAAGTTT ACTA. RA-R TGCTTAATTAAGGGTTTGTGTGTGTGTGTGTC AAGAGT. Subclone the LA and RA into the MCS of attB-P[acman]-ApR vector by triple ligation. The BamHI site will be used for linearization in step 3 (see Note 1).

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2. Transform the Bacterial Artificial Chromosome (BAC) RP98-1A14 carrying the 40 kb Notch genomic locus (2.5 kb putative promoter, 37.4 kb genomic sequence which includes all nine exons and introns, and 0.2 kb 3′-UTR) into electrocompetent SW102 E. coli cells [27]. Amplify SW102 cells carrying the RP98-1A14 BAC in LB chloramphenicol overnight at 30 °C to avoid premature induction of recombination competency (see Note 2). Dilute the overnight culture 1:50 in fresh LB chloramphenicol and incubate at 30 °C for 3–4 h to reach an OD600 of 0.4–0.6. Incubate the culture at 42 °C for 15 min in a water bath to induce recombination. Prepare electrocompetent cells by several washes in ice-cold 10 % glycerol. As negative control, prepare uninduced SW102/RP98-1A14 competent cells in parallel by keeping the cells at 30 °C at all times. Refer to the supplementary material of ref. 11 for a detailed protocol on this procedure. 3. Linearize the targeting vector by digestion with BamHI. Purify the linearized targeting vector and transform into recombination-induced SW102 competent cells carrying the RP98-1A14 BAC prepared in step 2 (see Note 3). Plate the transformed SW102 cells on LB ampicillin and incubate at 30 °C for 36 h. Only transformants/recombinants with successful “recombineering-mediated gap repair” will grow on LB ampicillin plates. Since recombination occurs between the LA and RA of the targeting vector and the corresponding homology regions of the Notch locus (Fig. 2c), the resulting recombinants will carry the 40 kb Notch locus in attB-P[acman]-ApR vector, from here on referred to as the wild-type Notch genomic construct, or Ngt-wt-attB-P[acman]-ApR. 4. Pick 4–6 colonies/recombinants and grow in selection medium (LB ampicillin) at 30 °C overnight. Isolate the wild-type Notch genomic construct by alkaline lysis and ethanol precipitation. Fingerprint by digestion with EcoRI. Transform positive clones into EPI300 competent cells (Epicentre, Inc), grow on LB ampicillin plates, and incubate at 37 °C overnight. The reason for transferring the wild-type Notch genomic construct to EPI300 cells is to keep the construct, which is now in a conditionally amplifiable vector, in a stable condition by maintaining it at a low copy number (1–2 copies per cell). 5. Grow the EPI300 cells carrying the wild-type Notch genomic construct overnight at 37 °C. Dilute the overnight culture 1:10, add 1× Copy Control solution (Epicentre, Inc.) to induce high copy number, and grow diluted culture for at least 5 h. Isolate the wild-type Notch genomic construct by alkaline lysis and ethanol precipitation as above. Fingerprint the wildtype Notch genomic construct once more by digestion with EcoRI. Once reverified, sequence the wild-type Notch genomic construct for final confirmation (see Note 4).

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3.2 Introduction of Serine-to-Alanine Mutations in the Rumi Target Sites of Notch

To mutagenize Rumi target sites in Notch, we devised the “gaprepair mutagenesis” strategy, a two-step recombineering method which allows DNA modification without introducing unwanted DNA sequences at the modification sites [20]. To generate templates with mutations in Rumi target sites, we have used either site-directed mutagenesis or gene synthesis. For constructs with mutations in a subset of EGF repeats, we have shuffled the corresponding regions of interest between wild-type and mutant plasmids generated by gene synthesis. We will provide an example of each strategy in this section.

3.2.1 Inducing Mutations in the Rumi Target Sites of EGF 4 and 5

1. Prepare a plasmid that consists of the 213 bp region containing EGF repeats 4 and 5 of Notch, flanked by 500 bp left and right homology arms (LA and RA, respectively) in pCR-Blunt II-TOPO-KmR vector (Fig. 2d). To this end, generate the LA-EGF4,5-RA by PCR, and subclone into the MCS of pCRBlunt II-TOPO-KmR vector. Induce serine-to-alanine mutations in the Rumi target sites of EGF 4 and 5 by performing two rounds of site-directed mutagenesis. 2. In parallel, prepare a fragment that contains the selection cassette CAT/SacB (chloramphenicol acetyl transferase/sucrose sensitivity) flanked by the same 500 bp left and right homology arms used in step 1. This fragment can be assembled using the two multiple cloning sites flanking the CAT/SacB cassette in the CAT/SacB-pCR-Blunt II-TOPO-KmR vector, which was generated by transferring the CAT/SacB cassette from the pEL04 vector [29, 30] to pCR-Blunt II-TOPO-KmR. A NotI restriction site exists between the CAT and SacB. Since the Notch genomic locus does not contain a NotI site, it will be used in step 7 to linearize the resulting construct (see Note 5). 3. Transform wild-type Notch genomic construct (Ngt-wt-attBP[acman]-ApR made in Subheading 3.1) into SW102 cells. Prepare recombination-induced SW102 competent cells carrying the wild-type Notch genomic construct by heat-shocking at 42 °C for 15 min, as explained in Subheading 3.1. If desired, prepare uninduced SW102 competent cells in parallel by keeping the cells at 30 °C at all times to be used as negative control. Prepare electrocompetent cells from recombination-induced and control SW102 cells by several washes in ice-cold 10 % glycerol. 4. Transform LA-CAT/SacB-RA prepared in step 2 into recombination-induced SW102 competent cells prepared in step 3. Plate the transformed SW102 cells on LB chloramphenicol and incubate at 30 °C for 36 h to select for recombinants. Upon recombination, the targeted region—in this case a 213 bp region containing the EGF 4 and 5 of Notch—will be

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replaced with the CAT/SacB cassette (Fig. 2e, f). The resulting construct is referred to as N4,5/CS-attB-P[acman]-ApR. 5. Isolate N4,5/CS-attB-P[acman]-ApR by alkaline lysis and ethanol precipitation. Verify positive clones by fingerprinting with EcoRI and transform into EPI300 cells to keep N4,5/CS-attBP[acman]-ApR construct at low copy number. Induce high copy number by the addition of Copy Control solution, similar to step 5 of Subheading 3.1. 6. Prepare recombination-induced SW102 competent cells carrying the mutated LA-EGF4,5-RA-pCR-Blunt II-TOPO-KmR construct made in step 1 as described in Subheading 3.1, step 2. 7. Linearize N4,5/CS-attB-P[acman]-ApR made in step 4 by digestion with NotI. Complete digestion is crucial to prevent false positives in step 8. Purify and transform into SW102 competent cells carrying the mutated LA-EGF4,5-RA-pCRBlunt II-TOPO-KmR made in step 6. Plate the transformants on LB ampicillin and incubate at 30 °C for 36 h. Upon recombination, the CAT/SacB cassette will be replaced with mutated EGF 4 and 5. The resulting construct is referred to as Ngt-4,5attB-P[acman]-ApR (see Note 6). 8. Isolate Ngt-4,5-attB-P[acman]-ApR by alkaline lysis and ethanol precipitation. Verify positive clones by fingerprinting with EcoRI. A mixture of Ngt-4,5-attB-P[acman]-ApR and LA-EGF4,5-RA-pCR-Blunt II-TOPO-KmR will be obtained. This is because LA-EGF4,5-RA-pCR-Blunt II-TOPO-KmR is present at high copy number in SW102 cells and only a small fraction of LA-EGF4,5-RA-pCR-Blunt II-TOPO-KmR plasmids has undergone recombination with linearized N4,5/ CS-attB-P[acman]-ApR. To separate the Ngt-4,5-attBP[acman]-ApR from LA-EGF4,5-RA-pCR-Blunt II-TOPOKmR, serially dilute the isolated Ngt-4,5-attB-P[acman]-ApR and transform into EPI300 cells. Start with 1:100 and 1:1,000 dilutions. A good dilution is when less than ten transformants are present on a plate. Isolate plasmids from multiple transformants to identify one that has Ngt-4,5-attB-P[acman]-ApR but lacks LA-EGF4,5-RA-pCR-Blunt II-TOPO-KmR. Induce high copy number by the addition of Copy Control solution. Fingerprint the Ngt-4,5-attB-P[acman]-ApR construct one more time by digestion with EcoRI. Once reverified, sequence the Ngt-4,5-attB-P[acman]-ApR construct for final confirmation, similar to steps 4 and 5 of Subheading 3.1 (see Note 7). 3.2.2 Inducing Mutations in the Rumi Target Sites of EGF 10–35 and EGF 4–35

Since all 16 Rumi target sites in Notch EGF repeat 10–35 are encoded by a single exon (Fig. 1), we used gene synthesis to generate a 3.1 kb fragment with serine-to-alanine mutations in all of the sites, flanked with 500 bp homology arms. Several companies offer

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this service. We then cloned the 3.1 kb fragment into pCR-Blunt II-TOPO-KmR vector to generate LA-EGF10-35-RA- pCR-Blunt II-TOPO-KmR. The subsequent steps to obtain the final Ngt-10_35attB-P[acman]-ApR construct involved two rounds of recombineering, similar to steps 2–8 of Subheading 3.2.1 (Fig. 2e–g). Briefly, in the first round of recombineering, corresponding LA-CAT/SacB-RA were used to replace the 3.1 kb region in Ngtwt -attB-P[acman]-ApR with a CAT/SacB cassette to obtain N10_35/ CS-attB-P[acman]-ApR (Fig. 2f). In the second round of recombineering, the CAT/SacB was replaced with the synthesized 3.1 kb fragment containing Notch EGF 10–35 with mutated Rumi target sites to obtain the final construct Ngt-10_35-attB-P[acman]-ApR (Fig. 2g, h). To obtain Ngt-4_35-attB-P[acman]-ApR, which contains serine-to-alanine mutations in all 18 Rumi target sites of Notch, the Ngt-4,5-attB-P[acman]-ApR construct made in Subheading 3.2.1 was used as the starting construct (Fig. 2e). The strategy to mutate EGF 10–35 in Ngt-4,5-attB-P[acman]-ApR is essentially the same as the one explained above. 3.2.3 Inducing Mutations in a Subset of Rumi Target Sites

The N10_35/CS-attB-P[acman]-ApR generated in Subheading 3.2.2 can be used as a platform to generate constructs with mutations in a subset of Rumi target sites. EGF 10–35 of Notch has already been replaced by a CAT/SacB cassette in this construct (Fig. 2f). Therefore, only one round of recombination with a targeting construct harboring the desired EGF repeat mutations, LA-EGF-RApCR-Blunt II-TOPO-KmR, is required to obtain the final construct (Fig. 2g). As mentioned above, we used gene synthesis to generate LA-EGF10-35-RA pCR-Blunt II-TOPO-KmR, which harbors serine-to-alanine mutations in all 16 Rumi target sites in this region. We also generated the corresponding wild-type plasmid by PCR from genomic DNA. Shuffling of a region harboring the EGF repeats of interest between these two plasmids will generate the appropriate targeting construct for recombination with linearized N10_35/CS-attB-P[acman]-ApR. For example, to obtain LA-EGF1015-RA pCR-Blunt II-TOPO-KmR, we have shuffled the EGF 10–15 region between the wild-type and mutant plasmids using unique restriction sites that flank the EGF 10–15 region. With one round of recombineering between LA-EGF10-15-RA- pCR-Blunt II-TOPO-KmR and linearized N10_35/CS-attB-P[acman]-ApR (Fig. 2g), we generated the final construct Ngt-10_15-attB-P[acman]ApR. A combination of site-directed mutagenesis and shuffling can be used to introduce virtually any mutation in this region of Notch by one round of recombination.

3.3 Generation of Wild-Type and Mutant Notch Transgenes

The embryo injections to obtain the transgenic lines can be performed in house, or can be outsourced to one of the several companies that have a collection of docking sites and perform ΦC31-mediated transgenesis. The choice of the docking site is

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based on the location of the endogenous gene to be rescued and other genes and transgenes with which one might plan to perform genetic interaction studies. Since Notch is on the X chromosome and the glycosyltransferases of our interest are located on the third chromosome, we chose the VK22 docking site, which is on the second chromosome [11]. We have used a genomic source for the expression of the ΦC31 integrase because this strategy has been shown to be more efficient than the co-injection of ΦC31 mRNA with the transgenic construct [12]. Given the site-specific nature of the integration event, in theory, one transgenic line is enough for each construct. We usually establish two or three lines in the beginning. Once we confirm correct integration (please see below), we maintain one stock for each transgene. Although the majority of the transgenes obtained via ΦC31mediated transgenesis are correctly integrated in the intended docking site, rare cases of insertion in incorrect genomic loci have been reported [11, 12]. Therefore, correct integration into an attP-based docking site needs to be verified. Recombination between attB and attP sites results in the generation of attL and attR sites and the loss of attP site (Fig. 2d, h). Therefore, the following three PCR reactions are performed on genomic DNA extracted from transgenic lines by using pairs of the following primers [11]: attP-F CTTCACGTTTTCCCAGGTCAGAAG. attP-R GTCGCGCTCGCGCGACTGACGGTC. attB-F GTCGACGATGTAGGTCACGGTC. attB-R TCGACATGCCCGCCGTGACCGTC. 1. attP PCR: use primer pair attP-F and attP-R (expected product size: 168 bp). 2. attL PCR: use primer pair attB-F and attP-R (expected product size: 163 bp). 3. attR PCR: use primer pair attP-F and attB-R (expected product size: 289 bp). We use the original strain harboring the blank docking site (in this case, y w; PBac{attP}VK22) as control. As mentioned above, correct integration events should lose the attP site and gain attL and attR sites (see Note 8). 3.4 Genetic Analysis of Notch Transgenes

Once the wild-type and mutant Notch transgenes are generated and their integration in the intended docking site verified, the effects of the mutations on the activity of the Notch receptor can be examined.

3.4.1 Assessment of the Gain-of-Function Confluens Phenotype

Adding an extra copy of the Notch gene results in increased wing vein material, which is called the Confluens phenotype (Fig. 3) [9]. Although the molecular mechanism underlying this phenotype is

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Fig. 3 O-glucose mutations decrease the activity of the Notch protein in a temperature-dependent manner. (a) Schematic of the EGF repeats of wild-type and mutant Notch genomic transgenes. (b–e”’) Shown are wings of adult males with wild-type Notch on the X chromosome and one or two copies of N gt-wt (b–c”’) or two copies of N gt-mut transgenes (d–e”’) inserted at the VK22 docking site on the 2nd chromosome. (b–b”’) One copy of N gt-wt results in a Confluens phenotype (extra vein, arrowheads) at 18–30 °C. (c’–c”’) Two copies of N gt-wt cause an enhancement of the Confluens phenotype at 18–30 °C. (d–d”’) At 18 °C, two copies of N gt-10_15 show a Confluens phenotype comparable to that caused by two copies of N gt-wt (compare (c) and (d)). The amount of extra vein tissue gradually decreases as the temperature is increased from low (18 °C) to high (30 °C). (e–e”’) The extra vein phenotype caused by two copies of N gt-10_35 at 18 °C and 23 °C is much milder than that caused by N gt-wt and N gt-10_15 (compare to (c, c’) and (d, d’)). At 25 °C (e”) and 30 °C (e”’), almost no Confluens phenotype is observed (reproduced from ref. 20)

not clear, it seems to be correlated with the level of Notch pathway activity, as adding two extra copies results in a more severe Confluens phenotype (Fig. 3c–c”’, compare to 3b–b”’) [20]. The Confluens phenotype is stronger in males compared to females with the same number of extra Notch copies [20], most likely because dosage compensation operates on X-linked genes even when they are inserted in an autosome [31]. As a first step in assessing the functionality of a mutant Notch transgene, we examine adult flies with two extra copies of a given transgene for the presence or absence of the Confluens phenotype, and compare the level of Confluens with flies harboring two extra copies of the wild-type Notch transgene inserted in the same genomic locus. In our hands, the

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amount of extra vein tissue caused by additional copies of the wildtype Notch transgenes is not temperature-dependent (Fig. 3b–c”’). If the posttranslational modification under study is suspected to regulate Notch signaling in a temperature-dependent manner, we recommend raising flies harboring two extra copies of the mutant transgene at different temperatures during development (e.g., 18, 25, 30 °C) and comparing the Confluens phenotype with that observed in animals harboring two extra copies of the wild-type Notch. This strategy allows one to determine the effects of a mutation of interest on the strength of signaling mediated by Notch (Fig. 3d–e”’) (see Note 9). 3.4.2 Rescue of Notch Haploinsufficient Phenotypes

Drosophila Notch, which is located on the X chromosome, is dosagesensitive. Females lacking one copy of Notch show characteristic haploinsufficient phenotypes, including “notches” in the wing margin and expansion of wing veins (Fig. 4a–b) [8, 20]. Providing one copy of the wild-type Notch transgene based on the following crossing schemes can rescue these phenotypes (Fig. 4c–d): ♀ N55e11/FM7c, Kr-GAL4 UAS-GFP sn+ X ♂ y w/Y; PBac{Ngt-wt}VK22 Whether integrated at PBac{attP}VK22 or another docking site, our wild-type Notch transgene was able to rescue the haploinsufficient phenotypes of various null alleles of Notch at a range of

Fig. 4 A 40 kb Notch genomic transgene (Ngt-wt) behaves similarly to an endogenous copy of Notch. Gene dosage experiments at 30 °C are shown. (a) A wild-type adult female wing. (b) A Notch+/55e11 haploinsufficient female wing with thickened veins (arrowhead) and wing margin loss (arrow). (c) The Confluens phenotype in a female with one copy of the Ngt-wt transgene inserted at the VK22 docking site. Arrowheads mark extra wing veins. (d) The Ngt-wt transgene rescues the Notch haploinsufficient phenotypes (compare to (b)) (reproduced from ref. 20)

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temperatures spanning from 18 to 30 °C [20]. To test how an engineered Notch mutation affects signaling, one can ask whether providing one copy of the transgene can suppress haploinsufficient phenotypes of Notch+/− heterozygous animals using the example crossing scheme: ♀ N55e11/FM7c, Kr-GAL4 UAS-GFP sn+ X ♂ y w/Y; PBac{Ngt-mut}VK22 The female N55e11/y w; PBac{Ngt-mut}VK22/+ progeny of this cross are scored for wing margin and wing vein phenotypes. These animals can be distinguished from their y w/FM7c, Kr-GAL4 UAS-GFP sn+; PBac{Ngt-mut}VK22/+ female siblings by the absence of the phenotypes associated with the FM7c, Kr-GAL4 UAS-GFP sn+ chromosome, namely GFP expression and the Bar eye phenotype. If the mutations under study are suspected to affect the function of Notch in a temperature-dependent manner (like O-glucose mutations), one should raise the animals at several temperatures to test whether the ability of the mutant transgene to rescue the Notch haploinsufficient phenotypes differs in these conditions. The wing vein thickening phenotype of Notch+/− animals is 100 % penetrant, but the wing margin loss phenotype is only partially penetrant, depending on the Notch allele used and the genetic background. Therefore, it is important to score a sufficient number of adult wings to ensure that a partial rescue phenotype is not missed. We score at least 50 wings for each genotype in this type of experiment. 3.4.3 Rescue of the Lethality and the Phenotypes of a Notch Null Allele

Males harboring a null allele of Notch die as embryos. Therefore, in Notch mutant stocks all of the males will have the balancer X chromosome, which is wild-type at the Notch locus. One copy of the wild-type Notch transgene (PBac{Ngt-wt}VK22) fully rescues the lethality of Notch null alleles in males and generates adults without Notch-related phenotypes (Fig. 5a–a’) (see Note 10). To test the functionality of a given mutant transgene in the absence of endogenous Notch, we ask whether providing a copy of that transgene can rescue the embryonic lethality associated with males harboring a null allele of Notch. The crossing scheme for this experiment is the same as the one used in the Subheading 3.4.2: (female) N55e11/FM7c, Kr-GAL4 UAS-GFP sn+ X (male) y w/Y; PBac{Ngt-mut}VK22 We score at least 100 progeny of this cross for the presence of adult males with an N55e11/Y; PBac{Ngt-mut}VK22/+ genotype. These animals can be distinguished from their FM7c, Kr-GAL4 UAS-GFP sn+/Y; PBac{Ngt-mut}VK22/+ male siblings by the absence of GFP expression and the Bar eye phenotype. Similar to the abovementioned experiments, if the mutation is suspected to affect Notch signaling in a temperature-dependent manner, we

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Fig. 5 Rescue of a null allele of Notch by genomic Notch transgenes. (a, a’) A Notch 55e11/Y hemizygous male rescued with one copy of the N gt-wt transgene inserted at VK22 at 30 °C, showing normal bristle pattern on the thorax (a) and normal legs (a’). Arrowhead marks the sex comb, and arrows mark the leg joints, which depend on Notch signaling. (b, b’) When raised at 21–23 °C (room temperature), N gt-10_15 rescues the lethality and phenotypes of Notch null mutants. (c, c’) At 25 °C, N–/Y; N gt-10_15/+ males show a mild loss of bristles and normal legs. (d, d’) At 30 °C, N–/Y; N gt-10_15/+ males show a severe loss of bristles and shortened legs with severe joint defects (reproduced from ref. 20)

incubate the crosses at various temperatures between 18 °C and 30 °C. Presence of adult N55e11/Y; PBac{Ngt-mut}VK22/+ males indicates that one copy of the mutant transgene is able to rescue the embryonic lethality of N55e11. The rescued adults are then scored for morphological features affected by decreased Notch signaling, like thoracic bristles and leg joints (Fig. 5b–d’). This will help us determine whether the induced mutation(s) affect Notch signaling in modulatory and/or tissue-specific manners, or whether the corresponding posttranslational modifications are dispensable for the activity of the Notch proteins. Although lack of adult N55e11/Y; PBac{Ngt-mut}VK22/+ progeny from the above cross indicates a significant decrease in the activity of Notch as a result of the induced mutation(s), it does not necessarily mean that the mutant Notch protein is fully inactive. Indeed, the mutant transgene may partially rescue the embryonic lethality of N55e11 through larval or pupal stages but fail to provide enough function to generate adults. This possibility can be examined by following the development of GFP[-] progeny during the larval and pupal stages.

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Notes 1. If the efficiency of triple ligation is low, the LA and RA can be subcloned sequentially. 2. SW102 cells harbor the λ prophage recombineering system in their genome. This function is suppressed at 30 °C but becomes activated at 42 °C. 3. It is important to have a complete digestion to avoid false negatives in step 4. Run the BamHI digested targeting vector on an agarose gel to visualize a linear fragment. Cut and gel purify the linear fragment. Alternatively, run an aliquot of the digested product on an agarose gel to ensure complete digestion. Column purify the remaining digested product to obtain higher yield. 4. Sequencing reactions should cover at least the LA and RA including the junctions and all nine exons of the Notch gene. 5. If the gene of interest contains NotI sites, one needs to incorporate another “rare-cutter” site between the CAT and SacB fragments. This can be performed by synthesizing partially overlapping oligonucleotides harboring the desired restriction site, which anneal to generate sticky ends compatible with a BamHI-digested construct. The resulting double-stranded DNA can be cloned into the BamHI site adjacent to the NotI site between CAT and SacB fragments in the CAT/SacB-pCRBlunt II-TOPO-KmR vector. 6. It is crucial that the LA and RA be the only homologous regions between the N4,5/CS-attB-P[acman]-ApR and LA-EGF4,5-RA-pCR-Blunt II-TOPO-KmR. This ensures that recombination occurs only between the LA and RA homology arms. Therefore, if you use a different vector to clone the targeting fragment, its backbone sequence should not have homology to attB-P[acman]-ApR. 7. Due to the high efficiency of this method, we do not need to use the SacB for negative selection, although it can be used if need be. 8. The genomic source of the ΦC31 integrase in the M{vas-int} ZH-2A; PBac{attP}VK22 stock, which is used for generating the Notch transgenes, was itself generated by ΦC31-mediated integration [12]. Therefore, it needs to be removed from the Notch transgenic lines before the attP, attL, and attR PCRs are performed. Since this insertion is on the X chromosome (cytological position 2A), the easiest way to remove it is to cross a G0 male with eye color (indicating the integration of the w+ Notch transgene) to y w females, and to use the w+ male progeny of this cross for the diagnostic PCRs. The absence of

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the M{vas-int}ZH-2A transgene can also be confirmed using a fluorescent stereomicroscope, as this transgene drives the expression of GFP and RFP in late pupal and adult eyes [12]. 9. Although increasing the Notch gene dosage results in additional wing vein tissue (Confluens), the so-called Abruptex gain-of-function mutations in Notch as well as mutations in a number of negative regulators of Notch result in wing vein gaps [21, 32, 33]. Discussions of the potential mechanisms underlying these seemingly paradoxical observations are outside the scope of this chapter. However, these observations indicate that if specific point mutations in Notch render the molecule more active, instead of the Confluens phenotype they can result in loss of wing vein tissue [21]. 10. One copy of this transgene also rescues the lethality of Notch null females. As expected, the resulting adult females exhibit morphological abnormalities associated with Notch haploinsufficiency. However, they are sterile. The sterility does not seem to result from decreased levels of Notch expression by our transgene compared to an endogenous copy of Notch, because even two copies of the wild-type Notch transgene inserted in VK22 or another docking site cannot rescue the sterility in Notch−/− females. We have concluded that our construct most likely lacks an enhancer specifically required for oogenesis. Accordingly, to study the contribution of a given posttranslational modification of Notch to oogenesis, one would need to start with a somewhat bigger wild-type Notch construct capable of rescuing the female sterility.

Acknowledgments This work was supported by the NIH grant R01GM084135. The gap-repair mutagenesis method was developed by Dr. Rodrigo Fernandez-Valdivia, a former postdoctoral fellow in our group. We thank Dr. Graeme Mardon and Dr. Barbara Jusiak for generously providing the CAT/SacB-pCR-Blunt II-TOPO-KmR construct. We thank Dr. Karen Schulze for critical reading of this chapter. References 1. Fortini ME (2009) Notch signaling: the core pathway and its posttranslational regulation. Dev Cell 16:633–647 2. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233 3. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689

4. Rubin GM, Spradling AC (1982) Genetic transformation of Drosophila with transposable element vectors. Science 218:348–353 5. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415 6. Venken KJ, Simpson JH, Bellen HJ (2011) Genetic manipulation of genes and cells in the

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Jessica Leonardi and Hamed Jafar-Nejad nervous system of the fruit fly. Neuron 72:202–230 Lei L, Xu A, Panin VM et al (2003) An O-fucose site in the ligand binding domain inhibits Notch activation. Development 130:6411–6421 Mohr OL (1919) Character changes caused by mutation of an entire region of a chromosome in Drosophila. Genetics 4:275–282 Lyman D, Young MW (1993) Further evidence for function of the Drosophila Notch protein as a transmembrane receptor. Proc Natl Acad Sci U S A 90:10395–10399 Ramos RG, Grimwade BG, Wharton KA et al (1989) Physical and functional definition of the Drosophila Notch locus by P element transformation. Genetics 123:337–348 Venken KJ, He Y, Hoskins RA et al (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314:1747–1751 Bischof J, Maeda RK, Hediger M et al (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A 104:3312–3317 Groth AC, Fish M, Nusse R et al (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166:1775–1782 Copeland NG, Jenkins NA, Court DL (2001) Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2:769–779 Thorpe HM, Smith MC (1998) In vitro sitespecific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/ invertase family. Proc Natl Acad Sci U S A 95:5505–5510 Venken KJ, Popodi E, Holtzman SL et al (2010) A molecularly defined duplication set for the X chromosome of Drosophila melanogaster. Genetics 186:1111–1125 Hase S, Kawabata S, Nishimura H et al (1988) A new trisaccharide sugar chain linked to a serine residue in bovine blood coagulation factors VII and IX. J Biochem 104:867–868 Moloney DJ, Shair LH, Lu FM et al (2000) Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules. J Biol Chem 275:9604–9611 Acar M, Jafar-Nejad H, Takeuchi H et al (2008) Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132:247–258 Leonardi J, Fernandez-Valdivia R, Li YD et al (2011) Multiple O-glucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling. Development 138:3569–3578

21. Lee TV, Sethi MK, Leonardi J et al (2013) Negative regulation of Notch signaling by xylose. PLoS Genet 9:e1003547 22. Harris RJ, Spellman MW (1993) O-linked fucose and other post-translational modifications unique to EGF modules. Glycobiology 3:219–224 23. Rana NA, Nita-Lazar A, Takeuchi H et al (2011) O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1. J Biol Chem 286:31623–31637 24. Shao L, Luo Y, Moloney DJ et al (2002) O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase. Glycobiology 12:763–770 25. Sethi MK, Buettner FF, Ashikov A et al (2012) Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of notch. J Biol Chem 287:2739–2748 26. Sethi MK, Buettner FF, Krylov VB et al (2010) Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats. J Biol Chem 285:1582–1586 27. Warming S, Costantino N, Court DL et al (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33:e36 28. Hoskins RA, Nelson CR, Berman BP et al (2000) A BAC-based physical map of the major autosomes of Drosophila melanogaster. Science 287:2271–2274 29. Lee EC, Yu D, Martinez De Velasco J et al (2001) A highly efficient Escherichia colibased chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73: 56–65 30. Thomason L, Court DL, Bubunenko M, et al. (2007) Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol Chapter 1, Unit 1.16 31. Kelley RL, Meller VH, Gordadze PR et al (1999) Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 98:513–522 32. Fostier M, Evans DA, Artavanis-Tsakonas S et al (1998) Genetic characterization of the Drosophila melanogaster Suppressor of deltex gene: A regulator of notch signaling. Genetics 150:1477–1485 33. De Celis JF, Barrio R, Del Arco A et al (1993) Genetic and molecular characterization of a Notch mutation in its Delta- and Serratebinding domain in Drosophila. Proc Natl Acad Sci U S A 90:4037–4041

Chapter 4 Overview of Genetic Tools and Techniques to Study Notch Signaling in Mice Thomas Gridley and Andrew K. Groves Abstract Aberrations of Notch signaling in humans cause both congenital and acquired defects and cancers. Genetically engineered mice provide the most efficient and cost-effective models to study Notch signaling in a mammalian system. Here, we review the various types of genetic models, tools, and strategies to study Notch signaling in mice, and provide examples of their use. We also provide advice on breeding strategies for conditional mutant mice, and a protocol for tamoxifen administration to mouse strains expressing inducible Cre recombinase-estrogen receptor fusion proteins. Key words Conditional mutations, Notch reporter lines, Notch receptor-Cre fusions, Fate mapping, Lineage analysis, Domain-swap mice, Tamoxifen administration

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Introduction The Notch signaling pathway is an evolutionarily ancient form of cell– cell communication. In vertebrates it plays a central role in the development and homeostasis of most major organ systems and has been implicated in many forms of hereditary and idiopathic diseases, including cancer. The many complexities of the Notch signaling pathway have been described extensively elsewhere [1–4]. In this chapter, we describe how the laboratory mouse has been developed to study many aspects of Notch signaling in development and disease. In addition to the huge number of different genetically modified mouse lines that have been generated to cause loss or gain of function of different components of the Notch signaling pathway, recent years have seen the advent of new mouse lines to visualize Notch-responsive cells in the intact animal, to follow the fates of cells that have experienced Notch signaling, and to probe the structure–function relationships between different mammalian Notch receptors. In this chapter, we describe the current status of transgenic mouse technology that has addressed these questions, and provide some basic protocols for breeding and working with conditional mutants of the Notch pathway.

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_4, © Springer Science+Business Media New York 2014

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Loss- and Gain-of-Function Notch Pathway Mutant Mice

2.1 Constitutive Loss-of-Function Mutant Mice

The many different components of the Notch signaling pathway [2] have led to the generation of a huge number of genetically modified and mutagenesis-induced mouse lines that have loss of function in Notch signaling. As described in Subheading 6 below, there are comprehensive online databases that allow the investigator to search for a wide variety of different alleles of genes in the Notch pathway. Historically, these alleles were generated piecemeal in individual laboratories. More recently however, an international project has aimed to systematically produce targeted mutations in ES cells for every mouse gene [5, 6] (www.knockoutmouse.org). Many of these ES cell lines have already been used to generate mouse lines, and an initial systematic analysis of these lines is ongoing [7]. Individual investigators may order targeted ES cells from repositories supporting these projects, and in addition to generating mutant mice from such cell lines, it will be possible to reengineer them for new purposes (for example, expression of fluorescent proteins, recombinases, optogenetic channels, calcium indicator proteins, or to add epitope tags) using technology such as recombination-mediated cassette exchange [8, 9].

2.2 Conditional Loss-of-Function Mutant Mice

The early embryonic lethality exhibited by many constitutive Notch pathway loss-of-function mutants is often a result of defects in formation of the embryonic vasculature [10]. This emphasizes the importance of generating conditional and/or inducible mutants to facilitate study of Notch pathway function later in embryogenesis and in the postnatal and adult mouse.

2.2.1 Generating Conditional Mutant Mice with the Cre-Lox System

The ability of the P1 bacteriophage Cre recombinase to recombine palindromic loxP sites at high efficiency [11] in mammalian cells [12], even when separated by large intervening DNA sequences, has made it the method of choice for creating spatially and temporally controlled gain- and loss-of-function mutations in mice [13–15]. A huge armory of transgenic and knock-in lines expressing Cre recombinase under the control of different promoters and enhancers allows manipulation of genes in most tissues. Temporal control of Cre activity is also possible using fusions of the Cre protein with modified forms of the estrogen receptor, which allow nuclear localization and activation of Cre following administration of the estrogen analog tamoxifen [16–20]. Cre has also been fused with the progesterone receptor in a similar fashion to yield mice in which the recombinase activity can be activated by Mifepristone (RU486) [21], although this strategy is used far less than Cre-estrogen receptor fusions [22]. Similarly, although the FLP-FRT recombination system has also been used to drive recombination in the mouse genome, its use has tended to be reserved for intersectional recombination strategies [23], or for the removal of selection cassettes following the construction of targeted alleles [24].

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Many conditional alleles have been generated for components of the Notch signaling pathway. Conditional alleles are available for the Notch1, Notch2, and Notch3 receptors, for the Dll1, Dll4, Jag1, and Jag2 ligands, as well as several noncanonical ligands such as DNER, Contactin1, and MAGP1 [25]. Conditional alleles have also been generated for modifying and processing enzymes of Notch and its ligands, for nuclear co-activators and for direct downstream targets of Notch signaling. Subheading 6 gives advice for identifying and locating mouse mutants for various Notch signaling pathway components. More recently, the advent of large-scale mouse targeting projects, such as the Knockout Mouse Project (KOMP) and the European Conditional Mouse Mutagenesis Program (EUCOMM) [5, 6], has generated a large number of new targeted mutations of genes in the Notch pathway. Many of these alleles have used the “knockout first” targeting strategy, in which a targeted null allele can be converted to a conditional allele by mating with ubiquitously expressing FLP lines [26]. In addition to conditional “floxed” alleles, some mouse lines have been developed to produce dominant-negative mutants of Notch pathway components. For example, ROSA26 dnMAML1GFP mice express a Cre-inducible, GFP-tagged, truncated form of Mastermind-like1 (MAML1) that is knocked into the ROSA26 locus [27, 28]. This fusion protein is capable of binding Rbpj/ CSL, but cannot recruit transcriptional co-activators to this complex and thus acts as a dominant-negative protein. The use of a dominant loss-of-function mutation can make breeding strategies easier; however, it is possible that two mutant alleles may be required to give a strong loss of Notch signaling in some tissues. 2.2.2 Selection and Verification of Cre Driver Lines

Several databases listing Cre driver lines have been developed [29]. The Mouse Genome Informatics database maintains and curates an extensive and rapidly growing list of published Cre driver lines (www.informatics.jax.org/recombinase.shtml) that target many different tissues, cell populations, and stages of development. The activity and recombination pattern of these lines can be monitored by crossing them with a variety of Cre reporter lines, in which the expression of a fluorescent or enzyme marker is activated after Cre recombination. Many of these lines are available from the Jackson Laboratory (cre.jax.org/crereporters.html), which also maintains a Cre driver line characterization pipeline [30]. Additional Cre driver databases are maintained in Canada and Europe [31, 32]. It is strongly recommended that each investigator independently verifies the activity of a Cre driver line imported into their mouse colony before starting a conditional knockout experiment. It is common for Cre lines to produce patterns of recombination that are somewhat different from their first published descriptions. This can be due to differences in genetic background, or to differing sensitivities of the Cre reporter lines used. This advice is doubly true for studies using tamoxifen-inducible versions of Cre recombinase.

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Fig. 1 A sample conditional breeding strategy to generate conditional loss-of-function mutations in Notch pathway genes. GOI = Gene of interest, for which a conditional null allele (GOInull), and “floxed” allele (GOIflox) are available. The Cre driver allele is referred to as DriverCre. This mating scheme will produce approximately 25 % offspring that carry a conditional loss-of-function mutation

Here, the degree and efficiency of recombination is exquisitely sensitive to the dose and route of administration of tamoxifen, and careful monitoring of recombination efficiency is essential for correct interpretation of results using such mice. If possible, it is recommended to include a Cre reporter allele alongside a conditional allele, so that recombination can be observed directly in the conditional mutant strain. A protocol for tamoxifen administration is given below (Subheading 2.2.4). 2.2.3 Breeding Strategies for Generation of Conditional Mouse Mutants

A typical breeding scheme to generate conditional loss-of-function mouse mutants using the Cre-Lox system is shown in Fig. 1. In brief, a Cre driver mouse is crossed with a mouse from a second line that is heterozygous for a null allele of the gene of interest. The offspring from this cross are screened for mice carrying both the Cre and null alleles. These mice are then crossed with mice carrying two alleles of the gene of interest that has all or part of its coding region flanked by loxP sites (“floxed” or “flox” alleles). Approximately 25 % of the resulting offspring will inherit the Cre allele, a null allele, and a floxed allele of the gene of interest. These mice will then display a loss of function of the gene of interest in cells in which the Cre recombinase is active. If a tamoxifeninducible version of Cre recombinase is used, recombination will not occur until the mice are dosed with this drug.

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1. Anecdotal evidence suggests that recombination is more efficient if a null allele is crossed on to the Cre driver line as opposed to a floxed allele. This is presumably due to the presence of fewer loxP sites in the target genome. A recent published study has verified these anecdotal findings [33]. A null allele can easily be generated from a floxed allele by mating with a ubiquitously expressed Cre line. 2. Since the maintenance of mouse lines heterozygous for both the Cre driver allele and the null allele of the gene of interest requires genotyping at each generation and does not yield 100 % of the desired genotype, it is common to use male studs carrying these alleles, as a single stud can be mated repeatedly to multiple females. Since the propagation of a homozygous floxed line is far more efficient, homozygous floxed females are typically used in this mating scheme. 3. Before commencing a conditional breeding strategy, it is always advisable to check the chromosomal location of the Cre driver allele and the gene of interest to be used in the project, as well as any reporter alleles that may be included in the breeding scheme. Although the probability of the Cre allele and the gene of interest occurring on the same chromosome is low in mice, a quick verification may prevent the investigator wasting months of unproductive breeding.

2.2.5 Protocol: Administration of Tamoxifen to Mice by Oral Gavage Introduction

Materials

As described above, it is strongly recommended that investigators perform preliminary experiments to verify the fidelity of their Cre lines, and to determine the speed and efficiency of Cre-mediated recombination after administration of tamoxifen. It is possible to observe the first evidence of recombination within 6–8 h of tamoxifen administration [34], although this will obviously depend on the strength of the regulatory sequences in each Cre driver line. It should also be noted that recombination may continue for days, or in some cases weeks, after cessation of tamoxifen administration [35], so studies that make use of pulses of tamoxifen should be interpreted with appropriate caution. 1. Warm 5–6 ml corn oil (Sigma, C8267) in a 15 ml tube at 42 °C for 30 min. Corn oil can be stored at room temperature, but replace every 3–6 months to prevent the oil from becoming rancid. 2. Weigh out 100 mg tamoxifen (Sigma T5648) into a second 15 ml tube (see Note 1). Tamoxifen is light-sensitive, and should be stored in the dark at 4 °C with a desiccant. Allow the tamoxifen to equilibrate to room temperature before weighing. 3. Add 5 ml of the warm corn oil to the tamoxifen, wrap the tube in foil to protect from light and incubate in a 37 °C water bath to dissolve. The solution should be vortexed frequently to aid

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solubilization, which usually takes several hours. There should be no precipitate left in the solution. 4. The tamoxifen solution can be stored in the dark at 4 °C wrapped in foil. Discard the solution after 1 month, or sooner if a precipitate starts to develop. Methods

1. Weigh each mouse on a balance before administering tamoxifen. Accurate recording of weight is important to obtain consistent dosing. 2. Curved reusable metal gavage needles with a ball tip are available from a number of suppliers (for example, Fine Science Tools 18060-20) or from institutional veterinary staff. A 20 gauge needle with a tip of between 1 and 2 mm is suitable for adult mice. Alternatively, disposable needles are also available (for example, Fine Science Tools 18061-20). 3. Connect the needle to a syringe and fill with tamoxifen solution. Grasp the mouse by the scruff of the neck, and secure the tail in the manner one would use for an intraperitoneal injection. It is important that the mouse be securely restrained but relatively relaxed. 4. Introduce the needle tip into the side of the mouse’s mouth. Pass the needle tip to the rear of the mouth along the roof. Push the mouse’s head back and pass the needle fully down the esophagus into the stomach to deliver the tamoxifen dose. A minimum of force is required and the entire procedure should be performed as gently as possible to avoid distress. If the animal attempts to struggle, it is possible that the windpipe is obstructed—this should be avoided.

Notes

1. Tamoxifen is a carcinogen and appropriate caution should be taken when handling the powder and solution. Dispose of materials that have come into contact with tamoxifen in a manner consistent with local environmental safety regulations. 2. Some studies have administered tamoxifen by intraperitoneal injection. While this may require less training to administer, many anecdotal reports suggest pregnant females show signs of discomfort from the added burden of corn oil in their abdomens. 3. Adult mice can tolerate doses of tamoxifen as high as 0.3 mg/g body weight. Pregnant females and their litters are more sensitive to tamoxifen, so dosage should be determined empirically for each mouse strain. Females past the 10th day of pregnancy can typically tolerate as much as 0.12–0.13 mg/g body weight administered twice a day. Administration of tamoxifen at earlier stages can cause developmental problems or resorption of embryos, and the dose may need to be reduced appropriately [36].

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4. As tamoxifen is an estrogen analog, it can cause complications with pregnancy, especially in the final few days before delivery. To prevent late fetal abortion, progesterone (Sigma P3972) can be administered with the tamoxifen, typically at 50 % of the dose of tamoxifen. 5. It is recommended that pregnant dams be disturbed as little as possible by laboratory and animal care staff for several days before and after delivery. 6. Tamoxifen can be administered to neonatal pups in several ways. Direct intraperitoneal injection of tamoxifen works effectively, although care must be exercised when handling the pups. Alternatively, tamoxifen is known to be transmitted in maternal milk [37], so continued administration of tamoxifen to the mother will provide dosing to neonates, albeit in a more variable fashion. 7. All handling and use of animals requires approval by local monitoring agencies and should adhere to local and national animal care guidelines. It is strongly recommended that users learning the gavage procedure be trained by a veterinarian or veterinary technician, ideally by first practicing on an animal that has been euthanized. 2.3 Conditional Gain-of-Function Mice

Transgenic mis-expression of Notch pathway components can have very strong biological effects, such that constitutive gain-offunction transgenic or knock-in alleles are often lethal or difficult to maintain. This has led a number of investigators to generate conditional gain-of-function mice whose expression is activated by the Cre-Lox system. These alleles contain a transcriptional stop sequence, flanked on either side by a loxP site, located between the promoter and the coding sequences for the Notch pathway gene that will be expressed. These conditional gain-of-function alleles have been generated either as transgenes or as knock-in alleles into the widely expressed ROSA26 locus. Commonly, the gene being mis-expressed is the transcriptionally active Notch intracellular domain (NICD). For example, mice that conditionally express NICD1 [38] or NICD2 [39] have been reported; many additional publications using these or similar mice exist. Aifantis and colleagues recently reported generation of four knock-in strains, one for each NICD (1–4) [40]. Each line used the same allele design, in which coding sequences for NICD were inserted into the ROSA26 locus behind a loxP-stop-loxP cassette. NICD coding sequence was followed by an internal ribosome entry site-yellow fluorescent protein (IRES-YFP) cassette to facilitate monitoring of expression. Interestingly, when activated by expression of an inducible Cre-estrogen receptor fusion protein driven by the human ubiquitin C promoter, only the NICD1 line was capable of causing T cell leukemia, despite comparable levels of expression of all four

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NICDs. This result demonstrates sequence-specific differences among the NICDs in vivo [40], similar to those previously described in vitro [41].

3

Notch Signaling Reporter Lines Signaling pathway-specific transgenic reporter lines are very useful in analyzing pathway activation in both wild-type and mutant genetic backgrounds. These lines generally contain a gene encoding a reporter protein, such as β-galactosidase or a fluorescent protein, under the control of a promoter that is transcriptionally regulated by the specific pathway being analyzed (e.g., the Notch pathway). The two most commonly used transgenic Notch reporters are the Transgenic Notch Reporter (TNR) EGFP reporter line [42] and the Notch Activity Sensor (NAS) nuclear lacZ reporter [43]. Neither of these Notch reporter lines, however, has gained the extremely wide utilization of the Wnt pathway transgenic reporter lines TopGal [44] and BatGal [45]. The TNR line utilizes a synthetic promoter consisting of four binding sites for the Rbpj protein (the primary transcriptional mediator of the Notch pathway) linked to the basal SV40 promoter to drive EGFP expression, while the NAS reporter uses 12 multimerized Rbpj binding sites upstream of a minimal promoter from the beta globin gene. Other Notch reporters use endogenous Notch target genes containing Rbpj binding sites to drive reporter gene expression. For example, transgenic or knock-in reporters utilizing promoters from the Notch target genes Hes1 [46] and Hes5 [46–48] to drive β-galactosidase or EGFP expression have been described. Novel transgenic Notch pathway reporters continue to be developed. Two groups have developed new reporters that utilize improved, brighter fluorescent proteins in order to better enable in vivo or ex vivo imaging at the single cell level, as well as flow cytometry analyses. The Hes1-emGFP line is a knock-in allele that places the emerald variant of the GFP protein under the transcriptional control of the endogenous Hes1 promoter [49]. Hes1-emGFP mice have been used in the analysis of Notch signaling in intestinal stem cells [49], during mammary gland development (including the sorting by flow cytometry of emGFP-expressing cells from the mammary gland) [50], and in the analysis of Notch pathway activation in specific hematopoietic cell lineages during both normal and stress hematopoiesis [40]. The CBF:H2B-Venus transgenic line (Cbf1 is an alternative symbol for the Rbpj gene) expresses a fusion protein comprising human histone H2B (to provide nuclear localization) sequences linked to the yellow fluorescent protein Venus [51], and uses the same synthetic Notch target promoter as the TNR reporter line [42]. The sensitivity of the CBF:H2B-Venus reporter facilitates

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identification of new sites of Notch signaling in embryos, particularly within the epiblast of the early postimplantation embryo, and can be used for live cell imaging in ES cells, primary mouse embryonic fibroblasts, and in cultured postimplantation embryos [51].

4

Notch Receptor-Cre Fusions for Fate Mapping and Functional Analyses One criticism of Notch reporter lines such as those described in the preceding section, which utilize promoters containing synthetic arrangements of Rbpj binding sites or natural Notch target promoters containing Rbpj binding sites, is that they do not distinguish signal reception mediated by the different Notch receptor proteins. Several groups have generated knock-in or transgenic lines expressing fusions of Cre recombinase or the Gal4VP16 transcriptional activator with various Notch receptors. These lines can be used for both fate mapping of Notch receptor-expressing cell lineages, or for functional analyses of these lineages. Two groups have developed bi-transgenic Notch1 reporter systems that are dependent on gamma secretase-mediated proteolysis of a modified Notch1 gene to identify cells that have undergone active (i.e., proteolysis-mediated) Notch1 signaling. Kopan and colleagues used homologous recombination to generate a Notch1 knock-in allele in which sequence encoding the Notch1 (N1) intracellular domain downstream of the gamma secretase cleavage site was replaced with sequence encoding Cre recombinase [52]. The line, currently named N1::CreLO (but referred to in the original publication as N1IP-Cre, for Notch1 Intramembrane ProteolysisCre), when crossed with a Cre reporter such as the ROSA26R strain [53], permits in vivo mapping of Notch1 activation by gamma secretase-mediated proteolytic cleavage. While not a Notch receptor-Cre fusion, Radtke and colleagues have developed a conceptually similar bi-transgenic Notch1 reporter system [54]. They constructed a bacterial artificial chromosome (BAC) transgenic line (N1-Gal4VP16) containing a modified Notch1 cDNA, in which the intracellular domain of the Notch1 receptor was replaced with sequence encoding the Gal4VP16 transcriptional activator. When crossed to a transgenic Gal4 reporter line, such as UAS-lacZ mice [55], the N1-Gal4VP16 line permits the identification in vivo of cells undergoing active Notch1 signaling [54]. Kopan and colleagues have generated several additional Notch receptor-Cre fusion knock-in lines, including N1::CreHI (which expresses higher levels of Cre recombinase than the N1::CreLO line) [56], N1::CreERT2 [57], and N2::CreLO [57, 58]. Like the N1::CreLO line, all of these lines have Cre coding sequences replacing the Notch receptor intracellular domain, and must be activated by gamma secretase-mediated proteolytic cleavage in order to

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generate Cre recombinase activity. In addition to fate mapping studies [52, 58–60], some of these lines have been used in functional assays. Since the Cre coding sequences replace the Notch receptor intracellular domain in these alleles, they are functional null alleles that must be analyzed as heterozygotes. In a clever utilization of this system, trans-heterozygous N1::CreLO/N1flox mice were generated. The low efficiency of the N1::CreLO allele created sporadic clones of cells with Notch1 loss of heterozygosity (LOH) in these mice, which subsequently experienced widespread vascular tumors and lethality secondary to massive hemorrhage [56]. Notch receptor fusion lines such as N1::CreLO and N1-Gal4VP16 identify cell lineages that have undergone proteolysis-mediated Notch1 signaling. Artavanis-Tsakonas and colleagues have developed four CreERT2-expressing lines that, when mated to Cre reporter lines, identify cell lineages expressing the Notch1 through Notch4 genes without requiring their activation by proteolytic cleavage [49]. These lines have been used recently to identify hematopoietic cell lineages that express each of the Notch receptors during hematopoietic differentiation [40]. By crossing each of the four Notch-CreERT2 lines to the ROSA26lslRFP reporter line [61], administering tamoxifen and waiting for various chase periods, the authors found that only Notch1-CreERT2 and Notch2CreERT2 were expressed in bone marrow. Notch1-CreERT2 primarily labeled cells with lymphoid potential, while Notch2CreERT2-labeled cells were found mostly in nonlymphoid progenitors [40]. The Notch2-CreERT2 line also was used to identify, by mating to conditional β-galactosidase and Tomato red fluorescent protein Cre reporter lines, two previously unrecognized mammary epithelial cell lineages that are distinct from classical luminal and myoepithelial cells [50]. The Notch2-CreERT2 line was used to functionally assess the role of these lineages during mammary gland development in virgin and lactating females, by mating the line to a conditional diphtheria toxin receptor line [62] to perform cell ablation of Notch2-CreERT2-expressing cell lineages. These experiments demonstrated that the Notch2-CreERT2-expressing cell lineages were critical for the formation of tertiary branches of mammary gland ductal trees [50].

5

Domain-Swap Mice In vivo structure–function studies are a powerful tool to dissect protein function in mice. Kraman and McCright generated a knock-in allele (Notch2N1in) in which sequences encoding the carboxy-terminal 426 amino acids of the Notch2 protein were replaced with the equivalent region of the Notch1 protein [63]. Despite the fact that the amino acid sequences of the replaced region are only 37 % identical, no aberrant phenotypes were

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detected in mice homozygous for the Notch2N1in allele, indicating that the carboxy-terminal region of the Notch1 intracellular domain is able to functionally replace that of the Notch2 protein in vivo. In a genetic engineering tour de force, Kopan and colleagues recently reported much more extensive, and reciprocal, intracellular domain swaps of the Notch1 and Notch2 proteins [58]. They described construction of two knock-in lines in which the genomic regions encoding the intracellular domains (ICD) of the Notch1 and Notch2 proteins were interchanged. The N12 allele encodes the extracellular domain (ECD) of Notch1 with the ICD of Notch2, while the N21 allele encodes the ECD of Notch2 with the ICD of Notch1. The swapped regions extended from exon 28, encoding the transmembrane domain, to the stop codon in exon 34. In order to retain transcript-specific regulation of mRNA stability and translation (by microRNAs, for example), the 3′ untranslated regions of the two genes were not interchanged. Mice homozygous for either allele (N12/N12 or N21/ N21) were viable. The initial publication was confined to an analysis of kidney development in these mice [58]. The authors found that just a single copy of Notch1-ICD could fully rescue the loss of Notch2-ICD, if it was expressed from the Notch2 locus. These results demonstrated that, at least for kidney development, the Notch1-ICD and Notch2-ICD were fully interchangeable. They further demonstrated that the Notch2-ECD increased Notch receptor localization to the cell surface during kidney development, and was cleaved more efficiently than the Notch1-ECD upon ligand binding [58].

6

Locating Mouse Genetic Resources for In Vivo Analyses of Notch Signaling One of the greatest factors contributing to how widely a mouse line is utilized by the research community is whether the line is available from a public strain repository [64]. Many of the lines summarized in this overview are available from such repositories. Researchers can search the web site of the International Mouse Strain Resource (IMSR; www.findmice.org) to locate mouse lines available from strain repositories around the world. The search options on the IMSR site permit one to filter the results in various ways, and it can be useful to exclude the ES cell category (which can contain hundreds of gene trap alleles that have never been made into mice) from the search (Fig. 2). Information on different alleles of particular genes of interest is also available on the Mouse Genome Informatics site (www.informatics.jax.org). Table 1 provides examples of Notch signaling reporter and Notch receptorCre lines available (or soon to be available) from public mouse strain repositories. We have not attempted to summarize individual

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Fig. 2 Results of a search on the IMSR web site (www.findmice.org) for Notch1-related targeted mutations. Not all of the returned results are shown. The results show examples of live mice and cryopreserved sperm or embryos, available from the Jackson Laboratory (JAX), Taconic (TAC), and the RIKEN BioResource Center (RBRC)

Table 1 Notch reporter and Notch-Cre fusion lines available from strain repositories Namea

Reporter/Cre

Repository

Stock number

Reference

TNR

EGFP

JAX

18322

[41]

NAS

β-galactosidase

EMMA

2412

[42]

Venus-YFP

JAX

20942

[50]

Cre

JAX

6953

[51]

CBF:H2B-Venus LO

N1::Cre

JAX Jackson Laboratory, EMMA European Mouse Mutant Archive a This is the name used in the author’s publications, and in this chapter. The repositories may have these strains listed under different names. We recommend searching repository stock lists with the stock number

Notch pathway mutants deposited in mouse repositories, as the number of these strains is too numerous, but encourage investigators to utilize the search procedures described here and elsewhere [5, 6, 29, 64]. Strains not deposited in repositories may be available directly from the investigator who produced them. Recovering cryopreserved strains from repositories generally costs several thousand dollars, or its equivalent in other currencies. It is not unusual to see requests from investigators on email list servers such as MGI-list (hosted by the Mouse Genome Informatics site), inquiring whether anyone willing to share breeder stock has a certain strain live on the shelf. It may also be useful to contact laboratories that have published recently using the desired strains.

Analyzing Notch Signaling in Mice

7

59

Concluding Remarks The number and variety of genetic reagents and tools to study Notch signaling in mice has increased dramatically in recent years, and is sure to continue. The creation of large-scale international programs to generate and distribute mouse genetic resources [5, 6], the ability to reengineer these alleles [8, 9], and the development of novel techniques for genome engineering and generation of genetically engineered mice [65, 66] all promise to keep mouse biologists interested in studying the Notch pathway busy for many years to come.

References 1. Guruharsha KG, Kankel MW, ArtavanisTsakonas S (2012) The Notch signalling system: recent insights into the complexity of a conserved pathway. Nat Rev Genet 13:654–666 2. Ilagan MX, Kopan R (2007) SnapShot: Notch signaling pathway. Cell 128:1246 3. Kopan R (2012) Notch signaling. Cold Spring Harb Perspect Biol 4:a011213 4. Andersson ER, Sandberg R, Lendahl U (2011) Notch signaling: simplicity in design, versatility in function. Development 138:3593–3612 5. Bradley A, Anastassiadis K, Ayadi A et al (2012) The mammalian gene function resource: the International Knockout Mouse Consortium. Mamm Genome 23:580–586 6. Bucan M, Eppig JT, Brown S (2012) Mouse genomics programs and resources. Mamm Genome 23:479–489 7. White JK, Gerdin AK, Karp NA et al (2013) Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes. Cell 154:452–464 8. Osterwalder M, Galli A, Rosen B et al (2010) Dual RMCE for efficient re-engineering of mouse mutant alleles. Nat Methods 7:893–895 9. Schnütgen F, Ehrmann F, Poser I et al (2011) Resources for proteomics in mouse embryonic stem cells. Nat Methods 8:103–104 10. Gridley T (2010) Notch signaling in the vasculature. Curr Top Dev Biol 92:277–309 11. Sternberg N, Hamilton D (1981) Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol 150:467–486 12. Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 85:5166–5170

13. Gu H, Marth JD, Orban PC et al (1994) Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265:103–106 14. Kwan KM (2002) Conditional alleles in mice: practical considerations for tissue-specific knockouts. Genesis 32:49–62 15. Orban PC, Chui D, Marth JD (1992) Tissueand site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A 89: 6861–6865 16. Danielian PS, Muccino D, Rowitch DH et al (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol 8:1323–1326 17. Feil R, Brocard J, Mascrez B et al (1996) Ligandactivated site-specific recombination in mice. Proc Natl Acad Sci U S A 93:10887–10890 18. Feil R, Wagner J, Metzger D et al (1997) Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 237:752–757 19. Feil S, Valtcheva N, Feil R (2009) Inducible Cre mice. Methods Mol Biol 530:343–363 20. Hayashi S, McMahon AP (2002) Efficient recombination in diverse tissues by a tamoxifeninducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 244:305–318 21. Kellendonk C, Tronche F, Monaghan AP et al (1996) Regulation of Cre recombinase activity by the synthetic steroid RU 486. Nucleic Acids Res 24:1404–1411 22. Rose MF, Ahmad KA, Thaller C et al (2009) Excitatory neurons of the proprioceptive, interoceptive, and arousal hindbrain networks share a developmental requirement for Math1. Proc Natl Acad Sci U S A 106:22462–22467

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23. Dymecki SM, Ray RS, Kim JC (2010) Mapping cell fate and function using recombinase-based intersectional strategies. Methods Enzymol 477:183–213 24. Meyers EN, Lewandoski M, Martin GR (1998) An Fgf8 mutant allelic series generated by Creand Flp-mediated recombination. Nat Genet 18:136–141 25. D’Souza B, Meloty-Kapella L, Weinmaster G (2010) Canonical and non-canonical Notch ligands. Curr Top Dev Biol 92:73–129 26. Skarnes WC, Rosen B, West AP et al (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474:337–342 27. Tu L, Fang TC, Artis D et al (2005) Notch signaling is an important regulator of type 2 immunity. J Exp Med 202:1037–1042 28. Weng AP, Nam Y, Wolfe MS et al (2003) Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. Mol Cell Biol 23:655–664 29. Murray SA, Eppig JT, Smedley D et al (2012) Beyond knockouts: cre resources for conditional mutagenesis. Mamm Genome 23:587–599 30. Heffner CS, Herbert Pratt C, Babiuk RP et al (2012) Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource. Nat Commun 3:1218 31. Nagy A, Mar L, Watts G (2009) Creation and use of a cre recombinase transgenic database. Methods Mol Biol 530:365–378 32. Chandras C, Zouberakis M, Salimova E et al (2012) CreZOO—the European virtual repository of Cre and other targeted conditional driver strains. Database 2012:bas029 33. Bao J, Ma HY, Schuster A et al (2013) Incomplete cre-mediated excision leads to phenotypic differences between Stra8-iCre; Mov10l1(lox/lox) and Stra8-iCre; Mov10l1(lox/Delta) mice. Genesis 51:481–490 34. Cai T, Seymour ML, Zhang H et al (2013) Conditional deletion of Atoh1 reveals distinct critical periods for survival and function of hair cells in the organ of Corti. J Neurosci 33:10110–10122 35. Reinert RB, Kantz J, Misfeldt AA et al (2012) Tamoxifen-induced Cre-loxP recombination is prolonged in pancreatic islets of adult mice. PLoS One 7:e33529 36. Park EJ, Sun X, Nichol P et al (2008) System for tamoxifen-inducible expression of crerecombinase from the Foxa2 locus in mice. Dev Dyn 237:447–453 37. Leone DP, Genoud S, Atanasoski S et al (2003) Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells. Mol Cell Neurosci 22:430–440

38. Murtaugh LC, Stanger BZ, Kwan KM et al (2003) Notch signaling controls multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A 100:14920–14925 39. Varadkar PA, Kraman M, McCright B (2009) Generation of mice that conditionally express the activation domain of Notch2. Genesis 47:573–578 40. Oh P, Lobry C, Gao J et al (2013) In vivo mapping of Notch pathway activity in normal and stress hematopoiesis. Cell Stem Cell 13:190–204 41. Ong CT, Cheng HT, Chang LW et al (2006) Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability. J Biol Chem 281:5106–5119 42. Mizutani K, Yoon K, Dang L et al (2007) Differential Notch signalling distinguishes neural stem cells from intermediate progenitors. Nature 449:351–355 43. Souilhol C, Cormier S, Monet M et al (2006) Nas transgenic mouse line allows visualization of Notch pathway activity in vivo. Genesis 44:277–286 44. DasGupta R, Fuchs E (1999) Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126:4557–4568 45. Maretto S, Cordenonsi M, Dupont S et al (2003) Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci U S A 100: 3299–3304 46. Ohtsuka T, Imayoshi I, Shimojo H et al (2006) Visualization of embryonic neural stem cells using Hes promoters in transgenic mice. Mol Cell Neurosci 31:109–122 47. Basak O, Taylor V (2007) Identification of selfreplicating multipotent progenitors in the embryonic nervous system by high Notch activity and Hes5 expression. Eur J Neurosci 25:1006–1022 48. Imayoshi I, Sakamoto M, Yamaguchi M et al (2010) Essential roles of Notch signaling in maintenance of neural stem cells in developing and adult brains. J Neurosci 30:3489–3498 49. Fre S, Hannezo E, Sale S et al (2011) Notch lineages and activity in intestinal stem cells determined by a new set of knock-in mice. PLoS One 6:e25785 50. Sale S, Lafkas D, Artavanis-Tsakonas S (2013) Notch2 genetic fate mapping reveals two previously unrecognized mammary epithelial lineages. Nat Cell Biol 15:451–460 51. Nowotschin S, Xenopoulos P, Schrode N et al (2013) A bright single-cell resolution live imaging reporter of Notch signaling in the mouse. BMC Dev Biol 13:15

Analyzing Notch Signaling in Mice 52. Vooijs M, Ong CT, Hadland B et al (2007) Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE. Development 134:535–544 53. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71 54. Smith E, Claudinot S, Lehal R et al (2012) Generation and characterization of a Notch1 signaling-specific reporter mouse line. Genesis 50:700–710 55. Govindarajan V, Harrison WR, Xiao N et al (2005) Intracorneal positioning of the lens in Pax6-GAL4/VP16 transgenic mice. Mol Vis 11:876–886 56. Liu Z, Turkoz A, Jackson EN et al (2011) Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice. J Clin Invest 121:800–808 57. Liu Z, Obenauf AC, Speicher MR et al (2009) Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS). Genome Res 19:2081–2089 58. Liu Z, Chen S, Boyle S et al (2013) The extracellular domain of Notch2 increases its cell-surface abundance and ligand responsiveness during kidney development. Dev Cell 25:585–598 59. Morimoto M, Liu Z, Cheng HT et al (2010) Canonical Notch signaling in the developing

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lung is required for determination of arterial smooth muscle cells and selection of Clara versus ciliated cell fate. J Cell Sci 123:213–224 Liu Z, Liu Z, Walters BJ et al (2013) In vivo visualization of Notch1 proteolysis reveals the heterogeneity of Notch1 signaling activity in the mouse cochlea. PLoS One 8:e64903 Luche H, Weber O, Nageswara Rao T et al (2007) Faithful activation of an extra-bright red fluorescent protein in “knock-in” Crereporter mice ideally suited for lineage tracing studies. Eur J Immunol 37:43–53 Saito M, Iwawaki T, Taya C et al (2001) Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice. Nat Biotechnol 19:746–750 Kraman M, McCright B (2005) Functional conservation of Notch1 and Notch2 intracellular domains. FASEB J 19:1311–1313 Donahue LR, Hrabe de Angelis M, Hagn M et al (2012) Centralized mouse repositories. Mamm Genome 23:559–571 Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405 Wang H, Yang H, Shivalila CS et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918

Chapter 5 Immunohistochemical Tools and Techniques to Visualize Notch in Drosophila melanogaster Emiliana Tognon and Thomas Vaccari Abstract The ability to accurately visualize proteins in Drosophila tissues is critical for studying their abundance and localization relative to the morphology of cells during tissue development and homeostasis. Here we describe the procedure to visualize Notch localization in whole-mount preparations of several Drosophila organs using confocal microscopy. The use of monoclonal antibodies directed to distinct portions of Notch allows one to follow the fate of the receptor during constitutive and inductive processes. The protocol described here can be used to co-label with antibodies recognizing markers of subcellular compartments in wild-type as well as mutant tissues. Key words Notch localization, Drosophila melanogaster, Imaginal discs, Whole-mount immunohistochemistry, Confocal microscopy

1

Introduction Immunohistochemistry is the method of choice in modern cell biology to study localization and expression of proteins in fixed tissues. Most Drosophila tissues are suitable for immunohistochemical techniques, such as immunolabeling of antigens using fluorescent-conjugated antibodies. Immunofluorescence protocols require the use of primary antibodies specific to the protein of interest and a secondary antibody directly conjugated with a fluorochrome to reveal the signal [1]. In the case of Drosophila Notch, multiple primary antibodies can be used to detect its localization. However, due to their great signal-to-noise ratio and to broad availability, two mouse monoclonal anti-Notch antibodies are for the most part used for immunofluorescence. These recognize epitopes in the Notch extracellular domain (NECD) or the intracellular domain (NICD) [2, 3] (Fig. 1a–c). Immunofluorescence methods have the advantage of allowing labeling with multiple antibodies raised in different animals and secondary antibodies coupled with different fluorophores for

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_5, © Springer Science+Business Media New York 2014

63

Fig. 1 Examples of Notch localization in Drosophila tissue. (a, b) Immunolocalization of Notch using the NICD antibody in wing imaginal discs of a third instar larva in single confocal sections. Panel b is a high magnification of an area corresponding to the region boxed in a. (c–f′) Immunolocalization of Notch using the NECD

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65

simultaneous double, triple, or quadruple staining (Fig. 1d–g; see also Tables 1 and 2 for a list of antibodies used for Notch studies). It is thus possible to localize Notch relative to factors associated to its trafficking and activation. One can also co-stain with antibodies recognizing proteins with a known localization (Fig. 1d–f).

Table 1 List of antibodies used in Notch studies

Name of antibody

Source

Dilution and species

Epitope

Example of use

(a) Notch ligands Anti-delta (Dl)

[26]

1:3,000 Guinea pig

Extracellular

[27]

Anti-delta (Dl)

DSHB C594-9B Supernatant

1:100 Mouse monoclonal

Extracellular

[27]

Anti-serrate (Ser)

[28]

1:200 Rabbit

Extracellular

[27]

(b) Notch pathway components Anti-deltex

[29]

1:25 Rat

Deltex protein

[30]

Anti-neuralized (Neur)

[31]

1:1,000 Rabbit

Amino acids 11–360

[32]

Anti-neuralized (Neur)

[33]

1:600 Rabbit

Amino acids 368–523

[27]

Anti-numb (Numb)

[34]

1:1,000 Rabbit

Numb protein

[35]

Anti-mindbomb (D-mib)

[27]

1:100 Rabbit

CYNERKTDDSELPGN peptide

[27] (continued)

Fig. 1 (continued) antibody in eye imaginal discs of a third instar larva in single confocal sections. (d–f′) Show high magnification of the area corresponding to the region boxed in c. (e–f) Show Notch localization in discs mutant for the endosomal sorting component Hrs (hrsD28 mutant [22]); or the lysosomal component V-ATPase subunit A (vha68-1R6 mutant [23]). Discs have been co-stained to detect the early endosomal marker Avl [24]. Note that while in wild-type discs, intracellular Notch localized to late endosomes and thus rarely colocalizes with Avl (d–d′), in hrsD28 mutant cells Notch accumulates in Avl-positive endosomes [4], and in vha68-1R6 mutant cells it fails to be degraded in lysosomes, causing accumulation in enlarged Avl-negative late endosomes [23, 25]. Single channel images of Notch localization are shown in d′–f′. (g–g″′) Immunolocalization of Notch using the NICD antibody in single confocal sections of stage 5 and 6 mosaic egg chambers. Clones of hrsD28 mutant cells in the somatic follicular epithelium enwrapping germline cells are GFP negative and accumulate Notch intracellularly. (g′–g″′) Show single NICD, phalloidin, and GFP channels, respectively

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Emiliana Tognon and Thomas Vaccari

Table 1 (continued)

Epitope

Example of use

1:1,000 Rabbit

Amino acids 11–232

[35]

[36]

1:200 Rat

Amino acids 198–431

[35]

Anti-mastermind (Mam)

[37]

1:1,000 Rat

Amino acids 245–1,565 and 124–755

[38]

Anti-suppressor of hairless [Su(H)]

[39]

1:1,000 Rat

Amino acids 10-594

[27]

Anti-cut (Cut)

DSHB 2B10 Supernatant

1:100 Mouse monoclonal

Cut protein

[27]

Anti-hindsight (Hnt)

DSHB 1G9 Supernatant

1:25 Mouse monoclonal

Hindsight protein

[4]

Anti-wingless (Wg)

DSHB 4D4 Supernatant

1:10 Mouse monoclonal

Wingless protein

[40]

Name of antibody

Source

Anti-sanpodo (Spdo)

[36]

Anti-sanpodo (Spdo)

Dilution and species

(c) Notch target genes

(d) Cell fate determinants associated with Notch signaling Anti-scute (Sc)

[41]

1:200 Rabbit

Proneural clusters

[40]

Anti-senseless (Sens)

[42]

1:1,000 Guinea pig

Sensory organ precursors

[40]

Anti-elav

DSHB 7E8A19 Supernatant

1:500 Rat Monoclonal

Neurons in sensory organ precursor (nuclei)

[35]

Anti-futsch

DSHB 22C10 Supernatant

1:100 Mouse monoclonal

Neurons in sensory organ precursor (membrane)

[43]

Anti-prospero (Pros)

DSHB MR1a Supernatant

1:2 Mouse monoclonal

Sheath cells in sensory organ precursors

[44]

The table provides sources, experimental conditions, and recent examples of use of a selected list of the most used antibodies that recognize Notch associated proteins DSHB Developmental Studies Hybridoma Bank, University of Iowa

Notch Localization in D. melanogaster Tissues

67

Table 2 List of antibodies used to label vesicular trafficking compartments

Name of antibody

Source

Dilution and species

Marks

Example of use

(a) Endocytic proteins Anti-avalanche/syntaxin 7 (Avl)

[24]

1:100 Rabbit

Early endosome

[45]

Anti-Rab5

ab31261 Abcam

1:500 Rabbit

Early endosome

[46]

Anti-Rab5

[47]

1:200 Rabbit

Early endosome

[47]

Anti-smad anchor for receptor activation (Sara)

[48]

n.d. Rabbit

SARA endosome

[32]

Anti-hepatocyte growth factor-regulated tyrosine substrate (Hrs)

[22]

1:600 Guinea pig

Sorting endosome

[27]

Anti-vacuolar protein sorting 2 (Vps2)

[46]

1:500 Guinea pig

Late endosome

[46]

Anti-Rab7

[49]

1:5,000 Rabbit

Late endosome

[46]

Anti-Rab11

[50]

1:1,000 Rat

Recycling endosome

[35]

(b) Plasma membrane polarity proteins Anti-atypical protein kinase C (aPKC)

Santa Cruz sc-216

1:100 Rabbit

Apical Par complex

[45]

Anti-crumbs (Crb)

DSHB CQ4 Supernatant

1:20 Mouse monoclonal

Subapical Crumbs complex

[51]

Anti-armadillo (Arm)

DSHB N2-7A1 Supernatant

1:100 Mouse monoclonal

Adherens junctions

[40]

Anti-DE-cadherin (DE-Cad)

DSHB DCAD2 Supernatant

1:25 Rat Monoclonal

Adherens junctions

[45]

Anti-discs large (Dlg)

DSHB 4F3 Supernatant

1:100 Mouse monoclonal

Lateral membrane

[45]

1:1,000 Mouse

Mono- and polyubiquitinylated conjugates

[45]

(c) Other membrane compartment components Anti-ubiquitinylated protein (FK2)

Enzo life sciences BML-PW8819

(continued)

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Emiliana Tognon and Thomas Vaccari

Table 2 (continued)

Marks

Example of use

1:1,000 Guinea pig

Cis-Golgi apparatus

[35]

Calbiochema

1:100 Mouse

Medial-Golgi apparatus [53]

[54]

1:2,000 Guinea pig

Exocyst complex

Name of antibody

Source

Anti-lava lamp (Lva)

[52]

Anti-GP120 Anti-Sec15

Dilution and species

[35]

The table provides sources, experimental conditions, and recent examples of use of a selected list of the most used antibodies to label vesicular trafficking compartments DSHB Developmental Studies Hybridoma Bank, University of Iowa a No longer commercially available

Such established subcellular markers provide useful points of reference for determination of Notch localization. In addition, a host of fluorescent compounds or compounds directly conjugated to fluorophores can be used to identify a number of cell structures, such as the nuclei or the actin cytoskeleton, with respect to Notch (Fig. 1g). One of the challenges of immunolabeling with multiple antibodies and fluorescent compounds is to find the right parameters to retain good preservation of the morphology of the tissue after fixation and permeabilization, while allowing good penetration of the antibodies throughout the tissue. This is crucial in the case of Notch, a transmembrane protein associated with membranes when inactive, and cytoplasmic and nuclear when active [4–8]. The protocol presented below ensures good preservation of tissues and optimal penetration of antibodies, as indicated by the correspondence between the localization obtained by immunodetection and the localization displayed by GFP-tagged functional forms of Notch [9]. The purpose of this chapter is to explain in detail the immunohistochemical procedure optimized to detect Notch localization in whole-mount preparations of a number of commonly studied Drosophila tissues by florescent confocal microscopy. The procedure is currently used in our lab and in most other labs that study Notch localization relative to the plasma membrane and the trafficking machinery of the cell, two locales that are increasingly found to determine and control signaling activation [10]. In the following sections, we provide materials and methods for sample fixation, immunostaining, and mounting. Due to the impossibility of providing detailed preparation procedures for the many Drosophila cell types, tissues, and organs amenable to immunolabeling with anti-Notch antibodies, we describe schematically only

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the dissection and whole-mount preparation of three organs as an example (larval imaginal discs, pupal nota, and adult ovaries), and refer the reader to other sources for detailed descriptions, and for dissections of other organs. Similarly, given the wide diversity of microscopy setups employed in cell biology labs, we do not provide guidelines for image acquisition.

2

Materials

2.1 Equipment for Sample Dissection

1. Two pair of Dumont forceps #5 and #55.

2.1.1 Imaginal Discs and Ovaries Dissection

3. Glass Pasteur pipettes, 150 mm.

2. Glass watch for dissection (Corning or similar). 4. Gentle specimen mixer for microcentrifuge tubes (BD Adams Nutator or similar). 5. Dissecting stereomicroscope. 6. Directed light source (ideally, with dual gooseneck light guides).

2.1.2 Equipment for Pupal Dissection

1. Cohan-Vannas spring scissors, 5 mm straight blade. 2. Stainless steel Minutien Pins, 0.1 mm diameter. 3. Sylgard® 184 silicone elastomer (Dow Corning), cured in a 60 × 15 mm polystyrene petri dish.

2.2

Solutions

2.2.1 Fixation Solution

4 % (v/v) paraformaldehyde (PFA) in phosphate-buffered saline pH 7.4 (PBS).

2.2.2 Washing Solution

0.1 % Triton X-100 in 1× PBS (PBT).

2.2.3 Blocking Solution

5 % (w/v) bovine serum albumin (BSA) in PBT.

2.2.4 Primary Antibody

Primary antibody at appropriate dilution in blocking solution.

Notch Antibodies

Mouse monoclonal anti-Notch intracellular domain (NICD) C17.9C6 (Developmental Studies Hybridoma Bank; Fig. 1a, b, g) [3]. Mouse monoclonal anti-Notch extracellular domain, EGF repeats # 12-20 (NECD), C458.2H (Developmental Studies Hybridoma Bank; Fig. 1c–f) [2].

2.2.5 Secondary Antibody

Fluorescently conjugated secondary antibody at appropriate dilution in PBT (e.g., Alexa Fluor conjugated polyclonal antibodies).

2.2.6 Other Markers (Optional)

1. DNA stain: DAPI (4′,6-diamidino-2-phenylindole), 100× solution. 2. F-actin stain: Phalloidin-TRITC (tetramethylrhodamine B isothiocyanate).

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2.3 Storage, Mounting, and Viewing of Samples

1. Mounting medium: 1.5 % DABCO (1,4-diazabicyclo[2.2.2] octane) and 70 % glycerol in 1× PBS. 2. Microscope slides. 3. Coverslips. 4. Nail polish fast dry. 5. Confocal microscope.

3

Methods

3.1 Preparation of the Solutions for Immunostaining

To prepare the 4 % PFA solution, dilute 10 % PFA in 1× PBS. To make 100 mL solution add 40 mL 10 % PFA with 60 mL 1× PBS (see Note 1).

3.1.1 Preparation of the Fixation Solution 3.1.2 Preparation of the Washing Solution

To prepare the PBT solution, dilute 1 mL of TritonX-100 in 1 L 1× PBS. Store at room temperature (see Note 2).

3.1.3 Preparation of the Blocking Solution

To prepare the blocking solution, dissolve 0.5 g BSA in 10 mL PBT (see Note 3).

3.1.4 Preparation of the Primary Antibody Solution

Dilute the primary antibody in freshly prepared blocking solution (see Note 4). When using anti-NICD dilute 1:40, or when using anti-NECD dilute 1:25 from the supernatants supplied by DSHB. Supernatant batches may vary in antibody abundance; thus it is useful to test new batches to find the appropriate dilutions.

3.1.5 Preparation of the Secondary Antibody Solution

Dilute the secondary antibody in PBT. Discard it after use. Be careful to match the secondary antiserum to the species of the primary antisera (see Note 5).

3.1.6 Preparation of the Mounting Medium

For 50 mL solution, add 0.75 g DABCO, 15 mL 1× PBS, and 35 mL glycerol and mix on rocking platform until the solution is homogeneous. Aliquot in 1.5 mL tubes and store at −20 °C (see Note 6).

3.2 Dissection of Organs 3.2.1 Dissection of Larval Imaginal Discs

1. Dissection is performed in 1× PBS on a glass well under the dissecting scope with side light (see Note 7). 2. The imaginal discs are located on the anterior portion of the larva, near the mouth hooks (see Note 8). Tear the larva in half and discard the posterior. Invert the anterior like a sleeve by pushing in delicately on the mouth hooks with the #55 forceps. Hold the body steady near the severed part with the #5 forceps. The wing and haltere discs are attached to tracheolae that branch

Notch Localization in D. melanogaster Tissues

71

off the two main tracheal tubes running below the cuticle along the body wall. The eye-antennal discs are located between the surface of the optical lobe of the larval brain and the mouth parts. (For dissection of other larval organs see Note 9). 3. Imaginal discs are kept attached to carcasses to facilitate handling. To prepare carcasses for fixation, clean them of the gut, fat tissue, and salivary glands using the #55 forceps while holding them with the #5 forceps. 4. Transfer carcasses to a 1.5 mL tube filled with 1× PBS. Dissect 10–12 larvae per genotype within no more than 20 min and proceed to fixation. For a detailed video description of dissection of imaginal discs see [11]. 3.2.2 Dissection of Pupal Nota

1. Collect pupae at the desired developmental time point (see Note 10). 2. The pupal case retains the overall shape of the larva, with mouth hooks at the anterior and spiracles on the posterior. Place the pupa with mouth hooks to the front, dorsal side up, on a petri dish half filled with polymerized silicone. Secure to the dish with a Minutien pin through the abdomen. 3. Add sufficient 1× PBS to cover the pupa. 4. Cut off the pupal spiracles at the posterior and split the pupal case longitudinally by slicing up the abdomen with a micro-scissor. 5. Using the forceps, gently peel back the pupal case. 6. On the anterior side, remove the head with the micro-scissors and insert the scissors into the opening you have made in the pupal case. Cut the lateral sides of the pupa down to the abdomen. Then cut off and remove the ventral side of the animal. Trim away unnecessary tissue. 7. Remove the fat body and gut by replacing the 1× PBS. 8. Remove the remaining fat bodies and the trachea that adhere to the notum with #55 forceps. 9. Immediately add the fixative directly in the petri dish using a glass pipet. Make sure that nota do not dry out. For detailed descriptions of pupal notum dissections, see [12, 13].

3.2.3 Dissection of Adult Ovaries

1. Feed mated flies in well-yeasted tubes or bottles to engorge the ovaries for 1–2 days prior dissection (see Note 11). 2. Anesthetize the flies (see Note 12). 3. Using a pair of forceps hold a female fly by the upper part of the abdomen, and pull the tip of the abdomen out with the other forceps. Internal organs including the gut, the two ovaries, and the oviducts will be exposed. Detach the pair of ovaries from other organs and collect them in a 0.5 mL tube

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containing 1× PBS while dissecting the next fly. It is better to dissect 15–20 flies over a period of no longer than 20 min to avoid tissue deterioration. Proceed with fixative and immunostaining protocol. For a detailed video description on how to dissect ovaries, see [14]. 3.3 Immunohistochemistry

1. Transfer the carcasses to 1.5 mL tube filled with 1× PBS (for pupal notum see Note 13).

3.3.1 Tissue Fixation

2. Remove as much as PBS as possible and replace with fixative (up to 450 μL). 3. Place the 1.5 mL tube onto a nutator for 20–30 min at room temperature. 4. Remove the tube from the nutator and allow the carcasses to settle to the bottom of the tube before removing the fixative. 5. After removal of the fixative, add PBT to the tube and place it on the nutator to rinse the tissues for 5–10 min. Repeat this step 3 times to remove all traces of fixative (see Note 14).

3.3.2 Blocking and Antibody Incubations

1. Remove the PBT and add blocking solution. Block carcasses at least 30 min at room temperature (see Note 15). 2. Remove blocking solution and add the desired mouse antiNotch antibody diluted as described above. Also add any nonmouse primary antibody with which you wish to co-stain (concentrations depend on the antisera used; see Note 16). 3. Incubate primary antibody overnight on the nutator at 4 °C or 2 h at room temperature (see Note 17). 4. Remove the primary antibody and store it at 4 °C if needed. Primary antibody can be reused. 5. Wash tissues 3 times in PBT for 5–10 min each. 6. Remove the PBT wash and add the fluorophore-conjugated mouse secondary antibody diluted in PBT. Other secondary antibodies can be added to match the primary antibodies used. At this step, together with the secondary antibody it is possible to add fluorophore-conjugated phalloidin, such as phalloidinTRITC, to mark F-actin and visualize the overall morphology of cells. 7. Incubate for 2 h at room temperature on the nutator in the dark. 8. Wash tissues 3 times for 10 min each with PBT. 9. (Optional) Perform a nuclear counter stain by incubating for 10 min with PBT solution containing 1× DAPI in the dark. 10. Wash tissues once in PBT for 10 min.

Notch Localization in D. melanogaster Tissues

3.4

Mounting

73

1. Transfer carcasses to the slide.

3.4.1 Fine Dissection of Organs and Transfer to Slides

2. Blot excess liquid with paper tissue or absorbing paper.

Wing Discs

4. Using fine forceps, carefully remove the imaginal discs from the carcasses. Wing discs appear as pear-shaped organs flapping on either side of the carcass, and should be gently detached from the tracheolae, which hold them. Avoid damaging the discs with the forceps.

Eye Antennal Discs

1. To remove eye-antennal imaginal discs, gently rip the nerves connecting the ventral ganglion with the carcass wall by sliding the forceps tips between them.

3. Quickly add a couple of drops of mounting medium on the tissue using a Pasteur pipette.

2. Grab the base of the mouth hooks with one forceps and pull the mouth parts away from the rest of the body with the other forceps. Eye-antennal imaginal discs and brain will be removed from the carcass as a single mass, together with the mouth hooks. To sever the brain from the eye-antennal discs, use one forceps to carefully pinch the nerve connecting each optic lobe to its disc. 3. Transfer discs to the slide by holding them by the attached mouth hooks. 4. Blot excess liquid with paper tissue or absorbing paper. 5. Quickly add a couple of drops of mounting medium on the tissue using a Pasteur pipette. 6. Detach the eye-antennal discs from the mouth hooks by pinching the narrow connection between the antennal disc and the mouth hooks. Discard the mouth hooks. Mounting Pupal Nota

1. Gently transfer the nota onto the slide using forceps. 2. Blot excess liquid with paper tissue or absorbing paper. 3. Quickly add a couple of drops of mounting medium on the tissue using a Pasteur pipette. 4. Orient them apical domain up (cuticle is glossy and should face the slide, while the epithelium is matte and should face up).

Mounting Adult Ovaries

1. Allow the ovaries to settle to the bottom of the tube. 2. Remove the most of the PBS or washing solution. 3. Add a couple of drops of mounting medium to the tube using a Pasteur pipette. 4. Dissociate ovaries into ovarioles and individual egg chambers by pipetting up and down several times, using a 200 μL pipet tip. Gently transfer the egg chambers by delicate resuspension to the slide using a glass pipet.

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Emiliana Tognon and Thomas Vaccari

3.4.2 Sealing of the Slides and Imaging

1. Make sure that the tissue is flat and unwanted tissue parts are removed from the slide, before covering with a coverslip. In particular, parts thicker than the tissue to be analyzed should be removed to avoid excess spacing between slide and coverslip which creates movement/vibration of the sample during imaging. Conversely, when z dimensions need to be preserved (i.e., for z-confocal sectioning), a Dakopen or other hydrophobic barrier marker can be used to ensure appropriate spacing between slide and coverslip. 2. Seal the edges with nail polish. Store the slide at 4 °C in the dark. 3. When using Mowiol-containing medium (see Note 6), allow 12–24 h before imaging to ensure hardening of the resin. 4. Analyze the sample at a fluorescent microscope. Whole-mount preparations are perfectly suited for confocal microscopy.

4

Notes 1. The PFA solution is essential to fix tissues by preserving the physical structures and preventing digestion by enzymes and bacteria. The PFA solution is toxic and it should be carefully manipulated under the hood. PFA is a suspected carcinogen and should be handled as such with appropriate PPE. Aliquot in 2 mL tubes and store at −20 °C where it is stable for few months. 2. Pure Triton X-100 is very viscous. 10 mL of a 10 % solution can be used to reduce pipetting errors due to viscosity. A low detergent concentration preserves membrane structures such as small endocytic vesicles for imaging. Moreover, a harsh treatment with detergents might affect tissue integrity. However, tissues such as ovaries and some antibodies may require high levels of detergents to permeabilize the membrane. 3. Other blocking reagents such as 5 % normal goat serum in PBT might be used to reduce background staining. Store both reagents at 4 °C for a short period of time. 4. Antibodies that bind different Drosophila antigens in addition to Notch can be used as long as they are not raised in mouse (Fig. 1d–f; Tables 1 and 2). In order to reduce resulting aspecific staining it is possible to pre-adsorb primary antisera. To this end, sera can be pre-adsorbed to fixed tissues which do not express the desired antigen. If this is not available, antigenexpressing tissue can be used. 5. Fluorescent labels for the secondary antibodies can be chosen depending on the availability of the microscope lasers. Note that the staining protocol preserves the properties of the fluorescently tagged proteins, a very important feature when one

Notch Localization in D. melanogaster Tissues

75

wants to immunolocalize Notch in tissues expressing luminescent proteins such as GFP (Fig. 1g). 6. DABCO is an antifade reagent. It acts as a reactive oxygen species scavenger and it prevents the bleaching process. The mounting medium can be supplemented with a clariant resin. In our lab, we use also Mowiol® 4-88 (Sigma-Aldrich). Mowiolcontaining mounting medium hardens and has the same refractive index as the immersion oil. The inclusion of Mowiol in the mounting medium depends on the experiment. For colocalization experiments it is better to mount the sample in glycerol since it maintains the 3D conformation of the tissue, while the hardening properties of Mowiol help long-term storage of samples. Commercial preparations are also available which achieve similar effects as the DABCO/Mowiol mixture. 7. By positioning incident light perpendicular to the path of view (side lighting), the contrast between the tissues and the environment increases and identification of the different tissues becomes easier. 8. The dissection protocol for imaginal discs refers to third instar (L3) larvae. For general fly husbandry and for protocols to obtain L3 larvae please refer to [15]. 9. Other organs that can be harvested for fixation and anti-Notch staining in larvae are the optic lobes and ventral ganglion, the salivary glands, the gut, the lymph gland, and the hemocytes. Detailed dissection protocols and examples of immunolocalizations are described in [16–21]. 10. The appearance of immobile white pupae marks the transition between larval and pupal stages and they are considered the 0 h time point of puparium formation. Using a paintbrush, one can place the white pupae into a new vial and wait until the pupae reach the desired developmental stage for dissection. 11. This procedure is important to maximize egg chamber production in female flies. 12. To anesthetize the flies use carbon dioxide. CO2 keeps the flies asleep during manipulation. Anesthesia longer than 10–15 min is lethal. If flies need to be recovered, keep manipulations under CO2 to a minimum. 13. For pupal nota, antibody staining is not performed in 1.5 mL tube but rather in a humid chamber to avoid dehydration. Transfer the notum in a glass dish filled with PBT and then proceed directly to incubation with primary antibodies. The blocking step is usually not required. Incubation in the primary antibody for 90 min can be also sufficient. 14. Fixed tissue can be stored at 4 °C for a week to 10 days at this stage. In this case, wash and store with 1× PBS instead of PBT.

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15. Pretreatment with 1× PBS, 1 % Triton X-100 for 30 min or 1 h may be needed to ameliorate the permeabilization of the membrane and increase the penetration of some antibodies. However, due to extraction of soluble proteins this treatment may reduce detection of some antigens. 16. The number of antibodies is dictated ultimately by nature of the lasers on your confocal system. In a typical setup up to three different primary antibodies can be used (Fig. 1g). 17. Overnight incubations return a sharper signal and allow the staining experiment to cover 2 days, with an image acquisition session in the afternoon of day 2.

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Drosophila germ plasm assembly. Development 135:1107–1117 Dollar G, Struckhoff E, Michaud J et al (2002) Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation. Development 129:517–526 Wang W, Li Y, Zhou L et al (2011) Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe. Biochem Biophys Res Commun 410:714–720 Sisson JC, Field C, Ventura R et al (2000) Lava Lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization. J Cell Biol 151:905–918 Emery G, Hutterer A, Berdnik D et al (2005) Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system. Cell 122:763–773 Mehta SQ, Hiesinger PR, Beronja S et al (2005) Mutations in Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst components. Neuron 46:219–232

Chapter 6 Antibody Uptake Assay and In Vivo Imaging to Study Intracellular Trafficking of Notch and Delta in Drosophila Lydie Couturier and François Schweisguth Abstract Notch signaling depends on regulated intracellular trafficking of the receptor and its ligands (Kopan and Ilagan, Cell 137:216–233, 2009; Le Borgne et al., Development 132:1751–1762, 2005). Here we describe two methods to study the intracellular trafficking of Notch and Delta in Drosophila. First, an ex vivo antibody uptake assay is used to monitor endocytosis of Notch and Delta by living cells in dissected explants (Le Borgne and Schweisguth, Dev Cell 5:139–148, 2003). Second, real-time imaging of fluorescent proteins that are expressed at physiological levels is used to study trafficking of Notch in living flies (Venken et al., Science 314:1747–1751, 2006; Couturier et al., Nat Cell Biol 14, 131–139, 2012). Key words Live imaging, Antibody uptake, Endocytosis assay, Notum dissection, Drosophila

1

Introduction Notch regulates binary fate decisions in various developmental contexts, including asymmetric cell divisions [1, 2]. In this environment, generation of cell fate diversity and self-renewal of stem cells may rely on the unequal segregation of regulators of the intracellular trafficking of Notch and Delta. In Drosophila, each bristle sensory organ consists of four distinct cells that are generated via a series of stereotyped asymmetric divisions from a single Sensory Organ Precursor cell (SOP) [3]. At pupal stages (during metamorphosis), SOPs divide asymmetrically to generate an anterior pIIb cell and a posterior pIIa cell in the notum (a single-layered epithelium forming the dorsal thorax). The binary pIIa versus pIIb decision is regulated by Notch; inhibition of Notch specifies pIIb whereas activation of Notch leads to pIIa specification. Two cell fate determinants, Numb and Neuralized (Neur), localize at the anterior cortex of mitotic SOPs, are specifically inherited by pIIb cells and act in parallel to set up directional Notch signaling [4, 5]. Numb has been proposed to inhibit the recycling of Notch indirectly via its interaction with Sanpodo (Spdo), a four-pass transmembrane

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protein that regulates Notch endocytosis [6–8]. The E3 ubiquitin ligase Neur regulates ubiquitin-dependent endocytosis and activity of the ligand Delta (Dl), thereby activating Notch in pIIa [4, 9]. To further elucidate the activity of Notch and its ligands during cell fate determination, we have developed two assays. The first (Subheading 3.1) is an antibody uptake assay which allows one to study endocytosis, i.e., internalization, of Notch and Dl in the pupal notum. Briefly, the single-layered epithelium corresponding to the pupal notum is dissected and cultured in presence of antibodies recognizing an extracellular epitope of Dl or Notch. Following medium changes and fixation, the uptake of anti-Dl or anti-Notch antibodies is revealed using secondary antibodies. Thus, this ex vivo assay monitors the internalization of Notch and its ligand Dl [4, 6] and can be used to genetically study the regulation of Notch and Dl endocytosis [10–12]. The second (Subheading 3.2) allows one to study the intracellular trafficking of Notch in living flies, using GFP-tagged versions of Notch and its regulators Numb and Spdo that we have generated [6, 7, 13]. Here, we describe how to study Notch intracellular trafficking by live imaging in Drosophila with these flies.

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Materials

2.1 Materials for Method A: Antibody Uptake Assay to Monitor the Trafficking of Notch and Its Ligand Delta in Drosophila

1. Dissection Petri dishes layered with Rhodorsil RTV-2 silicone (or similar cured silicone elastomer such as Sylgard®). 2. Dissection tools: Dumont #5 forceps, Vannas micro-scissors (75 mm straight), fine Minutien pins (0.15 mm diameter). 3. Binocular stereomicroscope and confocal microscope. 4. Small glass incubation dishes. 5. Schneider’s Drosophila medium containing 1 % fetal calf serum. 6. PBS 10× to prepare PBT (PBS 1× with 0.1 % Triton X100). 7. 4 % paraformaldehyde (PFA) in PBS 1× (1 ml aliquots kept at −20 °C; aliquots prepared from PFA powder). 8. Primary antibodies: mouse monoclonal anti-DeltaECD (C594.9B) directed against the extracellular domain of Dl [14] and anti-NotchECD (C458.2H) directed against EGF repeats 12–20 of the extracellular domain of Notch [15]; concentrates obtained from the Developmental Studies Hybridoma Bank, University of Iowa. 9. Fluorescently conjugated anti-mouse secondary antibodies. 10. Prepare 50 % glycerol (v/v), 50 % (v/v) PBS 1× solution. 11. Mounting medium: 20 % (v/v) PBS 1×, 80 % (v/v) glycerol, 2 % (w/v) N-propyl-galate (Sigma).

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1. Transgenic flies carrying P{acman} Bacterial Artificial Chromosome (BAC) transgenes encoding GFP-tagged Notch (N-iGFP, Notch intracellular Green Fluorescent Protein), Numb (Numb-GFP), or Spdo (Spdo-iGFP). These transgenes were obtained by modifying BAC clones generated by Venken and colleagues [16]. They encode fully active proteins expressed at physiological levels, as demonstrated using a genomic rescue assay [6, 7] (see also Chapter 3). 2. Transgenic flies carrying RFP-tagged markers that are expressed specifically in SOPs, such as neur-PH-RFP (expressing the PIP2 binding domain of PLCγ1 fused to mRFP1 under the control of a neur SOP-specific enhancer) or neur-H2B-RFP (expressing Histone2B fused to mRFP1 in SOPs) [6, 7]. 3. Forceps (Dumont #5), Vannas micro-scissors (75 mm straight), fine Minutien pins (0.15 mm diameter). 4. Binocular stereomicroscope (for dissection); fluorescent microscope or confocal microscope (for imaging). 5. Small Petri dishes (diameter 55 mm) and temperaturecontrolled fly incubator (25 °C). 6. Standard microscope slides. 7. Custom-made metallic spacers (10 × 10 × 0.7 mm); can be replaced by a stack of 4–5 standard coverslips (0.17 mm) glued together using nail polish. 8. Double-sided tape. 9. Voltalef oil 10S (VWR #24627-188); other high viscosity Halocarbon oil can also be used.

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Methods

3.1 Antibody Uptake Assay to Monitor the Trafficking of Notch and Its Ligand Delta in Drosophila 3.1.1 Selection and Staging of Pupae

3.1.2 Dissection of Staged Pupae

All steps are performed at room temperature unless otherwise specified. 1. Select pupae of the appropriate genotype at the onset of metamorphosis, i.e., at 0 h After Puparium Formation (0 h APF) by visual inspection of the vial every 30 min (check for eversion of the anterior spiracles in immobile larvae). Soft white pre-pupae can be gently removed from the vial using a soft-bristled brush. 2. Place collected pre-pupae in a petri dish with a moistened tissue paper and place in an incubator: SOPs divide around 16.5 h APF at 25 °C, at 24 h APF at 21 °C, and at 33 h APF at 18 °C. 1. Transfer staged pupae to the dissecting dish ventral side up. 2. Pin down pupae by inserting two fine needles through the abdomen (two needles are necessary to prevent rotation).

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3. Cover pupae with Schneider’s Drosophila medium containing 1 % fetal calf serum. 4. Using the micro-scissors, cut the anterior tip of the pupal case. Carefully insert one blade of the micro-scissor between the body of the immature fly and the pupal case and make a medial cut into the pupal case (along the ventral midline). Using forceps, tear and remove the pupal case. The body of the immature fly should then be free of the pupal case except over its abdomen [17]. 5. Using the micro-scissors, cut through the pupal head at the level of the eyes to remove the anterior part (from now on, pieces of tissue floating in the pupal body cavity will escape). 6. Carefully insert one blade of the micro-scissor inside the head opening, make a cut laterally at the level of the developing wing down to the abdomen, and repeat on the contralateral side. 7. Lift the ventral part of the immature fly and detach it by making a transverse cut at its base, i.e., at the level of the abdomen. 8. Using a Pasteur pipette, gently flush once the inside of the pupa. Speed (hence practice) is the trick, so dissect and process pupae one at a time. 3.1.3 Antibody Uptake Assay

1. Remove the medium and replace it with fresh medium containing the anti-DeltaECD (or anti-NotchECD) antibody (1:10– 1:100) to perform the antibody uptake assay of the freshly dissected notum (see Note 1). 1 ml of antibody solution is required to cover the dissected pupa in the dissecting dish. Alternatively, particularly if the antibody is precious, it is possible to wipe out the dissecting dish around the dissected pupa and to cover the pupa with a 0.1 ml drop of antibody solution. 2. Place the dissecting dish in a humidified box and incubate at 25 °C for the desired period (5–30 min) (see Note 2). Internalization of Dl can be detected within 3 min and a strong signal can be measured within 10 min in SOPs. In our hands, the internalization of Notch is less efficient than Delta and longer incubation (15 min) is required to detect the internalization of Notch. 3. Optional: the antibody-containing medium can be replaced by fresh medium for a defined period of chase (5–30 min) prior to fixation. 4. Remove the antibody-containing medium and wash once with Schneider’s Drosophila medium. 5. Fix the pupa directly in the dissecting dish for 20 min under a chemical fume hood by adding 1 ml of fixative.

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6. Wash 3 × 5 min in PBT. 7. Complete the notum dissection by trimming down the notum on both sides (dorsal to the pupal wings) using micro-scissors to keep only the dorso-central part of the dorsal thorax (see Note 3). Cut the notum away from the abdomen and transfer it using forceps into a small glass incubation dish. To avoid damaging the notum, grasp the dissected tissue by the head part. 8. To evaluate variability in the uptake, repeat the internalization of anti-DlECD (or anti-NECD) with 5–10 nota. Pool these nota after the post-fixation dissection step and before proceeding to the antibody detection step below. 9. To detect internalized mouse monoclonal antibodies, incubate the notum for 60 min with fluorescently labeled antimouse secondary antibodies (1:2,000) in PBT on a rotating platform. 10. Optional: to compare the distribution of internalized antiDlECD with endogenous Dl, primary guinea-pig anti-Dl antibodies (1:2,000; obtained from M. Muskavitch) [18] can be combined with the fluorescently labeled anti-mouse secondary antibodies. If so, proceed, and an additional secondary antibody incubation step will be required. 11. Optional: appropriate primary antibodies against sensory cell and/or endosome markers can also be combined with the fluorescently labeled anti-mouse secondary antibodies. If so, proceed, and an additional secondary antibody incubation step will be required. 12. Wash 3 × 10 min in PBT. 13. Wash and equilibrate in 50 % glycerol in PBS 1×. 14. Using forceps, transfer the nota into a drop of mounting medium deposited on a standard microscope slide, cover with coverslip and seal. 15. Under the binocular scope, mount nota dorsal up (i.e., apical up for the notum epithelium). 16. Analyze the results by confocal microscopy. 3.2 In Vivo Imaging of Intracellular Trafficking of Notch Using a GFP-Tagged Protein in Drosophila 3.2.1 Mounting Staged Pupae for Live Imaging

1. Place a piece of double-sided tape onto the microscope slide. Transfer pupae (staged as in Subheading 3.1.1; now brown and hard) from the petri dish and adhere the pupal case to the double-sided tape with the ventral side down (see Note 4). 2. Using forceps, grasp the anterior edge of the operculum (anterior-dorsal tip of the pupal case, just above the head of the immature fly), lift and remove. Carefully insert one blade of the micro-scissor between the body of the immature fly (at the level of the humerus) and the pupal case and make a cut into the pupal case. Tear apart the fragments of the pupal case

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covering the dorsal thorax (notum) using the forceps and/or micro-scissor. Stick the removed pieces onto the double-sided tape. It is not necessary to remove the part of the pupal case that covers the abdomen. Be careful not to puncture the pupal body as it is soft and very fragile. 3. Place spacers on the double-sided tape on both sides of the pupae. 4. Using a Pasteur pipette, spread a small amount of Voltalef oil into a thin layer covering a coverslip (see Note 5). Deposit the coverslip onto the spacers, oiled face down. Check that the notum is in direct contact with the coverslip. Tape the coverslip onto the slides using regular tape. Pupae are now ready for imaging. 3.2.2 Live Imaging

1. Perform live imaging at 20±2 °C using a microscope equipped with a 63× objective (HCX PL APO CS, N.A. 1.3). Inverted or upright microscopes may be used. Also, scanning or spinning disk confocal microscopes may be used. Using a PerkinElmer UltraVox Spinning Disk equipped with a CSU-X1 disk on an inverted Zeiss microscope, 488 and 561 lasers and two Hamamatsu ImagEM EM-CCD cameras for simultaneous acquisition in the green and red channels (see Note 6), movies of dividing SOPs and or pIIa/pIIb cell pairs can be obtained with high spatial and temporal resolution by acquiring a stack of 15–20 images with a Δz of 0.3 mm and an acquisition time of 100 ms every 3–5 s. Under these conditions, time-lapse series of 100–150 stacks can be acquired with reduced bleaching. Imaging conditions need to be adapted to the intensity of fluorescence as well as the size and mobility speed of the objects that are tracked over time. Microscope control and image acquisition is driven under Volocity. Fiji/ImageJ is used for post-acquisition processing of the images. 2. Following imaging, the microscope slide with the imaged pupae can be transferred into a humidified chamber to let pupae develop into adult flies. If adult flies need to be recovered, remove the coverslip and double-sided tape around the pupae.

4

Notes 1. The antibody uptake assay can be adapted to monitor the internalization of anti-DlECD/NECD antibodies in real time by forming in vitro complexes between anti-DlECD/NECD and fluorescently labeled anti-mouse F(ab′)2 fragments [19]. Additionally, the time-course of internalization can be studied by incubating the notum sequentially with two anti-DlECD/ anti-mouse F(ab′)2 fragments solutions that differ only by the

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fluorochrome coupled to the F(ab′)2 fragments (our unpublished results). 2. A 4 °C incubation step is not required for cell surface binding of the antibody prior to its internalization. A 4 °C incubation step may actually promote the antibody-mediated clustering of epitopes, thereby promoting their internalization. 3. The antibody uptake assay can be applied to genetically mosaic flies carrying clones of mutant cells, thereby permitting a genetic analysis of the process of Notch and/or Dl internalization. 4. Using fully functional tagged proteins expressed at physiological levels is highly desirable, since trafficking routes may depend on the association/dissociation kinetics of components of the endocytic machinery and, therefore, on cargo concentration. Overexpression of fluorescently tagged proteins should therefore be avoided to study intracellular trafficking. Similarly, overexpression of endosomal markers, such as FP-tagged Rab GTPases, should be avoided as this may impact upon endosomal dynamics. Hence we recommend using genomic tagged P{acman} BAC clones available at BACPAC Resources, Children’s Hospital Oakland Research Institute (http://bacpac.chori.org/) [16]. 5. It is important to minimize the amount of oil on the coverslip when using an upright microscope for live imaging. Indeed, too much oil will result in oil sliding along the side of the pupa. It is also important to ensure that the notum is in direct contact with the coverslip to optimize the light path, but do not apply too much pressure on the pupa to increase the size of the contact region between the pupa and the coverslip (i.e., to maximize the field of view) as the living tissue may react to physical pressure. The refraction index of the Voltalef oil is very similar to the refraction index of the oil used for the objective. 6. Using two EM-CCD cameras for fast simultaneous acquisition allows both channels to be acquired at the exact same time. This may be important to study the co-localization of red and green markers into rapidly moving endosomes. The spatial registration of the signals acquired by the two cameras should be verified prior to imaging by stopping the rotation of the spinning disk and imaging the two channels through the pinholes of the disk. While a spinning disk is best suited for the imaging of rapidly moving structures, laser scanning microscopes can also be used under fast scanning modes. Excitation should be minimized to limit both phototoxicity and bleaching.

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Acknowledgments This work was supported by the Fondation pour la Recherche Médicale (DEQ20100318284). The antibody uptake assay was initially developed together with R. Le Borgne. References 1. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233 2. Le Borgne R, Bardin A, Schweisguth F (2005) The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 132:1751–1762 3. Gho M, Bellaiche Y, Schweisguth F (1999) Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell. Development 126:3573–3584 4. Le Borgne R, Schweisguth F (2003) Unequal segregation of Neuralized biases Notch activation during asymmetric cell division. Dev Cell 5:139–148 5. Rhyu MS, Jan LY, Jan YN (1994) Asymmetric distribution of Numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76:477–491 6. Couturier L, Vodovar N, Schweisguth F (2012) Endocytosis by Numb breaks Notch symmetry at cytokinesis. Nat Cell Biol 14: 131–139 7. Couturier L, Mazouni K, Schweisguth F (2013) Numb localizes at endosomes and controls the endosomal sorting of notch after asymmetric division in Drosophila. Curr Biol 23:588–593 8. Cotton M, Benhra N, Le Borgne R (2013) Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor. Curr Biol 23:581–587 9. Yamamoto S, Charng WL, Bellen HJ (2010) Endocytosis and intracellular trafficking of Notch and its ligands. Curr Top Dev Biol 92:165–200 10. Giagtzoglou N, Yamamoto S, Zitserman D et al (2012) dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions. J Cell Biol 196:65–83

11. Daskalaki A, Shalaby NA, Kux K et al (2011) Distinct intracellular motifs of Delta mediate its ubiquitylation and activation by Mindbomb1 and Neuralized. J Cell Biol 195:1017–1031 12. Le Borgne R, Remaud S, Hamel S et al (2005) Two distinct E3 ubiquitin ligases have complementary functions in the regulation of Delta and Serrate signaling in Drosophila. PLoS Biol 3:e96 13. Venken KJ, He Y, Hoskins RA et al (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314:1747–1751 14. Qi H, Rand MD, Wu X et al (1999) Processing of the Notch ligand Delta by the metalloprotease Kuzbanian. Science 283:91–94 15. Diederich RJ, Matsuno K, Hing H et al (1994) Cytosolic interaction between deltex and Notch ankyrin repeats implicates deltex in the Notch signaling pathway. Development 120: 473–481 16. Venken KJ, Carlson JW, Schulze KL et al (2009) Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster. Nat Methods 6:431–434 17. Jauffred B, Bellaiche Y (2012) Analyzing frizzled signaling using fixed and live imaging of the asymmetric cell division of the Drosophila sensory organ precursor cell. Methods Mol Biol 839:19–25 18. Klueg KM, Parody TR, Muskavitch MA (1998) Complex proteolytic processing acts on Delta, a transmembrane ligand for Notch, during Drosophila development. Mol Biol Cell 9:1709–1723 19. Coumailleau F, Furthauer M, Knoblich JA et al (2009) Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division. Nature 458:1051–1055

Chapter 7 Tracking Trafficking of Notch and Its Ligands in Mammalian Cells Patricia Chastagner and Christel Brou Abstract The Notch receptor and its ligands are cell surface transmembrane proteins that are internalized. Endocytosis and vesicle trafficking play key roles in Notch signaling activation and modulation. In mammalian cultured cells it is possible to track these cell surface molecules by pulse-labeling these proteins in vivo. One labeling protocol consists in the covalent linkage of membrane-impermeable biotin followed by western blotting. An alternative protocol consists of using high affinity antibodies against the extracellular domains of the proteins followed by immunofluorescence, thereby allowing monitoring of the fate of the labeled proteins. In this chapter, we will describe these two approaches to study the dynamics of receptor and ligand trafficking. Key words Notch, Delta, Jagged, Biotinylation, Antibody uptake, Trafficking, Degradation, Recycling, Endocytosis, Internalization

1

Introduction Notch signaling activation and regulation heavily rely on intracellular trafficking events undergone by the Notch receptors and their ligands [1]. Endocytosis regulates the abundance and activity of the Notch receptors as well as the abundance and activity of its receptors at the cell surface. In mammalian cells, Notch receptors have a limited half-life at the cell surface in the absence of ligand binding. The Notch receptors are constantly internalized and either recycled [2] or degraded through the lysosomal pathway [3]. Upon ligand binding in trans, endocytosis of the ligand physically bound to Notch in the cell sending the signal stretches the Notch protein in the cell receiving the signal, thereby permitting proteolysis by the ADAM (A Disintegrin And Metalloproteinase) proteases and intramembrane cleavage of Notch [4–7]. However, we and others have shown that ligand binding to Notch is not sufficient to induce activation, and that signaling ensues only when the ligands have previously gone through a recycling journey [7–9]. This raises

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many questions: Is the detected Notch or ligand internalization preceding, following, directing, or negatively regulating Notch signaling? Are all vesicles the same or different? In contrast to whole organisms or tissues, cell cultures allow establishing cell lines that express a specific receptor or ligand in a context in which the binding partner is absent. Therefore it is possible to specifically address the fate of receptor or ligand trafficking in the absence of the ligand and activation. Activation can then be induced by co-culturing receptor and ligand expressing cells. The protocols described in this chapter allow the monitoring of cell surface molecules in different cellular compartments. The principle is to pulse-label a pool of molecules, either by the addition of a biotin tag or by binding of a high affinity antibody, and then to monitor their fate during a chase period. Both approaches are applicable to receptor or ligand-expressing cells. Biotin-based protocols allow monitoring of cargo endocytosis and degradation in a time-dependent manner. Furthermore the techniques permit quantification of the efficiency of recycling of the cargo, and assessing possible posttranslational modifications which lead to a shift in apparent molecular weights of the proteins [2, 7–10]. On the other hand, antibody uptake assays allow one to assess endocytosis and degradation kinetics, with the additional feature of visualizing the compartments through which the cargos traffic [2, 3, 11]. Hence, the two technical approaches are complementary.

2

Materials

2.1 Common Materials

1. Culture medium: Dulbecco’s Modified Eagles Medium (DMEM) containing GlutaMAX (Life Technologies), 4.5 g/ l D-glucose and pyruvate, and supplemented with 10 % fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin unless specified. Store at 4 °C. 2. Cell lines: Notch-expressing cell lines: human U2OS or murine embryonic fibroblasts (MEF), expressing full-length human Notch1 with an HA-epitope tag inserted between EGF 22 and 23 [12]. Dll1-expressing cell lines: human U2OS or murine OP9 cells, expressing murine Dll1 bearing a VSVepitope tag after amino acid (a.a.) 45 [13]. In all cases, cell lines were obtained by retroviral transduction. 3. 37 °C incubator containing 7 % CO2. 4. PBS (Phosphate Buffered Saline): 138 mM NaCl, 2.7 mM KCl, 8.2 mM Na2HPO4, 1.5 mM KH2PO4. Use sterile 10× PBS to prepare all the solutions.

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1. Biotin labeling solution: 0.5 mg/ml EZ-Link®Sulfo-NHS-SSBiotin (sulfosuccinimidyl-2-(biotinamido) ethyl-1.3-dithiopropionate) (Thermo Scientific) in PBS to be freshly prepared and chilled on ice. 2. Glycine solution: 0.15 % m/v glycine in PBS to be freshly prepared and chilled to 4 °C. 3. TNM buffer: 20 mM Tris pH 8, 150 mM NaCl, and 1 mM MgCl2. 4. MesNa stripping buffer: 50 mM MesNa (sodium 2-mercaptoethanesulfonate, Sigma-Aldrich) in TNM buffer. 5. Extraction buffer: 50 mM Tris, pH 7.9, 300 mM NaCl, 5 mM MgCl2, 1 % Triton X-100. Protease inhibitor cocktail (e.g., Roche Diagnostics) is added freshly. Keep on ice. 6. NeutrAvidin beads: Ultralink-NeutrAvidin (Thermo Scientific) beads are equilibrated by washing twice with PBS. Keep the 50 % slurry at 4 °C. 7. Reducing buffer: 2× Laemmli sample buffer (e.g., Biorad) supplemented with DTT to 40 mM. 8. Iodoacetamide solution: 5 mg/ml iodoacetamide in TNM buffer.

2.3 Antibody Uptake Assay

1. 12 mm diameter round glass coverslips, certified for immunofluorescence, is convenient for 24-well plates. Some cells (MEF cells for instance) will require treatment of the coverslips with collagen before seeding. Prepare the coverslips by incubating them in an aqueous solution of collagen 0.1 %, acetic acid 2 %, then dry 1 h under a fume hood and refrigerate overnight. 2. Antibodies: For Notch labeling: anti-hemagglutinin (HA), mouse monoclonal 16B12 (Developmental Studies Hybridoma Bank, University of Iowa), Alexa Fluor conjugates (488 or 594, e.g., Life Technologies), dilute 200- to 300-fold. Any antibody recognizing the Notch extracellular domain may be suitable as long as it does not provoke Notch activation (see Chapter 26). It is also possible to use nonconjugated antibodies (e.g., anti-HA.11 monoclonal antibody (Covance) diluted 1,000-fold); in this case cells are processed for immunostaining with an appropriate secondary antibody after fixation and a supplemental step of permeabilization after step 15 of Subheading 3.2. For Dll1 labeling: monoclonal anti-VSV glycoprotein, clone P5D4, conjugated to Cy3 (dilution 1: 5,000, Sigma). Other antibody directed against Dll1 extracellular part could probably be used after validation. 3. EGF conjugates: Alexa Fluor 555 dye-labeled EGF, use at a 200-fold dilution (Life Technologies).

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4. PFA solution: dilute paraformaldehyde 20 % solution, EM grade (Electron Microscopy Sciences) to 4 % in PBS. Work under chemical hood. Keep aliquots frozen, do not use them several times. Add CaCl2 to 0.1 mM and MgCl2 to 0.1 mM freshly from a 1,000× stock solution. 5. NH4Cl solution: 50 mM NH4Cl in PBS. 6. PBS/serum: 10 % fetal calf serum (filtered through 0.45 μm membrane) in PBS. The serum can be replaced by 1 % bovine serum albumin. This serves as a blocking reagent. 7. Humidified chamber: in a light-protected box, place damp Whatman paper covered with parafilm. The antibody solution (20–40 μl) is placed on the parafilm (as many drops as coverslips). 8. Hoechst solution: Hoechst 33342 (Promega): dilute stock solution (50 mg/ml in H2O, store at −20 °C) in PBS to a final concentration of 1 μg/ml just prior to use. It is possible to keep an intermediate stock at 1 mg/ml at −20 °C. 9. Glass slides. 10. Mounting medium: Tris-MWL 4-88 solution (Biovalley) is a solution of poly (vinyl alcohol) [Mowiol™ 4-88] in a water/ glycerol/Tris buffer mix. Following loss of water it forms clear films and is therefore used as a permanent mountant. Add an anti-fading reagent (e.g., 1,4-diazabicyclo-(2.2.2) octane, DABCO, 2.5 % or AF-100 (Biovalley)) to prevent photobleaching prior to use. 11. Epifluorescent or confocal microscope for visualizing cells.

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Methods

3.1 Cell Surface Labeling of Notch and Its Ligands with Biotin

Proteins on the cell surface can be labeled with membraneimpermeable biotin as long as they exhibit free ε-amine of lysine residues. The biotin derivative that we recommend (EZ-Link Sulfo-NHS-SS-Biotin) is water-soluble, enabling biotinylation to be performed in the absence of an organic solvent. In addition this biotin can be removed in reducing conditions (MesNa buffer), allowing one to discriminate internalized molecules from the molecules on the cell surface. Biotinylation is performed at a temperature that is nonpermissive to trafficking (4 °C), then internalization is allowed to proceed at 37 °C. After removing biotin from proteins still present at the cell surface, the amount of various internalized biotinylated cargo can be quantified in parallel from the same extracts (Subheading 3.1.1). If required, recycling of Notch1 or Dll1 can be monitored after a second 37 °C incubation and MesNa treatment round (Subheading 3.1.2). The main steps of these experiments are schematized in Fig. 1, and representative results are presented in Fig. 2.

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Fig. 1 Schematic representation of biotinylation experiments. (a) Endocytosis assay. At time 0, the membrane-impermeable biotin analog Sulfo-NHS-SS- biotin (red circles) labels cell surface cargo (black rectangle). Without MesNa treatment, the total surface biotinylation is quantified (pink background, T0−), whereas MesNa treatment should lead to a total disappearance of the expected western blot signal (T0+). When endocytosis proceeds (time points T1 to T4), increased signal is observed after MesNa treatment (T1– T3), and then signal decreases because of cargo degradation (T4). From this experiment the peak of endocytosis (green background) is determined as time point T3. (b) Recycling assay. Starting from T3 point of endocytosis determined in a, each time of recycling is treated with or without MesNa and analyzed by western blot. If a recycling step occurs (upper row), the MesNa-treated samples are weaker than the controls without MesNa. If no recycling happens (lower row), there is no difference between MesNa-treated and nontreated samples because the cargo is not sent back to cell surface

3.1.1 Endocytosis Assay

1. Seed cells at a density of 15,000 cells/cm2 in culture dishes (50 cm2) the day before the experiment. One dish is required for each time point to be investigated and three additional dishes are needed as controls (see Note1). 2. Prepare: 4 °C chilled solutions: PBS, biotin labeling solution, glycine solution, TNM buffer, MesNa stripping buffer, Extraction buffer (200 μl/dish should be provided); DMEM without serum at 37 °C, NeutrAvidin-agarose beads (washed in PBS) to have 20 μl/dish (1:1 beads: liquid).

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Fig. 2 Examples of endocytosis and recycling assays. (a) Endocytosis of Notch 1, analyzed by western blotting of NeutrAvidin-bound material (upper panel) and of the whole cell extracts (WCE, lower panel). NB is the non-biotinylated control, which is unable to bind NeutrAvidin-agarose. T0− is the total surface Notch, T0+ is the MesNa stripping control. Endocytosis occurs from time point 10′ and Notch degradation begins to be clearly detected at time 60′. (b) Recycling of Dll1, but not of the mutant Dll1 K17R. After internalization of biotinylated proteins for 20 min (lane 3), cells were re-incubated at 37 °C for 10, 20, or 30 min (indicated as +10′, +20′, +30′) and then subjected (+) or not (−) to a second MesNa treatment. WCE or NeutrAvidin-agarose-retained fractions are analyzed by western blot. In the two upper panels, Dll1 recycling is detected, and compared to the WCE. The lower panels show that the mutant Dll1, where all the intracytoplasmic lysine residues have been replaced by arginine (K17R), is unable to recycle, as compared to Cadherin as a control

3. Cool cells by placing dishes on ice in a large basin. Remove culture medium and wash three times with cold PBS (10 ml/dish). 4. At this step it is advisable to harvest cells from one dish to have a control extract of cells without application of any biotin (NB), which will serve to measure nonspecific binding to NeutrAvidin beads. 5. Biotinylate cells by chilled (4 °C) biotin labeling solution (5 ml/dish). Incubate for 1 h at 4 °C (see Note 2). 6. Quench residual biotin by washing the dishes with chilled glycine solution (10 ml/dish), three times each for 5 min at 4 °C. Then wash twice with cold PBS. 7. Collect cells from one dish, which serves to measure total surface biotinylation (T0−). Treat this dish as in step 10.

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8. A second sample plate is used to determine the efficiency of removal of biotinyl group on the cell surface (T0+, see Note 3). Wash cells once with cold PBS and twice with TNM. Incubate with chilled MesNa stripping buffer (10 ml/dish) for 20 min at 4 °C three times to remove the biotinyl group, i.e., 1 h of incubation in total while changing the buffer twice. Then go to step 10. 9. Incubate the other samples at 37 °C in pre-warmed DMEM without serum. At indicated time points, treat the samples as in step 8 (see Note 4). 10. Wash cells once with cold PBS and replace with 1 ml of cold PBS/dish. Harvest the cells with a rubber policeman and transfer to a 1.5 ml microcentrifuge tube. Pellet cells by spinning at 150 × g for 5 min. Lyse the cells in extraction buffer (200 μl/time point) for 20 min on ice. Centrifuge at 15,000 × g in a cooled (4 °C) benchtop microcentrifuge for 20 min. This process clears the lysate of any residual cellular debris. Transfer the supernatant to a new tube (see Note 5). 11. Mix equal amounts of protein lysates with 20 μl of NeutrAvidinagarose beads in 1.5 ml microcentrifuge tubes. Bring the volume to 300 μl with extraction buffer. Incubate for 1 h at 4 °C on a rotating wheel (see Notes 6 and 7). 12. Pellet the NeutrAvidin beads by centrifugation at 500 × g for 3 min and remove the supernatant by gentle aspiration (see Note 8). 13. Wash the pellets three times with extraction buffer (400 μl) to remove any nonspecifically bound proteins. 14. Elute the biotinylated proteins from the NeutrAvidin beads by adding 20 μl of reducing sample buffer to the washed NeutrAvidin bead pellets. Resuspend by gently tapping the tubes and incubate for 10 min at room temperature (see Note 9). 15. Heat samples at 95 °C for 3 min, then pellet the NeutrAvidin beads by centrifugation and resolve the eluted proteins by SDS-PAGE. 16. Analyze by western blotting using antibodies against the tag/ protein of interest. An example of the data obtained is shown in Fig. 2a. 3.1.2 Recycling Assay

1. Seed cells at a density of 15,000 cells/cm2 in culture dishes (50 cm2) the day before the experiment. One dish is required to measure total surface biotinylation (T0−), another dish for MesNa stripping control (T0+), and a third for the start point of recycling (see Note 10). Additionally prepare two dishes for each time point of recycling to be investigated. 2. Treat the dishes as in steps 2–8 of endocytosis assay.

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3. At this step the cells are biotinylated and the two controls (total surface biotinylation T0− and MesNa stripping control T0+) have been collected. 4. Incubate the dishes at 37 °C in pre-warmed DMEM without serum. After 20 min of endocytosis, prepare the cells for the first MesNa treatment. Wash cells one time in cold PBS and twice in TNM. Incubate with 10 ml/dish of chilled MesNa stripping buffer for 20 min at 4 °C three times. 5. Incubate cells for 10 min on ice with iodoacetamide solution (10 ml/dish) to quench free SH groups. 6. Wash cells once with TNM. 7. Replace TNM by pre-warmed DMEM without serum and incubate at 37 °C for various periods of time to allow transport through recycling endosomes (see Note 11). 8. At each time point remove two dishes from the incubator. Strip one set of cells of surface biotin in MesNa stripping buffer as in step 4. Treat a second set of cells in TNM without MesNa. This sample monitors the total amount of biotinylated molecules at a given time point. If there is a difference between MesNa treated and nontreated samples, it indicates that recycling happened (see Fig. 1b). 9. Wash twice with cold PBS. Replace with 1 ml of cold PBS/ dish. Harvest the cells with a rubber policeman and transfer to a 1.5 ml microcentrifuge tube. Pellet cells by spinning at 150 × g for 5 min. Lyse the cells in extraction buffer (200 μl/ dish) for 20 min on ice. Centrifuge at 15,000 × g in a cooled (4 °C) benchtop microcentrifuge for 20 min (see Note 12). 10. Mix equal amounts of protein lysates with 20 μl of NeutrAvidinagarose beads in 1.5 ml microcentrifuge tubes. Bring the volume to 300 μl with extraction buffer. Incubate for 1 h at 4 °C on a rotating wheel. 11. Pellet the NeutrAvidin beads by centrifugation at 500 × g for 3 min and remove the supernatant by gentle aspiration. 12. Wash the pellets three times with extraction buffer (0.4 ml). Elute the biotinylated proteins from the NeutrAvidin beads by adding 20 μl of reducing sample buffer to the washed NeutrAvidin bead pellets. Resuspend by gently tapping the tubes and incubate for 10 min at room temperature (see Note 9). 13. Heat samples at 95 °C for 3 min, then pellet the NeutrAvidin beads by centrifugation and resolve the eluted proteins by SDS-PAGE. 14. Analyze by western blotting. An example of the data obtained is shown in Fig. 2b.

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The fluorescent ligands or antibodies described in this section first bind to cell surface receptors, then are internalized and, in some cases, recycled to the cell surface or trafficked for lysosomal degradation. In most applications, the cell surface and internalized labeled populations are spatially resolved by imaging. It is often desirable to include internalized plasma membrane reference markers in these labeling protocols. We routinely use EGF receptor (EGFR) labeling with fluorescent EGF, since the dissociation constant (Kd) of the EGF conjugates in DMEM-HEPES medium is in the low nanomolar range, a value that approximates that of the unlabeled EGF. Activated EGFR is internalized through clathrin-dependent mechanisms, and EGF-receptor complexes are targeted to lysosomes for degradation [14], providing a control for the normal degradation machinery. Most epithelial cultured cells express EGFR. When using cells of hematopoietic origin or other cells devoid of EGFR, alternative controls have to be tested, e.g., Cadherin, or Transferrin receptor. An example is shown in Fig. 3. Here, we present a protocol for antibody uptake in living cells using anti-HA or anti-VSV antibodies in cells that are stably expressing tagged forms of Notch receptor or ligands. Other antibodies or cells can also be used if appropriate controls have been made (see step 2 of Subheading 2.3). Additional immunofluorescence steps can be incorporated after antibody uptake using specific markers to label compartments to assess a more precise subcellular distribution of the Notch receptors or its ligands. For example, EEA1 (Abcam or BD Transduction Laboratories), LAMP-1 (Developmental Studies Hybridoma Bank, University of Iowa), and rab5 (BD Transduction Laboratories) are often used to identify specific endocytotic compartments. In addition, chemical treatments (with leupeptin for example) can prevent degradation into the lysosomes, without impairing the cargo to reach this compartment. In this case, the co-labeling with LAMP-1 will increase. Culture cells on coverslips placed in 24-well plates without reaching confluence (see Note 13). 60–80 % confluency on the day of the experiment is optimal. At least one well is required for each time point to be investigated (see Note 14). 1. Prepare: 37 °C preheated DMEM (without serum), PBS at room temperature (RT), 4 °C chilled DMEM (without serum), DMEM (without serum) at RT. 2. Remove the culture medium and replace by preheated serumfree medium, 0.5 ml/well for 1 h at 37 °C. 3. Place the plates on ice for 10 min (see Note 15). 4. Prepare the antibody mix: dilute the anti-HA or VSV antibody, together with dye-labeled EGF if necessary, into serumfree medium (see Note 16). Keep the mixture on ice. Prepare 0.2 ml/well.

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Fig. 3 Notch receptor internalization and degradation monitored by antibody uptake. MEF cells stably expressing HA-tagged Notch were transfected either with non-targeting siRNA (si NT, first column), or with siRNA targeting USP8 (si USP 8, second column) and USP 12 (si USP 12, third column), respectively. Living cells were labeled with anti-HA antibody coupled to Alexa Fluor 488 (green) together with EGF coupled to Alexa Fluor 555 (red). Cells were incubated for 0, 30, or 120 min at 37 °C before fixation and analysis. USP8/UBPY invalidation impairs EGF receptor degradation [15, 16], resulting in persistent red EGF signal at T 120 min, whereas USP12 knock down specifically delays Notch degradation without affecting EGF receptor [11]. Images were acquired using an AxioImager microscope with ApoTome system, 63× magnification, and AxioVision software (Carl Zeiss MicroImaging Inc.)

5. Replace the medium with the antibody mix, leave on ice for 30 min (see Note 17). 6. Remove the plates from ice; leave them at RT (see Note 18). 7. Wash the cells twice with serum-free medium at room temperature (0.5 ml/well). 8. For time 0: wash twice with PBS (0.5 ml/well) at RT, and fix the cells directly (go to step 12). 9. For the other time points: replace the washing medium with preheated serum-free medium, 0.5 ml/well, and place the plates into the incubator for the desired period of time (see Note 19).

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10. Following the desired period, remove the culture medium and quickly rinse with PBS (at RT) twice. 11. Fix the cells by replacing PBS by PFA solution and incubate for 20 min at RT. Work under chemical hood and process the waste separately, since paraformaldehyde is a suspected carcinogen. 12. Quench the PFA by replacing the solution with 50 mM NH4Cl solution in PBS for 10 min at room temperature. From this step, keep the coverslips protected from light as much as possible (cover with aluminum foil). 13. Wash twice with PBS. At this point, coverslips can be stored in 1 ml PBS at 4 °C in dark for several weeks (see Note 20). 14. If no secondary antibody or additional staining is required, go to step 21. 15. Permeabilize cells by incubating with 0.2 % Triton (in PBS) for 5 min at room temperature. Then immediately wash three times with PBS, and replace PBS by PBS with serum (0.4 ml/well). 16. Incubate coverslips in primary antibody (for additional staining of an organelle of interest) diluted in PBS with serum for 45 min at room temperature. Use 0.2 ml/well as a minimal volume of incubation. If minimizing antibody volumes is required, place each coverslip cells facing down onto a drop of antibody solution (40 μl) into a humidified chamber (see Note 21). Be careful to use organelle markers antibodies from different species or with different isotypes than the internalized antibody you are following. 17. Gently wash coverslips 3 times in PBS with serum (0.4 ml/ well). If a humidified chamber has been used, put the coverslips back into a 24-well plate (cells up) before washing. 18. Dilute the secondary antibodies in PBS with serum. Each of the antibodies must be conjugated to a different fluor for independent visualization. Incubate coverslips for 30–45 min, protected from light, at RT. 19. Wash three times in PBS with serum, and three times in PBS. 20. Incubate coverslips in Hoechst solution for 3 min to stain the nuclei (see Note 22). Process the waste separately as this material is considered hazardous. Wash again three times with PBS. 21. Mount coverslips on glass slides by gently placing each coverslip cells facing down onto a small (5–20 μl) drop of mounting medium (see Note 23). Air-dry for several hours. 22. Visualize cells by epifluorescence or confocal microscopy. The mounted slides can be stored at 4 °C for months. Figure 3 shows images of Notch and EGF receptors internalization and degradation.

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Notes 1. 60–80 % confluency on the day of the experiment is optimal. If necessary, the cells can be previously transiently transfected. 2. Make sure to incubate the dishes on a flat surface (on ice or in a cold room). 3. This step is crucial. If biotin removal is not complete, the background signal will persist and preclude interpretation of the results. 4. The duration of the required internalization steps will vary between cell types and should be determined. For most applications 0, 15, 30, 60, and 120 min should be good starting points. 5. Cell lysis provides a convenient point to halt the experiment. If this is the case, lysates should be stored at −20 °C for several days. 6. Quantitate protein lysates. Dilute the lysates with extraction buffer so that they are all at the same concentration. However we observed that using a constant volume of each lysate generally gives good results. Keep some extracts for direct analysis by SDS-PAGE. 7. Streptavidin can also be used but NeutrAvidin protein yields the lowest nonspecific binding among the known biotin binding proteins. 8. Be careful not to aspirate to the point that the beads completely dry out. 9. At this step the samples can be kept at −20 °C for weeks. 10. Use the data from the endocytosis assay to determine the starting time point of the recycling assay. The time point in which endocytosis of the protein reaches the maximum is a good starting point. We often use 20 min of internalization of Dll1, as exemplified in Fig. 2b. 11. Three time points were tested for Dll1 recycling: 10, 20, 30 min. 12. It is possible to wait until the end of recycling steps to lyse all of the cells together. Keep the dishes on ice till then. 13. If necessary, the cells can be transfected with the siRNAs or plasmids of interest prior to the assay. We have found it to be convenient to transfect or treat the cells in 60 or 100 mm tissue culture dishes, then to count and distribute them on 24-well plates the day before the experiment. 14. Prepare as many plates as time points to be able to treat each time point without removing all plates from the incubator.

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15. Use a large dish filled with ice to place the plates. For longer incubations (step 6), place the dish in the cold room. 16. Briefly centrifuge the protein conjugate solutions in a microcentrifuge before using the supernatant. This eliminates the aggregates that may have formed during storage. This procedure reduces nonspecific background staining. 17. Gently apply the mix onto the cells (on the wall rather than on the bottom), in order not to detach them. When removing solutions from coverslips by aspiration, make sure not to aspirate directly from the center of the coverslip. Coverslips should never be aspirated to the point of being completely dry, as this will lead to alterations in cellular morphology. 18. We observed that temperature shift from 4 to 37 °C often provokes cell detachment (depending on the cell line); that is why we prefer a step at room temperature. In this case washing steps have to be quick to prevent initiation of endocytosis. As a last resort, the labeling can be performed at 37 °C for 2 min, followed by quick washing. 19. The duration of the internalization steps required will vary between cells and depends on the purpose of the experiment. For most applications 0, 30, 60, 120, 240 min should be good starting points. Note that at earlier time points (15 min) cells are often weakened by temperature shifts and can be in a bad shape. 20. It can be useful to prepare duplicates of each time point. Depending on the analysis on the first set of samples, antibodies for other intracellular compartments can be tested on the second set. 21. To remove the coverslip from the well, use a 1-ml syringe with a bent needle. 22. Hoechst, in particular 33342, is a permeable DNA dye, so no permeabilization step is necessary. This staining is very useful for counting the cells. Also, nuclear staining allows the identification of cells in case the specific signal is weak (such as after the degradation of the receptor for instance). 23. Mowiol has the advantage of giving a clear film, which holds the coverslip in place and obviates the need to seal with nail polish. Wait until hardening is complete before observing the samples under a microscope.

Acknowledgments We thank Loredana Puca for critical reading of the manuscript, J. Moretti, S. Heuss, and F. Logeat for discussion and materials. Financial support of Institut Pasteur, CNRS, and Ligue Nationale Contre le Cancer (LNCC RS11/75-21) is acknowledged.

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References 1. Yamamoto S, Charng W-L, Bellen HJ (2010) Endocytosis and intracellular trafficking of Notch and its ligands. Curr Top Dev Biol 92:165–200 2. McGill MA, Dho SE, Weinmaster G et al (2009) Numb regulates post-endocytic trafficking and degradation of Notch1. J Biol Chem 284:26427–26438 3. Chastagner P, Israël A, Brou C (2008) AIP4/ Itch regulates Notch receptor degradation in the absence of ligand. PLoS One 3:e2735 4. Parks AL, Klueg KM, Stout JR et al (2000) Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127:1373–1385 5. Nichols JT, Miyamoto A, Olsen SL et al (2007) DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur. J Cell Biol 176:445–458 6. Meloty-Kapella L, Shergill B, Kuon J et al (2012) Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin. Dev Cell 22: 1299–1312 7. Heuss SF, Ndiaye-Lobry D, Six EM et al (2008) The intracellular region of Notch ligands Dll1 and Dll3 regulates their trafficking and signaling activity. Proc Natl Acad Sci U S A 105:11212–11217 8. Shergill B, Meloty-Kapella L, Musse AA et al (2012) Optical tweezers studies on Notch: single-molecule interaction strength is independent of ligand endocytosis. Dev Cell 22: 1313–1320

9. Shah DK, Mohtashami M, Zúñiga-Pflücker JC (2012) Role of recycling, Mindbomb1 association, and exclusion from lipid rafts of δ-like 4 for effective Notch signaling to drive T cell development. J Immunol 189:5797–5808 10. Watanabe-Hosomi A, Watanabe Y, Tanaka M et al (2012) Transendocytosis is impaired in CADASIL-mutant NOTCH3. Exp Neurol 233:303–311 11. Moretti J, Chastagner P, Liang C-C et al (2012) The ubiquitin-specific protease 12 (USP12) is a negative regulator of Notch signaling acting on Notch receptor trafficking toward degradation. J Biol Chem 287:29429–29441 12. Moretti J, Chastagner P, Gastaldello S et al (2010) The translation initiation factor 3f (eIF3f) exhibits a deubiquitinase activity regulating Notch activation. PLoS Biol 8:e1000545 13. Six E, Ndiaye D, Laabi Y et al (2003) The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and ɣ-secretase. Proc Natl Acad Sci U S A 100:7638–7643 14. Sorkin A, Goh LK (2009) Endocytosis and intracellular trafficking of ErbBs. Exp Cell Res 315:683–696 15. Row PE, Prior IA, McCullough J et al (2006) The ubiquitin isopeptidase UBPY regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation. J Biol Chem 281:12618–12624 16. Alwan HA, van Leeuwen JE (2007) UBPYmediated epidermal growth factor receptor (EGFR) de-ubiquitination promotes EGFR degradation. J Biol Chem 282:1658–1669

Chapter 8 Visualizing Notch Signaling In Vivo in Drosophila Tissues Benjamin E. Housden, Jinghua Li, and Sarah J. Bray Abstract The ability to visualize Notch pathway activity in vivo is invaluable for studying the functions and mechanisms of Notch signaling. A variety of tools have been developed to enable monitoring of pathway activity in Drosophila, including endogenous Notch-responsive genes and synthetic transcriptional reporter constructs. Here we summarize some of the different Notch signaling reporters that are available, discuss their relative merits, and describe two methods for visualizing their expression (immunostaining and X-gal staining). These approaches are widely applicable to a range of tissues and stages in Drosophila development. Key words Notch signaling, Notch reporters, Transcriptional reporters, Antibodies, X-gal staining, Immunostaining, Fluorescent reporters

1

Introduction Monitoring Notch pathway activity in vivo is an essential part of elucidating the roles and mechanisms of Notch signaling in both development and adult homeostasis. The cascade of intracellular events that take place following Notch receptor activation is relatively simple [1], although attempts to specifically monitor these events have had limited success. In the case of translocation of NICD to the nucleus, for example, direct studies are most likely hindered by the low level and rapid turnover of NICD [2–4]. Instead, detection of pathway activation has relied on monitoring the transcriptional changes downstream of Notch activation, using either previously identified target genes or synthetic reporter constructs. One of the simplest ways to detect activation of the Notch pathway is to visualize expression of one or more target genes using specific antibodies. For example, many studies have taken advantage of Notch induced Cut expression at the dorsal-ventral (DV) boundary of the larval wing pouch to monitor changes in downstream signaling. In this case, such an approach is effective because loss of Notch signaling is sufficient to ablate cut expression, and increased pathway activity close to the DV boundary is

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_8, © Springer Science+Business Media New York 2014

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sufficient to induce ectopic cut transcription [5]. Although effective in the wing pouch, cut expression is regulated by many other inputs so expression and responsiveness do not always correlate with Notch activity. The other caveat is that Cut is only a reliable indicator of Notch activity in late stage wing discs. Each tissue analyzed thus requires the identification of a suitable directly regulated gene (preferably for which antibodies are available) and care must be taken to identify which aspects of expression are due to Notch and which are due to other inputs. Amongst the most widely expressed targets of the Notch pathway are the well-characterized Enhancer of split (E(spl)) complex genes [6–9]. This genomic region contains two types of Notchresponsive genes, the seven E(spl)basic Helix Loop Helix (bHLH) genes (members of the HES family) and four Bearded family genes. These genes are direct targets of Notch pathway activity, responding rapidly and robustly to Notch activity in many contexts, making them reliable reporters of pathway activity in many different tissues. However, the 11 Notch-responsive genes do not have identical patterns of expression and some respond much more widely than others [10–12]. One way to detect expression of E(spl)bHLH genes is to use an antibody that recognizes several members of this group. This valuable tool was used to visualize Notch activity in a wide variety of tissues [7]. However, the transient expression of the E(spl) proteins and the relatively low affinity of the antibody have tended to limit its use. As an alternative to visualize endogenous Notch regulated genes, several transgenic lines have been developed carrying reporter constructs. Many of these were generated using Notchresponsive regulatory regions from E(spl) genes and placing them upstream of easily monitored reporter proteins such as lacZ, GFP, or RFP (Table 1). As each individual E(spl) gene is responsive to Notch in only a subset of tissues, the appropriate reporter must be chosen depending on the context. Nevertheless, between them, these reporters cover a broad spectrum of different tissues and developmental processes. For example, good indicators of Notch activity include E(spl)mβ1.5-lacZ in the wing disc, mδ0.5-lacZ in photoreceptors, E(spl)mγ-GFP in postembryonic neuroblasts, and E(spl)mα-RFP during SOP development in the pupal notum (Table 1). To overcome some of the limitations from using endogenous gene reporters, including their tissue restrictions, several synthetic reporters were developed [13–16] (Table 1). Of those, the most sensitive and widely responsive reporter is Gbe-Su(H)-lacZ or NRE-lacZ (and its derivatives) (Fig. 1). This combines two copies of the paired Su(H) binding sites from a known target gene (E(spl) m8-HLH) with three binding sites from the widely expressed transcriptional activator, Grainyhead [13]. The NRE is kept silent by Su(H) in the absence of Notch activity and is able to respond to

Table 1 Summary of widely used Notch reporters Reporter name

Details

Relevant tissues

References

NRE-lacZ

NRE = two paired Su(H) binding sites (4 Su(H) sites total) combined with Grh binding sites Also called Gbe + Su(H)-lacZ

Many tissues including wing disc, eye disc, leg disc (various cell types), Adult intestine (EEs, ISCs), Germ line (polar cells)

[13]

NRE-GFP/mCherry/ Venus/lacZ

NRE as above was combined with different reporters and flanked by insulator sites

As above

[14]

NRE:EGFP

Variant combining the NRE with GFP, precise details unclear

Tested in wing and eye discs

[15]

p12XSu(H)bs-lacZ

Ten synthetic Su(H) sites upstream of the E(spl)mγ promoter region (containing another two Su(H) sites) in pCaspAUG-βgal

Eye discs

[16]

E(spl)mβ1.5-lacZ/CD2

Enhancer construct: 1.5 kb Psp1406I fragment, including the promoter of E(spl)mβ, was cloned upstream of CD2 or lacZ coding sequences in pWhite Rabbit or HZ50PL, respectively

Wing disc, eye disc, leg disc (various cell types), Adult intestine (EEs, ISCs), Germ line (polar cells)

[27, 28]

E(spl)m7-lacZ

P-element enhancer trap inserted just 5′ of E(spl) m7-bHLH

Follicle cells

[29, 30]

E(spl)mγ-GFP

Genomic fragment encompassing E(spl)mγ gene with GFP fused in frame in pWhiteRabbit

Larval brain neuroblasts

[31]

E(spl)mδ0.5-lacZ

Enhancer construct: 0.5 kb fragment from E(spl)mδ gene upstream of hsp70 minimal promoter in HZ50_PL (lacZ)

Eye disc, R4 and R7 photoreceptors

[32]

E(spl)mδ1.9-lacZ

Enhancer construct: 1.9 kb fragment from E(spl)mδ gene upstream of hsp70 minimal promoter in HZ50_PL

Eye disc, proneural clusters, R4 and R7 photoreceptors, cone cells

[27]

Synthetic Notch reporters

E(spl) gene reporters

(continued)

Table 1 (continued) Reporter name

Details

Relevant tissues

References

E(spl)m8-lacZ

Enhancer construct: 2.61 kb fragment from E(spl)m8 including the promoter in pWlac2B

Wing disc, D/V boundary, and proneural clusters. Neuroectoderm in embryos

[33]

E(spl)m8-GFP

Enhancer construct: 1.1 kb genomic EcoRI-XhoI DNA fragment (–1,174 to –72) into the MCS of pGreen H-Stinger

Wing disc, D/V boundary, and proneural clusters

[34]

E(spl)mα-RFP/GFP/lacZ

Enhancer construct: 1 kb genomic fragment (–1,083 to –71) from the E(spl)mα gene in pRed/ pGreen H-Stinger

Wing disc and pupal notum (proneural clusters)

[34–36]

E(spl)m6-GFP

Enhancer construct: 2.1 kb fragment from E(spl)m6 including the promoter in Green Pelican

Wing disc, adult muscle progenitors

[ 8]

E(spl)m4-lacZ

Enhancer construct: 0.5 kb genomic SacI-XhoI fragment from E(spl)m4 promoter in CaSpeRlacZ

Wing disc (proneural clusters)

[ 6]

Anti-E(spl)bHLH

Monoclonal antibody 323

Many tissues including wing disc, D/V boundary; eye disc proneural territory photo receptors and cone cells

[ 7]

vg[BE]-lacZ

Enhancer construct: 750 bp genomic EcoRI fragment upstream of a LacZ reporter gene

Wing discs D/V boundary

[37]

bib-lacZ

P-element enhancer trap in big brain

Leg disc, leg joint primordia

[28]

wg-GFP

Enhancer construct: 6.1 kb genomic fragment in pGreenRabbit

Wing disc D/V boundary

[38]

Anti-Cut

Mouse monoclonal antibody, 2B10; DSHB

Wing disc D/V boundary

[39]

Anti-Wg

Mouse monoclonal antibody, 4D4; DSHB

Wing disc D/V boundary

[40]

Anti-Hnt

Mouse monoclonal antibody, 1G9; DSHB

Follicle cells

[41]

Other reporters

DSHB Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA

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Fig. 1 Example of NRE-GFP reporter staining. Immunostaining of discs from third instar larvae carrying the NRE-GFP reporter. Upper panels show NRE-GFP expression in green and the posterior domain in red (marked with anti-Engrailed). Lower panels show NRE-GFP expression alone. (a-a′) NRE-GFP expression in wing disc (right ; note strong expression at D/V boundary, blue arrows), leg disc (upper left; note rings of expression at presumptive leg joints, white arrows), and haltere disc (lower left ; note stripe at D/V boundary, yellow arrows). (b-b′) Wing disc with Notch signaling reduced in the posterior domain by driving Notch-RNAi expression using en-Gal4. NRE-GFP expression is reduced/absent in this region (which is also reduced in size)

Notch signaling in a wide variety of tissues. Initially produced using lacZ as a transcriptional readout, it is now available with a variety of different reporters: EGFP, mCherry, lacZ, and VenusPEST [14]. Although in some cases it is possible to visualize Notch reporter expression directly (e.g., E(spl)mα-RFP in the pupal notum; fluorescent NRE reporters at the DV boundary of the wing disc), most often antibodies against reporter proteins are used to amplify the signal. This involves the incubation of dissected

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tissue with a primary antibody specific to the reporter protein and then incubation with a secondary antibody conjugated to a fluorescent molecule so that the expression can be visualized. An alternative approach, detecting the mRNA expression by in situ hybridization, is also feasible. Although less commonly used in recent times, histochemical (X-gal) staining provides an alternative method to detect the presence of the lacZ encoded β-galactosidase enzyme. This technique offers some advantages over immunostaining as it is very sensitive and requires a less time-consuming protocol. It is also very useful for beginners who are unfamiliar with Drosophila tissues (we routinely use this protocol when training students). However, resolution is considerably lower than that obtained using immunostaining and it is not easily combined with techniques to simultaneously visualize other markers. Table 1 provides a summary of frequently used Notch reporters. Key factors to consider in deciding which reporter to use are (1) whether there is prior evidence that the reporter is expressed in the tissue of interest—if investigating a novel developmental context it is worth testing a range of different reporters; (2) what type of experiments are planned (e.g., mCherry and RFP reporters are often less easy to detect than GFP but may be more suitable in genetic experiments where many chromosomes are marked with GFP); (3) whether it is important that the reporter reproduces temporal aspects of signaling—one with a long half-life may not reflect the dynamics well. In this case, the use of reporters expressing destabilized GFP [17], such as NRE-Venus-PEST [14], may be worth consideration. Finally, if investigating a novel site of Notch activity, it is important to keep in mind the caveat that some reporters may have Notch independent elements to their expression pattern. Combining the reporters with tools that perturb Notch signaling (Table 2) is essential to prove that the pattern of reporter expression is a bona fide indicator of Notch activity. Currently one of the simplest strategies is to use targeted expression of Notch RNAi [18] or of a dominant negative Mastermind [19] to inhibit pathway activity. These are effective in most instances where suitable Gal4 drivers are available and can be combined with Gal80ts to provide temporal control [20, 21]. Alternatively, mutant strains exist for most components of the pathway. However, as most Notch pathway mutants cause early embryonic or larval lethality, it is often necessary to examine the consequences on reporter genes in clones of mutant cells using strategies such as FLP/FRT-mediated somatic recombination [22] or MARCM [23].

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Table 2 Useful fly strains for modulating Notch activity Fly line

Description

Chromosome References/sources

Lines to repress Notch activity UAS-Notch RNAi

RNAi targeting the Notch receptor

X, II, III

BDSC: 7078

UAS-Mastermind-DN Dominant negative form of a Su(H) co-activator

II

[42]

Notch55e11

X

[16] BDSC: 28813

II, III

[43, 44]

Spontaneous null mutant of the Notch receptor

Lines to increase Notch activity UAS-NICD

Intracellular region of the Notch receptor, constitutively active, independent of gamma-secretase

UAS-NΔECD

Deletion of the Notch extracellular II domain, constitutively active in the presence of gamma-secretase complex

[45]

UAS-Su(H)-VP16

Su(H) transcription factor fused to a X, II VP16 transcriptional activator domain

[13]

BDSC Bloomington Drosophila Stock Center, Indiana University, Bloomington, IN

2

Materials

2.1 Suitable Reporter Fly Strains (See Table 1) 2.1.1 Immunostaining Materials

1. Phosphate Buffered Saline (PBS pH 7.4): KCl 2.68 mM, KH2PO4 1.76 mM, NaCl 136.89 mM, Na2HPO4 10.1 mM (see Note 1). 2. PBS-T: Triton X-100 0.2 % in PBS. 3. PBS-BT: Triton X-100 0.2 %, BSA 0.5 % in PBS. 4. Formaldehyde (e.g., Polysciences 16 % Formaldehyde, methanol free). 5. Primary antibody. 6. Secondary antibody. 7. Mounting medium (e.g., Vectashield (Vector Labs)).

Citifluor

8. Microscope slides. 9. Cover slips. 10. Clear nail polish for sealing slides.

(Agar

Scientific)/

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2.1.2 X-gal Staining Materials

1. Phosphate Buffered Saline (PBS pH 7.4): KCl 2.68 mM, KH2PO4 1.76 mM, NaCl 136.89 mM, Na2HPO4 10.1 mM (see Note 1). 2. Glutaraldehyde. 3. Glycerol. 4. Microscope slides. 5. Cover slips. 6. Clear nail polish for sealing slides. 7. Staining solution: 10 mM MgCl2, 0.3 % Triton X-100, 3.2 mM K4[Fe2+(CN)6], 3.2 mM K3[Fe3+(CN)6], 0.2 % 5-bromo-4chloro-3-indolyl-β-D-galactopyranoside (X-gal) in PBS. Note that X-gal should be added fresh from a 20 % solution in DMF (dimethylformamide).

3

Methods Perform the following procedures in 1.5 ml microcentrifuge tubes except where specified otherwise (see Note 2). Volumes of reagents are generally not critical as long as they are sufficient to keep the samples submerged throughout the process.

3.1 Immunostaining Method

1. To examine Notch activity in larval tissues, wandering 3rd instar larvae are collected from the walls of culture vials containing flies of a suitable Notch reporter strain (see Note 3). 2. Crudely dissect the larva in PBS at room temperature, being sure to completely remove the intestine and fat body. For analyzing most imaginal discs or larval brains the simplest approach is to separate the front (head) 1/3 of the larva from the rest and then turn the head fragment inside out to expose the attached internal tissues. This is a bit like turning a sock inside out. 3. Once dissected, place larval heads into cold PBS on ice while dissecting the remaining larvae. Samples can be maintained in this state for 30 min without noticeable changes in staining quality, but longer incubations should be avoided. 4. Remove PBS and add 4 % formaldehyde diluted in PBS. Incubate samples at 4 °C with gentle mixing for 20 min (see Notes 4 and 5). Be careful not to damage larval heads while changing solutions. 5. Wash larval heads twice in PBS-T on ice for 10 min each time (see Note 6). 6. Block samples in PBS-BT for 10–20 min on ice. 7. Incubate with primary antibody overnight at 4 °C with gentle mixing (see Notes 8 and 9). Antibody should be prepared by

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diluting in PBS-BT to an appropriate concentration (based on manufacturer’s recommendations). 8. Wash samples with PBS-BT three times for 10 min each time on ice. 9. Incubate with secondary antibody for 5 h at 4 °C with gentle mixing (see Notes 9 and 10). Antibody should be prepared by diluting in PBS-BT to an appropriate concentration (based on manufacturer’s recommendations). 10. Wash samples with PBS-BT three times for 20 min each time on ice. 11. Wash samples with 70 % glycerol for 20 min at room temperature. 12. Dissect specific tissues, e.g., imaginal discs, from larval heads in 70 % glycerol and place directly into mounting medium on a microscope slide. Once all tissues are dissected, gently separate them so that they do not overlap when the cover slip is put in place. Also, unfold any tissues that have become folded over during dissection. 13. Place cover slip gently over samples and wait for the mounting medium to fill the gap between microscope slide and cover slip. 14. Seal the edges of the cover slip using clear nail polish. 15. Image discs using standard fluorescence microscopy techniques. 3.2 X-gal Staining Method (See Note 1)

1. Perform steps 1–3 as described for immunostaining above. 2. Remove PBS and add 2.5 % glutaraldehyde diluted in PBS. Incubate samples at 4 °C with gentle mixing by rotation for 7 min (see Notes 11 and 12). 3. Wash larval heads three times in PBS on ice for 10 min each time. 4. Incubate samples with staining solution at 37 °C with gentle mixing until staining develops (see Note 13). 5. Stop the reaction by washing samples with PBS three times for 20 min each time on ice. 6. Wash samples with 70 % glycerol for 20 min at room temperature. 7. Dissect discs from larval heads in 70 % glycerol and place directly into 70 % glycerol on a microscope slide. Once all discs are dissected, gently separate them so that they do not overlap when the cover slip is put in place. 8. Place cover slip gently over samples and wait for the mounting medium to fill the gap between microscope slide and cover slip. 9. Seal the edges of the cover slip using clear nail polish. 10. Image discs using standard DIC microscopy techniques.

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Notes 1. Most reagents used in these protocols are not hazardous. However, safe handling of formaldehyde-containing solutions requires wearing safety glasses, lab coat, and gloves. Be aware that some antibodies may be stored in sodium azide, which is toxic. 2. Techniques are described for analyzing Notch activity in imaginal tissues. Protocols for other developmental stages are available, e.g., [24, 25]. 3. Fly strains are generally grown on standard cornmeal media at 25 °C; for further details of fly husbandry see [26]. 4. If many larvae must be dissected for an experiment, they can be split into smaller batches. As noted in the procedure above, unfixed larval heads should not be kept for more than 30 min before proceeding with the protocol. If it is not possible to dissect all larvae during this time, the first batch should be processed up to the end of step 5. These will be relatively stable for a long period of time and can be left on ice while the next batch is processed. 5. Changing the incubation time with fixative will affect the results. Over-fixing may reduce accessibility of substrates to the primary antibody. Under-fixing may lead to degradation of the tissue. The times given above work well for staining discs with anti-GFP but for other tissues and antibodies further optimization may be required. 6. Incubation times are generally not critical for wash and antibody steps. The values given should be considered as minimum times, as reducing them may diminish staining quality, but they can be increased considerably without affecting the results. However, when using a low affinity antibody wash times should be decreased. For example, if using anti-E(spl)bHLH mAb323 [7] it is important that washing times after the primary antibody are kept to a minimum (less than 1 h total). 7. The Triton X-100 present in the wash and staining solutions permeabilizes cell membranes, allowing antibodies to enter the cells. The concentrations given above should work for most applications but may be altered if required. For example, with denser tissues such as larval CNS, Triton X-100 concentration can be increased (e.g., 0.5 %). 8. Primary antibody solutions can be reused several times. After incubation of larval heads with the primary antibody, the solution can be kept at 4 °C for some time and reused to stain further samples. Due to reduction of nonspecific binding from previous incubations, staining quality can actually increase with subsequent uses.

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9. Results using antibodies that give high background signal can be improved by preincubating the antibody solution with larval heads lacking the antibody’s substrate. This will reduce the nonspecific binding capacity of the antibody solution. 10. Results can be improved by altering the concentration of primary or secondary antibody staining solutions. Concentrations should generally be lowered to reduce background staining and raised to improve signal. Note that poor quality staining can be caused by several factors so changing antibody concentrations may not always help and staining results are often limited by the quality of the primary antibody itself. 11. Safe handling of glutaraldehyde containing solutions requires wearing of safety glasses, lab coat, and gloves. 12. Changing the incubation time with glutaraldehyde will affect the results. Over-fixing may inhibit the enzymatic activity of β-galactosidase (lacZ) required to develop the stain in the X-gal procedure and under-fixing may lead to degradation of the tissues. The times given above work well for staining discs but for other tissues optimization may be required. 13. During the X-gal staining procedure, signal intensity will increase with time of incubation in the staining solution. However, this relationship is not linear. We find that maximum signal intensity occurs after about 24 h and the majority of this signal is present after the first few hours of incubation.

Acknowledgements We would like to thank Hamid Moosavi for contributing data used in Fig. 1. References 1. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689 2. Fryer CJ, Lamar E, Turbachova I et al (2002) Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes Dev 16:1397–1411 3. Fryer CJ, White JB, Jones KA (2004) Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol Cell 16:509–520 4. Schroeter EH, Kisslinger JA, Kopan R (1998) Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393:382–386

5. Micchelli CA, Rulifson EJ, Blair SS (1997) The function and regulation of cut expression on the wing margin of Drosophila: Notch, wingless and a dominant negative role for Delta and Serrate. Development 124:1485–1495 6. Bailey AM, Posakony JW (1995) Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity. Genes Dev 9: 2609–2622 7. Jennings B, Preiss A, Delidakis C et al (1994) The Notch signalling pathway is required for Enhancer of split bHLH protein expression during neurogenesis in the Drosophila embryo. Development 120:3537–3548

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8. Lai EC, Bodner R, Posakony JW (2000) The enhancer of split complex of Drosophila includes four Notch-regulated members of the bearded gene family. Development 127: 3441–3455 9. Lecourtois M, Schweisguth F (1995) The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling. Genes Dev 9:2598–2608 10. Wech I, Bray S, Delidakis C et al (1999) Distinct expression patterns of different enhancer of split bHLH genes during embryogenesis of Drosophila melanogaster. Dev Genes Evol 209:370–375 11. de Celis JF, de Celis J, Ligoxygakis P et al (1996) Functional relationships between Notch, Su(H) and the bHLH genes of the E(spl) complex: the E(spl) genes mediate only a subset of Notch activities during imaginal development. Development 122:2719–2728 12. Nellesen DT, Lai EC, Posakony JW (1999) Discrete enhancer elements mediate selective responsiveness of enhancer of split complex genes to common transcriptional activators. Dev Biol 213:33–53 13. Furriols M, Bray S (2001) A model Notch response element detects suppressor of hairless-dependent molecular switch. Curr Biol 11:60–64 14. Housden BE, Millen K, Bray SJ (2012) Drosophila reporter vectors compatible with phiC31 integrase transgenesis techniques and their use to generate new Notch reporter fly lines. G3 Bethesda 2:79–82 15. Saj A, Arziman Z, Stempfle D et al (2010) A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network. Dev Cell 18:862–876 16. Go MJ, Eastman DS, Artavanis-Tsakonas S (1998) Cell proliferation control by Notch signaling in Drosophila development. Development 125:2031–2040 17. Li X, Zhao X, Fang Y et al (1998) Generation of destabilized green fluorescent protein as a transcription reporter. J Biol Chem 273: 34970–34975 18. Presente A, Shaw S, Nye JS et al (2002) Transgene-mediated RNA interference defines a novel role for notch in chemosensory startle behavior. Genesis 34:165–169 19. Helms W, Lee H, Ammerman M et al (1999) Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function. Dev Biol 215:358–374

20. McGuire SE, Le PT, Osborn AJ et al (2003) Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302:1765–1768 21. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415 22. Xu T, Rubin GM (1993) Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117:1223–1237 23. Lee T, Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22: 451–461 24. Classen AK, Aigouy B, Giangrande A et al (2008) Imaging Drosophila pupal wing morphogenesis. Methods Mol Biol 420:265–275 25. Muller HA (2008) Immunolabeling of embryos. Methods Mol Biol 420:207–218 26. Stocker H, Gallant P (2008) Getting started : an overview on raising and handling Drosophila. Methods Mol Biol 420:27–44 27. Cooper MT, Tyler DM, Furriols M et al (2000) Spatially restricted factors cooperate with notch in the regulation of Enhancer of split genes. Dev Biol 221:390–403 28. de Celis JF, Tyler DM, de Celis J et al (1998) Notch signalling mediates segmentation of the Drosophila leg. Development 125: 4617–4626 29. Pines MK, Housden BE, Bernard F et al (2010) The cytolinker Pigs is a direct target and a negative regulator of Notch signalling. Development 137:913–922 30. Assa-Kunik E, Torres IL, Schejter ED et al (2007) Drosophila follicle cells are patterned by multiple levels of Notch signaling and antagonism between the Notch and JAK/STAT pathways. Development 134:1161–1169 31. Almeida MS, Bray SJ (2005) Regulation of post-embryonic neuroblasts by Drosophila Grainyhead. Mech Dev 122:1282–1293 32. Cooper MT, Bray SJ (1999) Frizzled regulation of Notch signalling polarizes cell fate in the Drosophila eye. Nature 397:526–530 33. Kramatschek B, Campos-Ortega JA (1994) Neuroectodermal transcription of the Drosophila neurogenic genes E(spl) and HLHm5 is regulated by proneural genes. Development 120:815–826 34. Castro B, Barolo S, Bailey AM et al (2005) Lateral inhibition in proneural clusters: cis-regulatory logic and default repression by suppressor of hairless. Development 132:3333–3344

Visualization of Notch Signaling In Vivo 35. Barolo S, Castro B, Posakony JW (2004) New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP. Biotechniques 36:436–440, 442 36. Afek Y, Alon N, Barad O et al (2011) A biological solution to a fundamental distributed computing problem. Science 331:183–185 37. Williams JA, Paddock SW, Vorwerk K et al (1994) Organization of wing formation and induction of a wing-patterning gene at the dorsal/ventral compartment boundary. Nature 368:299–305 38. Djiane A, Krejci A, Bernard F et al (2013) Dissecting the mechanisms of Notch induced hyperplasia. EMBO J 32:60–71 39. Blochlinger K, Bodmer R, Jack J et al (1988) Primary structure and expression of a product from cut, a locus involved in specifying sensory organ identity in Drosophila. Nature 333:629–635 40. Brook WJ, Cohen SM (1996) Antagonistic interactions between wingless and decapentaplegic responsible for dorsal-ventral pattern

41.

42.

43.

44.

45.

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in the Drosophila Leg. Science 273: 1373–1377 Yip ML, Lamka ML, Lipshitz HD (1997) Control of germ-band retraction in Drosophila by the zinc-finger protein HINDSIGHT. Development 124:2129–2141 Giráldez AJ, Pérez L, Cohen SM (2002) A naturally occurring alternative product of the mastermind locus that represses notch signalling. Mech Dev 115:101–105 Lieber T, Kidd S, Alcamo E et al (1993) Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev 7:1949–1965 Seugnet L, Simpson P, Haenlin M (1997) Requirement for dynamin during Notch signaling in Drosophila neurogenesis. Dev Biol 192:585–598 Rebay I, Fehon RG, Artavanis-Tsakonas S (1993) Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell 74:319–329

Chapter 9 Monitoring Notch Activity in the Mouse Swananda Marathe and Lavinia Alberi Abstract Several laboratories have developed genetic methods to monitor Notch activity in developing and adult mice. These approaches have been useful in identifying Notch signaling with high temporal and spatial resolution. This research has contributed substantially to our understanding of the role of Notch in cell specification and cellular physiology. Here, we present two protocols to monitor Notch activity in the mouse brain: (1) by intraventricular electroporation and (2) by intracranial viral injections of Notch reporter constructs. These methods allow monitoring of Notch signaling in specific brain regions from development to adulthood. In addition, using the appropriate modifications, the Notch reporter systems can also be used to monitor Notch activity in other organs of the mouse such as retina, skin, skeletal muscle, and cancer cells. Key words Notch activity reporter, CBF1, Hes genes, In utero electroporation, Intracranial viral injection

1

Introduction Notch signaling occurs early in development when cell to cell communication specifies and patterns organogenesis [1–3]. The array of Notch targets is context dependent. At present, it remains largely unsolved how Notch achieves its functional specificity. Therefore, identifying functional targets of Notch in a cellular context has been a priority. To restrict the analysis to cells with ongoing Notch signaling and explore its genetic signature and functional relevance, several laboratories have devised strategies to monitor Notch activity in cells. At present, several Notch reporter constructs and mouse lines are available. Based on their core responsive elements, we divide them into four groups: (I) the multimeric CSL (CBF1/RBPJK/Su(H)/Lag-1) reporters, CBFRE [4–6]; (II) the Hes reporters [7–11]; (III) the Notch1 proteolysis reporters [12, 13], and (IV) the Notch1:CSL association reporter [14] (Table 1). The reporters belonging to the first two groups are independent of the Notch paralog that is activated

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Group I: Multimeric CSL reporters

Reporter type

pAAV-CBFREwt-DOIEYFP-WPRE-pAa (Cre expressing mouse) pAAV-CBFREwt DOI Halo-EYFP-WPRE-pA(a) (Cre expressing mouse)

pCAG-CBF1DBMVP16 Luciferase pMAMLDN [15] activity GSI (DAPT) pCAGCBF1DBMVP16MAMLDN Fluorescence GSI (DAPT) A: 480 E: 509 pCAGCBF1DBMVP16MAMLDN Fluorescence GSI (DAPT) A: 512 nm E: 528 nm AAVEf1α Fluorescence MAML A: 514 nm E: 527 nm AAVEf1α Yellow light MAMLDN A: 570 nm

AAVEf1α NICD

pCAG-NICD pCAG-CBF1VP16 AAVEf1α NICD

pCAG-NICD pCAG-CBF1VP16 pCAG-NICD pCAGCBF1VP16

CBFREwt::Luc [4] CBFREmut::Luc (WT mouse) CBFREwt::EGFP [5] CBFREmut::EGFP (WT mouse) CBFRE::H2BVenus [6] (WT mouse)

Quantitation of Notch activity Fate mapping, genetic fingerprint Fate mapping, genetic fingerprint Mapping of Notch activity Inhibition of neurons with ongoing Notch activity

Visualization Application

Negative control

Construct (mouse strain) Positive control

Table 1 List of Notch activity reporter systems

Disadvantages Reliable readout Not paralog specific; of Notch/ readout of CBF1 CBF1 activity activation independently of Notch activation

Advantages

pCAG-NICD

Hes1::Luc [8] Hes5::Luc (WT mouse) Hes1::EGFP [9] Hes5::EGFP (WT mouse) Hes1::UbLuc [10] (WT mouse)

Notch1NtLuc CtLucCBF [14] (WT mouse)

N1ΔEN-Luc

CtLucCBFDN MAMLDN

MAMLDN GSI (DAPT) MAMLDN GSI (DAPT)

MAMLDN GSI (DAPT)

MAMLDN GSI (DAPT) MAMLDN GSI (DAPT) MAMLDN GSI (DAPT)

Luciferase activity

βGal activity

βGal activity

Luciferase activity Fluorescence: A: 480 E: 509 Luciferase activity

βGal activity

Quantitation of Notch/ CBF activity

Fate mapping

Fate mapping

Quantitation of Hes activity Fate mapping, Genetic fingerprint Quantitation of Hes activity, real time visualization

Fate mapping

Activated Notch1 can undergo degradation without signaling; restricted to Notch1 Reliable readout Restricted to Notch1 of Notch1/ CBF1 complex formation Reliable reporter of Notch1 activation through γ-secretase

Reliable readout Not paralog specific; of Notch/ Hes genes are CBF1 also under transcriptional regulation activity; of other Reliable readout signaling of Hes cascades expression

CBFRE C-promoter binding factor responsive element, pCAG chicken actin promoter, CBF1DBM CBF1 DNA binding mutant, DOI double flox, NICD Notch intracellular domain, MAMLDN dominant negative Mastermind, GSI (DAPT) gamma secretase inhibitors, Luc luciferase, EGFP enhanced green fluorescent protein, A Absorbance, E Emission, LacZ β-Galactosidase [1], Ub ubiquitin promoter, WT wild-type a Modified from pAAV-DOI-eNpHR-EYFP-WPRE-pA [16] b Destabilized Fluorescent Protein

Group IV:

N1ΔE-VP16Gal4

N1ΔENCre

pCAG-NICD

pCAG-NICD

pCAG-NICD

Hes1::LacZ [7]

Notch1PCre [12] Group III: Notch1 (ROSA26R mouse) proteolysis Notch1VP16Gal4 [13] reporters (UAS-lacZ mouse)

Group II: Hes reporters

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and may translate CSL activity independently of Notch activation. In contrast, the last two groups can only be used to monitor Notch1 activity. With the latter approaches, cells with Notch1 signaling can be distinguished and isolated. To gain further spatiotemporal specificity of the Notch signal, Cre/lox recombination can be adopted to monitor Notch signaling in cellular ensemble expressing Cre under a cell specific promoter [15] (Fig. 2a). In addition, we document how a Notch responsive Halorhodopsin can be used to repress the activity of neuronal ensembles with ongoing Notch signaling [16]. In this chapter, we describe how to achieve stable expression of fluorescent Notch activity reporters through genetic transfer of Notch responsive elements in the developing embryo via electroporation and in the adult mouse brain via intracranial viral injection of an adeno‐associated virus (AAV) encoding a Notch reporter. In principle, all the constructs containing the Notch reporters listed can be used with this protocol. Depending on the construct used, care should be taken in selecting the genotype of the host, as indicated in Table 1. Electroporation of the same constructs can also be employed for gene transfer in other organs such as retina, muscle, skin, kidney, and liver using different protocols.

2

Materials Here, we list materials and reagents needed for obtaining fluorescent labeling of cells with ongoing Notch activity through intraventricular electroporation and intracranial viral injection. The outlined protocols are based on genetic transfer of the CSL reporters (group I) [5]. Nevertheless, other Notch reporter constructs, Table 1, may be used following the same technical procedures.

2.1 Intraventricular Electroporation of E15.5 Mouse Embryos 2.1.1 Transfection Reagents

(A) Transfection quality plasmid DNA (group I): 1. 4xCBFREwt::EGFP (Addgene, USA; #17705) or 4xCBFREmut::EGFP (Addgene, USA; #26870). 2. pCAG-DsRed2 (Addgene, USA; #15777). Alternatively a CBFRE‐EGFP‐CAG‐DsRed2 can be requested and used [5]. 3. Positive control: CBF1-VP16 [5] or pCAG-NICD (Addgene, USA; #26891). 4. Negative control: CBF1DBM-VP16 [5] or MAMLDN [17]. The DNA is obtained using a Pure Yield™ Maxi Prep kit (Promega, USA; #A2392). Plasmids are resuspended in sterile PBS at a concentration of 2 μg/μl. (B) Fast green FCF (Sigma‐Aldrich, USA; #F7252) to be added at final concentration of 0.1 %.

Monitoring Notch Activity in the Mouse 2.1.2 Electroporation Equipment and Materials

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1. Borosilicate glass capillaries of 1 mm diameter and 10 cm length (Sutter Instruments, USA; #BF 100‐78‐10) are pulled using a vertical puller, Model 700C (David Kopf Instruments, USA). Capillaries are fixed with the tip facing the edge of the cabinet; a current of 20.5 A, with solenoid settings 20, is applied with highest speed to extend the capillary and form a tip. Capillaries are inspected under a microscope. The level of the capillary at which the tip has an inner diameter of 30 μm is labeled and is pinched off with a #5 Dumont forceps. Glass capillaries are inspected again under a microscope. Pointy uneven tips are discarded to avoid any tissue lesion. Sharp tips are desired. 2. Glass capillaries are mounted onto a silicone hose of 5 mm diameter connected with a mouth pipet (Sigma, Germany; #A5177). 3. Microdissection tools include straight 10 cm long dissecting scissors, Vannas microscissors, Dumont #5 fine forceps, Iris forceps, and ring forceps. These tools are sterilized in a dry glass bead sterilizer and sprayed down with 70 % ethanol. 4. Commercial heating pad with step regulator for thermoregulation during mouse surgery and anesthesia. 5. Commercial electrical razor to shave the hair from the mother’s abdomen. 6. Sterile saline solution (0.9 % NaCl in endotoxin free double distilled water). Penicillin and streptomycin (100 μg/ml) can be added. 7. Over the counter sterile gauze to protect the animal and perform surgery and electroporation of the embryos. 8. ECM 830 Electroporator (BTX Harvard Apparatus, USA; #45‐0052) with round tweezertrode (BTX Harvard Apparatus, USA; #45–0166) and foot pedal (BTX Harvard Apparatus, USA, #45‐0211). 9. Coated VICRYL Braided Monofilament (Ethicon Inc., USA; #K871). 10. Needle holder (World Precision Instruments, USA; #14109). 11. 9 mm Autoclip applier (Kent Scientific, USA; #INS500545). 12. 9 mm surgical Autoclips (Kent Scientific, USA; #INS500546). 13. Over the counter povidone‐iodine solution.

2.1.3 Animal Model

1. E12‐15.5 timed pregnant mouse. 2. Inhalant anesthetic for induction: 4–5 % isoflurane delivered to an induction chamber. Inhalant anesthesia for maintenance: 1–2 % isoflurane delivered with a pressure vaporizer (Kent Scientific, USA; #ACV‐1205SR) connected to the mouse’s snout through a nose holder. Alternatively, an injection anesthetic can be used: ketamine (80–120 mg/kg) and xylazine (5–10 mg/kg).

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2.2 Intracranial Viral Injection 2.2.1 Viral Reagents

2.2.2 Stereotaxic Equipment

1. Packaged viral particles of pAAV‐CBFREwt‐DOI‐EYFP‐ WPRE‐pA and pAAV‐CBFREmut‐DOI‐EYFP‐WPRE‐pA (as negative control). The constructs have been cloned from the pAAV‐doublefloxed‐eNpHR‐EYFP‐WPRE‐pA (Addgene, USA; #20949) [16]. AAV with serotype 9 efficiently infects post‐mitotic neurons [18]. The optimal viral titer for in vivo injection is 108–12 IU/ml. 1. Model 900 stereotaxic apparatus for small animals with single manipulator (David Kopf, USA). 2. Model 5000 Microinjection Unit (David Kopf, USA). 3. OPMI microscope (Zeiss, Germany). 4. 75RN Syringe (Hamilton, USA; #7634‐01) with exchangeable fine needle (33 gauge, Length: 51 mm, Needle point style: Sharp 10–12° beveled needle) (Hamilton, USA; #207434). 5. Conventional heating pad with step regulation for thermoregulation during mouse surgery and anesthesia. 6. Precision Drill (Proxxon, LU). 7. Sterile scalpel with removable blade, Dumont #5 fine forceps, Iris forceps. 8. Sterile saline solution (0.9 % NaCl). 9. Kimwipe tissue paper folded and cut in 0.75 cm2 squares.

2.2.3 Animal Model

1. Transgenic Cre mouse line: CamKIIT29‐1Stl/J::Cre (Jackson Laboratories, USA; #005359). 2. Injection anesthetic: ketamine (80–120 mg/kg) and xylazine (5–10 mg/kg).

3

Methods All procedures should be carried out in a room dedicated to animal surgery with continuous air circulation and a temperature of 20–22 °C. Surgery is always performed on a clean and ethanol sterilized surface.

3.1 In Utero Electroporation

1. Anesthetize the pregnant mother in an induction chamber using isoflurane at a concentration of 4–5 %. An injection anesthetic, ketamine/xylazine mixture, may be used. 2. After anesthetizing the mouse, place the animal on the operating platform with its back on a heating pad and the abdomen facing up. Secure the limbs with tape. 3. Maintain anesthesia by delivering 1–2 % isoflurane through a pressure vaporizer connected to the mouse’s snout through a nose holder.

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4. Clean the abdomen with 70 % ethanol and shave the skin of the abdomen using an electric razor. 5. Cover the abdomen with 3 cm gauze precut along the longitudinal axis and wet the gauze with sterile saline. 6. Make an incision on the abdominal skin by pulling up the abdomen gently using the Iris forceps and making a fine cut with the Vannas scissors of about 2 cm and continuing with cutting the underlying muscle for about 1.5–1.7 cm. 7. After the incision keep the cavity wet by supplying pre‐warmed (37 °C) sterile saline dropwise. 8. Aspirate by air suction into a pipette about 10 μl of solution containing 4xCBFREwt::EGFP or 4xCBFREmut::EGFP and pCAG-DsRed at a concentration of no less than 1 μg/μl. Plasmids are mixed in a 3:1 ratio for 4xCBFREwt::EGFP and pCAG-DsRed, respectively. Fast green should be added at a final concentration of 0.1 %. Two capillaries are prepared separately for the wild-type and the mutant construct. 9. With the Iris forceps extract the uterus carefully from the abdominal cavity by pinching the gap between the embryos; 3–4 embryos can be extracted at the time. Keep the exposed uterus wet using pre‐warmed (37 °C) sterile saline. 10. Hold one embryo gently by the head using the ring forceps and direct the forebrain towards you. The telencephalon should be clearly visible. Insert the pipette into the lateral ventricle and release 1–3 μl of DNA solution by air pressure. The ventricle should be visibly filled with the green dye FCF (Fig. 1a). 11. Place the tweezertrode across the head with the anode on the injected side and deliver the pulse using the footswitch at the preset settings: Voltage = 35 V, pulse length = 50 ms, Pulse Interval = 1 s, number of pulses = 5 (Fig. 1a). 12. When using the multimeric CBFRE plasmid, inject half of the centrally located embryos with 4xCBFREwt::EGFP/pCAGDsRed on the right ventricle and half with 4xCBFREmut:: EGFP/pCAG-DsRed on the left ventricle. The two embryos closer to the end of the placenta remain un‐injected to avoid miscarriage. 13. After injecting and electroporating the embryos, the uterus is carefully placed back in the abdominal cavity. 14. Close the abdominal incision by surgical suturing and join the skin of the abdomen with 9 mm surgical autoclips. Disinfect the abdomen by gentle streaking with a cotton swab immersed in povidone‐iodine. 15. Keep the animal on the heating pad until it fully recovers. 16. Place the animal back into a clean cage and inspect the health status 4 h following surgery and daily the next days.

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Fig. 1 Intraventricular electroporation of Notch activity reporter. (a) Scheme of electroporation steps in the developing forebrain. (b) Fluorescent microscopy showing an E15 forebrain co-transfected at E12 with CBFRE‐ EGFP‐CAG‐DSRed2 construct and CBF1VP16 or CBF1DBMVP16. Expression of EGFP reads out responsiveness of the multimeric CBF reporter construct in CAG-DsRed2 positive cells. Reproduced by permission of Nature Press, UK. (c) Section of the cortex from 4 months old mouse electroporated at E15 with CAG‐DsRed2 and CBFREwt::EGFP or CBFREmut::EGFP. Expression of EGFP reads out endogenous Notch activity in cortical networks. Scale bars in (b) and (c) are 50 μm

3.1.1 Sample Analysis

1. After electroporation, the embryos can be recovered one or more days later. In addition, the pups can also progress into postnatal life. The embryonic brains are recovered in ice‐cold PBS and placed overnight in 4 % paraformaldehyde (PFA) in phosphate buffer. Alternatively for postnatal brains, mice are flushed with sterile saline and transcardially perfused with 4 % PFA in phosphate buffer. Brains are then kept in 4 % PFA overnight. Fixed embryonic or postnatal brains are cryoprotected with 30 % sucrose and processed into slices for immunohistochemistry. 2. Since endogenous EGFP can be quenched by PFA fixation, an immunohistochemical reaction using a specific antibody against EGFP can be used.

3.2 Intrahippocampal Viral Injection

1. Anesthetize a 2–3 months old mouse using ketamine/xylazine mixture. 2. Place the animal back into a clean cage. 3. After 10–15 min (min) check the pinch reflex. If the mouse reacts to pain, inject 1/4th of the initial dose of anesthetic to induce deep anesthesia, and test the pinch reflex in 10 min.

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4. Carefully shave the head’s hair between the ears of the mouse. 5. Mount the mouse head using the ear bars and the mouth support on the stereotaxic setup, and proceed with a 1.5 cm incision of the head’s skin along the midline using the scalpel. Flap the skin open and clean the surface of the skull with a cotton swab. 6. Using the OPMI stereoscope identify the region of penetration using a millimeter paper and mark the region with a fine black permanent pen. 7. Drill around the black spot concentrically and clean every 5 circles the surface from the skeletal dust using a cotton swab. After 20 circles the borders of the circles should be thin enough to see the vasculature, test if the surface is loose and lift the drilled skull piece gently using a #5 Dumont forceps. 8. Deliver to the exposed brain surface, a drop of sterile saline and cover with a precut square of tissue paper impregnated with sterile saline. 9. Load the syringe with the virus by retrieving 2.5 μl of viral stock and test that the needle is free by delivering a minute amount of virus into the stock vial. 10. Mount the syringe on the syringe holder and position the needle perpendicular to the Bregma at 3 mm distance from the surface of the skull. Take the coordinates on the stereotaxic setup. Retract the syringe at 1 cm distance from the skull and move anterio‐caudally 2 mm from the Bregma (AP) and 2 mm lateral (ML). Test if the needle is still free by rotating the manipulator of the syringe holder for a couple of cycles clockwise until a drop is visibly forming. Clean the test drop with the tip of a Kimwipe and proceed until touching the surface of the brain. Advance the tip of the syringe into the brain by using the stereotaxic Z manipulator (DV). 11. Wait 1 min for the syringe to stabilize in the brain parenchyma. 12. Deliver using the syringe manipulator 1 μl of viral solution (titer 108–12 IU/ml) into hippocampal CA1 at a rate of 5–10 nl per sec. 13. After having injected the virus wait for 3 min to allow complete diffusion. 14. Retract the syringe, check that the needle is still unclogged, and proceed with unmounting the syringe. 15. Suture the skin using a re-absorbable thread. 16. Put the mouse in its cleaned home cage on a heating pad and let the mouse recover. 17. Place the mouse in a dedicated room and inspect daily for 2–3 days following surgery.

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Fig. 2 Intrahippocampal viral injection of a cell specific Notch responsive construct. (a) Strategy of the Cre/lox recombination used to obtain a cell specific Notch reporter. (b) Drawing of mouse brain section at which the injection is performed. The red lining indicates the CA region where Cre is expressed in the CamKIIT29‐1Stl/ J::Cre mouse line. (c) One week after injection, in response to endogenous Notch activity, EYFP is expressed specifically in hippocampal CA3. Scale bar in (c) is 25 μm 3.2.1 Sample Analysis

1. One week after intracranial viral injection, the mice are anesthetized with ketamine and xylazine. Deep anesthesia is tested by pinch reflex and mice are transcardially perfused with 0.9 % NaCl followed by 4 % PFA in phosphate buffer. The brains are recovered and kept in 4 % PFA overnight. Fixed embryonic or postnatal brains are cryoprotected using 30 % sucrose and processed into slices for immunohistochemistry. 2. Serial sections of the entire hippocampus are imaged using a Nanozoomer (Hamamatsu, Japan) using fluorescence excitation settings at 514 nm and absorbance of 527 nm (Fig. 2).

3.2.2 Critical Steps and Troubleshooting

4

For both procedures, it is very important to select the appropriate host according to the Notch activity reporter used. It is always advisable to test the constructs in vitro first, using either 3T3 cells or primary fibroblasts obtained from E14.5 mouse of a specific genotype, and then proceed to the in vivo application.

Notes on Intraventricular Electroporation The main challenge of the in utero electroporation is survival of the embryos and sometimes of the mother. Great care should be taken while operating using sterile measures. The other equally critical step is the handling of the embryo or the pup.

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The delivery of the DNA solution through the glass capillary is critical. 1. Before starting surgery, the shaved abdomen should be rubbed with 70 % ethanol. The mouse abdomen should be covered with sterile gauze to avoid any contact between the embryos and the surface of the abdomen. 2. The abdominal cavity is kept humidified using sterile saline (0.9 % NaCl). Saline can be supplemented with penicillin and streptomycin (100 U/ml). 3. All surgical tools should be sterilized and sprayed with 70 % ethanol. The other equally critical step is the handling of the embryo or the pup: 4. The embryos should be pulled one by one by grasping the uterine wall between the embryos with blunt forceps, to avoid any lesion. Two to three embryos can be taken out at a time. 5. The embryo’s forebrain can also be pressed against the uterine wall with two fingers. Gloves should be worn at all times and frequently rubbed with 70 % ethanol. 6. In order to prevent any lesion to the uterus, the two embryos closer to the end of the placenta should remain un‐injected to avoid miscarriage. The delivery of the DNA solution through the glass capillary is critical: 7. Mouth expiration should be exercised to deliver 1–3 μl of solution. 8. A test volume of approximately 1 μl should be extruded before injection to check for any clogging of the capillary. The tip is then wiped out using a Kimwipe before intraventricular injection. 9. The electroporation settings can be critical and depend on quality of the DNA and the age of the embryo and pup. 10. Commercially available kits with purification columns ensure endotoxin-free DNA but there is usually loss of DNA in the purification process. Prepare 0.5 l of maxiprep solution to obtain sufficient quantity of DNA. 11. When co‐transfecting 2 or 3 plasmids together, be aware of the molar ratio of the constructs, not their final concentration. 12. Check the settings suggested by the electroporator manufacturer. A standard setting of voltage = 35 V, pulse length = 50 ms, pulse interval = 1 s, number of pulses = 5 can be used for embryos of embryonic stage E12-15. An increment of 10 V is desired with each further embryonic day. Postnatal pups (P0–P4) can be injected at a voltage of 100 V. Mortality can be expected but should be lower than 20 % [19].

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13. After surgery, the mother’s abdomen should be sutured twice. The inner wall should be sutured with knots every 3–4 mm. The outer wall should be joined using Autoclips. The mouse should be allowed to recover on a heating pad and then transferred to a clean cage. 4.1 Notes on Intracranial Injection

The most critical part of this procedure is the mouse anesthesia, which can be lethal to the animal. 1. Weigh the mouse before injecting and calculate the dose of injectable anesthetic. 2. If after 10 min, the mouse responds to pain by pinch reflex, inject 1/4th of the dose and wait for additional 10 min. 3. If after 10 min sensitivity to pain is still present, do not reinject and wait 10 more min and try again. 4. If during anesthesia the mouse starts moving the whiskers, it’s an indicator that anesthesia is decaying. Extract the needle from the brain and administer ½ of the initial dose. Wait 10 min, check the absence of any pain reflex and then proceed with the viral injections. The second more critical step is the flow of the viral solution through the needle. Virus can be sticky and can clog the fine needle. 5. Flow is checked regularly by pressing the plunger using the syringe holder manipulator until a drop is visible. Check if the drop is forming shortly after having collected the virus into the syringe, before inserting the needle into the brain, and after injection. The drop should be wiped gently with a folded Kimwipe from the needle. 6. If the needle is clogged, or the flow is not continuous, change the needle and retry. 7. A clogged needle can be cleaned by placing the needle in a small beaker with double distilled water and sonicating it for 10–20 min using a bench top ultrasonic water bath (Branson, USA; #1200). 8. As a general rule after each injection, the needle should be cleaned thoroughly with double distilled sterile water on a clean metal surface to detect possible crystals which are extruded from the needle. In order to minimize any superficial cortical lesion during drilling and to avoid any postoperational sequelae such as bleeding, as well to ensure that the injection is done at the exact coordinates, the mouse head should be mounted correctly on the stereotaxic setup. 9. After the mouse head is mounted with the ear bars, the snout should not move and the stability should be further tested by lifting the animal so that the head rotates 45° on the ear bars axis.

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10. Pull the mouth holder and take great care that the two front teeth are fitted into the mouse holder hole and secure the mouth holder avoiding pressing heavily on the mouse’s nose. 11. If the mouse head moves during drilling, unmount the mouse and remount it. Try to be quick. 12. The drill should be removed before the surface of the brain is exposed. 13. The Dumont #5 forceps should be held parallel to the surface of the brain and is inserted carefully at the border of the drilled surface to lift the cranial window. 14. While preparing the syringe, the cranial surface is kept wet using a folded Kimwipe impregnated with sterile saline. If you are interested in injecting the virus in small nuclei of the brain of C57BL/6 mice, we recommend using the coronal view of the Allen Mouse Brain Reference Atlas (http://mouse.brain-map. org/static/atlas), which was constructed based on the C57BL/6 strain. Adeno‐associated virus can be used to infect a variety of organs in the mouse body such as cardiac muscles and skeletal muscles that are notoriously difficult to infect using lentiviruses.

5

Typical Protocol Results

5.1 Results of In Utero Electroporation

The method for intraventricular electroporation described above can be used with all listed constructs (Table 1) in wild-type and transgenic mice. This protocol should allow visualization of Notch signaling in the developing forebrain as well as in the mature nervous system. The Notch responsive elements can be used to visualize Notch activity by overexpression of positive or negative regulators (Fig. 1b) as well as to study endogenous Notch activity (Fig. 1c). Intraventricular electroporation can be performed as early as E12.5. In Fig. 1b, wild-type E12.5 embryos were transfected with a multimeric CBF‐based Notch responsive construct (CBFRE‐EGFP‐ CAG‐DsRed2) and either a transactivator (CAG‐nlsLacZ‐CBF1‐ VP16) or a repressor (CAG‐nlsLacZ‐CBF1DBM‐VP16) [5]. Using the transactivator CBFVP16 as a readout of Notch activity, an increase in EGFP expression is visible in the developing forebrain at E15. In addition, activation of Notch retains the cells in the more ventricular regions as indicated by expression of the CAG‐DsRed2 reporter. In contrast, transfection with a mutated form of CBF reduces CBFRE‐EGFP expression and transfected cells migrate radially from the ventricle. Figure 1c shows endogenous Notch activity in the postnatal mouse cortex. In utero electroporations were performed at E15 on half of the embryos on the right side (pCBFREwt:: EGFP and pCAG-DsRed2) and half of the embryos on the left side (CBFREmut::EGFP and pCAG-DsRed2). The pups were then

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allowed to mature until postnatal week 4. In the cortex of the mice, transfected with CBFRE::EGFP, only a small proportion of neurons displays EGFP expression with variable intensity. This suggests that Notch activity is ongoing in neuronal ensembles with different activation levels. On the other hand, neurons double‐ transfected with pCAG-DsRed2 and the mutant construct do not display any EGFP expression, validating the reliability of the system. Based on the numerous Notch reporter constructs available and the pleiotropic functions of Notch, electroporation can be carried out in a variety of organs of the mouse in order to study ongoing Notch signaling and its significance. 5.2 Results of Intracranial Viral Injections

Viral technology has expanded in order to achieve gene transfer in postnatal tissues and cell types that are typically difficult to transfect. AAV of serotype 9 has a selective tropism for neurons and can be used to study cellular processes in the mature brain [18]. AAV backbones with floxed cassettes allow for further spatial specificity based on neuronal specific promoters driving Cre expression. In Fig. 2a, we present our strategy to visualize Notch signaling in postmitotic neurons of the CA field using the construct pAAV‐CBFREwt‐doublefloxed‐EYFP‐WPRE. The cassette containing EYFP is flipped in the direction of transcription after recombination only in neurons expressing Cre driven by CamKII. Nevertheless, EYFP is only expressed in neurons where Notch activity is ongoing. The AAV is injected into the hippocampus of the CamKIIT29‐1Stl/J::Cre mouse at stereotaxic coordinates: AP = −2 mm from Bregma, ML = 2 mm, and DV = 2 mm (Fig. 2b). One week after injection, EYFP is localized to the CA3 field (Fig. 2c). Interestingly, a similar expression is observed in the Hes1GFP mouse line [9]. No expression is observed when injecting the pAAV‐CBFREwt‐doublefloxed‐EYFP‐WPRE in a RBPJKcKO (RBPJKflox/flox [20]; CamKIIT29‐1Stl/J::Cre) where RBPJK expression is deleted in the CA field (data not shown). Alternatively, a mutant version of the pAAV‐CBFREmut‐doublefloxed‐EYFP‐ WPRE can be used as negative control. The same strategy can be used to drive Halorhodopsin in response to Notch activity in neuronal ensembles and manipulate neuronal networks in learning and memory tasks.

Acknowledgments We would like to thank Nicholas Gaiano and Kenichi Mizutani for allowing us to reprint one of their results and for helping with the intraventricular electroporation technique. We would also like to thank Mauro Giacca and Lorena Zentilin for packaging the AAVs. This work is supported by the Swiss National Foundation, the Synapsis Foundation for Alzheimer’s Research, and Swiss Heart Association.

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References 1. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233 2. Guruharsha KG, Kankel MW, ArtavanisTsakonas S (2012) The Notch signalling system: recent insights into the complexity of a conserved pathway. Nat Rev Genet 13:654–666 3. Lathia JD, Mattson MP, Cheng A (2008) Notch: from neural development to neurological disorders. J Neurochem 107:1471–1481 4. Souilhol C, Cormier S, Monet M et al (2006) Nas transgenic mouse line allows visualization of Notch pathway activity in vivo. Genesis 44: 277–286 5. Mizutani K, Yoon K, Dang L et al (2007) Differential Notch signalling distinguishes neural stem cells from intermediate progenitors. Nature 449:351–355 6. Nowotschin S, Xenopoulos P, Schrode N et al (2013) A bright single-cell resolution live imaging reporter of Notch signaling in the mouse. BMC Dev Biol 13:15 7. Ishibashi M, Moriyoshi K, Sasai Y et al (1994) Persistent expression of helix-loop-helix factor HES-1 prevents mammalian neural differentiation in the central nervous system. EMBO J 13:1799–1805 8. Shimizu K, Chiba S, Saito T et al (2002) Functional diversity among Notch1, Notch2, and Notch3 receptors. Biochem Biophys Res Commun 291:775–779 9. Ohtsuka T, Imayoshi I, Shimojo H et al (2006) Visualization of embryonic neural stem cells using Hes promoters in transgenic mice. Mol Cell Neurosci 31:109–122 10. Masamizu Y, Ohtsuka T, Takashima Y et al (2006) Real-time imaging of the somite segmentation clock: Revelation of unstable oscillators in the individual presomitic mesoderm cells. Proc Natl Acad Sci U S A 103:1313–1318

11. Vilas-Boas F, Fior R, Swedlow JR et al (2011) A novel reporter of notch signalling indicates regulated and random Notch activation during vertebrate neurogenesis. BMC Biol 9:58 12. Vooijs M, Ong CT, Hadland B et al (2007) Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE. Development 134:535–544 13. Smith E, Claudinot S, Lehal R et al (2012) Generation and characterization of a Notch1 signaling-specific reporter mouse line. Genesis 50:700–710 14. Ilagan MX, Lim S, Fulbright M et al (2011) Real-time imaging of Notch activation using a luciferase complementation-based reporter. Sci Signal 4:rs7 15. Nagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26: 99–109 16. Gradinaru V, Thompson KR, Deisseroth K (2008) eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications. Brain Cell Biol 36:129–139 17. Maillard I, Weng AP, Carpenter AC et al (2004) Mastermind critically regulates Notchmediated lymphoid cell fate decisions. Blood 104:1696–1702 18. Aschauer DF, Kreuz S, Rumpel S (2013) Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310 19. Fernández ME, Croce S, Boutin C et al (2011) Targeted electroporation of defined lateral ventricular walls: a novel and rapid method to study fate specification during postnatal forebrain neurogenesis. Neural Dev 6:13 20. Tanigaki K, Han H, Yamamoto N et al (2002) Notch-RBP-J signaling is involved in cell fate determination of marginal zone B cells. Nat Immunol 3:443–450

Chapter 10 Notch Signaling Assays in Drosophila Cultured Cell Lines Jinghua Li, Benjamin E. Housden, and Sarah J. Bray Abstract Signaling assays in Drosophila cell lines are a valuable method for investigating whether other proteins influence the function of the Notch pathway and for assessing whether specific enhancers or genes are regulated by Notch. In this chapter, we will describe two different types of assays that can be used to monitor Notch activation in Kc167 and S2 cells. One involves activating Notch in cultured cells and measuring the change in endogenous gene expression levels. The other uses luciferase reporters and measures their response to Notch, by co-transfecting with NICD. Key words Notch, Drosophila cell line, EDTA/EGTA treatment, Real-time PCR, E(spl) genes, NRE, Enhancer, Dual-luciferase assay, Luciferase reporter, Cell transfection

1  Introduction The core Notch pathway is relatively simple: when NICD is translocated in the nucleus and binds to CSL, co-activators get recruited and hence the target gene is expressed [1–3]. Transcriptional read-­outs therefore provide a powerful method for monitoring Notch activity and for testing whether genes respond to Notch. An advantage of performing such experiments in Drosophila cells is that flies have a single type of Notch receptor. Furthermore, one can quantitatively investigate the response to Notch signal activation using two types of assays that are complementary to each other. One involves measuring, on transcriptional level, how the endogenous genes respond to Notch activation. The other is to analyze the capability of a regulatory element (enhancer) to activate reporter gene expression in the presence of activated Notch. In cell lines that express Notch, a calcium chelator (EDTA or EGTA) can be used to activate Notch, resulting in changes in target gene expression. Exposing cells to calcium chelators stimulates shedding of the Notch ectodomain [4]. This renders the residual transmembrane fragment a substrate for gamma-secretase cleavage, Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_10, © Springer Science+Business Media New York 2014

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releasing Notch intracellular domain (NICD), which will enter the nucleus and up-regulate expression from competent targets. The mRNA is then extracted under conditions with and without EDTA/EGTA treatment and the expression levels of genes of interest (GOI) are compared. The E(spl) genes are well-­ characterized Notch targets that are activated within 30 min of EDTA/EGTA treatment [5], and provide an assay for monitoring Notch pathway activity after knock-down or over-expression of putative regulators (Fig. 1). Details of primers that can be used to measure expression of these E(spl) targets by quantitative PCR are provided in Table 1. This assay can be combined with RNAi treatment of cells to assess the relevance of genes that are required for Notch dependent transcription [6].

Fig. 1 E(spl )mβ-HLH and E(spl )m3-HLH mRNA levels with EGTA induction in Kc167 cells. Cells were treated with PBS or 4 mM EGTA for 30 min. RNA levels were quantified by RT-QPCR as described in Subheading 2. Data were normalized to media only

Table 1 Primer sequences used in real-time PCR Primers

Sequences

Rp49_sense

TCTGCATGAGCAGGACCTC

Rp49_antisense

CGGTTACGGATCGAACAAG

mbeta_sense

CTGGAGCTTGAAGAGGCACT

mbeta_antisense

CCCAGCTGATGAGTCACCTT

m3_sense

AGCCCACCCACCTCAACCAG

m3_antisense

CGTCTGCAGCTCAATTAGTC

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The second assay relies on luciferase reporters, which provide a quantitative analysis of Notch activity or of putative Notch responsive enhancers. The enhancers are cloned upstream of a minimal promoter and then transiently transfected into Drosophila cells in the presence of NICD. The Schneider 2 (S2) cell line is commonly used because the cells are easy to transfect and Notch and its ligands, Delta and Serrate, are not expressed in this cell line [5]. Therefore the level of Notch can be controlled by co-­ transfection with pMT-NICD or pMT-Full length Notch, so in this case it is ligand independent Notch signaling. The pMT vector uses the Drosophila metallothionein gene promoter, which can be induced upon addition of copper sulfate or cadmium chloride to the culture medium. Endogenous levels of the core transcription factor, Suppressor of Hairless (Su(H)), and of the co-activator Mastermind are sufficient to achieve high levels of stimulation. By comparing the luciferase values between pMT-NICD and pMT only, one can analyze quantitatively the enhancer’s potential to regulate gene expression upon Notch activation. More complex assays using ligand stimulation by co-culturing receptor- and ligand-expressing cells are also plausible [7]. Standard Notch reporters, containing Notch responsive element (NRE), are available, along with a control mutated version that has compromised Su(H) binding (NME) (Fig. 2) [8, 9].

Fig. 2 pGL3-min vector map. Overview of the pGL3-min vector [12] indicating key regions. MCS contains cutting sites for enzyme KpnI, SacI, Xmal, Smal, XhoI, and BglII. Composition of the synthetic NRE is shown in the lower panel, blue indicates palindromic Grainyhead binding motifs, and violet indicates Su(H) paired motifs

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Fig. 3 Response of different enhancers to NICD overexpression in luciferase assays. E(spl)m3-NRE (m3-NRE) is an example of an enhancer cloned into pGL3-min plasmid that gives robust positive response to NICD, whereas the NME (synthetic NRE with Su(H) motifs mutated) gives little/no response and provides a useful negative control. Klu-NRE is an enhancer from klumpfuss (klu), one of the novel Notch targets [9], and loses its response when the Su(H) motifs are mutated (Klu-Su(H)-mut)

The NRE-luciferase contains two copies of the paired Su(H) binding sites from the E(spl)m8 gene, in combination with four binding sites for the widely expressed Grainyhead transcription factor [10], and is stimulated 10- to 20-fold by the presence of NICD. This response is eliminated in the NME-luciferase where the Su(H) motifs are mutated. Another positively responding reporter, which uses the E(spl)m3 regulatory region (m3-NRE), can be up-­regulated 20- to 60-fold by NICD (Fig. 3). Putative Notch responsive enhancers from other genes can be tested in the same way, as illustrated by the klumpfuss enhancer, which is stimulated 20-fold by NICD and loses its response when the Su(H) motifs are mutated (Fig. 3).

2  Materials 2.1  Materials for Part I

1. Drosophila Kc167 cell growth media: Schneider’s Drosophila Medium, 5 % heat-inactivated fetal bovine serum (FBS). Kc167 cells do not grow at 10 % FBS. 2. EDTA (ethylene diamine tetraacetic acid) solution or EGTA (ethylene glycol tetraacetic acid) solution: Dilute from 0.5 M EDTA or 0.5 M EGTA stock to PBS.

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3. TRIzol® Reagent (Invitrogen). Store at 2–25 °C (preferably in the fridge). 4. Chloroform. 5. Isopropyl alcohol. 6. 70 % ethanol (in diethylpyrocarbonate (DEPC)-treated water). 7. RNase-free water. 8. Tabletop microcentrifuge reaching up to 12,000 × g. 9. M-MLV Reverse Transcriptase and 5× buffer (Promega). 10. Oligo(dT) primers. 11. 10 mM dNTPs. 12. Recombinant RNasin Ribonuclease Inhibitor. 13. LightCycler® 480 SYBR Green I Master (Roche). 14. Suitable machine for real-time PCR. The analysis methods in this chapter are based on the LightCycler®480 System (Roche). 15. Primers for quantitative PCR against a control gene (e.g., Rp49) and Notch target genes (e.g., E(spl)mβ and E(spl)m3) (see Table 1). 2.2  Materials for Part II

1. Drosophila S2 cell growth media: Schneider’s Drosophila Medium, 10 % heat-inactivated fetal bovine serum (FBS). 2. Plasmid DNA for NRE-luciferase as positive control and NME-luciferase reporters as negative control, Renilla luciferase along with luciferase reporters for your gene of interest (GOI). 3. Plasmid DNA for pMT-NICD and pMT along with any additional factors for testing. 4. Plasmid Isolation Kit (e.g., Qiagen Mini-, Midi-, or Maxiprep). 5. Opti-MEM® I Reduced Serum Media (Invitrogen). Store at 2–8 °C in the dark. 6. FuGENE® 6 Transfection Reagent (Promega). Store at 4 °C. Do not freeze or store below 0 °C. 7. 0.5 M CuSO4. 8. 1× Passive Lysis Buffer (PLB, from Promega): Add 1 volume of 5× PLB to 4 volumes of distilled water. Mix well. Store at 4 °C (≤1 month). 9. Luciferase Assay Reagent II (LAR II) (Promega): Resuspend the lyophilized Luciferase Assay Substrate in Luciferase Assay Buffer II. Store at –20 °C (≤1 month) or −70 °C (≤1 year). 10. Stop & Glo Reagent (Promega): To the required amount of Stop & Glo® Buffer, add 50× Stop & Glo® Substrate to a final 1× concentration.

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3  Methods 3.1  Part I: EDTA/ EGTA Treatment Assay to Measure How Endogenous Genes Respond to Notch Activation

1. EDTA/EGTA treatment: Incubate Kc167 cells with 4 mM EDTA or EGTA in PBS for 30 min (see Note 1). Treat control cells with PBS without addition of EDTA/EGTA. 2. Total RNA extraction:  (a) Remove solution from culture plate. For 6-well plate (5–10 × 106 cells per well), add 0.75 mL TRIzol Reagent per well. Lyse the cells by pipetting the cells in TRIzol solution up and down several times. (b) Incubate the samples for 10 min at room temperature. (c) Collect the samples in 1.5 ml microcentrifuge tubes and add 0.15 mL of chloroform. Mix by vortexing for 10 s. (d) Leave the samples in microcentrifuge tubes on the bench for 5 min at room temperature. (e) Centrifuge at 12,000 × g for 15 min at 4 °C.  (f) Transfer the aqueous phase into a new tube. Avoid touching any material that is in the interphase. (g) Add same volume of 100 % isopropanol (~0.5 mL), incubate at −80 °C for 2 h or −20 °C overnight to precipitate the RNA (see Note 2). (h) Centrifuge at 12,000 × g for 15 min at 4 °C.   (i) Remove the supernatant, wash once with 0.75 mL of 75 % ethanol at 4 °C. Discard the supernatant.   (j) Dry the RNA pellet at room temperature for ~ 5 min. Do not over-dry the pellet. (k) Resuspend in ~100  μL RNase-free water (see Note 3). 3. Reverse-transcription: For a 25 μL reaction: (a) In a sterile and RNAse-free microcentrifuge tube, add 0.5 μg of oligo(dT) primer and 2 μg of mRNA in a total volume of ≤15 μL in RNase-free water. (b) Heat the samples at 70 °C for 5 min to melt any secondary RNA structure. (c) Cool down immediately on ice to prevent secondary structure reformation. (d) Add 5 μL of M-MLV 5× Reaction Buffer, 1.25 μL of 10 mM dNTPs, 0.5 μL Recombinant RNasin Ribonuclease Inhibitor, 200 units of M-MLV RT and Nuclease-Free Water to final volume 25 μL. (e) Mix gently by flicking the tube and incubate for 60 min at 42 °C (see Note 4).   (f) Incubate for 10 min at 70 °C to inactivate the enzyme. Purification of cDNA is not required.

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4. Quantitative (real-time) PCR: For a 10 μL reaction. Add 5 μL of SYBR Green I Master, PCR primers (see Note 5) at a final concentration of 0.5 μM, 0.5 μL of cDNA from the reverse transcription and top up to 10 μL with nuclease-free water (see Note 6). 5. Relative mRNA level calculation: In addition to running qPCR reactions using primers against GOI on EDTA/EGTA ­treatment and control treatment samples, another qPCR reaction using primers against an endogenous control, such as Rp49, must be performed in parallel (see Note 7). Use second derivative maximum to call Ct values for each primer set. More details about the protocol and data analysis can be found in Bustin et al. book chapter about qPCR [11]. Then use the following equation to calculate the relative fold change (N) upon EDTA/EGTA-mediated Notch activation (see Fig. 1). Primer efficiency (E) can be calculated by running standard curve.

( EGOI ) = ∆Ct ( ECalibrator )

∆Ct GOI( control− EDTA )

N 3.2  Part II: Transient Co-transfection of GOI Luciferase Reporter and NICD in S2 Cell Lines

Calibrator ( control− EDTA )

1. Plasmid DNA preparation: Grow E. coli cells transformed with plasmid DNA in appropriate volume of LB media with ampicillin overnight and isolate DNA using standard Qiagen Mini-, Midi-, or Maxiprep protocol (see Note 8). For testing novel enhancers, fragments should be cloned into an adapted version of pGL3 (Invitrogen) containing a Drosophila promoter (e.g., pGL3-min which contains a minimal hsp70 promoter [12]). 2. Cells: Culture S2 cells at 25 °C in Schneider’s Drosophila Medium containing 10 % FBS (see Note 9) in 100 mm plates. For maximal transfection efficiency, the number of cells when transfecting DNA should reach 80–90 % confluence. However, optimal cell density may vary for some cell lines. 3. Perform transient cell transfection of pMT-NICD/pMT vectors along with reporter constructs using Fugene 6 as follows. Other transfection reagents are also effective, but in our hands Fugene has worked reproducibly in many Drosophila cell types. (a) Split S2 cells one day before transfection so that the cell density is approximately 70–80 % confluent on the day of transfection (see Note 10). (b) On the day of transfection, plate 1–2 × 106 cells in 500 μL per well of a 24-well plate (see Note 11), then put the plate back into 25 °C incubator to allow at least 30 min for the cells to attach to the bottom before transfection. (c) Prepare a mix of Opti-MEM (see Note 12) and Renilla DNA solution (40 μL Opti-MEM/well + 200  ng Renilla DNA/well). Mix thoroughly by vortex (see Note 13).

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(d) For each reporter construct, 4 wells are needed—two technical replicates co-transfected with pMT and the other two with pMT-NICD plasmids. So for each reporter construct, add 160 μL of Opti-MEM plus Renilla in a ­microcentrifuge tube. (e) Add 1.2 μg (0.3 μg/well) of reporter constructs (luciferase plasmids) to the 160 μL mix. Mix by vortexing the microcentrifuge tube (see Note 14).  (f) Aliquot 80  μL of each mix into 2 microcentrifuge tubes. Then add 0.6 μg (0.3 μg/well) of pMT or pMT-NICD (see Note 15) to each tube. Mix well by vortexing. 4. In a separate tube, mix 25 μL of Opti-MEM/well with 3 μL of Fugene/well (see Note 16). Do not vortex (see Note 17). Leave for 5 min at room temperature. 5. Add Fugene mix to the plasmid mix from step 6 (80 μL ­mixture + 56  μL Fugene). Use the pipette to mix briefly and gently, do not vortex. 6. Incubate the mix for 30 min at room temperature. Avoid moving the tubes while incubating (see Note 18). 7. Take the 24-well plate from step 2 out from the incubator. Remove 250 μL of media from each well without disturbing cells. Then add 65 μL of transfection mix from step 9 to each well. (You should have enough volume for two wells for each condition as technical replicates.) 8. Put the cells back in the incubator for 6 h (see Note 19). 9. Replace the media with 0.5 mL fresh media containing 500 μM CuSO4. 10. Lyse and harvest cells after another 16–20 h (see Note 19). 11. Dual-luciferase reading.  (a) Cell Lysis: Remove all media from cultured cells, and then add 100 μL of 1× PLB Buffer per well of the 24-well plate. Incubate while gently shaking the plate for 15 min at room temperature (see Note 20). (b) Dual-luciferase assay with Manual or Single Injector Luminometer: Add 45 μL of LAR II and 5 μL of cell lysate into luminometer tube. Mix for 3 s and then measure firefly luciferase activity. Then add 45 μL of Stop & Glo Reagent. Mix for 3 s and measure Renilla luciferase activity (see Note 21). 12. Calculating Notch reporter responsiveness. (a) First, calculate the ratio of firefly luciferase activity to Renilla luciferase activity. If the two replicates have similar ratio value, take the average as one biological replicate result. Renilla luciferase activity is used to normalize the variation due to transfection efficiency.

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(b) Calculate the ratio of NICD (luciferase to Renilla value for pMT-NICD sample) to control (luciferase to Renilla value for pMT sample). This is the index to measure construct responsiveness to Notch (see Note 22). Repeat the entire experiment 3 times and calculate the mean and the standard error for each construct/condition (see example in Fig. 3).

4  Notes 1. As EDTA and EGTA chelate Calcium ions, either can be used successfully for activation of Notch. The fact that EGTA is relatively specific for calcium makes it the preferred reagent. However, it is recommended to test which is more efficient at activating Notch target genes in each cell type, as this can differ. The concentration and treatment time period of EDTA or EGTA should also be optimized for a particular cell type. It is not recommended to use growth media for EDTA/EGTA treatment, because the metal ions present in the media can compromise the effect. 4 mM EGTA in HBSS (Invitrogen) has been tested to work well in DmD8 and BG3 cell lines. To assess specificity, incubations with a gamma-secretase inhibitor can be included in parallel. 2. E(spl) genes are typically used as a read-out. These mRNAs are very short; therefore longer precipitation time is recommended here. 3. Measure the concentration and quality of the extracted total RNA. Partially dissolved RNA will have an A260/280 ratio laa

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Fig. 2 Applying the transcriptional reporter assay to probe mechanistic aspects of Notch signaling. Transcriptional reporter assays can be used for structure-function analyses (a), and to measure effects of inhibitory drugs (b) and polypeptides (c). (a) Substitution of key residues within the RAM domain abrogates Notch transcriptional activity on the Hes1-Luc reporter in 3T3 cells. Note that the measured loss in function can be affected by the amount of transfected Notch expression plasmid. (b) The γ-secretase inhibitor DAPT prevents ligand-dependent activation of the reporter: MEFs endogenously expressing Notch receptors were transfected with TP1-Luc and CS2 + βgal. The day after transfection, cells were co-cultured with CHO or CHOJag1 cells in medium containing DMSO vehicle or 5 μM DAPT. Reporter assays were performed 24 h later. (c) Expression of dominant negative MAML (DNMAML; [26]) prevents ligand-dependent activation of the reporter: MEFs expressing endogenous Notch receptors were transfected with TP1-Luc and CS2 + βgal along with EGFP or DNMAML-EGFP and co-cultured the next day with CHO or CHO-Jag1 cells. Reporter assays were performed after 24 h of co-culture. DAPT and DNMAML can be used to assess specificity of reporter activation to Notch signaling in cell lines of interest

Fig. 3 A fluorescent reporter allows monitoring of pathway activation at the single cell level. CHO cells stably expressing full-length Notch2 [20] and a 12XCSL-mCherry reporter were co-cultured with CHO cells expressing GFP or Jag1-IRES-GFP [21]. mCherry reporter expression occurs only in the presence of ligand-expressing cells

However, they may not always accurately reflect the transactivation activities on native promoters, which can differ in the number, orientation, and spacing of their CSL-binding sites [1, 2].

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Fig. 4 Target promoter considerations in designing a Notch signaling assay. (a) The TP1-Luc reporter provides a quantitative measure of a wide range of signaling activities in cell populations: Different forms of Notch were cotransfected with TP1-Luc and CS2 + βgal control into unstimulated 3T3 cells. Luciferase values were corrected for transfection efficiency and normalized to that of the vector control to obtain fold activation. (b and c) Luciferase reporters controlled by multimerized CSL-binding sites (4XCSL, TP1) and endogenous target promoters (Hes, Hey) vary in basal activities and dynamic ranges: 3T3 cells were cotransfected with CS2 + βgal and different Notch reporters, with and without N∆E. Luciferase values were corrected for transfection efficiency to obtain relative Luc activities across the different reporters (b). Normalization of the same data to the respective vector control determines the dynamic range for each reporter (c). (d) The amount of activated Notch should be titrated for every reporter construct and cell line combination to ensure that the measured response is in the linear range. Compared to TP1-Luc, Hes1-Luc reaches maximal response at a lower Notch concentration in 3T3 cells

Exogenous promoters or upstream activating sequences (UAS) coupled with chimeric Notch receptors have also been used to report receptor cleavage (Fig. 1b). Pros for this strategy include a boost in sensitivity through the use of the potent transcriptional activator GalVP16 [16, 17]. Moreover, the use of a heterologous DNA-binding domain or transcriptional activator enables the experimenter to assess paralog-specific activation [18], exclude signaling input from endogenous receptors [18], and avoid activating endogenous targets [10]. It is important to remember though that chimeric Notch receptors may have altered properties, such as trafficking and stability. Cleavage events involving the foreign sequences

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may also produce false positive or higher background signals. Therefore, these experiments should be adequately controlled and the results validated using other approaches. If cell type choices are not dictated by the question being asked, selecting one of the numerous available cell lines to investigate Notch transcriptional activation can be a daunting task. Key considerations include expression levels of Notch pathway components and of the transfected plasmids as well as transfectability of the chosen cell. Reporter assays typically utilize transient transfections of cells, enhancing flexibility in experimental design. However, viral delivery, stable integration, and episomal-based expression of reporters are advantageous for difficult-to-transfect lines or longterm studies. In such cases, it may be wise to test several independent clones. Depending on the experiment, the amount of endogenous signaling (Notch dependent and independent) present in the cell can also be an important factor. In order to confirm that an observed response from a transcriptional reporter is specific to Notch activation, it would be advisable to use established inhibitors of the pathway, such as γ-secretase inhibitors and/or dominant negative MAML (DNMAML) (Fig. 2b, c). In the following sections, we outline general protocols for performing transient luciferase-based transcriptional reporter assays with a special emphasis on the methods employed to monitor both ligand-independent and ligand-dependent Notch activation.

2 2.1

Materials Plasmids

1. Notch-responsive reporter plasmid (e.g., TP1-Luciferase [19]). 2. Control reporter plasmid (e.g., CS2 + βgal; see Note 1). 3. CS2+ plasmid or another appropriate empty vector. 4. Notch expression vector. 5. Ligand-Fc and Fc expression vectors (e.g., Dll1-Fc). 6. Optimal transfection reagent for target cells (e.g., Calcium phosphate or Lipofectamine 2000).

2.2

Cell Lines

1. Notch-expressing cells (e.g., CHO-Notch2 [20]). 2. Ligand-expressing cells (e.g., CHO-Dll1-IRES-GFP, CHOJag1-IRES-GFP [21]). 3. 293T cells (for generating ligand-Fc conditioned media). 4. Other cell lines of interest.

2.3 Calcium Chelation Reagents

1. EGTA (0.5 M stock solution in dH2O, pH 7.0; sterilized). 2. Hank’s balanced salt solution (HBSS), no calcium or magnesium (see Note 2).

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2.4 Ligand Immobilization Reagents

1. Ligand-Fc protein (see Note 3). 2. IgG control (see Note 4). 3. Conditioned media containing ligand-Fc or Fc (see Note 5). 4. Affinity-purified anti-Fc antibody. 5. Sterile PBS.

2.5 Reporter Assay Reagents

1. Lysis buffer (0.2 % Triton X-100, 100 mM potassium phosphate buffer, pH 7.8, 1 mM DTT, protease inhibitors) (see Note 6). 2. Luciferase assay reagent (30 mM Tricine pH 7.8, 3 mM ATP, 15 mM MgSO4, 10 mM DTT, 0.2 mM Coenzyme A, 1 mM D-luciferin) (see Note 7). 3. Galacton® Substrate (100×; Life Technologies). 4. Galacton® Diluent (100 mM sodium phosphate buffer, pH 8.0, 1 mM MgCl2). 5. Light Emission Accelerator (Life Technologies). 6. 96-well white assay plates. 7. Luminometer (see Note 8).

3

Methods

3.1 Transient Transfection of Reporters for Different Modes of Notch Activation

3.1.1 Transfection with Activated Forms of Notch

The protocols described below are based on a 24-well plate format; however, the conditions can be scaled up or down to other plate formats in proportion to their respective well surface areas. We generally run experiments in triplicate or quadruplicate. To test potential modifiers of Notch activation, additional expression plasmids can be cotransfected along with the reporter plasmids. It is important to include a control expression vector to normalize the total amount of DNA transfected across all assay plates and to rule out artificial effects simply due to protein overexpression or promoter squelching resulting from competition for cellular transcription factors. To help avoid these issues a titration of expression plasmids should be performed to determine the minimum amount required. 1. Day 1: Seed cells in 24-well plates in complete medium such that the cell density is optimal for transfection the next day (see Note 9). Incubate the cells overnight at 37 °C. 2. Day 2: Transfect each well with 200 ng Notch-responsive luciferase reporter, 10 ng CS2 + cytoβgal (as a transfection efficiency control), 0.5–10 ng Notch∆E or NICD expression plasmid (see Note 10) and empty vector as carrier DNA (to 500 ng total DNA per well). Incubate the cells overnight at 37 °C.

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3. Day 3: Feed the transfected cells with fresh medium (see Note 11). 4. Day 4: Proceed to Subheading 3.2 to measure reporter activities. 3.1.2 LigandIndependent Activation of Full-Length Notch: Calcium Chelation (See Note 12)

1. Day 1: Seed Notch-expressing cells (see Note 13) in 24-well plates in complete medium such that the cell density the next day is optimal for transfection. Incubate the cells overnight at 37 °C. 2. Day 2: Transfect each well with 200 ng Notch-responsive luciferase reporter, 10 ng CS2 + cytoβgal and empty vector (to bring to 500 ng total DNA per well). Incubate the cells overnight at 37 °C. 3. Day 3: Feed the transfected cells with fresh medium. 4. Day 4: Activate Notch via calcium chelation. Wash cells once with HBSS (without calcium or magnesium). Treat cells with 0.5 ml HBSS containing 0.5 mM EGTA (=1,000× dilution from 0.5 M stock) for 30 min at 37 °C (see Note 14). As a control, treat a parallel set of cells with HBSS alone. After the 30 min incubation, replace buffer with complete medium (see Note 15). Incubate the cells for 6 h at 37 °C (see Note 16). Proceed to Subheading 3.2 to measure reporter activities.

3.1.3 Ligand-Dependent Activation of Full-Length Notch: Immobilized Ligands

1. Day 1: Seed Notch-expressing cells in complete medium in 24-well plates that have been precoated with ligand (see Note 17) such that the cell density the next day is optimal for transfection. Incubate the cells overnight at 37 °C. 2. Day 2: Transfect each well with 200 ng Notch-responsive reporter, 10 ng CS2 + cytoβgal and empty vector (to bring to 500 ng total DNA per well). Incubate the cells overnight at 37 °C. 3. Day 3: Feed the transfected cells with fresh medium. 4. Day 4: Proceed to Subheading 3.2 to measure reporter activities.

3.1.4 Ligand-Dependent Activation of Full-Length Notch: Co-culture

1. Day 1: Seed Notch-expressing cells in 24-well plates in complete medium such that the cell density is optimal for transfection the next day. Incubate the cells overnight at 37 °C. 2. Day 2: Transfect each well with 200 ng Notch-responsive reporter, 10 ng CS2 + cytoβgal and empty vector (to bring to 500 ng total DNA per well). Incubate the cells overnight at 37 °C. 3. Day 3: Feed the transfected cells with fresh medium. Seed ligand-expressing cells (50,000–100,000 per well) on top of the transfected cells. Co-culture cells overnight at 37 °C. 4. Day 4: Proceed to Subheading 3.2 to measure reporter activities.

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Reporter Assays

1. Harvest the cells and prepare cell lysates: Aspirate the medium and wash the cells once with PBS (see Note 18). Add 100 μl of lysis buffer into each well. Incubate the culture plate(s) on a shaker for 15 min at room temperature. Spin down cell debris at maximum speed for 5 min. 2. Luciferase Assay: Transfer 50 μl of each lysate into wells of a 96-well assay plate. Measure luminescence using a luminometer equipped with automatic injectors: Inject 50 μl luciferase assay reagent (thawed and equilibrated to room temperature). After a 2 s delay, measure the luminescent activity using an integration time of 5 s. 3. β-galactosidase assay: Pipette 5–20 μl of each lysate into wells of a 96-well assay plate. Add 70 μl of 1× Galacton (prepared by diluting 100× Tropix® Galacton® Substrate in Galacton Diluent) into each well and mix by shaking the plate gently. Incubate the plate, covered, for 30–45 min at room temperature. Measure luminescence with a luminometer: Inject 100 μl Tropix® accelerator reagent (equilibrated to room temperature). After a 1 s delay, measure the luminescent activity using an integration time of 1 s. 4. Data analyses: To perform a background correction, subtract the average of the mock-transfected measurements from the test sample values (see Note 19). Divide the luciferase values by the β-galactosidase values to normalize for variation in transfection efficiencies among the samples (see Note 20). Relative luciferase activities can then be normalized to an appropriate control (e.g., HBSS alone for calcium chelationmediated activation, no ligand control for co-culture or immobilized ligand assays, or empty vector control for transiently expressed activated forms of Notch) to obtain fold activation. When normalized, results from assays done on different days can be better compared for statistical analyses.

4

Notes 1. This reporter controls for off-target effects and for variations in sample processing, transfection efficiency, cell number, and cell viability. It is typically expressed under a constitutive promoter that is not affected by the stimulus or experimental condition. This should be empirically confirmed for the specific conditions being studied, as it is important that reported effects on Notch reporter expression are not being driven by changes in the control reporter. Aside from β-galactosidase, Renilla luciferase is another popular choice since this allows the use of the dual luciferase assay (Promega) to sequentially measure firefly and Renilla luciferase activities in the same lysate sample.

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2. Sterile PBS can also be used as long as it does not contain calcium or magnesium. 3. Purified ligand-Fc fusions are available from commercial sources (e.g., R&D Systems, Enzo Life Sciences). They can also be expressed in mammalian (e.g., 293T, CHO) or insect (e.g., Sf9) cells and affinity-purified from conditioned media (for example, see refs. 22, 23). 4. Affinity-purified IgG control can be purchased. Use the IgG isotype and species that corresponds to the Fc fragment within the ligand-Fc protein used for experiments. 5. To generate conditioned medium, transfect 293T cells with ligand-Fc or Fc expression plasmids (10 μg plasmid/P100 plate) using Fugene 6. The next day, feed cells with fresh medium (10 ml/P100 plate) and incubate for 24–48 h. Collect supernatants and spin down or filter-sterilize to remove cell debris. Conditioned media can be stored at 4 °C for several months. 6. Add DTT and protease inhibitors to the lysis buffer just before using. 7. The luciferase assay reagent can be prepared as a stock solution, then aliquoted and stored at −20 °C. Thaw and equilibrate to room temperature prior to use. 8. The protocols described herein were optimized for the Tropix TR717 luminometer. Protocols and measurement settings may have to be optimized for other luminometers. 9. We have performed Notch reporter assays in many different cell lines (for example, see ref. 2). In addition, we have been able to monitor ligand-dependent activation of endogenously expressed Notch receptors in HeLa, C33A, MEFs, 3T3, and mK4 cells (Ilagan, M.X.G. and R. Kopan, unpublished). We recommend empirically determining the optimal seeding densities and transfection reagents and conditions for introducing reporter constructs into cells of interest. 10. The activated forms of Notch are very potent transcription factors (Fig. 4a). Because the expression level can influence the observed biological effects (for example, see Fig. 2a), we typically use a low concentration of Notch expression vector. The optimal amount can vary depending on the cell line and promoter (for both Notch expression and the reporter) (Fig. 4d); therefore we strongly recommend performing a titration experiment and choosing a concentration where the response is in the linear range and will likely be sensitive to inhibition/modification. 11. This step is more important when using transfection reagents that are particularly toxic or stressful to cells, such as calcium phosphate or Lipofectamine 2000. For some transfection reagents such as Fugene, this media change may not be necessary.

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12. Ca2+ chelation/addition will trigger a strong calcium response that may affect other cellular processes that can indirectly impact reporter gene expression. Additional controls should be used to confirm that any response is specific to Notch signaling. 13. Notch-expressing cells can be stable lines or cells that express Notch receptors endogenously. NotchFL expression vectors can also be transiently transfected into cells of interest. 14. EGTA, EDTA, and BAPTA can all be used to chelate Ca2+ and activate Notch in a ligand-independent manner [24]. We favor EGTA because it is specific towards Ca2+ and therefore does not inhibit Zn2+-dependent proteases. It also exhibits a slightly better dynamic range for activation than EDTA in our recent studies [24] (see also Chapter 12). 15. Calcium chelator treatment causes the cells to circularize and detach so buffer removal and media addition should be performed carefully to prevent the cells from coming off the wells. For cell types that are more sensitive to this issue, it may be helpful to shorten the chelator treatment and/or to seed the cells onto plates that have been pretreated with poly-lysine or other coatings that promote adhesion. 16. In the calcium chelation experiment, only a pulse of Notch activation is provided to the cells so downstream transcriptional reporter activities should be assessed within a few hours after chelator treatment. Depending on the reporter and cell line, a time course experiment may be helpful to establish the optimal timing for maximal activity (for example, see ref. 25). 17. To coat plates with purified ligands, incubate cell culture plates with a 5–10 μg/ml solution of ligand-Fc (or control IgG) in sterile PBS (50 μl/well for a 96-well plate; 250 μl/well for a 24-well plate) for 2 h at room temperature or overnight at 4 °C. Remove unbound ligand/antibody by washing the wells once with sterile PBS prior to cell seeding. This is an important step as soluble ligand can potentially be inhibitory. Ligands can also be immobilized from conditioned media using affinity-purified anti-Fc antibodies. Incubate plates with a 5–10 µg/ml solution of anti-Fc antibody in sterile PBS for 2 h at room temperature. Aspirate the antibody and then add conditioned media carrying ligand-Fc or Fc control. Incubate for another 2 h at room temperature. Remove unbound ligand by washing the wells once with sterile PBS prior to cell seeding. 18. Once the PBS wash has been completely removed, the cell plate can be frozen down and stored at −80 °C for analyses at a later time. In this case, the incubation time with the lysis buffer is extended to allow thawing.

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19. Some mammalian cells have endogenous β-galactosidase activities, which can be determined from the mock-transfected cell lysates and subsequently subtracted from the values of test samples. 20. It is important for the luciferase and β-galactosidase values to fall within the linear ranges of their respective assays. To establish the linear ranges under any given experimental condition, generate standard curves using serial dilutions of commercially available β-galactosidase and luciferase enzymes. If needed, various experimental parameters (e.g., lysate volume, plasmid amount, promoter strength, luminometer settings) can be adjusted to fit the linear range.

Acknowledgements We thank members of the Kopan lab for technical assistance and helpful discussions, most especially to Scott Boyle for providing the fluorescence images of the CHO co-cultures and to Matt Hass for reading the manuscript. This work was supported by NIH grant GM55479. References 1. Arnett KL, Hass M, McArthur DG et al (2010) Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nat Struct Mol Biol 17:1312–1317 2. Ong C, Cheng H, Chang LW et al (2006) Target selectivity of vertebrate Notch proteins. Collaboration between discrete domains and CSL binding site architecture determine activation probability. J Biol Chem 281:5106–5119 3. Schroeter EH, Kisslinger JA, Kopan R (1998) Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393:382–386 4. Tamura K, Taniguchi Y, Minoguchi S et al (1995) Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-J kappa/Su(H). Curr Biol 5:1416–1423 5. Hicks C, Johnston SH, diSibio G et al (2000) Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nat Cell Biol 2:515–520 6. Weng AP, Ferrando AA, Lee W et al (2004) Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306:269–271

7. Wang NJ, Sanborn Z, Arnett KL et al (2011) Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma. Proc Natl Acad Sci U S A 108:17761–17766 8. Moellering RE, Cornejo M, Davis TN et al (2009) Direct inhibition of the NOTCH transcription factor complex. Nature 462:182–188 9. Roti G, Carlton A, Ross KN et al (2013) Complementary genomic screens identify SERCA as a therapeutic target in NOTCH1 mutated cancer. Cancer Cell 23:390–405 10. Sprinzak D, Lakhanpal A, LeBon L et al (2010) Cis interactions between Notch and Delta generate mutually exclusive signaling states. Nature 465:86–90 11. Hansson EM, Teixeira AI, Gustafsson MV et al (2006) Recording Notch signaling in real time. Dev Neurosci 28:118–127 12. Masamizu Y, Ohtsuka T, Takashima Y et al (2006) Real-time imaging of the somite segmentation clock: revelation of unstable oscillators in the individual presomitic mesoderm cells. Proc Natl Acad Sci U S A 103:1313–1318 13. Ohtsuka T, Imayoshi I, Shimojo H et al (2006) Visualization of embryonic neural stem cells using Hes promoters in transgenic mice. Mol Cell Neurosci 31:109–122

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14. Duncan AW, Rattis FM, DiMascio LN et al (2005) Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance. Nat Immunol 6:314–322 15. Souilhol C, Cormier S, Monet M et al (2006) Nas transgenic mouse line allows visualization of Notch pathway activity in vivo. Genesis 44:277–286 16. Berechid BE, Kitzmann M, Foltz DR et al (2002) Identification and characterization of presenilin-independent Notch signaling. J Biol Chem 277:8154–8165 17. Struhl G, Adachi A (1998) Nuclear access and action of notch in vivo. Cell 93:649–660 18. Gordon WR, Vardar-Ulu D, L’Heureux S et al (2009) Effects of S1 cleavage on the structure, surface export, and signaling activity of human Notch1 and Notch2. PLoS One 4:e6613 19. Minoguchi S, Taniguchi Y, Kato H et al (1997) RBP-L, a transcription factor related to RBPJkappa. Mol Cell Biol 17:2679–2687 20. Shimizu K, Chiba S, Hosoya N et al (2000) Binding of Delta1, Jagged1, and Jagged2 to Notch2 rapidly induces cleavage, nuclear translocation, and hyperphosphorylation of Notch2. Mol Cell Biol 20:6913–6922

21. Ong CT, Sedy JR, Murphy KM et al (2008) Notch and presenilin regulate cellular expansion and cytokine secretion but cannot instruct Th1/Th2 fate acquisition. PLoS One 3:e2823 22. Varnum-Finney B, Purton LE, Yu M et al (1998) The Notch Ligand, Jagged-1, influences the development of primitive hematopoietic precursor cells. Blood 91: 4084–4091 23. Varnum-Finney B, Wu L, Yu M et al (2000) Immobilization of Notch ligand, Delta-1, is required for induction of Notch signaling. J Cell Sci 113:4313–4318 24. Ilagan MX, Lim S, Fulbright M et al (2011) Real-time imaging of notch activation with a luciferase complementation-based reporter. Sci Signal 4:rs7 25. Rand MD, Grimm LM, Artavanis-Tsakonas S et al (2000) Calcium depletion dissociates and activates heterodimeric notch receptors. Mol Cell Biol 20:1825–1835 26. Weng AP, Nam Y, Wolfe MS et al (2003) Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. Mol Cell Biol 23:655–664

Chapter 12 Monitoring Notch Activation in Cultured Mammalian Cells: Luciferase Complementation Imaging Assays Ma. Xenia G. Ilagan and Raphael Kopan Abstract Notch activation and cleavage releases the Notch intracellular domain (NICD), which translocates to the nucleus, where it associates with its DNA-binding partner CSL to recruit the coactivator MAML and additional cofactors to ultimately activate target gene expression. Taking advantage of the specific interaction between NICD and these factors, we have developed a luciferase complementation imaging (LCI)based reporter system to quantitatively monitor Notch activation in real time in live cells. In this chapter, we describe the use of Notch LCI reporters for measuring protein interactions and performing detailed kinetic analyses of receptor activation and its responses to various stimuli. Key words Notch, Protein complementation assay, PCA, Luciferase complementation imaging, LCI, Split luciferase, Molecular imaging, Bioluminescence, Protein–protein interaction

1

Introduction Elucidating the molecular mechanisms that control the timing and dose of Notch signaling requires sensitive tools for detecting Notch activity in real time. To address this need, we have recently developed and validated a reporter system for Notch activation based on optimized luciferase complementation imaging (LCI) (Figs. 1 and 2) [1, 2]. In this assay system, complementary N-terminal and C-terminal luciferase fragments (NLuc and CLuc), which lack activity on their own, reconstitute enzymatic activity when brought together by the interacting proteins to which they are fused [2]. By fusing NLuc to Notch and CLuc to CSL (specifically RBPjκ), we can then directly monitor the association of NICD and RBPjκ in real time (Fig. 1a). When CLuc is fused to MAML, we can use the assay to monitor ternary complex formation (Fig. 1b, c) [1]. The major advantages of the LCI reporter as a protein–protein interaction assay include (1) its sensitivity and high signal-to-background ratio due to enzyme-mediated signal amplification and the absence of background luminescence from cells, (2) the negligible binding

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_12, © Springer Science+Business Media New York 2014

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Fig. 1 Overview of the Notch luciferase complementation imaging (LCI) assay system. (a) To create a real-time reporter for Notch activation, the luciferase fragments NLuc and CLuc were fused to Notch and CSL (i.e., RBPjκ in our system), respectively. Upon receptor activation and cleavage, the specific interaction between the released NICD and RBPjκ brings together NLuc and CLuc to reconstitute luciferase enzymatic activity. (b and c) Fusing CLuc to MAML enables the reporter system to monitor ternary complex formation, i.e., the RBPjκ -independent interaction of NICD-NLuc and CLuc-MAML (b) or NICD-dependent interaction of CLuc-MAML and RBPjκ-NLuc (c). Different forms of Notch can be used to study different steps of the Notch activation process

energy between the NLuc and CLuc fragments, which makes complementation reversible and enables accurate quantification of protein interactions, and (3) a substrate that is nontoxic and readily permeable to cell membranes, allowing noninvasive, repeated imaging in live cells and in living animals. Notably, the luciferase complementation pairs constitute a complete assay system that does not rely on downstream activation of a reporter gene and therefore reconstituted luciferase activity can be measured in real time, facilitating dynamic analyses of signaling events [3–5]. Notch LCI is a specific, flexible, and robust method that can be utilized in several different modes [1]. First, it can be used to monitor protein association and dissociation with a high degree of sensitivity, enabling rapid structure-function analyses and determination of on/off rates in intact cells. The assay can quantify NICDRBPjκ binding and NICD-RBPjκ-MAML ternary complex formation. In addition, it can be adapted to study interactions involving other Notch paralogs as well as other signaling proteins to monitor cross talk and signal integration. The development of dual color complementation assays based on click beetle green and red luciferases (CBG and CBR) should even enable simultaneous monitoring of multiple protein interactions in the same cell [6, 7].

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Fig. 2 Domain organization of Notch, RBPjκ, and MAML and their respective luciferase fragment fusions. Different LCI reporter combinations can be used to interrogate different protein interactions and mechanistic aspects of Notch signaling. Available mutant variants for Notch (CC > SS—activating mutation in HD; ∆RAM—RAM domain deletion; WFP > LAA—mutation within RAM domain; V1744G—a TMD mutation; M1 and M2 are mutations in the ANK domain) and RBPjκ (dnRBP—a mutant deficient in DNA binding; R178H in human RBPjκ) are listed. A flexible Gly-Ser linker was included in all fusion constructs to minimize steric hindrance

Second, LCI can be used as an enzymatic reporter for nuclear translocation of NICD in a manner that is more sensitive and easier to measure compared to nuclear translocation assays with NotchGFP fusion proteins [8]. Lastly, a major application for Notch LCI stems from its capacity to quantitatively report on the relative amount of NICD-RBPjκ complexes in live cells with detailed temporal resolution. The LCI assay can, after some calibration experiments, replace labor-intensive western analyses of NICD levels while providing more quantitative information on the activation status of the receptor, making it an effective approach for dynamic analyses of the signaling pathway in response to various ligands, modifiers, and pharmacological stimuli [1, 9].

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In this chapter, we describe technical considerations and detailed methods for designing and performing Notch LCI assays in live mammalian cells. We provide protocols for different applications including transient and stable reporter expression, ligandindependent and ligand-dependent activation, and endpoint and kinetic LCI readouts.

2 2.1

Materials Plasmids

1. NLuc expression vector (e.g., Notch-NLuc). 2. CLuc expression vector (e.g., CLuc-RBPjκ). 3. CMV-Renilla (transient transfection control). 4. CS2+ or another appropriate empty vector. 5. Selectable marker plasmids (for stable line generation; e.g., pTK-Hyg). 6. pOG44 Flp expression vector (for use with Invitrogen’s Flp-In system). 7. Ligand-Fc and Fc expression vectors (for generating conditioned media).

2.2 Cell Lines and Culture Media

1. Cell lines of interest (for Notch LCI assays). 2. Notch LCI stable reporter lines. (a) HeLaTetON-based reporter lines [1] (i) Line FL2 (pBI Tet-inducible NotchFL-NLuc and CLuc-RBPjκ) (ii) Line E6 (pBI Tet-inducible N∆E-NLuc and CLucRBPjκ) (iii) Line IC1 (pBI Tet-inducible NICD-NLuc and CLucRBPjκ) These lines are maintained in DMEM containing 10 % FBS and Pen/Strep. They are G418- and Hygromycin-resistant but do not require these antibiotics to maintain Tetracycline (Tet)-inducible expression. Doxycycline (Dox), a more stable analog of Tet, is added to the medium at 0.5–1 μg/ml during cell seeding to induce reporter expression. (b) Flp-In TRex 293-based reporter lines [9] (see Note 1) (i) Lines D6 or D10 (pcDNA3 puro-CBG CLuc-RBPjκ) (ii) Lines D6-N1 or D10-N1 (Notch1-CBG NLuc Flp-In cells in D6 or D10 backgrounds) (iii) Lines D6-N21 or D10-N21 (Notch21-CBG NLuc Flp-In cells in D6 or D10 backgrounds; Notch21, which is a chimeric Notch receptor composed of

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the Notch2 extracellular domain and TMD fused to the Notch1 intracellular domain, allows quantitative comparison of the responsiveness of the Notch1 and Notch2 extracellular domains to ligands, agonists and modifiers) These lines are maintained in DMEM containing 10 % FBS and Pen/Strep. Puromycin (0.4 μg/ml) is added to maintain stable expression of CBG CLuc-RBPjκ. Hygromycin (100 μg/ml) is added to maintain stable expression of Notch1-NLuc from the Flp-In locus. For imaging experiments, we seed these cells onto plates coated with 100 μg/ml poly-lysine to help maintain cell adherence during media changes. 3. 293T (for generating ligand-Fc conditioned media). 4. Ligand-expressing stable lines (e.g., CHO-Dll1-IRES-GFP, CHO-Jag1-IRES-GFP [10]). 5. Transfection reagents (e.g., Lipofectamine 2000, Fugene 6, calcium phosphate). 2.3 Calcium Chelation Reagents

1. EGTA (0.5 M stock solution in dH2O, pH 7.0; sterilized) (see Note 2). 2. Hank’s balanced salt solution (HBSS), no calcium or magnesium.

2.4 Ligand-Fc Immobilization and Clustering Reagents

1. Sterile Dulbecco’s PBS. 2. Affinity-purified ligand-Fc protein (see Note 3). 3. Affinity-purified IgG control (see Note 4). 4. Conditioned media containing ligand-Fc or Fc (see Note 5). 5. Affinity-purified anti-IgG, Fc fragment specific (for immobilizing ligand-Fc). 6. Anti-IgG, Fc fragment specific (for clustering ligand-Fc).

2.5 Bioluminescence Assay Materials

1. D-luciferin (Biosynth; 200× stock = 30 mg/ml solution in dH2O, filter-sterilized, aliquoted, and stored at −20 °C). 2. Native Coelenterazine (Biotium; 1000× stock = 400 μM stock solution in ethanol, stored at −20 °C). 3. PBS++ (Dulbecco’s PBS supplemented with 0.1 % glucose, 1 mM MgCl2, 0.9 mM CaCl2). 4. Imaging medium (phenol red-free DMEM containing 10 % FBS) (see Note 6). 5. Black multi-well tissue culture plates. 6. IVIS imaging system (Caliper) and Living Image software.

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Methods

3.1 Transient Transfection of Notch LCI Reporter Plasmids

1. Day 1: Seed cells in complete medium in black cell culture plates such that the cell density will be optimal for transfection the next day. Incubate the cells overnight at 37 °C. 2. Day 2: Transfect each well with vectors expressing NotchNLuc and CLuc-RBPjκ fusion proteins along with appropriate carrier DNA, and if using, a transfection control and other test plasmids (see Note 7). Incubate the cells overnight at 37 °C. 3. Day 3: Feed the transfected cells with fresh medium (see Note 8). 4. Day 4: Proceed to Subheading 3.3 to measure bioluminescence.

3.2 Generation of Stable Lines Expressing Notch LCI Reporter Fusions

While transient transfection experiments can be flexible and address a multitude of biological questions, stable lines expressing the Notch LCI reporters can be particularly useful for some applications (e.g., high throughput screening for pathway modifiers and analyses of the dynamics of NotchFL activation). To this end, we have generated stable Notch LCI reporter lines using the Tet-inducible expression system (Clontech) [1] and the Flp-In system (Invitrogen) [9]. Below, we provide details on how these two sets of lines were established so that other users can adapt the system(s) for their own research purposes.

3.2.1 Tet-Inducible Notch LCI Reporter Lines

We have generated HeLaTetON-based cell lines expressing CLucRBPjκ and different forms of Notch1 (NotchFL-, N∆E-, or NICD-NLuc) (Figs. 1a and 2) [1]. Inducible expression allows the user to control the amount of reporter for different applications. This also prevents potential adverse effects due to prolonged constitutive expression of the activated forms of Notch. 1. Day 1: Seed cells in complete medium in 12-well plates at a density optimal for your chosen transfection method (e.g., for HeLaTetON cells, we use Lipofectamine 2000 reagent and hence, seed cells such that they are 90 % confluent at the time of transfection). Incubate the cells overnight at 37 °C. 2. Day 2: Transfect each well with 1 μg of the Tet-inducible expression vector carrying the NLuc and/or CLuc fusion constructs (e.g., pBI-NotchFL-NLuc/CLuc-RBPjκ). Cotransfect 100 ng of a selectable marker plasmid (e.g., pTK-Hyg), if needed (see Note 9). 3. Day 3: Expand cells to P100 cell culture dishes (see Note 10). 4. Day 4: Add selection antibiotic at the optimal concentration. 5. Feed cells with fresh antibiotic-containing medium every 5 days until isolated colonies appear. 6. After 2–4 weeks, pick healthy colonies and expand them for further analyses. See Subheading 3.3.4 for recommendations on screening stable clones using LCI.

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We have generated an Flp-In parental cell line that stably expresses CLuc-RBPjκ from a constitutively active promoter. The Flp-In system enables integration of Notch-NLuc variants into a single transcriptionally active locus to easily produce multiple isogenic lines for structure-function analyses [9]. 1. Day 1: Seed cells in complete medium in 12-well plates at a density optimal for the chosen transfection method. For instance, for Flp-In TRex 293 CBG CLuc-RBPjκ cells, we use Fugene 6 reagent and hence, seed cells such that they are 50 % confluent at the time of transfection. Incubate the cells overnight at 37 °C. 2. Day 2: Transfect each well with 100 ng pcDNA5/FRT-NotchNLuc (see Note 11) and 900 ng of the Flp expression vector pOG44. 3. Day 3: Expand cells to P100 cell culture dishes (see Note 10). 4. Day 4: Add selection antibiotic at the optimal concentration. 5. Feed cells with fresh antibiotic-containing medium every 5 days until isolated colonies appear. 6. After 2–4 weeks, pick healthy colonies and expand them for further analyses. See Subheading 3.3.4 for recommendations on using LCI to screen the stable clones.

3.3 Bioluminescence Assays

1. Set the imaging stage to the desired temperature. Live cell Notch LCI assays are typically performed at 37 °C.

3.3.1 General Procedure for IVIS Bioluminescence Imaging (See Note 12)

2. Launch the Living Image program and initialize the IVIS system according to the manufacturer’s instructions. Let the camera cool down to the appropriate temperature (−90 °C). 3. Load the cell culture plate that has been fed with D-luciferincontaining medium/buffer onto the stage, remove the plate lid, and then acquire the bioluminescent image. For Notch LCI, the typical acquisition parameters are as follows: exposure time, 1–5 min; binning, 4 or 8; no filter; f-stop, 1; field of view, 12 or 15 cm (see Note 13). 4. Using the Living Image software, measure the photon fluxes (photons/s) within the regions of interests (ROIs) for each image. Export the data to your software of choice for further analyses (see Note 14).

3.3.2 Endpoint LCI Assays

Endpoint LCI readouts are carried out for most transient transfection experiments (for example, see Fig. 3) as well as NotchFL activation setups using ligand co-culture (see Note 15) or immobilized ligand-Fc (see Note 16) [1]. 1. Aspirate medium and add prewarmed PBS++ supplemented with 150 μg/ml D-luciferin to cells. Incubate for 10 min at 37 °C.

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+ CLuc-RBPjK

Fig. 3 Notch LCI as a specific and sensitive assay for quantifying protein interactions and activation status of the Notch pathway. (a) Complementation activities of different Notch-NLuc fusions with CLuc-RBPjκ. The constitutively active forms of Notch (N∆E and NICD) exhibit the greatest complementation activity. (b) N∆ENLuc has robust complementation activity with CLuc-RBPjκ but not with CLuc. Deleting the high affinity RBPjκ interaction domain (RAM) or mutating the γ-secretase cleavage site (V1744G) diminishes complementation

2. Measure bioluminescence using the IVIS as described in Subheading 3.3.1. 3. OPTIONAL: If performing Renilla luciferase imaging on the same cells (e.g., as a transfection efficiency control for transient Notch LCI assays; for example, see Fig. 3a), aspirate the D-luciferin-containing PBS++ and replace with buffer containing 400 nM coelenterazine. Measure bioluminescence within 1 min using the IVIS with the filter set at 96 %). 3. Iodoacetamide (>99 %). 4. Ammonium bicarbonate (>99.0 %). 5. Methanol (mass spectrometry grade). 6. Acetonitrile (mass spectrometry grade). 7. Glacial acetic acid (99.99 %). 8. Formic acid (mass spectrometry grade). 9. Acetone (HPLC-UV grade). 10. Water (HPLC grade). 11. Nonreducing SDS 2× sample buffer: 2.4 % SDS, 20 % glycerol, 3.6 % DTT, 0.002 % bromophenol blue in 150 mM Tris– HCl, pH 6.8. 12. Standard SDS-polyacrylamide gels of appropriate percentage for the proteins being analyzed. 13. 100 mM Iodoacetamide in 50 mM Tris–HCl, pH 8.0 (must be made freshly). 14. Gel Code Blue Staining Kit (Thermo Scientific). 15. 1.5 ml Protein LoBind Tubes (Eppendorf).

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16. Destaining solution: 30 % Acetonitrile in 25 mM ammonium bicarbonate (must be made freshly). 17. Protease stock solutions diluted as follows and stored at −80 °C (see Note 1): Trypsin (Promega), 0.5 μg/μl in 20 mM diammonium phosphate, pH 8.0. Chymotrypsin (Princeton), 0.01 μg/μl in 20 mM ammonium bicarbonate, pH 8.0. GluC (V8) (Sigma-Aldrich), 0.2 μg/μl in 20 mM ammonium bicarbonate, pH 8.0. 18. Gel wash solution: 50 % Methanol in 20 mM diammonium phosphate, pH 8.0. 19. ZipTip microcolumn (Millipore). 20. ZipTip solution A: 95 % Acetonitrile, 0.1 % acetate. 21. ZipTip solution B: 0.1 % Acetate. 22. ZipTip solution C: 50 % Acetonitrile, 0.1 % acetate. 23. In-solution digestion buffer: 8 M Urea, 10 mM TCEP, 4.5 mM CaCl2, 225 mM Tris–HCl, 7.5–8.0. 24. Bath sonicator. 2.3 Analysis of Peptides by Mass Spectrometry

1. Nano-LC-MS/MS ion trap mass spectrometer equipped with an HPLC Chip-Cube interface (Agilent 6340) or equivalent. 2. Zorbax 300SB-C18 chip with a 40 nl enrichment column and a 43 mm × 75 μm separation column (Agilent) or equivalent. 3. Buffer A: 0.1 % Formic acid. 4. Buffer B: 95 % Acetonitrile in 0.1 % formic acid.

3

Methods

3.1 Expression and Purification of Soluble Mouse Notch1 (mN1) ECD Fragments

1. Prepare transfection mixture by mixing 2 μg of plasmid DNA (pSecTag-mN1 EGF29-36MycHis6 or EGF1-36Myc-His6) with 12 μl PEI reagent and 200 μl of Opti-MEM I at RT for 15 min for each 10 cm plate of cells. Transiently transfect subconfluent HEK293T cells and culture for ~96 h in 10 ml of Opti-MEM I. Typical preparations use four plates of cells. 2. Collect and clarify the medium by centrifugation (1,500 rpm for 5 min). Incubate the supernatant with Ni-NTA-agarose overnight at 4 °C (0.1 ml of Ni-NTA-agarose for 40 ml of medium). 3. Wash the Ni-NTA-agarose with wash buffer (1 ml), and elute the protein with 0.6 ml of elution buffer.

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3.2 In-Gel Protease Digestions (for Samples Containing Contaminating Protein Species)

1. Concentrate a portion of the eluted protein (0.2 ml) by precipitation with 4 volumes of ice-cold acetone for 2 h at −20 °C. Collect precipitated protein by centrifugation for 10 min at 10,000 × g at 4 °C. 2. Resuspend acetone precipitate in 8 μl nonreducing 2× SDS sample buffer and 8 μl of 20 mM TCEP in nonreducing SDS 2× sample buffer. Heat at 100 °C for 5 min, and cool to room temperature. 3. Add 8 μl of 100 mM iodoacetamide in 50 mM Tris–HCl, pH 8.0, and incubate the mixture in the dark at RT for 30 min. 4. Run the sample on SDS-PAGE appropriate for the molecular weight of the protein being analyzed (see Note 2). 5. Stain the gel with Gel Cold Blue Staining reagent, and cut out the protein band using a clean scalpel. The gel should be cut into small pieces (90 % pure by this method can be digested in solution. 6. LC-MS/MS systems utilize electrospray ionization (ESI) to generate ions (see [23] for more details). During ESI, individual peptides pick up different numbers of protons depending on the sequence of the peptide, so peptides can exist in 2+, 3+, 4+, etc. charge stages. Typically, the more basic amino acids in a peptide (Lys, Arg, His), the more protons it can pick up during ionization.

Acknowledgements We would like to thank Haltiwanger lab members for helpful comments. Primary work introduced here was supported by NIH grants GM061126 and CA12307101. References 1. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233 2. Rana NA, Haltiwanger RS (2011) Fringe benefits: functional and structural impacts of O-glycosylation on the extracellular domain of Notch receptors. Curr Opin Struct Biol 21: 583–589 3. Moloney DJ, Shair LH, Lu FM et al (2000) Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules. J Biol Chem 275:9604–9611 4. Shao L, Moloney DJ, Haltiwanger RS (2003) Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand-binding site and the Abruptex region. J Biol Chem 278:7775–7782 5. Matsuura A, Ito M, Sakaidani Y et al (2008) O-linked N-acetylglucosamine is present on

6.

7.

8.

9.

the extracellular domain of notch receptors. J Biol Chem 283:35486–35495 Rana NA, Nita-Lazar A, Takeuchi H et al (2011) O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1. J Biol Chem 286: 31623–31637 Foltz DR, Santiago MC, Berechid BE et al (2002) Glycogen synthase kinase-3beta modulates notch signaling and stability. Curr Biol 12:1006–1011 Gupta-Rossi N, Six E, LeBail O et al (2004) Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor. J Cell Biol 166:73–83 Coleman ML, McDonough MA, Hewitson KS et al (2007) Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor. J Biol Chem 282:24027–24038

Post-Translational Modifications of Notch 10. Campbell ID, Bork P (1993) Epidermal Growth Factor-like Modules. Curr Opin Struct Biol 3:385–392 11. Sakaidani Y, Nomura T, Matsuura A et al (2011) O-linked-N-acetylglucosamine on extracellular protein domains mediates epithelial cell-matrix interactions. Nat Commun 2:583 12. Acar M, Jafar-Nejad H, Takeuchi H et al (2008) Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132: 247–258 13. Sethi MK, Buettner FF, Ashikov A et al (2012) Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of notch. J Biol Chem 287:2739–2748 14. Sethi MK, Buettner FF, Krylov VB et al (2010) Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats. J Biol Chem 285:1582–1586 15. Wang Y, Shao L, Shi S et al (2001) Modification of epidermal growth factor-like repeats with O-fucose. Molecular cloning of a novel GDPfucose protein O-fucosyltransferase J Biol Chem 276:40338–40345 16. Moloney DJ, Panin VM, Johnston SH et al (2000) Fringe is a glycosyltransferase that modifies Notch. Nature 406:369–375

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17. Sakaidani Y, Ichiyanagi N, Saito C et al (2012) O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1. Biochem Biophys Res Commun 419:14–19 18. Shi S, Stanley P (2003) Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways. Proc Natl Acad Sci USA 100:5234–5239 19. Fernandez-Valdivia R, Takeuchi H, Samarghandi A et al (2011) Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi. Development 138:1925–1934 20. Lee TV, Sethi MK, Leonardi J et al (2013) Negative regulation of notch signaling by xylose. PLoS Genet 9:e1003547 21. Panin VM, Papayannopoulos V, Wilson R et al (1997) Fringe modulates Notch-ligand interactions. Nature 387:908–912 22. Yamamoto S, Charng W-L, Rana NA et al (2012) A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands. Science 338:1229–1232 23. Liebler DC (2002) Introduction to proteomics tools for the new biology. Humana Press, Totowa 24. Unwin RD, Evans CA, Whetton AD (2006) Relative quantification in proteomics: new approaches for biochemistry. Trends Biochem Sci 31:473–484

Chapter 17 Assay to Probe Proteolytic Processing of Notch by γ-Secretase Lutgarde Serneels, Ina Tesseur, and Bart De Strooper Abstract With the increasing appreciation of the role of Notch in development and disease, measuring its cleavage and signaling activity in cellular systems has become important. Here we describe a cell-based method to analyze the cleavage of Notch at the S3 site by γ-secretase. HEK cells are transfected with an N-terminal truncated and myc-labeled mNotchΔE construct which can be easily and quantitatively detected by western blotting. Key words Notch signaling, Notch cleavage, NICD fragment, Cell-based assay, S3 cleavage, γ-Secretase, Immunoblot

1

Introduction Notch signaling plays an important role in numerous cellular and developmental processes (reviewed in [1, 2]). Accurate, reliable, and easy measurements of Notch cleavage and signaling have become more relevant because of the ongoing efforts to develop γ-secretase modulators as disease-modifying treatments for Alzheimer’s disease (AD) and cancer. For example, a recent clinical trial by Eli Lilly revealed severe side effects of the γ-secretase modulator semagacestat, which presumably could be due to altered Notch signaling [3], highlighting the need for better assays. A large array of Notch signaling assays have been described. Many measure downstream transcriptional activation of Notchactivated genes, such as the HES and HEY genes (see Chapter 9, Alberi and Marathe, 2014; Chapter 13, Shimojo et al. 2014; and Chapter 19, Bailis et al. 2014). However, it has been challenging to detect the endogenous Notch intracellular domain (NICD) by conventional immunological or biochemical methods due to lack of good NICD antibodies and the fact that NICD is extremely short lived. Indeed, phosphorylation and acetylation tune the stability and activity of the Notch activator complex [4], and

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* As we are using infrared dye-labeled secondary antibodies (green for Notch cleaved and red for Myc-tag Ab), this fragment will be detected as yellow in an overlay with both antibodies. Fig. 1 Schematic representation of the mNotchΔE construct used to analyze Notch processing. γ-Secretase will recognize the S3 cleavage site and release NICD. This generates a neo-epitope (Valine 1744) that is recognized by a specific Notch-cleaved antibody. The C-terminal myc-tag is recognized by the myc antibody 9E10. Bands detected by these antibodies and their respective molecular weights are indicated. The 45 and 20 kDa bands are generated by additional caspase cleavage of the Notch fragments and are not always visible

phosphorylation of the C-terminal conserved proline-, glutamic acid-, serine-, and threonine-rich (PEST) domain of Notch largely controls its stability [5, 6]. The method described here makes use of ectopic expression in HEK cells of a membrane-spanning murine Notch1 construct called mNotchΔE [7]. This assay was originally developed by the Kopan laboratory and has been used by many research groups. The construct contains a C-terminal myc-tag inserted at amino acid 2183 that replaces part of the C-terminus. It also has an N-terminal deletion of the entire extracellular domain except for the 20 amino acid signal sequence (Fig. 1). To eliminate the alternative initiation of translation at methionine 1726 in mNotchΔE, the methionine was mutated to a valine (M1726V) without loss of activity [7, 8]. Due to deletion of the N-terminal sequence, mNotchΔE is devoid of the TACE/Adam-10 cleavage site, which precludes measuring α-secretase/S2 cleavage but also avoids interference of α-secretase

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activity with the measurement of γ-secretase/S3 cleavage. To analyze the effects on Notch S3 cleavage, HEK cells can be treated with compounds or co-transfected with a protein of choice. Immunoblot analysis of the cleaved fragments in combination with red and green fluorescence allows for reliable, accurate, and quantitative analysis of NICD generation in a cell-based assay.

2

Materials Tissue culture reagents 1. 12-Well plates. 2. DMEM/F12. 3. FBS. 4. Trypsin–EDTA (1×). 5. Sterile serological pipettes. 6. Cell counter. 7. Lactacystin. DNA constructs 1. pCS2 empty vector (negative control). 2. pCS2NotchICV-6MT (NICD), plasmid 41730 from Addgene. 3. pCS2NotchDEMV-6MT (mNotchΔE), plasmid 41737 from Addgene. Antibodies and dilutions 1. Cleaved Notch1(Val1744) from Cell Signaling Technology (1:1,000). 2. B-actin from Sigma (1:20,000). 3. Goat Anti-Mouse IgG (H + L), DyLight 680 Conjugated from Pierce (1:5,000). 4. Goat Anti-Rabbit IgG (H + L), DyLight 800 Conjugated from Pierce (1:5,000). Additional reagents and equipment 1. Shaker. 2. Bio-Rad colorimetric Protein Assay kit or bicinchoninic acid (BCA) Protein Assay Reagent (Pierce). 3. Li-Cor Odyssey infra-red imaging system, Westburg. 4. Equipment for running SDS-PAGE and western blotting experiments. 5. TransIT-LT1 transfection reagent, Sopachem.

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Methods Perform all procedures at room temperature (RT) unless otherwise specified.

3.1 Day 1: Plating of Cells

1. Obtain a nearly confluent and exponentially growing plate of wild-type HEK cells (see Notes 1 and 2). 2. Trypsinize cells with trypsin–EDTA, and count the number of cells obtained. 3. Plate 180,000 cells/well in a 12-well plate in DMEM/F12 supplemented with 10 % FBS (see Note 3). 4. Allow cells to attach at least overnight and form a 60–70 % confluent layer (see Note 4).

3.2 Day 2: Transfection of Cells

1. Transfect one well with pCS2 vector only (negative control), one well with NICD (positive control), and the required amount of conditions with mNotchΔE (experimental conditions) using TransIT-LT1 transfection mix according to the manufacturer’s instructions (see Note 5) (Fig. 2).

3.3 Day 3: Treatment of Cells with Inhibitors or Incubation

1. When testing soluble compounds make dilutions of compounds in 500 μl DMEM/F12 containing 10 % FBS total per well and supplement with 10 μM lactacystin (see Note 6). 2. When testing transfected constructs prepare 500 μl DMEM/ F12 containing 10 % FBS total per well and supplement with 10 μM lactacystin (see Note 6). 3. Remove spent medium, and incubate cells overnight with medium prepared in Subheadings 3.3, step 1 or 2.

3.4 Day 4: Cell Extract Preparation and Western Blot Detection

1. Remove medium, and wash cells with 1 ml Dulbecco’s PBS (DPBS). 2. Add 100 μl STE buffer containing 1 % Triton-X100 and proteinase inhibitors to each well (see Note 7). 3. Place on a shaker at 4 °C for 20 min. 4. Collect supernatant in prelabeled microcentrifuge tubes and spin for 15 min at 15,000 × g at 4 °C. 5. Transfer supernatant to a new clearly labeled tube. 6. Measure protein concentration with the BCA protein measurement kit (see Note 8). 7. Load 20 μg protein onto 12 % polyacrylamide gels, and run the gel for 50 min at 150 V in MES buffer (see Note 9). 8. Transfer protein onto a 0.2 μm nitrocellulose membrane, and block with 1 % BSA in TBST buffer (see Note 10).

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Fig. 2 Immunoblot analysis of Notch-cleaved products in HEK cells. (a) Extracts of HEK cells transfected with NICD (lane A), control plasmid (lane B), or mNotchΔE (lanes C, D) were detected with the anti-myc antibody (red signal) and the neoepitope antibody (green signal). Actin was used as a loading control (red signal). Lanes A–C were treated with DMSO, and lane D was treated with 10 μM Notchsparing γ-secretase inhibitor. (b) Extracts of HEK cells transfected with control plasmid (lane E) or mNotchΔE (lanes F–K) were treated with DMSO (lanes E and F) and a serial dilution of γ-secretase inhibitor (lanes G–K). Bands are detected with the same antibodies as in (a). The yellow band is Notch cleaved at the S3 site (overlay of the green neo-epitope specific signal and the red myc-epitope signal is indicated with a *)

9. Incubate with primary antibodies overnight. 10. Detect with secondary antibodies, and quantify bands with the Odyssey software package (see Note 11).

4

Notes 1. HEK cells are maintained in DMEM/F12 medium supplemented with 10 % FBS. We routinely avoid using antibiotics while maintaining cells.

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2. HEK cells can be substituted by other cells. This may require optimization of the transfection protocol. 3. We routinely dilute cells to 180,000 cells/ml and plate 1 ml total volume per well, as this allows for homogeneous distribution of the cells. 4. Depending on the growth rate of the cell line used, the initial seeding confluency may need to be adjusted so as to obtain a confluent layer of cells on day 4 of the protocol. 5. For TransIT-LT1 transfection, mix per well 100 μl serum-free DMEM/F12 medium with 3 μl TransIT-LT1 and incubate for 5 min at RT. Next add 1 μg DNA (pCS2 empty vector, NICD, or mNotchΔE), mix gently, and incubate for 30 min at RT. Then add 100 μl of this mix to the cells. We routinely do not refresh medium before or after the transfection procedure. 6. NICD is very unstable and rapidly broken down into the proteasome (5, 6). In order to be able to detect NICD on western blot it is crucial to supplement the incubation medium with lactacystin, a proteasome inhibitor. We routinely prepare a 2 mM lactacystin stock in DMSO and dilute from this stock into the medium. As lactacystin is toxic to cells the ideal incubation time would be 4–6 h. In our experience with HEK cells, we can incubate with lactacystin for up to 12 h without significant effects on cell viability, and therefore we now add this inhibitor to our compound mixes as it significantly reduces work load. Lactacystin toxicity may vary depending on the cell line and needs to be tested prior to starting experiments. To test for toxicity an optional cell viability assay can be performed. To measure cell viability add 20 μl cellTiTer-blue reagent (Promega) to each well during the last hour of incubation at 37 °C. Measure OD of supernatant at 560/590 nm. 7. Method to prepare STE buffer: Prepare sterile stock solutions of 0.5 M EGTA and 1 M Tris–HCl pH 7.4. For 500 ml STE buffer mix 1 ml 0.5 M EGTA, 42.8 g sucrose, and 2.5 ml 1 M Tris–HCl pH 7.4 in 400 ml distilled water. After solubilization of the sucrose, adjust volume with distilled water to 500 ml and filter sterilize. This solution can be stored for several months at 4 °C. 8. We routinely use the Bio-Rad Protein Assay kit with satisfactory results; however if the protein extraction buffer is high in detergent this assay can be substituted by a BCA protein assay. 9. Alternatively a 7 % NuPage Tris–acetate gel with Tris–acetate running buffer and conventional ECL detection can be used. In this case the NICD and NotchΔE bands will be sufficiently

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resolved to allow for individual detection at 69 kDa and 61 kDa, respectively. The disadvantage of Tris–acetate gels is that often fuzzy bands are obtained. 10. For TBST buffer we mix 1 volume of 10× TBS with 9 volumes distilled water and add 0.5 % Tween-20. Stir well for at least 30 min to completely dissolve the Tween. This solution can be kept at RT for about 1 week. Just before use we add 1 % BSA to this buffer and block membranes for a minimum of 1 h at RT to reduce background staining of the antibodies. 10× TBS buffer is prepared with 24.2 g Tris, 87.6 g NaCl, and 15 ml 12.5 M HCl. 11. The use of infrared-labeled antibodies enables simultaneous two-color NICD and NotchΔE analysis. The wide linear range of fluorescent bands on the same blot increases quantification accuracy.

Acknowledgements This work was supported by VIB and a Methusalem grant from KU Leuven and the Flemish Government. We would like to thank Tine Vanhoutvin for useful technical discussions and Veerle Vulsteke for drafting Fig. 1. References 1. Borggrefe T, Liefke R (2012) Fine-tuning of the intracellular canonical Notch signaling pathway. Cell Cycle 11:264–276 2. Kopan R (2012) Notch signaling. Cold Spring Harb Perspect Biol 4(10) 3. Doody RS, Raman R, Farlow M et al (2013) A phase 3 trial of semagacestat for treatment of Alzheimer's disease. N Engl J Med 369: 341–350 4. Guarani V, Deflorian G, Franco CA et al (2011) Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase. Nature 473:234–238 5. Fryer CJ, White JB, Jones KA (2004) Mastermind recruits CycC:CDK8 to phosphorylate the

Notch ICD and coordinate activation with turnover. Mol Cell 16:509–520 6. Tsunematsu R, Nakayama K, Oike Y et al (2004) Mouse Fbw7/Sel-10/Cdc4 is required for notch degradation during vascular development. J Biol Chem 279:9417–9423 7. Kopan R, Schroeter EH, Weintraub H et al (1996) Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain. Proc Natl Acad Sci U S A 93:1683–1688 8. De Strooper B, Annaert W, Cupers P et al (1999) A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398:518–522

Chapter 18 Analyzing the Nuclear Complexes of Notch Signaling by Electrophoretic Mobility Shift Assay Kelly L. Arnett and Stephen C. Blacklow Abstract An electrophoretic mobility shift assay (EMSA) is a sensitive technique for detecting protein–DNA and protein–protein interactions in which complexes are separated by native (non-denaturing) gel electrophoresis. EMSAs can provide evidence for specific binding between components prepared from a wide range of sources, including not only highly purified proteins but also components of crude cellular extracts. EMSA experiments were critical in identifying the minimal protein requirements for assembly of transcriptionally active nuclear Notch complexes as well as the DNA sequence specificity of Notch transcription complexes. Here, we describe a radioactive EMSA protocol for detection of Notch transcription complexes. Key words Electrophoretic mobility shift assay (EMSA), NICD, CSL, MAML1, RAMANK, Protein– DNA interactions, Bacterial protein expression and purification

1

Introduction Upon activation of the Notch signaling pathway, the intracellular portion of Notch (NICD) is released from the membrane, translocates to the nucleus, and assembles into a transcriptional activation complex [1–4]. In the nucleus, NICD interacts with the DNAbinding protein called CSL (gene name RBPJ in mammals) and a member of the Mastermind family, such as mammalian Mastermindlike-1 (MAML1). The minimal regions of Notch required for assembly of the core Notch transcription complex are a highly conserved region immediately internal to the cell membrane called RAM and an ankyrin-repeat domain (ANK; together, this part of NICD is referred to as RAMANK). CSL has three structured domains, an N-terminal Relhomology region (NTD), a β-trefoil domain (BTD), and a C-terminal Rel-homology region (CTD). The Notch RAM peptide binds to the isolated β-trefoil domain with high affinity [5, 6], and the ANK domain of Notch, which has only weak intrinsic

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_18, © Springer Science+Business Media New York 2014

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Kelly L. Arnett and Stephen C. Blacklow NICD(ICN)

a NRR

Human NOTCH1

RAM

ANK

1760

EGF-like repeats

13 MAML1

TAD PEST 2555 2126

74 p300- CDK8-binding binding

9

1016

435

RBPJ

486 NTD BTD CTD

b

Fig. 1 Assembly of the Notch transcription complex. (a) Domain organization of human Notch1, MAML1, and CSL proteins and schematic drawing of the core components of the NTC. The initial and terminal residues of the constructs described in this protocol are numbered, and the binding regions and domains are colored: Notch1 RAM (blue); ANK repeats (cyan); CSL N-terminal domain (NTD), β-trefoil domain (BTD), and C-terminal domain (CTD) (shades of green); and MAML1 (red ). (b) The X-ray crystal structure of the human NTC, colored as in (a), showing a complex of CSL, Notch1 RAM, Notch1 ANK, and the MAML1 polypeptide bound to a segment of the HES1 promoter

affinity for CSL, engages the Rel-homology domains of CSL to create a composite binding surface for MAML1 [7, 8]. Only a very short region of 1,016 amino acid MAML1 (13–74) is required for cooperative binding to CSL and Notch-RAMANK (Fig. 1) [9]. Transcriptional activation of Notch-responsive genes is also dependent on the recruitment of additional coactivators linked to the general transcription machinery, such as p300 (E1A-binding protein p300) or CREB-binding protein (CBP) [10, 11]. A number of co-repressors have been shown to interact with CSL in order to repress Notch target gene expression, including MINT/SHARP, SMRT, SKIP, CIR, and KyoT2 in mammals [12–17].

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Initial findings about the composition of Notch nuclear complexes came from organismal and cellular systems. Many of the details of the interactions involved in assembly of the NTC have been elucidated by experiments involving highly purified components using experimental approaches that include X-ray crystallography, isothermal titration calorimetry, circular dichroism, analytical ultracentrifugation, and hydrogen exchange-coupled mass spectrometry. The electrophoretic mobility shift assay (EMSA) is a useful experimental tool for detecting protein–DNA and protein–protein interactions from a wide range of sources, from crude cellular extracts to highly purified proteins. A number of the key findings about the minimal requirements for nuclear Notch complex assembly were derived from EMSA experiments [5, 9, 10, 13, 18–23]. An EMSA, or gel shift assay, is a sensitive method for detecting molecular interactions. EMSAs are based on the observation that under native gel electrophoresis conditions, higher order complexes tend to migrate more slowly than the individual components. Rates of mobility in a non-denaturing native gel are determined by both molecular weight and charge, so the EMSA approach is particularly applicable in detecting protein–DNA interactions, as DNA carries a negative charge due to the phosphate backbone and its mobility is strongly correlated with molecular weight. Therefore, a protein–DNA complex will almost invariably migrate more slowly than free DNA. DNA is very easy to label specifically, allowing detection of DNA binding from a crude nuclear extract, without the need for extensive protein purification. Protein–protein EMSAs are also possible but can be complicated by the wide range of molecular charges that proteins adopt, which more significantly modulate electrophoretic behavior. Most EMSAs performed today are closely based on the original assays described over 30 years ago by Fried and Crothers [24] and Garner and Revzin [25]. Extensive review, updated protocols, and troubleshooting of protein–DNA EMSAs are also available [26, 27]. Several labeling strategies are commonly used in protein–DNA EMSA protocols. The original labeling method, and still most commonly employed method due to its high sensitivity, is radioactive labeling with α-32P-dNTP. Several nonradioactive DNA detection methods are also routinely used in EMSA protocols, including chemiluminescent detection of biotinylated DNA, detection of fluorescently labeled DNA, and staining with intercalating fluorescent dyes [28–31]. Unlike isotopic DNA labeling, which has little or no effect on protein binding affinity, use of non-isotopic labels including fluorophores or biotin can interfere with protein–DNA interactions. Higher protein and DNA concentrations are typically required for methods using fluorescent detection and DNA staining. The requirement for transfer to nitrocellulose is a complicating factor in chemiluminescence-based methods for detecting biotin-DNA.

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Equivalent transfer of disparately sized protein–DNA complexes is difficult, making this method less quantitative than direct detection methods. Here we outline an EMSA protocol that has been optimized for detection of purified nuclear Notch complexes bound to 32 P-labeled DNA. The protocol takes 2–4 days to complete with purified proteins in hand. We have added, as an appendix, protocols for purification of bacterially produced constructs encoding the minimal human NTC components, Notch1-RAMANK (1760–2126), CSL(9–435), and MAML1(13–74).

2

Materials Ultrapure water should be used for all protein purification, DNA labeling, and protein/DNA binding steps. Deionized water is sufficient in the gel electrophoresis buffers.

2.1

Reagents

1. Protein of interest at a concentration sufficient to bind DNA. For the method outlined here, 100–300 ng human CSL(9–435), 500–1,000 ng human Notch-RAMANK(1760–2126), and 100–1,000 ng human MAML1(13–74) per binding reaction are used. See Appendix (Subheading 4) for expression and purification protocols for each of these proteins. If the protein concentration is near the estimated Kd, then a significant fraction of DNA will be shifted by bound protein. 2. DNA oligonucleotides to be annealed (Fig. 2a): Purchase or synthesize single-stranded DNA oligonucleotides designed with at least 18 base pairs of overlap and 3–5 nucleotide overhangs at each 5′-end. Include at least three nucleotides complementary to the 32P-nucleotide being incorporated (see Note 1). If protein concentrations are significantly below the Kd, then more DNA or more 32P incorporation into the labeled DNA duplex may be required for a detectable DNA shift. 3. α-32P-dCTP 250 mCi, 6,000 Ci/mmol (Perkin Elmer). 4. 10× DNA labeling buffer: 500 mM NaCl, 100 mM Tris–HCl, 100 mM MgCl2, 10 mM DTT, pH 7.9. This can be made or purchased (NEB Buffer 2, New England Biolabs). 5. 20× dNTPs: 670 μM each of dATP, dTTP, dGTP. Store at −20 °C. 6. Klenow Enzyme, 5 U/l (New England Biolabs). 7. Siliconizing agent (Sigmacote, Sigma-Aldrich). 8. 10× Running buffer: 250 mM Tris base, 2.5 M glycine, (10 mM EDTA, optional) in 1 L. Eliminate EDTA if metal-coordinating proteins will be used in the assay. For each experiment, prepare a sufficient volume of 1× running buffer (see Note 2).

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5’-AGTTACTGTGGGAAAGAAAGTTTGGGAAGTTTCACACGAGC TGACACCCTTTCTTTCAAACCCTTCAAAGTGTGCTCGGCAAG-5’

b

RBPJ

-

-

N1-RAMANK

-

-

MAML1

-

-

WT mutant

-

-

-

-

WT

mutant

+

+

+

+

Fig. 2 Electrophoretic mobility shift assay illustrating cooperative dimerization of Notch transcription complexes on SPS DNA. (a) Human HES1 promoter oligonucleotide duplexes used for EMSA. (b) EMSAs performed on HES1 SPS element using wild-type Notch1-RAMANK or mutant R1984A form (prior literature refers to this mutant as R1985A; the change to R1984A reflects current Genbank numbering for human Notch1). A low (50 nM) and a high concentration (200 nM) of CSL were incubated with labeled DNA (40 nM), with an excess of wild-type or mutant Notch1-RAMANK (1.2 μM) in the presence or the absence of an excess of MAML1 (2.6 μM). In the absence of MAML1, wild-type and mutant forms of Notch both supershift CSL–DNA complexes. In the presence of MAML1, wild-type Notch1RAMANK supershifts to a higher order dimeric complex and the R1984A mutant does not. The gel is a 10 % acrylamide gel made as described in Table 1

9. 10 % (w/v) Ammonium persulfate (APS): May be stored at 4 °C for 1 month or frozen. 10. N,N,N′,N′-tetramethylethylenediamine (TEMED). 11. 30 (w/v) Acrylamide solution (AcrylaGel, National Diagnostics). 12. 2 % (w/v) Bis-acrylamide solution (bis-AcrylaGel, National Diagnostics). 13. 2.5× Binding/loading buffer: 25 % Glycerol, 50 mM HEPES pH 7.9, 150 mM KCl, 12.5 mM MgCl2, 25 mM DTT, 25 μg/ml poly-dIdC, 0.5 mg/ml BSA. May be frozen for a month. 14. Marker dye containing bromophenol blue, such as 6× DNA loading buffer 30 % (v/v) glycerol, 0.25 % (w/v) bromophenol blue, 0.25 % (w/v) xylene cyanol FF.

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Equipment

1. Tabletop microcentrifuge. 2. Microspin gel filtration columns (Illustra Microspin G25 or G50 column, GE Healthcare): Choice of resin will be guided by size of DNA being purified. 3. Water bath, set to 30 °C. 4. Gel electrophoresis apparatus (Protean II xi, Bio-Rad) (see Note 3). 5. Siliconized gel electrophoresis plates (see Note 4), 0.75 mm or 1 mm spacers and wide-tooth combs. 6. Cold room or gel-cooling apparatus. 7. Gel dryer. 8. Film and developer or phosphorimager.

3

Methods The protocol described here calls for the use of 32P, which is a source of harmful β-radiation. Follow the safety protocols outlined by your institution for the safe handling and disposal of this isotope.

3.1 Anneal and Label DNA

1. Mix complementary oligonucleotides to a final concentration of 10−5 M of each in 25 mM Tris pH 8 (mix 5 μl of each oligo at 10−4 M in 2 μl 0.5 M Tris pH 8 and 38 μl water). 2. Heat to 100 °C for 10 min. Slowly cool to room temperature for 4–16 h. 3. Radiolabel double-stranded DNA probes with a 3′ fill-in reaction using Klenow fragment. Mix 2 μl annealed DNA duplex, 2 μl 10× DNA labeling buffer (NEB Buffer 2), 13.5 μl water, 1 μl 20× dNTP mix (670 μM), 1 μl α-32P-dCTP, and 0.5 μl Klenow enzyme (see Note 5). 4. Incubate at room temperature for 15 min. Stop reaction with 10 mM EDTA, and dilute to 50 μl for a final concentration of 0.4 μM (mix 1 μl 0.5 M EDTA and 29 μl water, and add to 20 μl reaction). 5. Remove excess α-32P-dCTP on a microspin gel filtration column. For each labeling reaction, spin a new column at 735 × g for 1 min in a microcentrifuge to remove the preservative fluid from the resin. Add reaction mix to the column, and let sit for 1 min. Place the column in a clean microcentrifuge tube and spin at 735 × g for 2 min to recover the labeled DNA in the flow-through. The excess nucleotide will remain on the resin.

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3.2 Protein/DNA Binding

1. Mix 6 μl binding/loading buffer and proteins (100–300 ng CSL, 500–1,000 ng Notch1-RAMANK, 100–200 ng MAML1). Dilute to 14 μl. 2. Add 1 μl 32P-labeled DNA duplex (0.4 pmol) for a total reaction volume of 15 μl. 3. Incubate for 30 min at 30–32 °C in a water bath. 4. Spin down briefly before loading onto the gel.

3.3 Gel Electrophoresis

1. Prepare 10× running buffer. 2. Assemble plates for gel electrophoresis with 0.75 or 1 mm spacers, and partially insert combs of the same thickness. 3. Prepare an acrylamide solution with 40:1 ratio of acrylamide to bis-acrylamide in 1× gel running buffer (see Table 1). Add the APS and TEMED last, and mix quickly (see Note 6). 4. Pour the gel, and then insert the combs fully (see Note 7). Allow the gel to polymerize for at least 30 min. 5. Prepare 1× gel running buffer. For a large format gel, 3 L is sufficient. 6. After polymerization, remove comb from the gel while holding upside down. Rinse wells thoroughly to remove unpolymerized acrylamide (see Note 8). 7. Assemble gel apparatus, and test for leaks (see Note 9). Fill with 1× gel running buffer. 8. Load samples onto gel. The binding/loading buffer contains no dyes (as dyes such as bromophenol blue can interfere with some protein/DNA interactions). Load marker dye in an empty lane (see Note 10). 9. Run the gel at 4 °C for 1–4 h, at a voltage appropriate for the gel apparatus, being careful to avoid overheating the gel as this may denature protein/DNA complexes (see Note 11). Table 1 Preparation of polyacrylamide gel mixture for one 16 × 16 cm gel Acrylamide

4%

8%

10 %

Bis-acrylamide

0.10 %

0.20 %

0.25 %

ddH2O (ml)

21.17

15.67

12.92

10× Running buffer (ml)

3

3

3

30 % Acrylagel (ml)

4

8

10

2 % Bis-acrylagel (ml)

1.5

3

3.75

10 % APS (0.1 g/ml) (ml)

0.3

0.3

0.3

TEMED (ml)

0.03

0.03

0.03

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3.4 Detection of Bands on Gel

1. After electrophoresis is complete, remove gel and plate assembly. Carefully pry apart gel plates, allowing gel to remain attached to one of the plates. 2. Place Whatman paper on the gel to remove it from the glass plate. Cover with plastic wrap. 3. Dry the gel using a vacuum gel dryer, applying heat (70–75 °C) and vacuum for up to 2 h (see Note 12). 4. Autoradiography can be performed using film or phosphor screen (Fig. 2b). Expose for 30 min to overnight before development depending on the amount and age of 32P used and the concentrations of protein and DNA in the binding reaction.

4

Appendix: Expression and Purification of Nuclear Notch Proteins

4.1 Reagents for Protein Expression and Purification

1. Competent bacterial cell lines suitable for IPTG-inducible expression: BL21(DE3), BL21(DE3)pLysS, and Rosetta2 (DE3)pLysS (EMD Millipore). 2. LB media and agar. 3. Antibiotics: Ampicillin (100 mg/ml, sterile filtered), kanamycin (50 mg/ml, sterile filtered), chloramphenicol (34 mg/ml in ethanol). 4. Isopropyl-β-D-thiogalactopyranoside (IPTG). 5. Glutathione sepharose. 6. Ni-NTA agarose. 7. Ethylenediaminetetraacetic acid (EDTA). 8. Protease inhibitors: Either one Roche Complete tablet with EDTA per cell lysis or 1:1,000 each of 150 mM PMSF, 0.7 mg/ml pepstatin A, 2 mg/ml aprotinin, 0.5 mg/ml leupeptin. 9. Reducing agents: β-Mercaptoethanol (βME) and dithiothreitol (DTT). 10. Buffers and solution components: Tris, sodium phosphate monobasic, sodium phosphate dibasic, imidazole, sodium chloride, guanidinium · HCl. 11. Acetic acid. 12. Specific protease for cleavage of tags (e.g., tobacco etch virus (TEV) protease).

4.2 Equipment for Protein Expression and Purification

1. Incubator 37 °C, 18 °C. 2. Protein gel electrophoresis apparatus. 3. Centrifuge (4,000 × g) and rotors to hold 1 L bottles and 50 ml tubes.

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4. Centrifuge (15,000 × g) and rotor and appropriate 40–50 ml tubes. 5. Rockers at 4 °C and room temperature. 6. FPLC with anion exchange, cation exchange, and S200 gel filtration columns. 7. Spin concentrators, 30 and 3.5 kDa molecular weight cutoff. 8. Lyophilizer. 4.3 Protocol for Bacterial Expression and Purification of GST-TEV-RAMANK

1. Plasmid: pGEX-4 T or pDEST15, ampicillin resistant, with an insert encoding a TEV cleavage site and human Notch1 (1760–2126). 2. Transform competent BL21(DE3) cells with Notch1RAMANK plasmid and grow on LBA plates (LB agar with 100 mg/L ampicillin) (see Note 13). 3. Grow 20–50 ml overnight culture from a single colony in LBA media (LB with 100 mg/L ampicillin). 4. Inoculate 2 L with 20 ml overnight culture, and grow 37 °C until culture reaches OD600 of 0.5–0.8 AU. 5. Take aliquots before and after induction and at each step during purification (see Note 14). 6. Induce protein expression with 1 mM IPTG. Grow for 4 h post-induction. 7. Harvest cells by centrifugation in 1 L bottles at 4,000 × g for 25 min. 8. Resuspend cell pellet in 40 ml lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl) and freeze. 9. While thawing cells, add 5 mM βME and protease inhibitors (see Note 15). 10. Sonicate thoroughly to lyse cells and shear genomic DNA. Take care to cool between sonication steps to prevent overheating and possibly denaturing protein (see Note 16). 11. Centrifuge at 14,000–18,000 × g for 30 min, and collect supernatant. 12. Apply supernatant to 4 ml pre-equilibrated glutathione sepharose beads, and rock at 4 °C for 1 h. 13. Wash beads in a column with 50–100 volumes or in batch at least three times with 10 column volumes wash buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM DTT). 14. Remove GST-tag with TEV protease (100–200 μg) overnight at 4 °C (see Note 17). 15. Collect supernatant from cleavage reaction by spinning down beads and rinsing once with wash buffer.

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16. (Optional) Remove TEV protease by applying to 0.5 ml Ni-NTA agarose (TEV is His-tagged) for 1 h and collecting supernatant (see Note 18). 17. (Optional) Purify further on MonoQ anion-exchange column: Buffer A: 20 mM Tris pH 8.0, 5 mM DTT, 1 mM EDTA, 100 mM NaCl; buffer B: 20 mM Tris pH 8.0, 5 mM DTT, 1 mM EDTA, 1 M NaCl. 18. (Optional) Purify further on S200 size-exclusion column: Buffer: 20 mM Tris pH 8.5, 150 mM NaCl, 5 mM DTT. 19. Store at −80 °C at concentrations of 1 mg/ml. Expected yield is about 5 mg/L. The molecular weight of Notch1RAMANK(1760–2126) after TEV cleavage is 40.7 kDa. The molecular weight of the GST-fusion is 69.2 kDa. 4.4 Protocol for Bacterial Expression and Purification of CSL-6H

1. Plasmid: pET28a, kanamycin resistant, with insert encoding human CSL(9–435)-6H (see Note 19). 2. Transform competent Rosetta2(DE3)pLysS cells with CSL plasmid and grow on LBKC plates (LB agar with 50 mg/L kanamycin, 34 mg/L chloramphenicol). 3. Grow overnight cultures from a single colony in LBKC media (LB with 50 mg/L kanamycin and 34 mg/L chloramphenicol). 4. Inoculate each 2 L flask with 20 ml overnight culture, grow at 37 °C until culture reaches OD600 of 0.3–0.5 AU, then lower temperature to 18 °C, and allow density to reach OD600 of 0.7–1.0 AU. 5. Induce protein expression with 0.5 mM IPTG, and grow overnight at 18 °C. 6. Harvest cells by centrifugation in 1 L bottles at 4,000 × g for 25 min (see Note 20). 7. Resuspend cell pellet in 40–80 ml lysis buffer (50 mM Tris pH 8.5, 500 mM NaCl) and freeze at −80 °C. 8. While thawing cells, add 5 mM βME and protease inhibitors. 9. Sonicate thoroughly to lyse cells (e.g., three times for 30 s, cooling between sonication steps). 10. Centrifuge at 14,000–18,000 × g for 30 min, and collect supernatant. 11. Batch bind to 5 ml Ni-NTA agarose resin (pre-equilibrated with lysis buffer) at 4 °C for 1 h. 12. Wash with 20–50 column volumes wash buffer (50 mM Tris pH 8.8, 0.5 m NaCl, 5 mM βME, 10 mM imidazole). 13. Elute with 2–5 column volumes of elution buffer (50 mM Tris pH 8.8, 0.5 m NaCl, 5 mM βME, 250 mM imidazole) (see Note 21).

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14. Prepare buffers for anion exchange: Buffer A (20 mM Tris pH 8.8, 5 mM DTT) and buffer B (20 mM Tris pH 8.8, 1 M NaCl, 5 mM DTT). Wash and equilibrate column with 15 % B (150 mM NaCl). 15. Dilute protein solution with buffer A to reduce the NaCl concentration to 150 mM, and load this sample onto the column immediately. Elute with a 20 column volume gradient to 40 % B (400 mM NaCl). 16. Buffer exchange into storage buffer (50 mM Tris pH 8.5, 0.5 M NaCl, 5 mM DTT) by size-exclusion chromatography (Superdex 200) if further purification is needed or in a 30 kDa molecular weight cutoff spin concentrator. 17. Store at 1 mg/ml or less at −80 °C in 20 % glycerol for longterm storage or at 4 °C for short-term storage (up to several months if the protein is free of protease contamination). Expected yield is low, up to 0.5 mg/L. The molecular weight of CSL(9–435)-6His is 49.3 kDa. 4.5 Protocol for Bacterial Inclusion Body Expression and Purification of MAML1

1. Plasmid: pRSETa, ampicillin resistant, with insert encoding 6H-TEV-MAML(13–74). 2. Transform BL21(DE3)pLysS with plasmid (or streak from glycerol stock). 3. Grow 50 ml overnight culture from a single colony in LBAC in 100 ml flask. 4. Inoculate 2 × 2 L with 20 ml overnight culture, and grow at 37 °C to OD600 0.8 AU (about 6 h). 5. Induce with 0.5 mM IPTG for 4 h. 6. Recover cells by centrifugation and resuspend in 40 ml lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 6 M guanidinium · HCl, 5 mM βME, with 150 μM PMSF). 7. Sonicate three times for 30 s, cooling between sonication steps. 8. Centrifuge at 14,000 × g for 30 min. 9. Batch bind to 5 ml Ni-NTA agarose resin (pre-equilibrated with lysis buffer) at 4 °C for 1 h. 10. Wash with 10 column volume wash buffer (50 mM Tris pH 8, 150 mM NaCl, 6 M guanidinium · HCl, 5 mM βME, 10 mM imidazole). 11. Elute with 1 column volume elution buffer (50 mM Tris pH 8, 150 mM NaCl, 6 M guanidinium · HCl, 5 mM βME, 250 mM imidazole). 12. Dialyze against 5 % acetic acid for 24 h, and lyophilize for at least 24 h. 13. Dissolve in 1–2 ml water.

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14. Slowly adjust pH to 6.2 with phosphate buffer (50 mM sodium phosphate, pH 6.2, 5 mM DTT, 1 mM EDTA). 15. Slowly add TEV protease, and cleave overnight at room temperature. 16. (Optional) Remove TEV protease by applying to 0.5 ml Ni-NTA agarose (TEV is tagged with 6His) for 1 h and collecting the supernatant (see Note 22). 17. (Optional) Purify on cation-exchange column: Buffer A (20 mM sodium phosphate buffer, pH 6.8, 5 mM DTT, 1 mM EDTA), buffer B (20 mM sodium phosphate buffer, pH 6.8, 2 M NaCl, 5 mM DTT, 1 mM EDTA). 18. Important note: Test fractions for binding activity because there is a frequent contaminant that has the same apparent molecular weight as the MAML1 peptide but does not bind Notch and CSL. EMSA is a good method for this test. 19. Store at a concentration of 5–10 mg/ml at −80 °C. Expected yield is 5–10 mg/L. The molecular weight of MAML1 (13–74) after TEV cleavage is 7.5 kDa.

5

Notes 1. We design a 3–5 nucleotide overhang at the 5′-end of each oligo with a total of 3 dGTP (typically 2 on one 5′-end and 1 on the other 5′-end) for incorporation of 3 32P-dCTP nucleotides for EMSAs performed with 0.2–1 pmol DNA per lane. 2. For a large format gel apparatus, 3 L of 1× running buffer is sufficient. 3. A large format is recommended in order to maximize resolution. This is especially true if proteins are dilute when loaded, and therefore they must be loaded in a large volume. 15–20 μl Load volumes on a large format gel with a 15-well comb will generally result in well-resolved bands of a wide range of sizes. 4. Siliconizing the inside of each plate before every use will facilitate transfer of the wet gel after electrophoresis. 5. DNA can be labeled up to a week before the EMSA. As the 32 P-dNTP or the DNA probe decays, exposure times will need to be increased. 6. We usually use a 10 % acrylamide gel for EMSAs of Notch nuclear complexes. The high percentage gel results in the sharpest bands and a gel that is resistant to damage during transfer. 7. It is critical to avoid forming air bubbles in the polymerizing gel. Make sure that the plates are very clean and that the gel is poured

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along one spacer edge so that it fills from the bottom. We pour gels using a 30 ml syringe with a 16–18 gauge needle. 8. One effective method of removing unpolymerized acrylamide is to rinse with water and then gently insert thin strips of Whatman paper into each well, allowing liquid to wick up the paper before removing carefully. 9. Test for leaks very carefully. Even a very slow leak that does not empty the upper buffer chamber will decrease the rate of electrophoresis. 10. DNA duplexes that are 20–30 base pairs in length will typically run near the bromophenol blue band. In order to see the free DNA on the gel, run until the bromophenol blue dye band is about 1 cm from the bottom of the gel. 11. Using a large-format gel apparatus such as the Bio-Rad Protean II, run for 1 h at 180 V, and then increase voltage to 190– 200 V for up to 3 more hours. 12. Removing the gel from the dryer before it has completely dried may lead to cracking of the gel. 13. Notch1-RAMANK and -ANK proteins can also be expressed in BL21(DE3)pLysS cells. 14. Dilute cell pellets in a volume of SDS-loading buffer proportional to cell density at the time the aliquot was taken, and load equal amounts. 15. Notch1-RAMANK is proteolytically sensitive. It is important to work fast and keep protease inhibitors in buffers until the protein is pure. 16. Sonicating three times for 30 s with 30 s on ice between sonication steps is usually sufficient. 17. This protocol describes removal of the GST-tag by protease cleavage. For purification of the GST-Notch1-RAMANK fusion, skip the TEV cleavage step and elute with 20 mM glutathione in wash buffer. 18. Removal of TEV protease is not necessary if following with anion exchange. 19. The protein yield of CSL-6H is low after expression and purification. With large-scale expression (6–12 L), yields can reach 0.5 mg/L, but may be lower for smaller scale preparations. 20. CSL yield is very low. An induced band is often not visible on a Coomassie gel. 21. CSL protein is not very pure at this stage and benefits greatly from a further step of purification. 22. Removal of the TEV protease is not necessary if following with another column purification step.

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Acknowledgements This work was supported by NIH grants CA-092433 and CA-119070 (to S.C.B.). References 1. Artavanis-Tsakonas S, Rand MD, Lake RJ (1999) Notch signaling: cell fate control and signal integration in development. Science 284:770–776 2. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689 3. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233 4. Kovall RA, Blacklow SC (2010) Mechanistic insights into Notch receptor signaling from structural and biochemical studies. Curr Top Dev Biol 92:31–71 5. Tamura K, Taniguchi Y, Minoguchi S et al (1995) Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-J kappa/Su(H). Curr Biol 5:1416–1423 6. Kovall RA, Hendrickson WA (2004) Crystal structure of the nuclear effector of Notch signaling, CSL, bound to DNA. EMBO J 23: 3441–3451 7. Nam Y, Sliz P, Song L et al (2006) Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes. Cell 124:973–983 8. Wilson JJ, Kovall RA (2006) Crystal structure of the CSL-Notch-Mastermind ternary complex bound to DNA. Cell 124:985–996 9. Weng AP, Nam Y, Wolfe MS et al (2003) Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. Mol Cell Biol 23:655–664 10. Fryer CJ, Lamar E, Turbachova I et al (2002) Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes Dev 16:1397–1411 11. Wallberg AE, Pedersen K, Lendahl U et al (2002) p300 and PCAF act cooperatively to mediate transcriptional activation from chromatin templates by notch intracellular domains in vitro. Mol Cell Biol 22:7812–7819 12. Kuroda K, Han H, Tani S et al (2003) Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. Immunity 18:301–312

13. Oswald F, Kostezka U, Astrahantseff K et al (2002) SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. EMBO J 21:5417–5426 14. Kao H-Y, Ordentlich P, Koyano-Nakagawa N et al (1998) A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev 12:2269–2277 15. Zhou S, Fujimuro M, Hsieh JJ et al (2000) A role for SKIP in EBNA2 activation of CBF1repressed promoters. J Virol 74:1939–1947 16. Hsieh JJ, Zhou S, Chen L et al (1999) CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. Proc Natl Acad Sci U S A 96:23–28 17. Taniguchi Y, Furukawa T, Tun T et al (1998) LIM protein KyoT2 negatively regulates transcription by association with the RBP-J DNAbinding protein. Mol Cell Biol 18:644–654 18. Tun T, Hamaguchi Y, Matsunami N et al (1994) Recognition sequence of a highly conserved DNA binding protein RBP-J kappa. Nucleic Acids Res 22:965–971 19. Jarriault S, Brou C, Logeat F et al (1995) Signalling downstream of activated mammalian Notch. Nature 377:355–358 20. Wu L, Aster J, Blacklow S et al (2000) MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nat Genet 26:484–489 21. Nam Y, Weng AP, Aster JC et al (2003) Structural requirements for assembly of the CSL.intracellular Notch1.Mastermind-like 1 transcriptional activation complex. J Biol Chem 278:21232–21239 22. Nam Y, Sliz P, Pear WS et al (2007) Cooperative assembly of higher-order Notch complexes functions as a switch to induce transcription. Proc Natl Acad Sci U S A 104:2103–2108 23. Arnett KL, Hass M, McArthur DG et al (2010) Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nat Struct Mol Biol 17:1312–1317 24. Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9:6505–6525

Analyzing the Nuclear Complexes of Notch Signaling… 25. Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res 9:3047–3060 26. Buratowski S, Chodosh LA (2001) Mobility shift DNA-binding assay using gel electrophoresis. Curr Protoc Mol Biol 36:12.2.1–12.2.11 27. Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2:1849–1861 28. Berger R, Duncan MR, Berman B (1993) Nonradioactive gel mobility shift assay using

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chemiluminescent detection. Biotechniques 15:650–652 29. Pagano JM, Clingman CC, Ryder SP (2011) Quantitative approaches to monitor proteinnucleic acid interactions using fluorescent probes. RNA 17:14–20 30. Rye HS, Drees BL, Nelson HC et al (1993) Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat shock transcription factor. J Biol Chem 268:25229–25238 31. Jing D, Agnew J, Patton WF et al (2003) A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels. Proteomics 3:1172–1180

Chapter 19 Identifying Direct Notch Transcriptional Targets Using the GSI-Washout Assay Will Bailis, Yumi Yashiro-Ohtani, and Warren S. Pear Abstract Genetic gain- and loss-of-function studies have traditionally been used to study transcriptional networks regulated by the Notch signaling pathway; however these techniques lack the ability to resolve primary and secondary transcriptional events. In contrast, the γ-secretase inhibitor (GSI) washout assay takes advantage of the reversibility of GSI, a pharmacological inhibitor of Notch signaling, along with the ability of cycloheximide to prevent secondary transcriptional effects to identify direct Notch pathway targets. Here we review this technique and the technical considerations for adapting this assay to a cell type of choice. Key words Notch, Target identification, GSI washout, Transcription, Signaling

1

Introduction Notch signaling regulates a wide network of target genes in multiple tissues. The relative accessibility of a given target gene is highly context dependent, and even conserved targets show variability in their responsiveness to Notch signaling. The HERP family genes Hey1, Hey2, and HeyL are all conserved targets of the Notch pathway [1]. Despite their sensitivity to changes in Notch signaling, all three Notch targets display distinct temporal and spatial expression patterns during embryonic development, highlighting the limitation of using a small set of canonical targets as universal surrogates for Notch signaling [2–4]. This dynamism of Notch target regulation is further illustrated by the ability of Notch to exert both proand anti-oncogenic functions depending on the tissue of origin [5–8]. Not only does the Notch pathway display context-specific activity, but many Notch targets are regulated by additional inputs, further curtailing the utility of canonical targets as faithful reporters for Notch signaling. For example, E-proteins are capable of regulating the expression of the conserved Notch targets Hes1, Notch1, Notch3, and Ptcra [9]. Thus, a reliable Notch target identification method will not only verify a given gene’s sensitivity to

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the modulation of Notch signaling but also stringently control for signaling kinetics as to exclude confounding secondary events and signals from parallel pathways. Since the Notch pathway was first discovered, multiple approaches have been taken to identify target genes of Notch signaling. Seminal studies performed in Drosophila relied heavily on the use of mutant Notch constructs in combination with complementation studies in order to parse out the progression of signal transduction and target activation in the Notch pathway [10–13]. These studies yielded the first bona fide Notch targets, the Enhancer of Split gene family, setting the foundation for future investigation. Similar studies performed in mammalian cells, utilizing retroviral expression of gain-of-function Notch mutants and reporter assays, corroborated Drosophila findings and demonstrated that Hes family proteins are highly conserved targets of the Notch pathway in multiple tissues [14]. Despite these advances in target identification, both gain- and loss-of-function models relied on correlative relationships and were limited in their ability to exclude secondary effects and place putative targets directly downstream of Notch. The ability to identify direct Notch transcriptional targets was greatly facilitated by the establishment of the chromatin immunoprecipitation (ChIP) assay and the development of pharmacological Notch inhibitors, in particular gamma secretase inhibitors (GSI). ChIP provides the ability to show interaction between a protein and a transcription factor-binding site. The development of ChIP-grade anti-Notch and anti-CSL (encoded by the RBPJ gene in mammals) antibodies provided the ability to verify the direct interaction between the Notch complex and endogenous target gene loci (see Chapter 20, Borggrefe and Liefke 2014, for more on Notch ChIP assays). Despite revealing this interaction of Notch/CSL and DNA, an important limitation to ChIP is that Notch and CSL binding does not always indicate that a particular locus is expressed [15]. Another important tool for identifying direct Notch transcripts are GSI, as these compounds provide a method for rapidly modulating Notch signaling. GSI were developed as Alzheimer’s therepeutics against gamma secretase-mediated cleavage of amyloid precursor protein [16–20]. The finding that these same drugs could be used to prevent Notch signaling equipped the field with an acute and reversible inhibitor that allowed for more precise control of transcriptional kinetics and the ability to readily manipulate the Notch pathway both in vivo and in vitro [21, 22]. In particular, GSI inhibits the gamma secretase-dependent S3 cleavage that releases the Notch intracellular domain from the Notch extracellular domain [23, 24]. Thus, both full-length forms of Notch and Notch mutants that contain the gamma secretase cleavage site are susceptible to GSI blockade. This includes the majority of gain-of-function mutations identified in T-ALL and other malignancies [25, 26].

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The GSI-washout assay takes advantage of these drug features and allows for the definitive identification of direct Notch targets [27]. In particular, the Notch S3 cleavage complex appears to be long lived at the cell membrane so that even after prolonged exposure to GSI, S3 cleavage, nuclear translocation, and transcriptional activation rapidly occur after removal of the GSI. The major limitation to the length of GSI treatment is the potential detrimental effects on the cells resulting from Notch inhibition. After pretreating cells with GSI, drug is washed off and cells are briefly returned to culture, permitting the recovery of Notch signaling; target genes are identified as transcripts that rebound in the washout sample, when compared to vehicle and mock-wash controls, and the activity can be ascribed as direct if the washout culture is performed in the presence of cycloheximide to prevent secondary effects. The protocol and technical considerations for the GSI-washout assay are reviewed in the sections below.

2

Materials

2.1 Reagents and Material for Cell Culture and Washout

1. T6E cells [28] (or a cell line that contains a form of Notch that is GSI sensitive). 2. T6E media (RPMI, 10 % FBS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate). 3. Phosphate-buffered saline (PBS). 4. 10 mM DAPT, GSI (Sigma, 208255-80-5). 5. Dimethyl sulfoxide (DMSO). 6. Cycloheximide (20 mg/ml stock solution). 7. Qiagen RNeasy Mini Kit (or equivalent). 8. Invitrogen SSIII reverse transcriptase kit (or equivalent). 9. 2× Sybr green master mix. 10. 10 μM Quantitative real-time PCR (qPCR) primers.

2.2 Equipment for Cell Culture and Washout

1. Incubator 37 °C. 2. Centrifuge (4,000 × g) and rotors to hold 15 ml conical tubes. 3. 15 ml Disposable polystyrene conical tube with screw cap. 4. 1.5 ml Microcentrifuge tubes. 5. 384-Well qPCR plate. 6. 6-Well flat-bottom tissue culture plate. 7. Bio-Rad C1000 Touch Thermal Cycler (or equivalent). 8. Applied Biosystems ViiA 7 (or equivalent).

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Methods The following protocol is optimized for a GSI-washout assay on T6E cells. Different cell populations and Notch targets within those cells will display varying levels of sensitivity to Notch inhibition. In order to obtain maximum sensitivity using the GSIwashout assay, it is critical to determine the optimum kinetics for the GSI pretreatment period and for the washout itself. Failure to do so may limit the dynamic range of the assay if the duration of either the GSI pretreatment or the washout is too short. Moreover, if these periods are too long, then secondary and/or off-target effects can occur that increase the potential for false positives. These potential limitations are discussed in greater detail in Subheading 4.

3.1 GSI Pretreatment of Cells

1. Prepare T6E cells in single-cell suspension (see Note 1). 2. Count viable cells by trypan blue exclusion. Adjust cell concentration to 5 × 105 cells/ml, and plate three wells of a 6-well plate with 3 ml of cell suspension each. 3. Dilute 1 μl of 10 mM GSI into 100 μl of T6E media. Add 30 μl of diluted GSI to two of the wells containing T6E cells so that the final concentration of GSI is 1 μM. Add 30 μl of identically diluted DMSO to the remaining well of T6E cells (see Note 2). 4. Place cells into the 37 °C incubator overnight.

3.2

GSI Washout

1. Harvest cells and transfer into 15 ml conical tubes, bringing up the total volume to 10 ml with PBS. 2. Spin cells at 500 × g for 10 min. Remove supernatant, and wash with PBS two additional times. 3. Replate cells into 3 ml of T6E media containing 20 μg/ml cycloheximide (see Note 3). Replate one of the GSI-pretreated samples so that the final concentration of GSI is 1 μM; see above (mock wash). Replate the other GSI-pretreated sample into media containing identically diluted DMSO (washout). Replate cells that were pretreated with DMSO into media containing identically diluted DMSO. 4. Place cells into the 37 °C incubator for 4 h. 5. Remove cells from incubator and transfer to 15 ml conical tubes, bringing up the total volume to 10 ml with PBS. 6. Spin cells at 500 × g for 10 min. Add 1 ml of PBS, transfer cells into 1.5 ml microcentrifuge tube, and then remove supernatant. Cells can be kept at −80 °C.

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Table 1 Primers Target gene

Forward primer

Reverse primer

Hes1

GAAAGATAGCTCCCGGCATT

GTCACCTCGTTCATGCACTC

Hes5

CCGACATCCTGGAGATGG

TAGCCCTCGCTGTAGTCCTG

Hey1

GGTACCCAGTGCCTTTGAGA

ACCCCAAACTCCGATAGTCC

Notch1

ATGGGCCGTACTCCGTTACA

TAGGTCATCCACGGCATTGA

Notch3

AGCAGTGGAGCGACTTGATT

GAACCAGAGGGTGCTGTG

Nrarp

CTACACATCGCCGCTTTCG

CGCGTACTTGGCCTTGGT

Dtx1

ATCAGTTCCGGCAAGACACAG

CGATGAGAGGTCGAGCCAC

EF1alpha

CACTTGGTCGCTTTGCTGTT

GGTGGCAGGTGT TAGGGGTA

3.3 RNA Isolation, Reverse Transcriptase PCR, and qPCR Analysis

1. Proceed to RNA purification following the protocol provided with the Qiagen RNeasy Mini Extraction Kit. Elute with 50 μl of RNase-free water. 2. Prepare cDNA using 10 μl purified RNA, following protocol provided with the Invitrogen SSIII reverse transcriptase PCR kit. 3. Prepare qPCR master mix. For 1-well: 5 μl 2× Sybr green mix, 0.1 μl forward primer (10 μM), 0.1 μl reverse primer (10 μM), 3.8 μl water. Use primers from Table 1 (note: these are primers for murine studies (see Note 4)). 4. Plate 9 μl of qPCR master mix per well onto a 384-well plate. 5. Add 1 μl of sample cDNA to each well containing master mix. 6. Analyze in Applied Biosystems Vii7 with EF1a as an internal control. 7. Notch dependent targets are then identified as those that display a significant decrease in transcript in the mock-wash sample, as compared to the DMSO control, and a recovery of transcript in the mock-wash sample to levels comparable to the DMSO control [19, 29].

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Notes 1. If using a cell type other than T6E cells, it will be necessary to optimize the pretreatment and GSI-washout kinetics. The first parameter to consider is the tolerance of the target cell popula-

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tion to Notch inhibition, as many cell lines and some cell types will undergo apoptosis after prolonged withdrawal from Notch signaling. Cell viability after GSI treatment should be tracked over time and determined before proceeding to the GSI-washout assay. We have used times ranging from 4 h to 3 days. For pretreatment, the longest tolerated treatment period should be used in order to decrease background and increase the dynamic range for target identification. For GSI washout, the optimal kinetics will be determined by the incubation period that provides the most robust recovery of target transcript in the washout sample, as compared to the DMSO and mock-wash controls. While longer culture periods during the washout will permit a greater recovery of transcripts, shorter incubation periods are desirable as they will limit any potential confounding effects of cycloheximide treatment. 2. Some cell types may exhibit poor Notch signaling when placed into in vitro culture, such that target genes are insensitive to GSI treatment. To overcome this problem, it may be necessary to culture experimental cells on OP9-delta cultures or purified Notch ligands (see Chapter 11 (Ilagan and Kopan 2014a), Chapter 12 (Ilagan and Kopan 2014b), and Chapter 25 (Koga and Aikawa 2014) for more information) in order to boost Notch signaling and to detect GSI sensitivity of target genes. It should be noted that this technique may result in false positives during target identification; while many genes are accessible to the Notch transcriptional complex, they may not be regulated by Notch at biological levels of Notch signaling for a given cell population. 3. Different cell populations will display differing levels of sensitivity to cycloheximide treatment. While some populations will require doses above 20 μg/ml in order to inhibit the translation of new protein, other populations will require lower doses in order to prevent toxicity or confounding effects on transcription. 4. The primers listed in Subheading 3.3, step 3, are designed against several highly conserved and sensitive Notch target genes. These are to be used as a means to determine the efficacy of the GSI-washout assay. While these genes are Notch targets in multiple contexts, they may not be sensitive to inhibition of Notch signaling in every cell type or culture condition. It will be necessary to determine which of these canonical target genes are accessible Notch targets for each experimental cell type used.

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14. Artavanis-Tsakonas S, Rand MD, Lake RJ (1999) Notch signaling: cell fate control and signal integration in development. Science 284:770–776 15. Wang H, Zou J, Zhao B et al (2011) Genomewide analysis reveals conserved and divergent features of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells. Proc Natl Acad Sci U S A 108: 14908–14913 16. Esler WP, Kimberly WT, Ostaszewski BL et al (2000) Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1. Nat Cell Biol 2:428–434 17. Zhang Z, Nadeau P, Song W et al (2000) Presenilins are required for gamma-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1. Nat Cell Biol 2: 463–465 18. De Strooper B, Annaert W, Cupers P et al (1999) A presenilin-1-dependent gammasecretase-like protease mediates release of Notch intracellular domain. Nature 398:518–522 19. Tsai JY, Wolfe MS, Xia W (2002) The search for gamma-secretase and development of inhibitors. Curr Med Chem 9:1087–1106 20. De Strooper B, Vassar R, Golde T (2010) The secretases: enzymes with therapeutic potential in Alzheimer disease. Nat Rev Neurol 6: 99–107 21. Hadland BK, Manley NR, Su D et al (2001) Gamma -secretase inhibitors repress thymocyte development. Proc Natl Acad Sci U S A 98:7487–7491 22. Geling A, Steiner H, Willem M et al (2002) A gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep 3: 688–694 23. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233 24. Gordon WR, Arnett KL, Blacklow SC (2008) The molecular logic of Notch signaling–a structural and biochemical perspective. J Cell Sci 121:3109–3119 25. Weng AP, Ferrando AA, Lee W et al (2004) Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306:269–271 26. Aster JC, Blacklow SC, Pear WS (2011) Notch signalling in T-cell lymphoblastic leukaemia/ lymphoma and other haematological malignancies. J Pathol 223:262–273

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27. Weng AP, Millholland JM, Yashiro-Ohtani Y et al (2006) c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma. Genes Dev 20:2096–2109 28. Pear WS, Aster JC, Scott ML et al (1996) Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing

activated Notch alleles. J Exp Med 183: 2283–2291 29. Bailis W, Yashiro-Ohtani Y, Fang TC et al (2013) Notch simultaneously orchestrates multiple helper T cell programs independently of cytokine signals. Immunity 39: 148–159

Chapter 20 Probing the Epigenetic Status at Notch Target Genes Robert Liefke and Tilman Borggrefe Abstract Chromatin-based mechanisms significantly contribute to the regulation of many developmentally regulated genes, including Notch target genes. After specific ligand binding, the intracellular part of the Notch receptor is cleaved off and translocates to the nucleus, where it binds to the transcription factor CSL (encoded by the RBPJ gene in mammals), in order to activate transcription. In the absence of a Notch signal, CSL represses Notch target genes by recruiting a co-repressor complex. Both NICD co-activator and CSL co-repressor complexes contain chromatin modifiers such as histone acetyltransferases and methyltransferases, which dynamically regulate chromatin marks at Notch target genes. Here we provide protocols for ChIP (chromatin immunoprecipitation) to analyze the chromatin status of dynamically regulated Notch target genes. Furthermore, an example is presented how to perform a primary analysis of ChIP-Seq data at Notch target genes using the Cistrome platform. Key words Notch, Transcription, Epigenetics, ChIP, ChIP-Seq, Histone modifications, Histone methyltransferase, Histone deacetylases, Cistrome

1

Introduction Though the Notch signaling cascade appears remarkably simple with no second messengers involved [1], the activation of downstream genes in a given tissue often remains complex and poorly understood. Specificity of a given Notch target gene is often set up long before the actual Notch signal is received. This is due to chromatin-based mechanisms that shape the specific epigenetic state of Notch-responsive genes. Notch target genes can be kept in a permissive (or “poised”) state reflected by a combination of positive and negative chromatin marks being able to respond to a Notch stimulus at the right time. Alternatively, certain Notch target genes can be fully shut off by the presence of multiple negative histone marks, DNA methylation, and eventually chromatin compaction. Since Notch target genes are themselves often master regulators, gene regulation of Notch target genes is of major importance to understand molecular control of cell differentiation and carcinogenesis.

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_20, © Springer Science+Business Media New York 2014

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Histone modifications affect chromatin structure either directly, by altering the electrostatic interactions between histones and DNA, or indirectly, by providing a specific platform recognized by chromatin-binding proteins which promote distinct cellular events. Histone acetylation leads to opening of the chromatin fiber, which allows binding of transcription factors and is generally associated with transcriptional activation. Histone methylation can occur at different lysine or arginine residues and correlates either with activation or repression. Well-known methylation marks are H3K4 methylation for gene activation, while H3K27 methylation is associated with gene repression, as reviewed in [2]. The transcription factor CSL plays a central role in transducing Notch signals into changes in gene expression [3–5]. Following activation, the formation of a ternary complex containing CSL, NICD, and Mastermind is essential for the upregulation of Notch target genes. Upon binding of NICD to CSL, CSL switches from repression into activation mode and promotes gene expression. The interaction of NICD with CSL creates an interface that is recognized by the essential co-activator Mastermind [6]. The CSL/ Notch/Mastermind co-activator can subsequently recruit the histone acetyltransferase p300 [7]. Interestingly, CSL was originally identified as a repressor of transcription [8]. The CSL activator/ repressor paradox was resolved with the finding that repression and activation via CSL involve recruitment of distinct protein complexes. So far, a model has emerged in which NICD displaces corepressors to convert DNA-bound CSL to an activator, as reviewed in [3, 4, 9, 10]. Notch target genes are regulated by a plethora of chromatin modifiers (reviewed in [10]). A role for histone acetyltransferase p300 [7] as well as histone deacetylase HDAC1 [11] and SIRT1 [12, 13] in Notch signaling has been proposed early on. More recently, dynamic regulation of H3K4 methylation has been demonstrated, regulated by histone demethylases KDM5A [14] and LSD1 [15]. The Polycomb complex, which regulates H3K27 methylation, has been implicated in repression of Notch target gene expression. Genetically, Drosophila polyhomeotic, a polycombcomplex component, suppresses Notch signaling [16] and mutations in polycomb complex PRC2 are found in chronic T-ALL [17]. A direct interactor of CSL, FHL1 (also known as KyoT2), may form the bridge between CSL and Polycomb [18]. The epigenetic status at Notch target genes is commonly addressed via chromatin immunoprecipitation (ChIP) experiments. Here, we provide protocols for cross-linking ChIP (X-ChIP) and native ChIP (N-ChIP). The major difference between those two approaches is that during the X-ChIP the chromatin-bound proteins are chemically cross-linked to the chromatin, whereas for native ChIP no such reaction is performed. Cross-linking ChIP is especially suitable for DNA-bound proteins, like CSL, and proteins

257

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Table 1 Suitable antibodies to probe the epigenetic status at Notch target genes Location

Recommended antibodies

X-ChIP

N-ChIP

H3



Abcam, ab1791

+

+

H3K4me1

Enhancers

Millipore, 07-436

+

+

+

+

+

+

+/−

+

+

+

+

+

Abcam, ab8895 H3K4me2

Enhancers/promoters

Millipore, 05-790 Millipore, 05-1338

H3K4me3

Active promoters, CpG islands

Millipore, 04–745 Millipore, 05-1339 Diagenode, pAb-003-050

H3K27me3

Repressed regions

Millipore, 07–449 Abcam, ab6002

H3K27ac

Enhancers

Active Motif, 39135 Diagenode, pAb-174-050

H3K9me3

Heterochromatin

Millipore, 07-523 Millipore, 05-1250

that are only weakly bound to chromatin. Cross-linking ChIP also works for most histone modifications. Native ChIP is mostly used for histone modifications but is also suitable for some strong histone-binding proteins. In Table 1 we summarize histone modifications, their occurrence, as well as the recommended ChIP antibody and approach. In both ChIP approaches the chromatin is cut in smaller pieces. For X-ChIP this is done physically using ultrasound sonication, while in the case of native ChIP, digestion with the enzyme micrococcal nuclease (MNase) is performed (Fig. 1). Subsequently, the DNA fragments are enriched by immunoprecipitation using a specific antibody. The fragments are isolated and further analyzed either by real-time PCR of selected Notch target genes or by high-throughput sequencing revealing genome-wide binding sites (ChIP-Seq). The obtained ChIP-seq data are usually further processed by a computational biologist. We provide here an introductory guide for a primary analysis of these data using the Cistrome platform [19]. This approach also allows an analysis of individual promoters using publically available datasets. To date, there are a few genome reports describing CSL- and NICD-binding sites in T-cells [20], in B-cells [21], and most recently in muscle cells [22]. These studies can be partially taken as reference points for further

258

Robert Liefke and Tilman Borggrefe Nucleosome

H4 Me

H3

Ac

K4

K27

DNA Transcription factor

H4

K27

H3

Formaldehyde crosslinking/ Sonication

MNase Treatment

Immunoprecipitation

Antibody

DNA Purification DNA

qPCR, ChIP-Seq

Fig. 1 Schematic outline of a chromatin immunoprecipitation (ChIP) experiment

studies looking at dynamic histone modifications in different systems and setups. To decipher the function of the CSL co-repressor complex as well as the Notch co-activator complex, the chromatin status at the target genes are ideally investigated in a dynamic system, where Notch signaling is either turned off or turned on. In chapters 11 (Ilagan and Kopan 2014a), 12 (Ilagan and Kopan 2014b), 19 (Bailis et al. 2014), 23 (De Kloe and De Strooper 2014), 24 (Gordon and Aster 2014), and 25 (Koga and Aikawa 2014), methods and tools for manipulating Notch signaling in mammalian cells are described.

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2

259

Materials

2.1 Chemicals and Reagents

1. Glycine. 2. Sucrose. 3. Tris base. 4. EGTA. 5. EDTA. 6. Nonidet P-40 (NP-40) or IGEPAL CA-630. 7. Glycerol. 8. DTT. 9. NaCl. 10. LiCl. 11. MgCl2. 12. CaCl2. 13. KCl. 14. SDS. 15. Ethanol (100 %). 16. Phenol/chloroform/isoamyl alcohol (25:24:1). 17. Chloroform. 18. Formaldehyde (37 %). 19. Sodium butyrate. 20. Proteinase K (10 mg/ml). 21. RNase A (10 mg/ml). 22. Protein A/G Sepharose Beads. 23. Glycogen (20 mg/ml). 24. Protease inhibitors (e.g., cOmplete Protease Inhibitor Cocktail Tablets from Roche). 25. MNase (Micrococcal nuclease, Sigma-Aldrich). 26. MilliQ water.

2.2

Equipment

1. Sonicator (for X-ChIP only). 2. Heat block or water bath at 37 °C, 45 °C, and 67 °C. 3. Ultracentrifuge with swing-out rotor (for N-ChIP only). 4. Refrigerated tabletop centrifuge. 5. Agarose gel electrophoresis apparatus.

2.3

X-ChIP Buffers

X-ChIP Lysis Buffer 1 50 mM Tris–HCl, pH 8.0. 2 mM EGTA.

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0.1 % NP-40 (v/v). 10 % Glycerol (v/v). 1 mM DTT (freshly added prior to use). Protease inhibitors (freshly added prior to use). X-ChIP Lysis Buffer 2 50 mM Tris–HCl, pH 8.0. 5 mM EGTA. 1 % SDS (w/v). 1 mM DTT (freshly added prior to use). Protease inhibitors (freshly added prior to use). X-ChIP Dilution Buffer 50 mM Tris–HCl, pH 8.0. 5 mM EGTA. 200 mM NaCl. 0.5 % NP-40 (v/v). Protease inhibitors (freshly added prior to use). NaCl Washing Buffer 20 mM Tris–HCl, pH 8.0. 500 mM NaCl. 2 mM EGTA. 0.1 % SDS (w/v). 1 % NP-40 (v/v). LiCl Washing Buffer 20 mM Tris–HCl, pH 8.0. 500 mM LiCl. 2 mM EGTA. 0.1 % SDS (w/v). 1 % NP-40 (v/v). X-ChIP Elution Buffer 10 mM Tris–HCl, pH 7.9. 1 mM EGTA. 2 % SDS (w/v). TE 10 mM Tris–HCl, pH 8.0. 1 mM EDTA.

Epigenetic Status at Notch Target Genes

Proteinase K Buffer (5×). 50 mM Tris–HCl, pH 7.5. 25 mM EGTA. 1.25 % SDS (w/v). 2.4

N-ChIP Buffers

N-ChIP Lysis Buffer 1 15 mM Tris–HCl, pH 7.5. 0.3 M Sucrose. 60 mM KCl. 5 mM MgCl2. 0.1 mM EGTA. 0.5 mM DTT. Protease inhibitors. N-ChIP Lysis Buffer 2 15 mM Tris–HCl, pH 7.5. 0.3 M Sucrose. 60 mM KCl. 5 mM MgCl2. 0.1 mM EGTA. 0.5 mM DTT. 0.4 % NP-40 (v/v). N-ChIP Lysis Buffer 3 15 mM Tris–HCl, pH 7.5. 1.2 M Sucrose. 60 mM KCl. 5 mM MgCl2. 0.1 mM EGTA. 0.5 mM DTT. MNase Digestion Buffer 50 mM Tris–HCl, pH 7.5. 0.32 M sucrose. 4 mM MgCl2. 1 mM CaCl2. Protease inhibitors. Stop Solution 20 mM EDTA pH 8.0.

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Resuspension Buffer 1 mM Tris–HCl, pH 7.5. 0.2 mM EDTA. Protease inhibitors. N-ChIP Dilution Buffer 50 mM Tris–HCl, pH 7.5. 50 mM NaCl. 5 mM EDTA. Protease inhibitors. N-ChIP Washing Buffer A 50 mM Tris–HCl, pH 7.5. 10 mM EDTA. 75 mM NaCl. N-ChIP Washing Buffer B 50 mM Tris–HCl, pH 7.5. 10 mM EDTA. 125 mM NaCl. N-ChIP Washing Buffer C 50 mM Tris–HCl, pH 7.5. 10 mM EDTA. 175 mM NaCl. N-ChIP Elution Buffer 50 mM Tris–HCl, pH 7.5. 50 mM NaCl. 5 mM EDTA. 1 % SDS.

3

Methods

3.1 CrossLinking ChIP

Day 1 1. To the media, containing about 2 × 107 cells (see Note 1), directly add formaldehyde (37 %) to a final concentration of 1 %. 2. Incubate cells for 10 min at room temperature (RT), shaking. 3. Add 1 M glycine pH 7.5 to a final concentration 0.125 M, and shake cells for 5 min at room temperature. 4. Collect cells, and centrifuge cells at 400 × g at 4 °C.

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5. Resuspend cell pellet with ice-cold PBS, and centrifuge cells at 400 × g at 4 °C. 6. Resuspend the cell pellet in 1 ml ice-cold X-ChIP lysis buffer 1 for 10 min on ice. 7. Centrifuge at 400 × g at 4 °C for 5 min. 8. Resuspend the pellet in 600 μl ice-cold X-ChIP lysis buffer 2 for 10 min on ice (see Note 2). 9. Sonicate cells 9 × 10 s with low energy (65 mA) on ice, wait for 30 s between each sonication step, to cool down. Upon sonication the cell suspension should turn clearer. Do not keep the sample too long on ice (>30 min), since the SDS will start to precipitate. 10. Centrifuge at 20,000 × g for 5 min; the pellet should be very small and is often blackish (see Note 3). 11. Transfer supernatant into a 15 ml Falcon containing 5,400 μl X-ChIP dilution buffer (see Note 6). 12. To reduce nonspecific binding to Sepharose beads, preclear the solution by adding 100 μl of washed (with X-ChIP dilution buffer) Protein G Sepharose Beads (see Note 7). 13. Rotate for 1 h at 4 °C. 14. Centrifuge at 400 × g at 4 °C for 5 min. 15. Transfer supernatants into new tubes (prevent taking any beads). Here split up the sample into several tubes (e.g., 1 ml)—for each antibody one tube. Save 10–50 μl as an “Input” sample, and keep it at −20 °C. 16. Add antibody to be tested (2–50 μg), and incubate rotating overnight at 4 °C. Day 2 17. Add 20 μl of washed (in dilution buffer) Protein A/G beads to each tube, and rotate for another hour at 4 °C. 18. Centrifuge at 400 × g at 4 °C for 5 min. 19. Wash beads twice with 1,000 μl NaCl washing buffer. 20. Wash beads twice with 1,000 μl LiCl washing buffer (see Note 9). 21. Wash beads 1× with ice-cold TE. 22. Add 150 μl X-ChIP elution buffer to beads. 23. Leave the tubes for 15 min at RT, vortex occasionally, centrifuge (400 × g, 5 min), and take the supernatant into a new tube. 24. Repeat previous step, and combine the supernatants of both steps (300 μl together).

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25. Include the input sample to each of the following steps (see Note 10). 26. Add 1 μl of RNase A (10 mg/ml). 27. Add 5 M NaCl to a final concentration of 0.3 M (18 μl). 28. Incubate for 4–5 h or overnight at 67 °C (reverse cross-linking). 29. Add 750 μl ethanol, mix, and let precipitate at −20 °C overnight. Day 3 30. Centrifuge at 20,000 × g for 15–20 min at 4 °C. 31. Remove supernatant, and let the pellet air-dry completely. 32. Dissolve pellet in 100 μl TE. Add 25 μl 5 × Proteinase K buffer and 1.5 μl Proteinase K (10 mg/ml), and incubate for 1 h at 45 °C, shaking. 33. Add 175 μl TE. 34. Add 300 μl phenol/chloroform/isoamyl alcohol (25:24:1), and vortex the tubes vigorously. 35. Centrifuge at 20,000 × g for 5 min, and take upper phase into a new tube, without touching the lower phase. Discard the lower phase. 36. Add 300 μl chloroform, and vortex the tubes vigorously. 37. Centrifuge at 20,000 × g for 5 min, and take upper phase into a new tube. Discard the lower phase. 38. Add 18 μl of 5 M NaCl and 5 μg glycogen (helps to precipitate small amounts of DNA), and mix. 39. Add 750 μl 100 % ethanol, and precipitate overnight at −20 °C. Day 4 40. Centrifuge at 20,000 × g for 15–20 min at 4 °C, remove supernatant, and let pellet air-dry completely. 41. Add 20–30 μl TE. Optional steps: 42. Quantify DNA (see Note 11). 43. Analyze DNA of suitable target genes with quantitative PCR. 44. Create DNA library for deep sequencing using Library Preparation kits (see Notes 12 and 13). 3.2

Native ChIP

Day 1 1. Harvest 1–5 million cells, and wash twice with PBS (see Note 1). 2. Resuspend cell pellet in 2 ml N-ChIP lysis buffer 1.

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3. Add 2 ml of N-ChIP lysis buffer 2 (total 4 ml), and keep it on ice for exactly 10 min (see Note 8). 4. Prepare two polypropylene tubes containing 8 ml N-ChIP lysis buffer 3. Layer on each of them 2 ml of the suspension. 5. Centrifuge at 10,000 × g in a swing-out rotor at 4 °C for 20 min; the nuclei form a pellet at the bottom, while the cytoplasmic fraction remains in the top layer. 6. Remove the supernatant using vacuum. The NP-40 containing top layer should not get into contact with the nuclear pellet (see Note 8). 7. Resuspend the pellet in 1 ml MNase digestion buffer. The DNA content of the resuspended nuclei may be quantified at 260 nm. The ratio OD260/OD280 should be around 1.1, due to the high protein proportion. 8. Add 2 U/ml MNase and incubate for 10 min in a 37 °C water bath (see Note 4). 9. Stop digestion by adding EDTA to a final concentration of 5 mM, and put samples on ice. 10. Centrifuge at 9,000 × g at 4 °C. 11. Save the supernatant (S1). 12. Resuspend pellet in 1 ml resuspension buffer, and incubate it at 4 °C overnight. The nucleosomes diffuse out of the nuclei into the solution. Day 2 13. Centrifuge at 9,000 × g at 4 °C. 14. Save the supernatant (S2). 15. Check the S1 and S2 on via agarose gel electrophoresis. Typically the S1 contains only mono- and di-nucleosomes, while S2 also has longer chains (see Note 3). 16. Dependent on the results from step 15, either use S1 or S2 or merge S1 and S2. 17. Dilute solution 1:10 in N-ChIP dilution buffer. 18. Divide the sample into several microcentrifuge tubes (e.g., 1 ml)—for each antibody one tube. Save 10–50 μl as an “Input” sample. 19. Add the antibody (2–50 μg) of interest, and incubate rotating for 4 h or overnight at 4 °C, dependent on the antibody. Day 3 20. Add 20 μl of washed (in N-ChIP dilution buffer) Protein A/G beads to each tube, rotate for another hour at 4 °C (see Note 7). 21. Centrifuge at 400 × g at 4 °C for 5 min.

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22. Wash beads 1× with 10 ml N-ChIP washing buffer A. 23. Wash beads 1× with 10 ml N-ChIP washing buffer B. 24. Wash beads 1× with 10 ml N-ChIP washing buffer C. 25. Resuspend beads in 300 μl N-ChIP elution buffer, and incubate for 30 min at room temperature. 26. Centrifuge (400 × g, 5 min, 25 °C), and transfer supernatants into new tube. 27. Proceed as described in X-ChIP protocol, step 34.

4

Analysis of ChIP-Seq Results Using Cistrome Analysis of ChIP-Seq data is often a challenge for non-computational biologists. Here we provide a step-by-step guide for how to extract the most crucial information from ChIP-Seq data. All steps presented here do not require any deep bioinformatics knowledge, special software, or strong computer power. It only requires an Internet connection and a free account at the Cistrome project (http://cistrome.org/ap/) [19]. We will use the publically available data from [20] as example. For advanced analyses, collaboration with a bioinformatics lab or usage of Bioconductor and R (http://www.bioconductor.org/) is recommended. Throughout this guide, standard settings are used, if not otherwise mentioned. 1. As first step we need to upload the data into Cistrome (if not yet done, first create an account). As starting material we use publically available Bed files (Table 2), which already contain the mapped reads of a ChIP-Seq experiment (see Notes 1–3). (a) Go to Import Data/Upload File. (b) Insert the URL to the BED files containing the mapped reads, into the field “URL/Text:” To obtain this URL go to the desired dataset in the GEO depository. Right click on the http link to the Bed file, and copy the address of the link. Paste this URL into the text field using right click, or CTRL-V. (c) Set the field “File Format” to “bed”. (d) Set the field “Genome” to “Human Mar 2006 (NCBI36/ hg18) (hg18)”. (e) Press “execute”. (f) Upload the bed files of the datasets presented in Table 2 into Cistrome. (g) For simplicity reasons we merge duplicate samples. Use Text Manipulation/Concatenate two datasets to merge

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267

Table 2 Datasets used for bioinformatic analysis GEO ID

Sample

GSM732903

Notch1-1

GSM732904

Notch1-2

GSM732905

CSL-1

GSM732906

CSL-2

GSM732907

ZNF143

GSM732908

Input-1

GSM732909

Input-2

GSM732910

H3K4me1

GSM732911

H3K4me3

GSM732912

H3K27me3

Input-1 and Input-2, CSL-1 and CSL-2, as well as Notch1-1 and Notch1-2, respectively. Afterwards, the unmerged files of CSL, Notch1, and Input can be deleted. We recommend renaming datasets in order to prevent later confusion. This can be done using the pen tool. 2. MACS (Model-based Analysis of ChIP-Seq): Next we perform MACS analysis to identify genomic regions where ChIP-Seq tags are enriched. Under Data Preprocessing/MACS use the dataset for Notch1 (and subsequently CSL, ZNF143, H3K4me3, H3K4me1 and H3K27me3) under “Treatment file:” and Input under “Input file:”. As settings, use Effective Genome Size: Human (hg18); File format: Bed; P-value: 1e–06. 3. Visualization in UCSC browser: The MACS analysis creates also a Wiggle file which can be used to visualize the ChIP-Seq data at the UCSC genome browser (Fig. 2). For practical reasons, we reduce the file size of the wiggle by using Liftover/Others/Standardize wig file with a span of 64 or 128 bps. For visualization in the UCSC browser perform the following steps: (a) Download standardized Wiggle. (b) Download Bed file containing called peaks (has been created by MACS). (c) If possible compress the file using gzip.

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Fig. 2 Visualization of ChIP-Seq data in the UCSC browser allows one to get a general idea about the data. For example here it can be seen that Notch1 and CSL occupancy positively correlate with H3K4me3, while Notch1 and CSL peaks are hardly present at H3K27me3-enriched regions

(d) Go to the UCSC browser (http://genome.ucsc.edu/). (e) Click on “Genome” and Select Group: Mammalian, Genome: Human; assembly: “Mar 2006 (NCBI36/hg18)”. (f) Upload the downloaded (and compressed) files using “add custom track”. (g) If you want to upload multiple files, please see Note 4. 4. Venn diagrams: Venn diagrams are a simple but efficient way to show the overlap between certain ChIP-Seq datasets. To create a Venn diagram of, e.g., Notch1, CSL, and ZNF143 Peaks use the Integrative Analysis/Venn Diagram tool. Use the MACS peaks results created by MACS (Fig. 3a). 5. Genomic distribution: To find out how a certain factor is distributed in the genome, we perform Integrative Analysis/CEAS: Enrichment on chromosome and annotation. As wiggle, use the wiggle made by MACS, and as Bed file use MACS Peaks or MACS Summits of the same factor. Transcription factors like CSL are often found enriched at promoters (Fig. 3b) (see Note 5). 6. Motif search: To search for enriched motifs at specific regions use Integrative Analysis/SeqPos motif tool. Use either the MACS peaks or the MACS summits as input Bed file. We generally recommend to search for known motifs but to also perform a de novo search. A maximum of 5,000 regions can be analyzed by SeqPos. If your Bed file contains more than 5,000 regions, you can

269

Epigenetic Status at Notch Target Genes

b

Genome

Notch1 ChIP

CSL ChIP

a Notch1

CSL 6070

4377

3393 2994 4917

ZNF143

c

z-Score

ETS family

CTCF

-35.2255

-24.1474

d

CSL

-23.775

-18.6977

H3K27me3 2.4 2.2 1.4

1.6

20

1.8

30

2.0

40

50

CREB

-21.8593

H3K4me3 CSL only sites Notch1/CSL sites

1.2

10

Average Profile

ZNF143

−2000

−1000

0

1000

2000

Relative Distance from the Center (bp)

CSL only sites Notch1/CSL sites

−2000

−1000

0

1000

2000

Relative Distance from the Center (bp)

Fig. 3 Using Cistrome, crucial information can be extracted from ChIP-Seq data. (a) Overlap of CSL, Notch1, and ZNF143 in CUTLL1 cells. (b) Genomic distribution of CSL and Notch1. (c) Enriched transcription factor motifs at CSL/Notch1-occupied sites. (d) Enrichment of histone modifications at CSL-only and CSL/Notch1occupied sites

randomly select 5,000 regions by using Text Manipulation/Select random lines. If you want to analyze specifically regions that are co-occupied by two factors use the Operate on Genomic Intervals/Intersect tool. For example, when using regions that are occupied by CSL and Notch1, many different transcription factor-binding motifs are found enriched, suggesting that formation of a CSL/Notch1-activating complex binding could be dependent on the presence of other transcription factors, beyond CSL. Some selected ones are shown in Fig. 3c (see Note 6).

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7. Profile of histone marks at CSL-binding sites: To address how histone marks correlate with the binding of Notch1 to CSL, we use Integrative Analysis/SitePro: Aggregation plot tool for signal profiling. For this purpose, we create summit datasets that contain regions that are bound by CSL, but not by Notch1 or regions that are bound by CSL and Notch1, respectively. To do this, we use the Operate on Genomic Intervals/Intersect or Subtract tool. For CSL-only regions subtract Notch1 MACS Peaks from CSL MACS Summits. For CSL- and Notch1-bound sites Intersect CSL MACS Summits (first dataset) and Notch1 MACS Peaks (second dataset). Afterwards set in SitePro the “SitePro behavior mode” to “multiple Bed vs 1 wiggle”. Use the MACS-created wiggle for H3K4me3 or H3K27me3 as wiggle and both Bed files created above as Bed. Use 2,500 bps as span. The outcome shows that Notch1 binding to CSL positively correlates with the presence of H3K4me3. The opposite is the case for H3K27me3 (Fig. 3d). 8. Heatmap: The heatmap tool under Integrative Analysis/Heatmap is useful to visualize ChIP-Seq data in a highly condensed way. When we use CSL MACS Peaks as Bed, and the wiggles for CSL, Notch1, H3K4me1, and H3K4me3, clustering by kmeans (with kmeans = 3), and an upstream and downstream span (under advanced option) of 2,500 bps, a heatmap can be obtained, as shown in Fig. 4a. A subset of CSL-bound regions are specifically enriched for H3K4me1, suggesting that these are enhancer sites. 9. Combining ChIP-Seq with microarray results: Lastly, we want to elucidate genes that are occupied by Notch1/CSL and their transcription is activated by Notch and hence are direct Notch1 target genes in CUTLL1 cells (see Note 7). (a) As first step we need to upload microarray data into Cistrome. For this purpose, we use Import data/Expression CEL file packager. Use as Control dataset GSM731503, GSM731504 and GSM731505 (GSI-treated cells) and as Sample Dataset GSM731515, GSM731516, GSM731517 (GSI washed off in the presence of cycloheximide). (b) Then perform Gene Expression/Gene expression index with standard setting on the uploaded dataset. Here the data will be normalized. (c) Subsequently, identify genes differently expressed in both dataset by using Gene Expression/Calculate differential expression on the normalized refseq value dataset, with a twofold cutoff.

Epigenetic Status at Notch Target Genes

a

b Gene

CSL

-2.5

0

Notch1

2.5 -2.5

0

H3K4me1

2.5 -2.5

0

H3K4me3

2.5 -2.5

0

HES1 HEY1 NRARP JUN APCDD1 SLC30A1 FOS BHLHE23 BMP4 DTX1 ZRANB3 C11orf96 PRR5 CPA4 LRP4 DDB2 RUNX3 GADD45A CD244 PFKFB2 NR4A3 ARHGEF3 COQ2 ICOS HES5

271

Fold Change upon Notch1 activation (log2) 5.02057501 4.60188118 4.57030821 3.9203584 3.70484386 3.52980369 3.00227784 2.90971876 2.84473854 2.80876668 2.77441408 2.73170015 2.69301734 2.60365478 2.57451069 2.55962378 2.510723 2.45534732 2.37930761 2.29667305 2.26805165 2.20189313 2.17405869 2.14497144 2.14252305

2.5 kbs

Fig. 4 (a) Heatmap of CSL, Notch1, H3K4me1, and H3K4me3 clustered by kmeans. (b) The 25 most Notch1 upregulated genes, which are occupied by CSL and Notch1, and are therefore likely direct Notch1 target genes in CUTTL1 cells

(d) Two files are created, a txt file and an HTML file. We continue to work with the txt file. It contains a table of the Refseq ID (e.g., NM_000043) and the log2 fold change. Next we want to convert the Refseq IDs into Gene Symbols. For this, we first need to remove the first line, by using Text Manipulation/Remove beginning. (e) Afterwards use the file created in (d) in Liftover/ Others/Convert between RefSeq, Gene Symbols to Entrez IDs with Conversion “Refseq IDs to Gene Symbol”. A new file containing the original RefSeq ID and the corresponding Gene Symbol is made. For further processing this file must be converted to a table. Use Text Manipulation/Convert delimiters to TAB and “whitespaces” for conversion. (f) The created file from (e) also lacks the expression data. Therefore we need to merge this file with the file obtained from (d) (after removal of the first line). For merging use Text Manipulation/Paste two files side by side. Use file from (e) as first file and the file created in (d) as second file. Now we have a file containing genes that are affected by Notch1 activation, with their respective fold expression change. (You can sort this with the Filter and Sort/Sort tool.)

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(g) Next we want to identify genes that are occupied by Notch1 and CSL. We use here a Peak dataset of overlapping peaks of CSL and Notch1, created by Operate on Genomic Intervals/Intersect. To find nearby genes for those peaks, we use Integrative Analysis/peak2gene: Peak Center Annotation, with standard settings. (h) Two files are created: one file containing annotation for each peak, and a second one containing the annotations for each gene. We continue to work with the latter one. First we remove the first eight lines using Text Manipulation/Remove beginning. (i) Then we want to join the results from gene expression analysis with the genes that are occupied by CSL/Notch1. To do this we use Join, Subtract and Group/Join two Datasets. Join the dataset from (f) (using “c2” as column) and the data from (h) (with “c4” as column). The outcome contains genes that are occupied by CSL/Notch1 and their expression is at least twofold affected upon activation of Notch. We recommend to download this file and further process this data with Excel (remove duplicate Columns/Genes, and Sort the data according to their expression change). Individual validation of each gene of interest is crucial. The top 25 upregulated genes, occupied by CSL/Notch1, are shown in Fig. 4b.

5

Notes

5.1 Notes for ChIP Experiments

1. Typically 20 million cells are suitable for most cell types and antibodies when doing cross-linking ChIP. However, the optimal amount of cells has to be individually determined for each cell type and antibody. A range from 500,000 to 50 million cells is recommended for testing. For native ChIP, best results are typically obtained with about 1–5 million cells, since too many cells increase the background. 2. If using a sonication tip, in our hands 600 μl volume works best, in most cases. Too large volume reduces the shearing efficiency, while lower volume can lead to formation of foam. 3. For X-ChIP the sonication should lead to DNA fragments of 300–800 bps (Fig. 5). To check and optimize the sonication we recommend analyzing the material before and after sonication via agarose gel electrophoresis. However, since proteins are still cross-linked to the DNA the results may not reflect the true size of the DNA. Therefore, analyzing the DNA size after decross-linking (4 h at 67 °C) is more reliable.

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bps 1000 500 200

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N-ChIP

Multi-nucleosomes

sheared DNA

Di-nucleosomes Mono-nucleosomes 3 6

9 min

Fig. 5 DNA after sonication or MNase treatment. Optimal are DNA fragments from 300 to 800 bps for X-ChIP or mainly mono- and di-nucleosomes for N-ChIP (shown are samples after 3, 6, and 9 min of MNase digestion)

4. For native ChIP the MNase digestion is crucial for a successful experiment. Optimal are mainly mono- and di-nucleosomes, and to a lower extent larger fragments. For optimization, we recommend to split the sample into three fractions and perform the MNase digest for, e.g., 3, 6, and 9 min. Afterwards choose the condition with the best ratio between lower and larger nucleosomal fragments (Fig. 5). 5. When performing ChIP against histone-acetylation marks, sodium butyrate (an HDAC inhibitor) should be added to all buffers (final concentration: 5 mM). 6. Do not use DTT during the immunoprecipitation step, since DTT can destroy the disulfide bonds of the antibody and impair the immunoprecipitation efficiency. 7. Do not use Protein A/G beads saturated with salmon sperm DNA for ChIP experiments that will be analyzed by deep sequencing. Since this DNA will be amplified together with the precipitated DNA during the library preparation, it will decrease the quality of the ChIP-Seq results. 8. The cell lysis at this step is achieved by a combination of a hypotonic buffer and NP-40. It is critical not to incubate the cells too long with this buffer, since NP-40 will also start to lyse the nuclei, which would reduce the quality of the ChIP experiment. 9. The buffers used here are relatively stringent. If the ChIP does not lead to enrichment on target genes, the buffer stringency should be reduced. Alternatively, ChIP protocols from antibody vendors like abcam and Upstate may be tested, as well. 10. During the DNA purification steps, we strongly recommend to always keep the input sample separate from the immunoprecipitation samples. Since the DNA content in the input sample

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is a thousandfold higher, any cross-contamination would impair the quality of the experimental results. Usually at each step we first process the immunoprecipitation samples, and afterwards the input sample. 11. To quantify the DNA, a fluorescence-based method is recommended, since this is more sensitive for small concentrations of DNA (e.g., Qubit). 12. If performing a ChIP experiment on an uncharacterized protein, the expected results are open. Therefore, it can be useful to create a cell line stably expressing a Flag- or a GFP-tagged version of the protein of interest. The ChIP should be performed with Flag-M2 beads (Sigma) or GFP antibody. A ChIP-Seq analysis will allow one to judge the general binding pattern of the protein and to select the best qPCR targets for optimization of the ChIP on the endogenous protein. 13. Multiplex ChIP-Seq allows merging several separate samples together. It saves money but reduces the number of reads per sample. 5.2 Notes for Bioinformatic Analysis

1. Most ChIP-Seq raw data are in a fastq format. This file format contains the sequence information of each read, but it does not contain the information, at which place in the genome a specific read maps. For mapping the reads we recommend using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) [23]. The output can be uploaded to Cistrome. See also Notes 2 and 3. 2. For uploading a large file to Cistrome and UCSC genome browser we recommend to compress the file beforehand using gzip (.gz extension). 3. In the GEO database many ChIP-Seq data are deposited as SRA files and not as fastq. However, most of these data are also available as fastq at the DRASearch database (http://trace. ddbj.nig.ac.jp/DRASearch/); for example the ChIP-Seq of ZNF143 in CUTLL1 cells (GSM732907/SRX070885) can be found as fastq, when searching for “SRX070885”. Alternatively, SRA files can be converted to fastq using the sratoolkit from NCBI (http://www.ncbi.nlm.nih.gov/Traces/ sra/?view=software). 4. MACS does not create an appropriate header for the UCSC track information, which leads to problems when uploading more than one dataset, since the Track names must be unique for each dataset. To correct the header we propose two ways:

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(a) Load the Wiggle/Bed locally into a suitable text editor, which is able to handle larger files (e.g., Ultraedit). In the wiggle change the header to, e.g., track type = wiggle_0 “Notch1”

name = “Notch1”

description =

In the Bed insert a first line as header: track name = “Notch1 Peaks”

Peaks”

description = “Notch1

(b) If a suitable text editor is not available, the headers can be directly changed in Cistrome: For the wiggle, the inappropriate header must first be removed using the Text Manipulation/Remove beginning (1 line). Afterwards use Graph/Display Data/Build custom track to add a new header to the wiggle or the Bed file. This program automatically creates a suitable header. Use for each dataset a unique track name. 5. A genomic distribution of ChIP-Seq peaks that is similar to the normal genomic distribution suggests that the ChIP did not work well, and many of the called peaks could be false positives. In such cases, a careful evaluation of the data is recommended. 6. Results from motif search should generally be handled very cautiously, because some motifs might get significantly enriched due to bioinformatic artifacts. Specifically, repeat sequences like GCGCGCG or ATATATAT are in most cases artifacts. 7. During preparation of this manuscript a new feature has been implemented into Cistrome, which allows performing this analysis directly. It can be found under Integrative Analysis/BETA. However, currently this feature is only available for the human genome hg19 and therefore cannot be applied for the example dataset used here.

Acknowledgments We thank Drs. K. Hein and B.D. Giaimo for critical reading of the manuscript and testing the bioinformatics guide. This work was supported by the Heisenberg program (BO 1639/5-1) of the DFG, the Max-Planck society, and the Excellence Cluster CardioPulmonary System (ECCPS) to T.B. R.L. has been supported by a DFG postdoctoral fellowship (LI 2057/1-1).

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References 1. Fortini ME (2009) Notch signaling: the core pathway and its posttranslational regulation. Dev Cell 16:633–647 2. Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705 3. Bray S, Bernard F (2010) Notch targets and their regulation. Curr Top Dev Biol 92: 253–275 4. Borggrefe T, Oswald F (2009) The Notch signaling pathway: transcriptional regulation at Notch target genes. Cell Mol Life Sci 66: 1631–1646 5. Schwanbeck R, Martini S, Bernoth K et al (2011) The Notch signaling pathway: molecular basis of cell context dependency. Eur J Cell Biol 90:572–581 6. Kovall RA (2008) More complicated than it looks: assembly of Notch pathway transcription complexes. Oncogene 27:5099–5109 7. Oswald F, Täuber B, Dobner T et al (2001) p300 acts as a transcriptional coactivator for mammalian Notch-1. Mol Cell Biol 21: 7761–7774 8. Dou S, Zeng X, Cortes P et al (1994) The recombination signal sequence-binding protein RBP-2 N functions as a transcriptional repressor. Mol Cell Biol 14:3310–3319 9. Kopan R (2012) Notch signaling. Cold Spring Harb Perspect Biol 4(10) 10. Borggrefe T, Liefke R (2012) Fine-tuning of the intracellular canonical Notch signaling pathway. Cell Cycle 11:264–276 11. Kao HY, Ordentlich P, Koyano-Nakagawa N et al (1998) A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev 12:2269–2277 12. Guarani V, Deflorian G, Franco CA et al (2011) Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase. Nature 473:234–238 13. Mulligan P, Yang F, Di Stefano L et al (2011) A SIRT1-LSD1 corepressor complex regulates

14.

15.

16.

17.

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21.

22.

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Notch target gene expression and development. Mol Cell 42:689–699 Liefke R, Oswald F, Alvarado C et al (2010) Histone demethylase KDM5A is an integral part of the core Notch-RBP-J repressor complex. Genes Dev 24:590–601 Wang J, Scully K, Zhu X et al (2007) Opposing LSD1 complexes function in developmental gene activation and repression programmes. Nature 446:882–887 Martinez AM, Schuettengruber B, Sakr S et al (2009) Polyhomeotic has a tumor suppressor activity mediated by repression of Notch signaling. Nat Genet 41:1076–1082 Ntziachristos P, Tsirigos A, Van Vlierberghe P et al (2012) Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia. Nat Med 18:298–301 Qin H, Du D, Zhu Y et al (2005) The PcG protein HPC2 inhibits RBP-J-mediated transcription by interacting with LIM protein KyoT2. FEBS Lett 579:1220–1226 Liu T, Ortiz JA, Taing L et al (2011) Cistrome: an integrative platform for transcriptional regulation studies. Genome Biol 12:R83 Wang H, Zou J, Zhao B et al (2011) Genomewide analysis reveals conserved and divergent features of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells. Proc Natl Acad Sci U S A 108:14908–14913 Zhao B, Zou J, Wang H et al (2011) EpsteinBarr virus exploits intrinsic B-lymphocyte transcription programs to achieve immortal cell growth. Proc Natl Acad Sci U S A 108: 14902–14907 Castel D, Mourikis P, Bartels SJ et al (2013) Dynamic binding of RBPJ is determined by Notch signaling status. Genes Dev 27: 1059–1071 Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25

Chapter 21 Notch-Ligand Binding Assays in Drosophila Cells Aiguo Xu and Kenneth D. Irvine Abstract Activation of the Drosophila transmembrane receptor protein Notch is induced by association with its transmembrane ligands, Delta and Serrate. The ability to assay binding between Notch and its ligands has been essential for characterizing the influence of posttranslational modifications, such as glycosylation, as well as for characterizing structural motifs involved in receptor–ligand interactions. We describe here a simple, widely used method for assaying receptor–ligand binding. This method involves expression of soluble forms of either Notch or its ligands, comprising the extracellular domains fused to an easily assayed tag, the enzyme alkaline phosphatase. These soluble proteins are then incubated with their binding partners, either as transmembrane proteins expressed on the surface of cultured cells or as extracellular protein domains attached to agarose beads. After washing, the amount of bound protein can be readily assayed by measuring alkaline phosphatase activity. Key words Notch, Serrate, Delta, Receptor, Ligand, Binding, Alkaline phosphatase, S2 cells

1

Introduction Binding between receptor and ligand is a crucial step in signal transduction pathways and often a key point of pathway regulation. Quantitative measures of binding can be used to determine physical parameters of binding interactions. In addition they can be used to map the contributions of different domains to the physical interactions and to characterize the influence of modulators of receptor–ligand interactions or of posttranslational modifications. Here, we describe a simple approach that has been used to characterize the strength of binding between the Notch receptor and its Drosophila ligands, Delta and Serrate. The basic approach is based on expression of a soluble, tagged form of one binding partner and a physically immobilized version of the other binding partner. This technique was first applied to binding studies between receptors and ligands of the Eph/ephrin family [1]. It was then adapted to studies of Notch-ligand binding in a characterization of the influence of Fringe on Notch-Delta binding [2] and has been employed in a variety of subsequent studies [3–8].

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_21, © Springer Science+Business Media New York 2014

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The soluble tagged binding partner in this approach comprises the extracellular domain of either Notch or one of its ligands fused to alkaline phosphatase. Alkaline phosphatase serves as a tag that can be readily quantified using a spectrophotometer to measure conversion of a colorless substrate into a colored product. This soluble binding partner is then mixed with an immobilized binding partner which is either expressed on the surface of cultured cells or attached to beads. Having one binding partner on the surface of a cell or a bead effectively allows the bound protein to be coprecipitated. Keeping the other binding partner soluble and fused to alkaline phosphatase facilitates accurate quantitation of input protein concentration. Thus, the choice of which binding partner (i.e., Notch or one of its ligands) is expressed in a soluble form is generally dictated by which protein is being experimentally manipulated, as it is important to use identical protein concentrations between samples being compared. For example, a soluble Notch protein could be produced either in the presence or the absence of the glycosyltransferase Fringe [2, 5]. These two different Notch samples could then be mixed with identical populations of Delta- or Serrate-expressing cells and the binding properties of the modified and unmodified Notch compared. The assay is performed using proteins expressed in cultured Drosophila S2 cells, which normally do not express detectable amounts of Notch or its ligands. Thus, cells transfected with vector or an irrelevant protein can serve as controls for these binding assays. An unrelated protein fused to alkaline phosphatase is used as a control soluble protein, and for historical reasons published experiments have often used the constant region (Fc domain) of immunoglobulin. Important variations in the assay include the use of purified proteins attached to beads instead of proteins expressed on the surface of cultured cells. Using proteins expressed on cells simplifies the assay as there is no need for purification of this binding partner; however it is difficult to know how much of the fixed binding partner is being used in the assay. Another variation is that stable cell lines expressing proteins of interest could first be generated rather than expressing proteins after transient transfection of cultured cells.

2

Materials 1. Hanks’ balanced salt solution (HBSS): 1.26 mM CaCl2, 5.33 mM KCl, 0.44 mM KH2PO4, 0.50 mM MgCl2, 0.41 mM MgSO4·7H2O, 138 mM NaCl, 4.00 mM NaHCO3, 0.3 mM Na2HPO4, 5.6 mM D-glucose. 2. S2 cell lysis buffer: 10 mM Tris–HCl pH 8.0, 1 % Triton-X 100.

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3. Shields and Sang M3 Insect medium (Sigma, commercially available from several sources). 4. M3 Complete medium: Shields and Sang M3 Insect Medium, supplemented with 5 % fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. 5. Schneider’s Drosophila media (Life Technologies, commercially available from several sources). 6. CCM3 medium: A commercial serum-free insect cell culture medium developed by Hyclone, currently available from Thermo Scientific. 7. Cellfectin: A commercially available transfection reagent. 8. AP assay solution: 6.25 mM p-Nitrophenyl phosphate (substrate), 5 mM MgCl2, in 1 M diethanolamine. 9. Ezview Red Anti-FLAG M2 Affinity Gel (Sigma). 10. Tris-buffered saline (TBS): 50 mM Tris-Cl, pH 7.5, 150 mM NaCl. 11. 70 mM CuSO4 solution: 100× Stock solution (final concentration 0.7 mM CuSO4). Used to induce expression of proteins controlled by the metallothionein promoter. 12. Rotary shaker.

3

Methods

3.1 Preparation of Soluble Proteins for Binding Assays

1. Culture Drosophila Schneider line 2 (S2) cells in M3 complete medium in 60 × 15 mm culture dishes to a density of 2–3 × 106 cells/mL (~80 % confluency). 2. Transfect cells with DNA constructs expressing soluble alkaline phosphatase (AP)-tagged fusion protein (see Note 1). For example, mix 4 μg Notch:AP-expression construct (see Note 2) with 500 μL serum-free medium (CCM3). Mix 45 μL Cellfectin (see Note 3) with 500 μL CCM3. A control protein, such as Fc:AP, should also be separately transfected. Then mix the DNA and Cellfectin solutions and let stand at room temperature for 10–15 min. Meanwhile, resuspend S2 cells to be transfected in 5 mL CCM3. Add the DNA + Cellfectin solution to the cells. Incubate for 6 h, and then replace CCM3 with M3 complete media (see Note 4). 3. Culture transfected cells for 24 h in M3 complete medium (see Note 5). 4. Induce expression of AP-tagged fusion proteins in M3 complete medium by addition of CuSO4 to 0.7 mM (from 1:100 dilution of 70 mM CuSO4 stock solution) for 48 h (see Note 6). 5. Collect conditioned medium and centrifuge at 14,000 × g for 10 min at 4 °C to precipitate cell debris.

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6. Assay conditioned medium for AP activity by mixing 25 μL of conditioned medium with 25 μL AP assay solution at 37 °C for 30 min (see Note 7). Stop the alkaline phosphatase reaction by addition of 50 μL 1 M NaOH. Then detect alkaline phosphatase activity by the formation of the colored reaction product at 405 nm using a spectrophotometer. The alkaline phosphatase activity detected is typically expressed as milliOD/min (mOD/min). For example, an absorbance reading of 0.208 after a 30-min alkaline phosphatase reaction would be expressed as 0.208 × 1,000/30 = 6.93 mOD/min (see Note 8). A mock assay, in which S2 cell conditioned medium is incubated with S2 cells expressing the transmembrane binding partner, is used for the spectrophotometer blank. 3.2 Preparation of S2 Cells for Binding Assays

1. Culture Drosophila Schneider line 2 (S2) cells in M3 complete medium in 60 × 15 mm culture dishes to a density of 2–3 × 106 cells/mL (~80 % confluency). 2. Transfect cells with DNA construct expressing transmembrane protein, as described in step 2 of Subheading 3.1 above. For example, transfect 4 μg plasmid expressing full-length Delta. A separate transfection with empty vector should also be performed to prepare control cells. 3. Culture cells to a density of ~5 × 106/mL in 4 mL CCM3. 4. Induce expression with 0.7 mM CuSO4 in CCM3 for 2 days. 5. Rinse transfected cells in CCM3 by centrifugation at 1,500 × g at 4 °C for 1 min, and then resuspend the cells in 0.3 mL of fresh CCM3 supplemented with 0.5 % BSA.

3.3 Assaying Binding of Proteins in Medium Conditioned to Cells

1. Mix 0.3 mL conditioned medium containing AP fusion protein (see Note 9) with 0.3 mL resuspended cells in CCM3 and incubate for 60 min at room temperature with gentle agitation on a rotary shaker. 2. Wash cells four times at room temperature in 0.5 mL HBSS containing 0.05 % BSA and 0.1 % azide. For each wash cells should be gently pelleted by centrifugation at 1,500 × g at 4 °C for 1 min, and then resuspend the cells in HBSS containing 0.05 % BSA and 0.1 % azide (see Note 10). 3. Lyse the cells by adding 50 μL of S2 cell lysis buffer at 4 °C for 30 min. 4. Heat the lysates at 65 °C for 10 min to inactivate endogenous S2 cell alkaline phosphatase. 5. Centrifuge cells at 12,000 × g at 4 °C for 10 min to remove cell debris. 6. Assay bound alkaline phosphatase activity by mixing 25 μL of supernatant from lysed cells with 25 μL AP assay solution at 37 °C for 30 min (see Note 11).

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7. Stop the alkaline phosphatase reaction by addition of 50 μL 1 M NaOH. 8. Detect alkaline phosphatase activity by the formation of the colored reaction product at 405 nm using a spectrophotometer. The alkaline phosphatase activity detected is typically expressed as milli-OD/min (mOD/min). For example, an absorbance reading of 0.208 after a 30-min alkaline phosphatase reaction would be expressed as 0.208 × 1,000/30 = 6.93 mOD/min. A mock assay, in which S2 cell conditioned medium is incubated with S2 cells expressing the transmembrane binding partner, is used for the spectrophotometer blank. 3.4 Preparing Beads for Binding Assays

1. Culture Drosophila Schneider line 2 (S2) cells in M3 complete medium in 60 × 15 mm culture dishes to a density of 2–3 × 106 cells/mL (~80 % confluency). 2. Transfect cells with DNA construct expressing extracellular domains of binding partners (e.g., Notch) fused to a FLAG epitope tag for affinity purification, as described in step 2 of Subheading 3.1 above. For example, transfect 4 μg plasmid expressing Notch:FLAG. 3. Culture cells to a density of ~5 × 106/mL in 4 mL CCM3 per culture well. Typically we prepare enough wells to allow collection of 50 mL conditioned medium. 4. Induce expression with 0.7 mM CuSO4 in CCM3 for 2 days. 5. Collect the conditioned media, and remove cell debris by centrifugation at 12,000 × g at 4 °C for 30 min in a 10 mL of centrifuge tube. 6. Prepare 50 μL of Ezview Red Anti-FLAG M2 Affinity Gel by washing these beads with 30 mL of TBS. Then mix 50 mL of conditioned medium with 50 μL of Ezview Red Anti-FLAG M2 Affinity Gel. Incubate overnight at 4 °C with gentle agitation to allow FLAG-tagged proteins to bind to beads. 7. Wash the beads three times using 30 mL of TBS supplemented with 5 mM CaCl2. For each wash, precipitate the beads by centrifugation at 10,000 × g for 1 min, and then exchange the supernatant. Following these washes the beads are suspended in 50 μL of TBS containing 5 mM CaCl2 and stored at 4 °C (see Note 12).

3.5 In Vitro Binding Assay

1. Block 5 μL of N-EGF:FLAG beads, or control beads, for 1 h at room temperature with 1 mL of TBS, supplemented with 1 % BSA and 5 mM CaCl2. 2. Precipitate beads by centrifugation at 10,000 × g for 1 min, and then resuspend beads in 50 μL conditioned media containing 3,000 mOD/min AP fusion protein for binding assays

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(e.g., Dl:AP). Incubate at room temperature for 1 h with gentle agitation on a rotary shaker. 3. Wash beads five times in 1 mL HBSS containing 0.05 % BSA and 0.1 % azide. For each wash, precipitate beads by centrifugation at 10,000 × g for 1 min, and then resuspend beads in HBSS. 4. Resuspend beads in 25 μL HBSS. 5. Assay bound alkaline phosphatase activity by mixing 25 μL beads with 25 μL AP assay solution at 37 °C for 30 min. 6. Stop the alkaline phosphatase reaction by addition of 50 μL 1 M NaOH. 7. Detect alkaline phosphatase activity by the formation of the colored reaction product at 405 nm using a spectrophotometer. A mock assay, in which S2 cell conditioned medium is incubated with beads, is used for the spectrophotometer blank.

4

Notes 1. While the protocol here expression constructs, an experiments could be to would then be induced subheading 3.1, step 4.

describes transient transfection of alternative that simplifies repeated first make stable cell lines, which as described in the beginning in

2. DNA amounts can be varied depending upon the amount of expression desired and the number of constructs expressed. The total amount of DNA between different conditions in an experiment should be kept constant by including vector DNA where necessary. 3. While the protocol described here uses Cellfectin as a transfection reagent, alternative transfection methods could be employed instead. 4. If knockdown of an endogenous gene by RNAi is to be performed, then at this point (6 h after transfection of expression constructs) 40 μg of dsRNA could be added and the cells cultured for 4 days in M3 before proceeding to subheading 3.1, step 4 for induction of transgene expression. 5. The transfection must work well for the assay to be successful. Transfection efficiency is most easily estimated in control experiments by transfecting a GFP-expressing plasmid. 6. Transgenes are often expressed under the control of the metallothionein promoter in cultured Drosophila cells, which provides strong inducible expression [9]. However, if constructs are expressed under the control of a constitutive promoter, like actin, then this step could be omitted. A plasmid

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vector commonly used for metallothionein-inducible expression in these binding assays is pRmHa-3 [9], which is available from the Drosophila Genomics Resource Center. 7. The incubation time for the AP reaction can be shortened if the AP activity is high. This will be evident by the appearance of a strong yellow color in the reaction. 8. In initial experiments with a new construct, it is recommended to perform western blotting to confirm that AP activity is proportional to amounts of the AP-tagged fusion protein. 9. Typical experiments would employ 1,000–8,000 mOD/min [5, 7]. The AP fusion protein in the media should be equalized for comparisons within the same experiment by addition of conditioned medium from untransfected S2 cells. If necessary to obtain a stronger signal, the AP fusion in the conditioned media can also be concentrated by centrifugation through Amicon Ultra centrifugal filters, 10,000 MWCO (EMD Millipore). 10. It is important that washing is sufficient to remove all nonspecific binding. This will be determined by the control binding reactions. If significant binding signal is observed using the control Fc:AP protein, or binding to mock-transfected S2 cells, then additional washes should be performed. 11. The incubation time can be adjusted if necessary, but incubation times longer than an hour should be avoided as potential instability of alkaline phosphatase over long periods may lead to a nonlinear response. 12. The amount of protein bound to beads should be evaluated by western blotting. Quantities can be normalized between different experiments by diluting N-EGF:FLAG-loaded beads with beads mock-loaded with S2 cell conditioned medium.

Acknowledgments Research in KDIs lab is supported by the Howard Hughes Medical Institute. References 1. Bergemann AD, Cheng HJ, Brambilla R et al (1995) ELF-2, a new member of the Eph ligand family, is segmentally expressed in mouse embryos in the region of the hindbrain and newly forming somites. Mol Cell Biol 15:4921–4929 2. Brückner K, Perez L, Clausen H et al (2000) Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406:411–415

3. Lei L, Xu A, Panin VM et al (2003) An O-fucose site in the ligand binding domain inhibits Notch activation. Development 130:6411–6421 4. Okajima T, Irvine KD (2002) Regulation of Notch signaling by O-linked fucose. Cell 111: 893–904 5. Okajima T, Xu A, Irvine KD (2003) Modulation of Notch-ligand binding by protein

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O-fucosyltransferase 1 and Fringe. J Biol Chem 278:42340–42345 6. Xu A, Lei L, Irvine KD (2005) Regions of Drosophila Notch that contribute to ligand binding and the modulatory influence of Fringe. J Biol Chem 280:30158–30165 7. Xu A, Haines N, Dlugosz M et al (2007) In vitro reconstitution of the modulation of Drosophila Notch-ligand binding by Fringe. J Biol Chem 282:35153–35162

8. Yamamoto S, Charng W-L, Rana NA et al (2012) A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands. Science 338: 1229–1232 9. Bunch TA, Grinblat Y, Goldstein LS (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res 16: 1043–1061

Chapter 22 Modeling Notch Signaling: A Practical Tutorial Pau Formosa-Jordan and David Sprinzak Abstract Theoretical and computational approaches for understanding different aspects of Notch signaling and Notch dependent patterning are gaining popularity in recent years. These in silico methodologies can provide dynamic insights that are often not intuitive and may help guide experiments aimed at elucidating these processes. This chapter is an introductory tutorial intended to allow someone with basic mathematical and computational knowledge to explore new mathematical models of Notch-mediated processes and perform numerical simulations of these models. In particular, we explain how to define and simulate models of lateral inhibition patterning processes. We provide a Matlab code for simulating various lateral inhibition models in a simple and intuitive manner, and show how to present the results from the computational models. This code can be used as a starting point for exploring more specific models that include additional aspects of the Notch pathway and its regulation. Key words Mathematical modeling, Simulations, Lateral inhibition, Notch signaling, Cis-interactions, Cell-to-cell communication, Pattern formation

1  Introduction The Notch signaling pathway has been shown to exhibit a great variety of complex behaviors in different developmental contexts [1, 2]. For instance, Notch signaling drives mutual inhibitory feedback between cells, known as lateral inhibition. This behavior leads to prototypical salt-and-pepper differentiation patterns with alternating fates in different animal tissues [3, 4]. Lateral inhibition involves competition between neighboring cells, where one cell within a group of initially equivalent cells “wins” the competition, differentiates first, and inhibits all its neighbors from differentiating themselves. The Notch-mediated inhibitory signal between the neighbors can be described by the following simplified regulatory feedback loop: Delta ligand in one cell binds to the Notch receptor on the membrane of a neighboring cell, a process that has been termed trans-interaction. Then, a proteolytic cleavage occurs, which releases the Notch intracellular domain (NICD) in the cell harboring the receptor. NICD serves as a co-transcription factor Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_22, © Springer Science+Business Media New York 2014

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that activates repressors of the Delta ligand which in turn can ­activate Notch signaling and its downstream targets in the neighboring cell. Different systems may vary from one to the other by having different regulatory circuits (e.g., through different target genes, or through different ligands) or tissue morphologies, what can lead to different spatiotemporal organizations in a tissue. What are the implications of different regulatory network architectures on Notch-mediated patterning? Interpreting biological experiments is sometimes very difficult, since genetic ­regulatory networks can become very complex and involve counterintuitive feedback mechanisms. In the past few years, different modeling approaches have provided novel insights on how different elements in the Notch regulatory network might be operating and on the implications of these architectures in patterning (see for instance [5–24]). These in silico approaches can often provide a complementary understanding of the experimental studies, enabling the formulation of new predictions that can be experimentally tested. This chapter is an introductory tutorial on how to start modeling some of the characteristic circuitry elements of Notchmediated patterning. We will focus on modeling the basic elements driving lateral inhibition. This tutorial is intended for readers coming from a more biological background, with some basic mathematical and computational knowledge, that are willing to get introduced into the world of modeling Notch signaling in a practical way. Some examples of Matlab code are provided so that the reader can use it as a starting point for exploring Notchmediated patterning. We strongly recommend though not to “copy and paste” the code from here to matlab, but to download it directly from https://github.com/dsprinzak. After reading this chapter, one should be able to model some of the basic components of Notch signaling in different kinds of cell lattices, perform numerical simulations in Matlab, and visualize the results. The structure of the chapter is as follows. First, in Subheading 1, we present the basic mathematical model developed for studying lateral inhibition in two cells. We then generalize it to lateral inhibition in regular cell lattices. Afterwards, in Subheading 2, we introduce a more realistic model in which proteolytic cleavage of receptors and ligands occurs and take into account interactions between receptors and ligands within the same cell, what is know as cis-interactions. We also show an example in which cell-to-cell interactions are mediated by longer range interactions (e.g., through filopodia). Additionally, we will provide an example where Notch signaling is modulated by an external morphogen gradient in the tissue. In Subheading 3 we will briefly discuss different sources of cell-to-cell variability that are being implemented in recent models of Notch signaling, comment on modeling additional Notch intracellular regulatory elements, and finally in Subheading 4 we provide additional references for further reading.

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2  Methods

2.1.1  Lateral Inhibition in Two Cells

One of the first theoretical models for lateral inhibition dynamics was proposed by Collier and coworkers in 1996 [5]. This is a simplified model of two ordinary differential equations per cell. In each cell (cell i) one equation models the dynamics of Delta concentration, Di, and the other equation accounts for the dynamics of repressor concentration, Ri. The Collier model basically assumes that lateral inhibition feedback is mediated by two regulatory processes: (1) Delta in each cell activates, through Notch signaling, the repressor in the neighboring cell, and (2) the repressor in each cell downregulates Delta expression in the same cell (Fig. 1a). This model uses Hill-type functions [25] to describe the activation and repression. Hill functions are monotonically increasing or decreasing sigmoidal functions which are widely used for modeling regulatory networks [25]. Apart of the Hill-type functions, each equation contains a normal linear degradation term, accounting for the typical half-life of every species (Di and Ri). The lateral inhibition circuit for the two-cell system is therefore given by dD1 = dt

ad h

æR ö 1+ ç 1 ÷ è qr ø

- g d D1



(1)

a Dm dR1 = m r 2 m - g r R1 dt qd + D 2



b Di Ri

Rj Dj

concentration [a.u]

a

(2)

cell #1 50 40

d r

30 20 10 0 0

20 40 time [a.u]

cell #2 concentration [a.u]

2.1  A Phenomenological Approach to Lateral Inhibition: The Collier Model

50 40 30 20 10 0 0

20 40 time [a.u]

Fig. 1 Lateral inhibition between two cells: the Collier model. (a) A scheme ­showing the lateral inhibition feedback loop in a model of two species per cell; the Delta ligand levels, D, and the repressor levels, R. Normal arrows denote activation, blunt arrows denote inhibition. A cell expressing Delta ligand activates the production of repressor in its neighboring cell, which represses the production of further ligand in such cell. (b) Simulation results for the Collier model in a two cell system (Eqs. 5–6). [a. u.] denotes arbitrary units. Further simulation details can be found in the text. Parameter values are written in the corresponding param function

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for cell #1 and dD 2 = dt

ad æR ö 1+ ç 2 ÷ è qr ø

h

- g d D2



m 1



(3)

aD dR 2 = mr - g r R2 dt qd + D1m

(4)

for cell #2, where τ is time, γx and αx are the degradation and maximal production rates for the x-species, θr is the threshold of repressor for inhibiting the ligand production to its half-value and θd is the threshold of Delta concentration for inducing half production of repressor in the neighboring cell, and m and h are the exponents for the activatory and inhibitory functions, respectively. In order to reduce the number of parameters, it is worth to nondimensionalize the system of Eqs. 1–4. We perform the change of variables by doing τ = T0t, Di = D0di, and Ri = R0ri, where T0, D0, and R0 are characteristic dimensional quantities of time, ligand, and repressor concentration, and t, di, and ri are the nondimensional time, ligand, and repressor concentration, respectively. The nondimensionalization (i.e., the particular choice of T0, D0, and R0) can be performed in different ways [26], and the modeler has to chose the one that is more convenient in relation to the questions to be answered. By choosing T0 = 1/γr, D0 = θd and R0 = θr we obtain the following nondimensionalized system of equations for cell i, (5)



ü ì b ddi = n í d h - di ý dt î1 + ri þ

(6)



brd j m dri = - ri , dt 1 + d j m



with i,j = 1,2 i ≠ j, and where βd = αd/γdθd and βr = αr/γrθr, so βd is related to the ligand production and βr to the strength of trans-­ activation due to cell-to-cell interactions. ν is a ratio of the ligand and repressor degradation rates, i.e., ν = γd/γr, or equivalently, the typical timescale of repressor dynamics with respect to the timescale of ligand dynamics. Note that different nondimensionalizations have been used in other studies (see for instance [5, 23]). After the nondimensionalization, we have just five parameters. We can easily investigate the behavior of two of them, βd and βr, and relate it with experimental perturbations where Delta expression is varied, and in which the processing rate of trans-interactions is disrupted, for instance, through Notch inhibitor treatment [17].

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In the simulation, we want to numerically solve these four equations. For doing that, we use a code written in Matlab, made of different functions (see the code below). There is a main function, in this case twocell_LI function, which calls other functions to perform the simulations. This function has the following structure: 1. Define the parameters of the system. Parameters are set through params structure. 2. Call the connectivity matrix M that indicates which cells are neighbors. This is a k × k symmetric matrix, where k is the number of cells. Position ij in the matrix (i.e., in the ith column and jth row) gets a value of 1 if cell i is a neighbor of cell j, and 0 otherwise. In the case of two cells (k = 2), the connectivity matrix reads



æ0 1ö M =ç ÷. è1 0ø

(7)

In this case this matrix describes a simple situation where cell #1 is the neighbor of cell #2, and vice versa. In the code below the connectivity matrix is defined in getconnectivityM function. Note that the Delta levels in the neighboring cell(s) to cell i, denoted by 〈di〉, can be represented by the following algebraic equation:



æ d1 ö æ d1 ö ç ÷ = M ç ÷, è d2 ø è d2 ø

(8)

This notation simplifies the code in the next sections. 3. Set the initial conditions. Here we choose initial repressor ­levels to be zero, while initial Delta levels are set to low values with some small variability or noise;

di (t = 0 ) =Îbd (1 + sU i ) ,



(9)

where ∊ being a small number (∊ = 10−5), σ is the noise amplitude (σ = 0.2) and Ui being a uniform random number between −0.5 and 0.5. This is set through getIC function. 4. Numerically solve the differential equations by using a standard numerical equation solver of Matlab, ode23. Function li in the code contains the differential equations for Delta and repressor concentration levels for each of the cells. This solver gets as an argument the li function, the time span for simulation, the initial conditions, and the parameters. 5. Plot the results. Function plot2cells plots Delta and repressor levels as a function of time.

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Table 1 Model variables and parameters used in the text (first column), and its correspondence in the Matlab code (second column) if any, followed by a brief explanation (third column) Model nomenclature

Code nomenclature Definitions and comments

βd, βn, and βr

betaD, betaN, and betaR

Delta, Notch, and repressor nondimensional productions

ν and μ

nu and mu

Delta and Notch degradation ratios with respect to repressor degradation

h and m

m and h

Cooperativity for Delta inhibition and repressor activation

kt and kc

kt and kc

Trans-annihilation and cis-inactivation nondimensional strengths

di, ri, and ni

D, R, and N

Levels of Delta, repressor, and Notch receptor concentration for the i cell (model) and for all cells (code)

〈di〉 and 〈ni〉

Dneighbor and Nneighbor

Average of Delta and Notch receptor concentration for the i cell (model) and for all cells (code)

di(t = 0), ri(t = 0), and ni(t = 0)

D0, R0, and N0

Initial conditions for Delta, repressor, and Notch levels for the i cell (model) and for all cells (code)

∊ and σ

Epsilon and sigma

Parameters related to the noise in the initial conditions

P, Q, k, w, and M P, Q, k, w, and M

Cell lattice parameters and connectivity matrix

Tmax

Maximum time for a simulation

l

Length scale of the gradient

The code is the following (see also Table 1):

function [yout,tout,params] = twocell_LI(params) %  Twocell_LI simulates lateral inhibition between two cells. The %  structure params contains the model parameters of the system. %  TOUT is a vector containing the time points of the solution %  between 0 and Tmax. YOUT is a matrix containing the numerical %  solution for each variable for each time point. Each row in %  YOUT is a vector of the size of TOUT. Tmax=40; tspan=[0 Tmax]; % set time for simulation k=2; % number of cells % get the default parameters if none provided if(nargin < 1) params=defaultparams; end

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% get the connectivity matrix params.connectivity=getconnectivityM;

% setting the initial conditions (IC) + noise y0=getIC(params,k); % run simulation with lateral inhibition [tout,yout] = ode23(@li,tspan,y0,[],params);

% show time traces of two cells with lateral inhibition plot2cells(tout,yout,k) function dy = li(t,y,params) nu=params.nu; betaD=params.betaD; betaR=params.betaR; h=params.h; m=params.m; M=params.connectivity; k=length(M);

D = y(1:k); % levels of Delta in cells 1 to k R = y(k+1:2*k); % levels of repressor in cells 1 to k Dneighbor=M*y(1:k);% Delta level in the neighboring cells % differential equations for Delta and repressor levels dD = nu * (betaD.*1./(1 + R.^h)-D); dR = betaR.*Dneighbor.^m./(1 + Dneighbor.^m)-R; dy = [dD;dR];

function params=defaultparams params.nu=1; params.betaD=50; production params.betaR=50; production params.h=3; function params.m=3; function params.sigma=0.2; conditions

% ratio of degradation rates % non-dimensional Delta %

non-dimensional repressor

% Hill coefficient repression % Hill coefficient activating % noise amplitude in initial

function M=getconnectivityM

M=[0 1;1 0]; % 2 cell connectivity matrix function y0=getIC(params,k)

U=rand(k,1) 1/2; % a uniform random distribution epsilon=1e-5; % multiplicative factor of Delta initial condition

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D0=epsilon*params.betaD.*(1 + params.sigma*U); % initial Delta levels R0=zeros(k,1); % initial repressor levels y0=[D0;R0]; % vector of initial conditions function plot2cells(tout,yout,k) figure(21); clf for i=1:2 subplot(1,2,i) plot(tout,yout(:,i),'-r','linewidth',2) % plot Delta levels hold on plot(tout,yout(:,k+i),'-b','linewidth',2) % plot  repressor levels title(['cell #',num2str(i)]) xlabel('time [a.u]');  ylabel('concentration [a.u]') legend('d','r') end

This code can be expanded to larger systems and other dynamics (see code examples in the next sections). Running the code results in Fig. 1b. Both cells start expressing Delta and the repressor, and pass transiently through a homogeneous state, i.e., a state in which both cells have the same levels in each of its variables. This transient homogeneous state matches with the homogeneous steady state of the dynamics, i.e., the solution of ddi/dt = dri/dt = 0 with 〈di〉 = di for every i-cell. The homogeneous state can be either stable or unstable. In the represented case, it is unstable, and the two cell system becomes patterned when Delta concentration in one cell goes up, inhibiting Delta concentration of its neighbor. In different parameter ranges, the system could stay in the unpatterned homogeneous steady state, for instance, if there is no cooperativity in the Hill functions, namely h = 1 and m = 1 [17]. 2.1.2  Lateral Inhibition in a Regular Cell Lattice



Lateral inhibition often occurs over extended regions of a tissue containing many cells. It is therefore interesting to model lateral inhibition on regular cell lattices. In this case, the repressor in each cell is activated by the average ligand concentration of its neighboring cells, so now the repressor dynamics reads dri br di = dt 1 + di

m m

- ri ,

(10)

where 〈di〉 has the following expression: di =

1 w

åd.

j Înn (i )

(11)

j



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Here j∊nn(i) refers to all j-cells that are nearest neighbors to cell i, and w is the number of nearest neighbors to cell i. For a one dimensional line of cells, the average ligand concentration (Eq. 11) will read di =



1 (di +1 + di -i ). 2

(12)

For squared and hexagonal two dimensional cell lattices this averaged term takes the form di =



1 d + d (i 2 ) + d (i 3 ) + d (i 4 ) 4 (i 1)

(

)

(13)

and

di =

1 d + d (i 2 ) + d (i 3 ) + d (i 4 ) + d (i 5) + d (i 6 ) , 6 (i 1)

(

)

(14)

where (ij) in Eqs. 13 and 14 represents the index of the jth neighbor of cell i. Herein, we choose the hexagonal cell lattice since this is the most similar to the natural cell packing. In the multicell_LI code we are simulating multicellular lateral inhibition in a hexagonal cell lattice of P rows and Q columns. The code is very similar to twocell_ LI except that now the connectivity matrix accounts for the six neighbors of each cell. In this case it is necessary to define an indexing scheme that easily allows tracking all the cells and their neighbors. Here, we switch between two indexing schemes—one that numbers the cells from 1 to k (i, Fig. 2a), and one that keeps the row and column of each cell (p, q, Fig. 2b). Each element of the connectivity matrix is multiplied by 1/w, with w being the number of nearest neighbors (e.g., w = 6). In order to avoid boundary effects in the simulations (e.g., cells at the edge may behave differently than cells in the middle), we normally use periodic boundary conditions. For example, in a line of cells, we define that the two cells at the two ends of the line become nearest neighbors, so instead of a line of cells we get a ring of cells. In such a ring with P cells, every x species (Delta or repressor) satisfies xi+P = xi. Similarly, for a two dimensional lattice of P × Q cells, periodic boundary conditions imply xp+P,q+Q = xp,q, so the cell lattice can be represented on a torus. The code for the multicellular system becomes (see also Table 1; copy functions from earlier code where indicated): function [yout,tout,params,F] = multicell_LI(params) %  multicell_LI simulates lateral inhibition in a hexagonal lattice.

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a

i=2P

b i=PxQ

i=P

i=P+2 i=2 i=P+1 i=1

p=P q=Q

p=P q=1

p p=2 q=1

p=1 p=1 q=2 q=1

p=1 q=Q

q

Fig. 2 Labeling schemes in a regular hexagonal cell lattice. (a) One index labeling scheme. (b) Two indices labeling scheme. Having two indices per cell facilitates the computation of the neighboring cell indices and the implementation of the periodic boundary conditions

% T  he structure params contains the model parameters of the system. % TOUT is a vector containing the time points of the solution %  between 0 and Tmax. YOUT is a matrix containing the numerical %  solution for each variable for each time point. Each row in %  YOUT is a vector of the size of TOUT. F is a movie of the % simulation. Tmax=40; tspan=[0 Tmax]; % set time for simulation % get the default parameters if none provided if(nargin < 1) params=defaultparams; end P=params.P; Q=params.Q; k=P*Q;

% number of cells per column % number of columns - MUST BE EVEN % number of cells

% get the connectivity matrix params.connectivity=getconnectivityM(P,Q);

% setting the initial conditions (IC) + noise y0=getIC(params,k); % run simulation with lateral inhibition [tout,yout] = ode23(@li,tspan,y0,[],params);

% show time traces of two cells with lateral inhibition plot2cells(tout,yout,k) % show lattice simulation F=movielattice(tout,yout,P,Q,k);

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function dy = li(t,y,params)

[USE THE SAME FUNCTION AS TWOCELL_LI] function params=defaultparams params.nu=1; % ratio of degradation rates params.betaD=50; % normalized Delta production params.betaR=50; % normalized repressor production params.h=3; % Hill coefficient repression function params.m=3; % Hill coefficient activating function params.sigma=0.2; % noise amplitude in initial conditions params.P=18; % number of cells per column params.Q=18; % number of columns - MUST BE EVEN function M=getconnectivityM(P,Q) k=P*Q; M=zeros(k,k); w=1/6;

% number of cells % connectivity matrix % weight for interactions

% calculating the connectivity matrix for s=1:k kneighbor=findneighborhex(s,P,Q); for r=1:6 M(s,kneighbor(r))=w; end end function y0=getIC(params,k)

[USE THE SAME FUNCTION AS TWOCELL_LI] function plot2cells(tout,yout,k)

[USE THE SAME FUNCTION AS TWOCELL_LI]

function out = findneighborhex(ind,P,Q)

% This function finds the 6 neighbors of cell ind [p,q] = ind2pq(ind,P); % above and below: out(1) = pq2ind(mod(p,P)+1,q,P); out(2) = pq2ind(mod(p-2,P)+1,q,P); % left and right sides: qleft = mod(q-2,Q)+1; qright = mod(q,Q)+1;

if q/2~=round(q/2), pup = p; pdown = mod(p-2,P)+1; else pup = mod(p,P)+1; pdown = p; end;

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out(3) out(4) out(5) out(6)

= = = =

pq2ind(pup,qleft,P); pq2ind(pdown,qleft,P); pq2ind(pup,qright,P); pq2ind(pdown,qright,P);

function ind=pq2ind(p,q, P) ind = p + (q-1)*P;

function [p,q]=ind2pq(ind, P) q = 1+floor((ind-1)/P); p = ind - (q-1)*P; function plotHexagon(p0,q0,c)

% This function plots a hexagon centered at coordinates p,q s32 = sqrt(3)/4; q = q0*3/4; p = p0*2*s32; if q0/2 == round(q0/2), p = p+s32; end; x(1)=q-.5; x(2)=q-.25; x(3)=q+.25; x(4)=q+.5; x(5)=q+.25; x(6)=q-.25; y(1)=p ; y(2)=p+s32; y(3)=p+s32; y(4)=p; y(5)=p-s32; y(6)=p-s32; patch(x,y,c,'linewidth',2);

function F=movielattice(tout,yout,P,Q,k)

% T  his function generates a movie of patterning in a hexagonal % lattice. The color represents the level of Delta. It also %  saves the movie as an AVI file.

figure(22) Cmax=max(yout(end,1:k)); % finds max(Delta) at the end point frameind=0; for tind = 1:5:length(tout), % shows every 5th frame clf; for i = 1:P, for j = 1:Q, ind = pq2ind(i,j,P); mycolor = min([yout(tind,ind)/Cmax,1]);  plotHexagon(i,j,[1-mycolor,1-mycolor,1]); end; end; axis image; axis off; box off; frameind=frameind+1; F(frameind) = getframe; % generates a movie  variable end;

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% save movie in avi format movie2avi(F,'movielattice','compression','none');

In Fig. 3 we can see how patterning spontaneously emerges in a hexagonal cell lattice from an initially uniform state. Note that the final simulation state exhibits domains of ordered patterns separated by gaps or defects. This is typically the case for Collier type models [5]. Often, apart from performing simulations with a single set of parameters, one is interested in performing extensive simulations across a two dimensional parameter space, i.e., to perform simulations by varying two parameters while maintaining the rest fixed. In these explorations it is useful to define an observable that allows characterization of the resulting phenotype. A possible observable would be the density of high ligand cells in the cell lattice [23], which provides an idea of the ratio between number of cells from each type, or the logarithm of the ratio between high Delta cells and low Delta cells, to distinguish patterned regions from homogeneous regions in the parameter space. Another interesting observable is a measure of the time required for patterning, which can reveal how the dynamics of the system is affected by the different parameters. The following code calls multcell_LI function in a new βd and βr parameter set each time and plots a phase diagram of the last two aforementioned observables in the βd and βr parameter space (Fig. 4). It is suggested to comment out the plotting functions in multicell_LI to shorten the running time of the code and to change Tmax to a larger value (to also capture slower dynamics).

Fig. 3 Snapshots at different time points of a simulation for the Collier model (Eqs. 5, 10 and 14) in a hexagonal cell lattice with periodic boundary conditions. Blue intensity denotes the Delta levels. Dark blue corresponds to di = 50, while white corresponds to di = 0. From left to right, the time points shown are t = 0, t = 16.0, t = 16.7, t = 18.1, and t = 29.6 in arbitrary units. Further simulation details can be found in the text. Parameter values can be found in the corresponding param function

log(βr )

patterning

1

6 4

0 −1 −1

120

8

2

2

no patterning 0

1

2

3

0

3 2

100 80

1

60 40

0

Time for patterning [normalized time]

b

3 log(dmax /dmin)

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log(βr )

298

20 −1 −1

log(βd )

0

1

2

3

0

log(βd )

Fig. 4 Parameter space analysis for the Collier model (Eqs. 5, 10, and 14). (a) A phase diagram showing log(dmax/dmin) for different values of the parameters βr and βd, where dmax and dmin are the maximal and minimal nondimensional Delta levels in the steady state. The color bar indicates the color code corresponding to the value of log(dmax/dmin). This figure shows the region in parameter space where the homogeneous solution is found (dark blue region with the label “no patterning”) and the region where patterning emerges (corresponding to the remaining colored region with the label “patterning”). (b) A phase diagram showing the time required for patterning for each parameter set where patterning occurs. The color bar indicates the color code corresponding to patterning time values (see text for definition of patterning time). Simulations were performed with Tmax = 200. This figure was generated by running paramsearch_LI code given in the text function paramsearch_LI %  This code plots the log(Dmax/Dmin) and the time required for patterning for different betaD and betaR. %  FOR A FASTER RUN, COMMENT OUT PLOT2CELLS AND MOVIELATTICE within multicell_LI function % fixed parameters params.nu=1; params.h=3; params.m=3; params.sigma=0.2; params.Q=12; params.P=12; k=params.Q*params.P;

% variable parameters betaD=logspace(-1,3,20); % creates a series of betaD from 0.1 to 1000 betaR=logspace(-1,3,20); % creates a series of betaR from 0.1 to 1000

ind=0; h=waitbar(0,'% of progress'); % generates a waitbar for i=1:length(betaD) for j=1:length(betaR) params.betaD=betaD(i); params.betaR=betaR(j);  ind=ind+1; waitbar(ind/(length(betaD)*length(betaR)))

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 [yout,tout] = multicell_LI(params); % calling the LI solver % finding max and min values of D Dmax(i,j)=max(max(yout(end,1:k))); Dmin(i,j)=abs(min(min(yout(end,1:k))));

 % finding cases where patterning occurs (when Dmax/Dmin>1.2) % and getting the patterning time if Dmax(i,j)/Dmin(i,j)>1.2  T(i,j)=getPatterningTime(tout,yout,... k,Dmax(i,j),Dmin(i,j)); else  T(i,j)=NaN; % patterning time is not % set for the no patterning case end

end end close(h) figure(23) imagesc(log10(betaD),log10(betaR),... log10(Dmax./Dmin)); set(gca,'YDir','normal') xlabel('log(\beta_d)','fontsize',14); ylabel('log(\beta_r)','fontsize',14); title('log(d_{max}/d_{min})','fontsize',14) colorbar figure(24) imagesc(log10(betaD),log10(betaR),T); set(gca,'YDir','normal') xlabel('log(\beta_d)','fontsize',14); ylabel('log(\beta_r)','fontsize',14); figtit0='Time for patterning' figtit1='[normalized time]' figtit=[figtit0,figtit1] title(figtit,'fontsize',14) colorbar

function T=getPatterningTime(tout,yout,k,Dmax,Dmin)

% This function estimates the time required for patterning. % This is done by the following 3 steps:

% 1. find all the high D cells ('onCells') % 2. find the time it takes for each 'on cell' to reach 90% of its final level ('TonCells') % 3. get median value of the times calculated in stage 2 onCells=find(yout(end,1:k)>0.5*(Dmax+Dmin)); for i=1:length(onCells)

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ind=find(yout(:,onCells(i))>0.9*yout(end,onCe T lls(i)),1,'first'); TonCells(i)=tout(Tind); 

end T=median(TonCells);

In Fig. 4 we can see that patterning occurs in a wide range of βd and βr values, which means that it is a very robust process. Figure 4b shows how patterning time varies with βd and βr values. The patterning time is significantly increased at the edge of the patterning region, a behavior known as critical slowing down [27]. The code can be easily adapted to plot the dependence on any two parameters of the model and to be used in any of the models provided in this tutorial. 2.2  Extensions to the Collier Model 2.2.1  Adding Trans-­Annihilation and Cis-Inactivation



So far we have simulated a simplified model, which does not take into account some of the biochemistry of Notch signaling. More kinetic-based models can also be used, which takes into account the cleavage of Notch, the endocytosis of Delta, and the cis-­ interaction between Notch and Delta [28]. The latter interaction has been shown to lead to mutual inactivation of both Notch and Delta [17, 18, 29]. To account for these processes we need to add to our model the level of Notch receptor concentration in a cell, given by the variable Ni. These processes modify the lateral inhibition model, which leads to the following differential equations: dN i = a n - K tN i Di - K cN i Di - g NN i dt dDi = dt

ad h

(15)

- K t Di N i - K cN i Di - g D Di



æR ö 1+ ç i ÷ è qr ø



æK N D ö ar ç t i i ÷ g nd dRi è ø -g R, = R i m dt æ K tN i Di ö m qnd + ç ÷ g nd è ø

(16)

m

(17)

where 〈Ni〉 and 〈Di〉 are the average receptor and ligand concentrations in the neighboring cells (see Eq. 11), so terms Kt Ni 〈Di〉 and Kt Di 〈Ni〉 denote trans-annihilation (cleavage of Notch and ­endocytosis of Delta), while Kc Ni Di denote cis-inactivation. The strength of these interactions is parameterized by Kt and Kc, respectively. γnd−1 is a typical timescale of the trans-complex, and θnd represents a typical amount of trans-complex for activating the

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repressor. More details about the derivation of these equations can be found in [17, 18]. We perform the nondimensionalization of Eqs. 15–17 by following the same steps as we did in a precedent section, with now Ni = N0ni, where N0 is a characteristic dimensional quantity of receptor concentration, and ni is the nondimensional receptor concentration in the i cell. Now we set T0 = 1/γr, N0 = θnd γnd/γn, D0 = θnd γnd/γd, and R0 = θr, so the resulting nondimensional system reads

dni = m {bn - ktni di - kcni di - ni } dt

(18)

(19)



ì b ü ddi = n í d h - kt di ni - kcni di - di ý dt î1 + ri þ



br (ktni di dri = dt 1 + ( ktni di

) )

m m

- ri ,

(20)

where μ = γn/γr, ν = γd/γr, kt = Ktγndθnd/(γdγn), kc = Kcγndθnd/(γdγn), βr = αr/γrθr, βd = αd/γndθnd, and βn = αn/γndθnd. Therefore, parameters μ and ν account for the timescale of receptor and ligand with respect to the repressor, respectively, kt and kc are the effective nondimensional strengths for cis and trans-interactions, and βn, βd, and βr are effective productions of receptor, ligand, and repressor. The code implementing Eqs. 18–20 for two cells is as follows (see also Table 1; copy functions from earlier code where indicated): function [yout,tout,params] = ... transcis2cell_LI(params) %  transcis2cell_LI simulates trans-annihilation with cis-inactivation %  between two cells. The structure params contains the model %  parameters of the system. %  TOUT is a vector containing the time points of the solution %  between 0 and Tmax. YOUT is a matrix containing the numerical %  solution for each variable for each time point. Each row in %  YOUT is a vector of the size of TOUT. Tmax=100; tspan=[0 Tmax]; % set time for simulation k=2; % number of cells % get the default parameters if none provided if(nargin < 1)

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end

params=defaultparams;

% get the connectivity matrix params.connectivity=getconnectivityM;

% setting the initial conditions + noise y0=getIC(params,k);

% run simulation with lateral inhibition [tout,yout] = ode23(@li,tspan,y0,[],params);

% show time traces of two cells with lateral inhibition plot2cells(tout,yout,k) function dy = li(t,y,params)

nu=params.nu; betaD=params.betaD; betaN=params.betaN; betaR=params.betaR; m=params.m; h=params.h; M=params.connectivity; k=length(M); mu=params.mu; kc=params.kc; kt=params.kt; D = y(1:k); % levels of Delta in cells 1 to k R = y(k+1:2*k); % levels of repressor in cells 1 to k N = y(2*k+1:3*k); % levels of repressor in cells 1 to k Dneighbor=M*y(1:k); % Delta level in the neighboring cells Nneighbor=M*y(2*k+1:3*k); % Notch level in the neighboring cells % differential equations for Delta, repressor, and Notch levels dN = mu * (betaN - kt.*N.*Dneighbor-kc.*N.*D-N); dD =nu * (betaD.*1./(1 + R.^h)-kt.*D.*... Nneighbor-kc.*N.*D-D); dR = betaR.*(kt.*N.*Dneighbor).^m./(1 + (kt.*N.*... Dneighbor).^m)-R; dy = [dD;dR;dN]; function params=defaultparams params.nu=1; params.betaD=50; params.betaN=1; params.betaR=200; params.m=1;

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params.h=1; params.sigma=0.2; params.mu=1; params.kc=10; params.kt=1;

function M=getconnectivityM

M=[0 1;1 0]; % 2 cell connectivity matrix function y0=getIC(params,k)

U=rand(k,1) 1/2; % a uniform random distribution epsilon=1e-5; % multiplicative factor of Delta initial condition D0=epsilon*params.betaD.*(1 + params.sigma*U); % initial Delta levels R0=zeros(k,1); % initial repressor levels N0=params.betaN.*ones(k,1); % initial Notch levels are betaN y0=[D0;R0;N0]; % vector of initial conditions function plot2cells(tout,yout,k)

[USE THE SAME FUNCTION AS TWOCELL_LI]

By including cis-inactivation we can get patterning even without cooperativity, i.e., when h = 1 and m = 1 (data not shown, see refs. 17, 18). It is also easy to demonstrate (for example by running the paramsearch_LI code) that the dynamics are strongly affected by cis-interactions [18]. A recent work proposing an alternative more Collier-based mathematical model of cis-interactions can be found in [30]. 2.2.2  Simulations with Longer Range Interactions

Recent work has shown that filopodia and cellular protrusions can take place during lateral inhibition, giving rise to sparser patterns [16]. To include these effects in our modeling framework, we have to take into account cell-to-cell interactions that can also reach cells that are further apart in the cell lattice, for example by allowing interactions between a cell and its next nearest neighbors. In the Collier model formulation, a very simple way of taking it into account would be by extending the cell-to-cell interaction in the following way: di =



ö 1æ çç å d j + å d j ÷÷ , w è j Înn (i ) j Înnn (i ) ø

(21)

where now nn(i) and nnn(i) refer to nearest and next nearest neighbors to cell i, and w is the total number of nearest and next nearest neighbors (w = 18 in a regular hexagonal lattice). A more realistic cell-to-cell coupling can be found in [16]. In this case, to compute the connectivity matrix and the indices of cells contributing

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in Eq. 21 we have the following code (see also Table 1; copy functions from earlier code where indicated): function [yout,tout,params,F] = ... largespacing_LI(params) [USE THE SAME FUNCTIONS IN multicell_LI REPLACING ONLY THE getconnectivityM FUNCTION] function M=getconnectivityM(P,Q) k=P*Q; % number of cells M=zeros(k,k); % connectivity matrix w=1/18; % weight for interactions % calculating the next nearest neighbor connectivity matrix for s=1:k % find the neighbors of cell s kneighbor=findneighborhex(s,P,Q); nn_neighbor=kneighbor; % find the neighbors of the neighbors of cell s for i=1:length(kneighbor)  nn_neighbor=[nn_neighbor;... findneighborhex(kneighbor(i),P,Q)]; end % find all the unique neighbors of cell s nn_neighbor=unique(nn_neighbor); for r=1:length(nn_neighbor); M(s,nn_neighbor(r))=w; end M(s,s)=0; % removing cell s from the connectivity matrix end From running this code we can see that lateral inhibition with longer range cell-to-cell interactions drives a sparser salt-and-­ pepper pattern of high Delta cells (see Fig. 5a) than the pattern obtained from the Collier model (see Fig. 3). 2.2.3  Adding External Gradients

Notch-mediated patterning often involves cues from other signaling systems, which may introduce long-range spatial modulation of Notch pathway components. The Notch pathway has been shown to be modulated by morphogens like Wnt, Hegdehog, EGF, among others [31, 32]. To study the effect of long-range morphogen gradients on lateral inhibition patterning, we consider a situation where a radial exponential gradient of a certain morphogen drives Delta production on a two dimensional hexagonal lattice.1 As a first approximation, we can omit the diffusing 1

 Here we are introducing the steady state profile of a radially diffusing morphogen in two dimensions that is linearly degraded. Note that the corresponding steady state of the morphogen would follow a modified Bessel function of second kind [33], but here we use an exponential decay for simplicity.

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Fig. 5 Simulations with models taking into account longer range interactions and a spatial gradient. (a) Collier model with additional next nearest neighbors interactions (Eqs. 5, 10 and 21) drives sparser patterns of higher Delta cells. (b) Collier model with an exponential spatial modulation of the Delta production parameter (βd) in the tissue. This situation emulates a scenario in which Delta is activated downstream a radial morphogen gradient that exponentially decays from the center of the tissue. This enables the creation of a localized patterning domain in the tissue. Color codes as in Fig. 3. Dark blue color corresponds to d = 50  in panel a while d = 2.96  in panel b. In panel b, the darkest blue intensity has been assigned to the 95th percentile of the Delta levels in the cell lattice at the steady state, so Delta levels larger than d = 2.96 also have been depicted by the same dark blue color. Further simulation details can be found in the text. Parameter values can be found in the corresponding params structure

­ orphogen and focus directly on modeling its downstream effect m as a spatial modulation of the Delta production parameter in the Collier model. In the following simulation code, βd is multiplied by an exponential function with lengthscale l, so that the production of Delta varies from cell to cell (see also Table 1; copy functions from earlier code where indicated): function [yout,tout,params,F] = morphogen_LI(params) %  morphogen_LI simulates lateral inhibition in a hexagonal lattice. %  The morphogen is introduced through a gradient on betaD. %  The structure params contains the model parameters of the system. %  TOUT is a vector containing the time points of the solution %  between 0 and Tmax. YOUT is a matrix containing the numerical %  solution for each variable for each time point. Each row in %  YOUT is a vector of the size of TOUT. F is a movie of the simulation. Tmax=30; tspan=[0 Tmax]; % set time for simulation

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%  get the default parameters if none provided if(nargin < 1) params=defaultparams; end

P=params.P; % number of cells per column Q=params.Q; % number of columns - MUST BE EVEN k=P*Q; % number of cells % get the connectivity matrix params.connectivity=getconnectivityM(P,Q);

% apply morphogen controlling betaD. % params.l is the lengthscale set by a morphogen params.l=1.5; Morph=getMorph(params.l,P,Q); % params.betaD becomes a vector describing the local production % of Delta controlled by a morphogen params.betaD=params.betaD*Morph;

% setting the initial conditions + noise y0=getIC(params,k);

% run simulation with lateral inhibition [tout,yout] = ode23(@li,tspan,y0,[],params);

% show time traces of two cells with lateral inhibition plot2cells(tout,yout,k) % show lattice simulation F=movielattice(tout,yout,P,Q,k);

[USE THE SAME FUNCTIONS AS IN multicell_LI ADDING ONLY getMorph FUNCTION] function Morph=getMorph(l,P,Q)

% This function generates an exponential morphogen profile with lengthscale params.l center=[floor(P/2) floor(Q/2)]; MorpPQ=zeros(P,Q); Morph=zeros(P*Q,1); for p=1:P for q=1:Q d i s t p q = s q r t ( ( ( ( p - c e n t e r ( 1 ) ) / l ) . . . ^2)+(((q-center(2))/l)^2)); MorphPQ(p,q)=exp(-distpq); ind=pq2ind(p,q, P); Morph(ind)=MorphPQ(p,q); end end

In Fig. 5b we can see that such radial morphogen gradient upstream of Delta can restrict the lateral inhibition pattern to a

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certain tissue domain. This could be a plausible mechanism to set the size of the domain of lateral inhibition patterns. Other examples of parameter modulation across a cell lattice in lateral inhibition dynamics can be found in [34, 35]. More complex models that explicitly take into account a diffusing morphogen that affects the patterning process can be found in the context of differentiation wavefronts [23, 36–38].

3  Notes 3.1  Adding Cell-to-­ Cell Variability

Cell-to-cell variability can be manifested in different ways in a ­tissue during the patterning process. Herein we will just mention some examples that have already been considered in models for lateral inhibition. Cells in a tissue can have different number of neighbors, so working with cell lattices with a certain degree of irregularity, e.g., Voronoi tessellations, could capture such heterogeneity in the number of first neighbors [9, 10, 16, 23, 39, 40]. One step further is to consider the connectivity matrix as a dynamic one [16, 41]. This has already been used for modeling the highly dynamic nature of filopodia, and it has been shown to have an effect in the refinement of the final pattern [16]. This dynamic cell-to-cell connectivity has been referred as structured noise [41]. Another source of cell-to-cell variability is cells having different contact areas among them due to heterogeneity in its shape. This can be set through an irregular cell lattice in which the strength of trans-interactions is proportional to each cell-to-cell contact area [9, 10, 23]. Cell-to-cell variability can also be taken into account through static heterogeneity in the model parameters [18]. Another source of variability may come from fluctuations in the levels of the molecular components of the pathway [42], e.g., receptors and ligands, and other molecular components in the cell. One can consider this effect by using stochastic differential equations in the Itô approximation [43]. This kind of dynamical noise has been implemented in different models of Notch signaling in different ways [23, 38, 44].

3.2  Modeling Additional Intracellular Regulatory Elements

Recent theoretical works have modeled downstream targets of Notch, or upstream regulators of Notch and its ligands [7, 11, 24, 45–48]. These elements have been modeled as separate small modules, and also have been embedded in larger models of Notch signaling. Note that adding more variables or degrees of freedom to the model increases the complexity of the system very rapidly. A classical challenge for the modelers is to find a trade-off between realism in the modeling framework—for capturing the essence of the question—and simplicity—for being able to solve the question with the available tools and knowledge.

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4  Further Reading More extended background on modeling genetic regulatory networks can be found in [25, 49]. Explanations about different analytical tools such as solving differential equations, nullcline analysis, and linear stability analysis can be found in [5, 8, 13, 26, 50, 51, 52].

Acknowledgements The code used in this manuscript was loosely based on a code written together with Michael Elowitz, Amit Lakhanpal, and Jordi GarciaOjalvo. We would like to thank Iftach Nachman and Oren Shaya for their instructive comments of the manuscript. D. S. would like to thank the support of the Community under a Marie Curie European Reintegration Grant and a grant from the Israel Science Foundation (grant 1021/11). References 1. Andersson ER, Sandberg R, Lendahl U (2011) Notch signaling: simplicity in design, versatility in function. Development 138: 3593–3612 2. Hori K, Sen A, Artavanis-Tsakonas S (2013) Notch signaling at a glance. J Cell Sci 126: 2135–2140 3. Neves J, Abelló G, Petrovic J et al (2013) Patterning and cell fate in the inner ear: a case for Notch in the chicken embryo. Dev Growth Differ 55:96–112 4. Formosa-Jordan P, Ibañes M, Ares S et al (2013) Lateral inhibition and neurogenesis: novel aspects in motion. Int J Dev Biol 57:341–350 5. Collier JR, Monk NA, Maini P et al (1996) Pattern formation by lateral inhibition with feedback: a mathematical model of deltanotch intercellular signalling. J Theor Biol 183:429–446 6. Owen M, Sherratt J (1998) Mathematical modelling of juxtacrine cell signalling. Math Biosci 153:125–150 7. Meir E, von Dassow G, Munro E et al (2002) Robustness, flexibility, and the role of lateral inhibition in the neurogenic network. Curr Biol 12:778–786 8. Webb SD, Owen MR (2004) Oscillations and patterns in spatially discrete models for developmental intercellular signalling. J Math Biol 48:444–476

9. Webb S, Owen M (2004) Intra-membrane ligand diffusion and cell shape modulate juxtacrine patterning. J Theor Biol 230:99–117 10. Podgorski GJ, Bansal M, Flann NS (2007) Regular mosaic pattern development: a study of the interplay between lateral inhibition, apoptosis and differential adhesion. Theor Biol Med Model 4:43 11. Buceta J, Herranz H, Canela-Xandri O et al (2007) Robustness and stability of the gene regulatory network involved in DV boundary formation in the Drosophila wing. PLoS One 2:e602 12. Morelli LG, Ares S, Herrgen L et al (2009) Delayed coupling theory of vertebrate segmentation. HFSP J 3:55–66 13. Formosa-Jordan P, Ibañes M (2009) Diffusible ligand and lateral inhibition dynamics for pattern formation. J Stat Mech Theor E 2009, P03019 14. Momiji H, Monk N (2009) Oscillatory Notch-­ pathway activity in a delay model of neuronal differentiation. Phys Rev E 80:21930 15. Barad O, Rosin D, Hornstein E et al (2010) Error minimization in lateral inhibition circuits. Sci Signal 3:ra51 16. Cohen M, Georgiou M, Stevenson NL et al (2010) Dynamic filopodia transmit intermittent Delta-Notch signaling to drive pattern refinement during lateral inhibition. Dev Cell 19:78–89

Modeling Notch Signaling 17. Sprinzak D, Lakhanpal A, Lebon L et al (2010) Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature 465:86–90 18. Sprinzak D, Lakhanpal A, Lebon L et al (2011) Mutual inactivation of Notch receptors and ligands facilitates developmental patterning. PLoS Comp Biol 7:e1002069 19. Shaya O, Sprinzak D (2011) From Notch signaling to fine-grained patterning: modeling meets experiments. Curr Opin Genet Dev 21:732–739 20. Stamataki D, Holder M, Hodgetts C et al (2011) Delta1 expression, cell cycle exit, and commitment to a specific secretory fate coincide within a few hours in the mouse intestinal stem cell system. PLoS One 6:e24484 21. Yaron A, Sprinzak D (2012) The cis side of juxtacrine signaling: a new role in the development of the nervous system. Trends Neurosci 35:230–239 22. Matsuda M, Koga M, Nishida E et al (2012) Synthetic signal propagation through direct cell-cell interaction. Sci Signal 5:ra31 23. Formosa-Jordan P, Ibañes M, Ares S et al (2012) Regulation of neuronal differentiation at the neurogenic wavefront. Development 139:2321–2329 24. Okubo Y, Sugawara T, Abe-Koduka N et al (2012) Lfng regulates the synchronized oscillation of the mouse segmentation clock via trans-repression of Notch signalling. Nat Commun 3:1141 25. Alon U (2007) An introduction to systems biology: design principles of biological circuits. CRC Press, Boca Raton, FL 26. Murray JD (2002) Mathematical biology. Springer, New York 27. Cross M, Greenside H (2009) Pattern formation and dynamics in nonequilibrium systems. Cambridge University Press, Cambridge 28. Sakamoto K, Ohara O, Takagi M et al (2002) Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification. Dev Biol 241: 313–326 29. Fiuza U, Klein T, Martinez Arias A et al (2010) Mechanisms of ligand-mediated inhibition in Notch signaling activity in Drosophila. Dev Dyn 239:798–805 30. Formosa-Jordan P, Ibañes M (2014) Com­ petition in Notch Signaling with Cis Enriches Cell Fate Decisions. PLoS One 9:e95744 31. D’Souza B, Miyamoto A, Weinmaster G (2008) The many facets of Notch ligands. Oncogene 27:5148–5167 32. Guruharsha K, Kankel M, Artavanis-Tsakonas S (2012) The Notch signalling system: recent

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insights into the complexity of a conserved pathway. Nat Rev Genet 13:654–666 33. Bollenbach T, Pantazis P, Kicheva A et al (2008) Precision of the Dpp gradient. Development 135:1137–1146 34. O’Dea RD, King JR (2011) Multiscale analysis of pattern formation via intercellular signalling. Math Biosci 231:172–185 35. O’Dea RD, King JR (2012) Continuum limits of pattern formation in hexagonal-cell monolayers. J Math Biol 64:579–610 36. Pennington MW, Lubensky DK (2010) Switch and template pattern formation in a discrete reaction-diffusion system inspired by the Drosophila eye. Eur Phys J E Soft Matter 33:129–148 37. Lubensky DK, Pennington MW, Shraiman BI et al (2011) A dynamical model of ommatidial crystal formation. Proc Natl Acad Sci U S A 108:11145–11150 38. Simakov DS, Pismen LM (2013) Discrete model of periodic pattern formation through a combined autocrine-juxtacrine cell signaling. Phys Biol 10:046001 39. Tanemura M, Honda H, Yoshida A (1991) Distribution of differentiated cells in a cell sheet under the lateral inhibition rule of ­differentiation. J Theor Biol 153:287–300 40. Eglen SJ, Willshaw DJ (2002) Influence of cell fate mechanisms upon retinal mosaic formation: a modelling study. Development 129: 5399–5408 41. Cohen M, Baum B, Miodownik M (2011) The importance of structured noise in the generation of self-organizing tissue patterns through contact-mediated cell-cell signalling. J R Soc Interface 8:787–798 42. Eldar A, Elowitz MB (2010) Functional roles for noise in genetic circuits. Nature 467: 167–173 43. Gardiner C (2004) Handbook of stochastic methods: for physics, chemistry & the natural sciences, vol 13, Series in synergetics. Springer, New York 44. Rudge T, Burrage K (2008) Effects of intrinsic and extrinsic noise can accelerate juxtacrine pattern formation. Bull Math Biol 70:971–991 45. Lewis J (2003) Autoinhibition with transcriptional delay: a simple mechanism for the zebrafish somitogenesis oscillator. Curr Biol 13:1398–1408 46. Agrawal S, Archer C, Schaffer DV et al (2009) Computational models of the Notch network elucidate mechanisms of context-dependent signaling. PLoS Comput Biol 5:e1000390 47. Petrovic J, Formosa-Jordan P, Luna-Escalante JC et al (2014) Ligand-dependent Notch signaling strength orchestrates lateral induction

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and lateral inhibition in the developing inner ear. Development 141:2313–2324 48. Schröter C, Ares S, Morelli LG et al (2012) Topology and dynamics of the zebrafish segmentation clock core circuit. PLoS Biol ­ 10:e1001364 49. Garcia-Ojalvo J (2011) Physical approaches to the dynamics of genetic circuits: a tutorial. Contemp Phys 52:439–464

50. Plahte E (2001) Pattern formation in discrete cell lattices. J Math Biol 43:411–445 51. Strogatz S (2001) Nonlinear dynamics and chaos: with applications to physics, biology, chemistry and engineering. Westview Press, Cambridge 52. Formosa-Jordan P (2013) Pattern formation through lateral inhibition mediated by Notch signaling. University of Barcelona, Barcelona

Chapter 23 Small Molecules That Inhibit Notch Signaling Gerdien E. De Kloe and Bart De Strooper Abstract The proteolytic processing of Notch receptors plays a central role in the transduction of Notch signaling, which is involved in a variety of important processes in the body. Abnormal Notch processing has been implicated in a variety of cancers. γ-Secretase is responsible for the third and last cleavage step of Notch receptors. Since γ-secretase plays an important role in Alzheimer’s disease, great effort has been spent to develop γ-secretase inhibitors (GSIs). The majority of these inhibitors block γ-secretase nonselectively, which means that these compounds can be used to block Notch cleavage and thereby regulate Notch signaling. In this review we give an overview of the most-used GSIs in the Notch field, together with examples of their use. It is a huge advantage that these drug-like compounds are already optimized for γ-secretase, and some are already being used in clinical trials. However, their nonspecificity has disadvantages as well, since four Notch receptors exist with different sites of expression and different roles in cell signaling and at least four different γ-secretase proteases are involved in their cleavage. It would be worth the effort to screen many GSIs for their selectivity for the different Notch receptors and γ-secretases, in order to obtain interesting tools for further research and—in the end—to develop safer drugs. Key words Notch, γ-secretase inhibitors, Transition-state analogs, Allosteric inhibitors, Selectivity

1

Introduction Proteolytic processing plays a central role in the transduction of Notch signals from the extracellular to the intracellular side of the cell [1]. The proteolytic processing of Notch receptors occurs in three steps. First, a furin-like convertase matures the protein. However the receptor remains inactive until Notch ligands (Delta and Jagged) bind and induce a second cleavage (S2 cleavage) by a membrane-tethered metalloprotease (ADAM10) [2] which cleaves the ectodomain a second time close to the membrane. The remaining membrane-bound fragment becomes by default a γ-secretase substrate [3, 4]. γ-Secretases are tetrameric aspartyl type proteases that cleave substrates in the plane of the cell membrane [5]. This cleavage (S3) releases the Notch Intracellular Domain (NICD), which translocates to the nucleus to activate expression of Notch target genes. In principle inhibitors for all three proteases will block

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Notch signaling. However, most research and pharmaceutical development has focused on γ-Secretase inhibition. The reason is that such inhibitors could block the production of the amyloid (Aβ) peptides that cause Alzheimer’s disease [6]. Obviously from the perspective of that field, Notch inhibition is a severe side effect, and actually major clinical trials have been stopped because of Notchrelated toxicity [7]. γ-Secretases are membrane bound, multiprotein complexes, consisting of four components: presenilin 1 or 2 (PS1/2), nicastrin (NCT), anterior pharynx 1 A or B (Aph 1A/B), and presenilin enhancer 2 (Pen2) [5]. In total the complex has at least 19 transmembrane domains (TMDs), which make its structural and functional characterization extremely challenging. Presenilin has to be endoproteolytically cleaved to form a heterodimer in order to become an active protease. The active site of this complex consists of two aspartate residues which are found at the interface of the PS heterodimer [8]. The role of the three other proteins Nicastrin, Aph1, and Pen2, while clearly needed to generate an active complex, remains insufficiently understood. Because two different presenilin subunits and two Aph1 subunits are found in the genome of human, while mice have three Aph1 subunits, different combinations yield at least four γ-secretases in human and even six in mouse. Not all combinations have similar roles in Notch signaling [9]. The first link between γ-secretase and Notch signaling was made in a genetic screen for Notch signaling modifiers in C. elegans [10]. Presenilin knock-outs in mouse and Drosophila showed developmental abnormalities corresponding with altered Notch signaling [11, 12]. Efficient Notch1 processing to produce NICD requires PS1 [3, 4]. A γ-secretase inhibitor (GSI) was able to block Notch signaling in zebrafish and caused a severe neurogenic phenotype in this species [13]. Other γ-secretase inhibitors MCL 28170, MG132, and MW167 are able to inhibit Notch processing [3]. All these inhibitors were originally developed to block the proteolytic processing of the Amyloid Precursor Protein (APP) to the infamous Aβ peptide, but a good correlation has been found between cleavage of β-APP and NotchΔE (β-APP and NotchΔE are direct substrates for γ-secretase, i.e., they do not need the S2 cleavage to become activated). These cleavages are blocked by inhibitors from six structurally different classes [14], strongly arguing that a similar enzymatic activity is responsible for cleavage of both Notch and APP. All γ-secretase inhibitors (GSIs) that have been developed until now were shown to give Notch-related side effects. Chronic treatment of mice with γ-secretase inhibitor LY-411575 had a dose-dependent effect on thymus, spleen, and intestine [15, 16]. Skin cancer is another observed side effect of these drugs and might be well a consequence of Notch signaling [17]. While these side effects have considerably complicated drug efforts in the Alzheimer’s field, γ-secretase inhibitors are now further developed for certain types of cancer. Four inhibitors have actually been evaluated in clinical trials [18].

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Overview of γ-Secretase Inhibitors When we scanned the literature using the keywords Notch and inhibitor, ~700 papers were found. In Fig. 1a we represent the top-ten most-used GSIs in Notch research. DAPT is used in more

a DAPT MRK-003 z-IL-CHO GSI II L-685,458 DBZ LY-411,575 Cmpd E RO4929097 GSI I

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Fig. 1 (a) Diagram of the ten most-used GSIs in vitro and in vivo. (b) Chemical structures of eight of the ten most-used GSIs (for MRK-003 and RO4929097 see Fig. 2)

Gerdien E. De Kloe and Bart De Strooper

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Fig. 2 Five GSIs in (pre)clinical trials for Notch signaling in cancer

than 50 % of the studies. In Fig. 1b the structures of eight compounds are presented; the structures of MRK-003 and RO4929097 are shown in Fig. 2. γ-Secretase inhibitors are further divided into three groups. The classical, transition state type of inhibitor is supposed to bind the active site in presenilin. Presenilin further has an initial substrate docking site that is distinct from the active site, for which also inhibitors were designed. A third class of GSIs binds to an allosteric site in the γ-secretase complex and affects its activity in that way. The three classes of inhibitors will be discussed in detail, including their applications in Notch signaling.

3

Active-Site Binders or Transition-State Analogs The development of the first class of γ-secretase inhibitors was guided by the knowledge gained from active-site inhibitors of HIV protease, which is also a complex aspartyl protease. The best known compound from this class is L-685,458 (Fig. 1), which is extensively used as tool compound. It was shown that L-685,458 binds to the N-terminal part of PS by photo-crosslinking studies [19]. Lewis et al. showed that active-site inhibitors do not discriminate pharmacologically between APP and Notch processing [14]. Due to these nonselective properties, this class of inhibitors is not suitable for AD and none of them have entered AD clinical trials [20]. Nevertheless, a couple of these peptide compounds are used in Notch-related studies. Figure 1 shows Z-IL-CHO, GSI I, and GSI II (MW167).

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Substrate Docking-Site-Binding Compounds The first substrate docking-site inhibitors were helical peptides mimicking the β-CTF substrate conformation [21]. The best substrate docking-site inhibitor has an IC50 for Aβ40 of 140 pM in a cell-free assay [22]. However, equal to the active-site inhibitors, no Notch-sparing selectivity was obtained and therefore none of the compounds have entered AD clinical trials [20]. To the best of our knowledge, none of these inhibitors have been used in any Notch signaling study.

5

Allosteric Binders The allosteric inhibitors form the largest and most diverse set of γ-secretase inhibitors. Several have entered clinical trials, both for AD and Notch-related cancers. The first orally active GSI and today the most important tool compound is DAPT (Fig. 1b), with an IC50 for Aβ of 20 nM and a comparable IC50 for NICD generation [23, 24]. DAPT is used in more than 50 % of the Notch signaling studies (Fig. 2a). This is remarkable, as in the meantime many inhibitors have been developed with much higher activities. It is also remarkable how little attention is given to the fact that these inhibitors do not discriminate between the different complexes. Nowadays good inhibitors are available that inhibit more than tenfold more efficiently presenilin 1 complexes versus presenilin 2 complexes [25] but those have been used very little by the Notch research field. The so-called compound E (CmpdE) is a classic example of the allosteric inhibitor series. It is an analog of the benzodiazepine series developed by Merck [26, 27]. The highly active compound has an IC50 of 300 pM for Aβ42 and 320 pM for NICD. Dibenzazepine (DBZ) is another example but developed by Hoffman-LaRoche and has an IC50 for Aβ40 of 2.0 nM. This compound has a low metabolic stability [20]. An almost identical compound is LY-411,575. RO4929097 (Fig. 2) is a close analog from the same inhibitor series. The displacement of the 3,5-difluorobenzyl group with a pentafluoropropyl group has improved the metabolic stability of the compound, making it suitable for clinical and biological studies. BMS-906024 is an analog of the benzoazepinone series developed by Bristol-Myers Squibb [28, 29]. BMS-906024 has an IC50 of 1.6 nM for Notch1 and 3.4 nM for Notch3 [29]. The compound is in phase I clinical trials for advanced metastatic solid tumors [30]; in parallel a phase I clinical trial is performed in combination with chemotherapy [31].

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Pfizer developed PF-03084014, structurally completely different from the other allosteric site binding inhibitors [32]. This inhibitor had an IC50 of 6.2 nM for Aβtotal and 13.3 nM for Notch1. The compound is currently in a phase 1B clinical study in combination with Docetaxel, for metastatic or locally recurrent/advanced triple negative breast cancer [33]. A novel class of allosteric inhibitors is the arylsulfonamides. Quite some compounds of this class display a certain degree of selectivity for Aβ over Notch cleavage. Begacestat for example was reported to have a 14-fold Notch-sparing selectivity, and was taken forward for clinical studies in AD [34]. However, the Merck arylsulfonamide series failed to show Notch-sparing selectivity. MRK003, a compound with picomolar activity, has progressed into preclinical studies and is used regularly in Notch signaling studies (Fig. 2). Merck also developed an arylsulfone for which a couple of clinical trials were started, MRK-0752. Phase I trials were performed against T-cell acute lymphoblastic leukemia (T-ALL), refractory solid tumors [35], and recurrent or refractory CNS cancer. A phase I/II study was started with gemcitabine to treat pancreatic cancer, and one against breast cancer, followed by treatment with docetaxel [18]. In general, we can conclude that the approach to take compounds that have been developed for γ-secretase inhibition of APP processing and apply them to study Notch processing works well. The designed compounds have drug-like properties and display high activities for γ-secretase. However, we found not a single structure-activity relationship study focusing on Notch processing. It is simply not known if Notch specific compounds exist, as they seem to exist for APP processing. Although Chen et al. reported no influence of MRK-003 on Notch3 processing, indicating that selectivity is possible [36], no further follow-up is available to confirm that compounds selective for one of the Notch receptors can be made. For example since Notch4 is specifically expressed in developing blood vessels, in particular in tumor vasculature in adults [37], specific inhibition of Notch4 cleavage could theoretically yield a safe anti-angiogenic drug. An interesting example is the development of a Notch1 selective antibody to block Notch signaling in alloreactive T-cells, preventing the severe intestinal side effects that were observed upon Notch inhibition with a GSI (DBZ) [38]. In our opinion, it would greatly benefit the field if an activity study was performed with a diverse set of γ-secretase inhibitors to test their activities on γ-secretase processing of the four different Notch receptors and probably as well on the processing of Notch ligands. As discussed, another selectivity issue is the four different γ-secretase complexes [9]. A systematic study is needed to identify tool compounds that block selectively the different enzymes.

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Examples of the Use of GSIs in Notch Signaling Models GSIs have been used in a variety of Notch signaling cell systems and animal models. In Table 1 we give a few examples for each GSI presented in Fig. 1, the top-ten most-used GSIs. We also give an overview of the activities of these GSIs for Notch cleavage by γ-secretase whenever possible. Unfortunately, the IC50’s for all GSIs are not always reported in the literature. It is also important to note that the IC50’s that are reported are not easy to compare, since the activity of a GSI in a cell system is largely dependent on the cell type [39]. The expression of the Notch receptors probably differs from one cell type to another, and the expression of the different γ-secretase complexes in different cell lines adds to this complexity. In Drosophila the situation is simpler as only a homogeneous population of γ-secretase proteases is expressed [40]. The examples in Table 1 should be seen as a starting point to develop a protocol that is specific for your cell system or animal model.

7

Use of Notch Inhibitors in Cancer Abnormal Notch signaling has been implicated in a variety of cancers, T-ALL being the best-studied example. Abnormal Notch signaling was also observed in solid tumors including cancers of the breast, kidney, pancreas, prostate, cervix, endometrium, brain, intestine, lung, and skin [37]. Over 50 % of all patients with T-ALL have activating mutations in Notch1 [64]. GSIs induce cell cycle arrest in T-ALL cell lines that expressed wild-type or mutant Notch [65]. However, animal studies already had shown that Notch inhibition would result in severe gastrointestinal toxicity, which was confirmed by a phase I study of MK-0752 in relapsed and refractory T-ALL [65]. Glucocorticoids are often used as first chemotherapy in T-ALL, but resistance towards this therapy is easily developed. Because GSIs have a profound effect on the homeostasis of T-ALL lymphoblasts, it was hypothesized that GSIs might sensitize T-ALL cells to chemotherapy. Therefore a combination therapy of dexamethasone, a glucocorticoid, and DBZ was tested in a mouse model. Indeed, inhibition of Notch1 by a GSI sensitizes glucocorticoid-resistant cells towards glucocorticoid-induced apoptosis. Unexpectedly, glucocorticoid treatment seemed also to have a protective effect against GSI-induced intestinal toxicity in mice [66]. This interesting result should be confirmed in clinical studies to see if a safe combination therapy can be found with a GSI and dexamethasone. The role of Notch signaling in breast cancer is reviewed by Shi et al. [37]. GSI PF-03084014 is co-administered with docetaxel in a preclinical study to enhance docetaxel activity and reverse

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Table 1 Activities and concentrations of GSIs used to regulate Notch signaling Name

Alternative name

Examples of use

GSI-I

Z-Leu-Leu-Nle-CHO (Nle = Norleucine)

GSI-I inhibits Notch signaling in eight hepatocellular carcinoma cell lines at 1.2 μM concentration [39] Marked proapoptotic effect in primary cutaneous anaplastic large cell lymphoma [41]

GSI-II

MW167/DFK167

IC50 = 13.5 nM in cell-based assay in Drosophila melanogaster S2 cells [40] CD4+ T cells (asthma model) [42]

GSI-IX

DAPT

IC50 = 788 nM in cell-based assay in D. melanogaster S2 cells [40] IC50 = 300 nM measured in Sup-T1 cells [24] Estrogen activated breast cancer stem cells [43] T-ALL (C3G-F+ SPA-1−/−) cell lines [44] Against T-ALL orally administered in mice [45]

GSI-X

L-685,458

IC50 = 135 nM measured in Sup-T1 cells [24] Angiogenesis, HUVEC cells [46] Rat lymphatic endothelial cells [47]

GSI-XII

Z-Ile-Leu-CHO

Human chronic lymphocytic leukemia cells, blocking of survival and chemoresistance [48] T-ALL, CD4+ T cells (for in vivo experiments LY-411,575 was used) [49]

GSI-XX

Dibenzazepine (DBZ)

IC50 = 2.92 nM in cell-based assay in D. melanogaster S2 cells [40] Intestine, in mouse model DBZ increases goblet cell numbers in gut [50, 51] CD4+ T cells [52]

GSI-XXI

Cmpd E

IC50 = 33.1 nM in cell-based assay in D. melanogaster S2 cells [40] Human T-ALL cell lines [53, 54]

LY-411,575

IC50 = 0.32 nM in HEK293 cells expressing NΔE [16] Regeneration of hair cells. Of DAPT, L-685,458, MDL28170, and LY-411,575, the latter gave the highest potency. In vivo orally administered in mice, 50 mg/kg for 5 days [55] Primary and immortalized KS cells [56] Breast cancer mouse model, 5 mg/kg, 3 days on, 4 days off [57] Colitis mouse model, oral administration [58]

RO4929097

IC50 = 5 nM in a Notch1 reporter assay in HEK293 cells [59] (Pre)clinical studies in breast cancer, 3D clonogenic assay [60] Metastatic melanoma cell lines, inhibitor tested in two in vivo xenograft models [61]

MRK-003

Breast cancer mouse model, 100 mg/kg [57] Non-small cell lung cancer, in vitro and in vivo (mouse models) [62] Bone metastatic cell lines, JAG1 OE tumor cells, and MC3T3-E1 cells [63]

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chemoresistance in breast cancer [33]. A clinical study is currently ongoing based on these preclinical results. Notch3 is being overexpressed in non-small cell lung carcinoma (NSCLC). GSI MRK-003 was tested for in vitro effects on lung cancer lines and in vivo effects on mouse xenografts. The results were promising: MRK-003 was able to inhibit Notch3, elevate the levels of apoptosis, and reduce tumor cell proliferation [67]. Gastrointestinal toxicity is one of the major side effects of Notch inhibition. This is likely due to an on-target toxicity. It has been reported that Notch drives gastrointestinal precursor cells toward an epithelial fate and away from a secretory cell fate; Notch inhibition causes an imbalance in this process resulting in an overproduction of secretory goblet cells [50]. This toxicity might be problematic for any Notch inhibitor. Two possible solutions have been proposed: the co-administration of GSIs with glucocorticoids as described above, and intermittent dosing schedules of GSI administration. The latter have been shown to largely spare the gut toxicity while maintaining antitumor efficacy [68]. Inhibition of Notch impacts also on the immune system [16]. Although the clinical studies have not investigated this aspect, this could become problematic when treating cancer patients. Finally, as mentioned in the introduction, other side effects could occur due to the ambivalent nature of Notch signaling. While in most tissues Notch can be considered a proto-oncogene, it acts as a tumor suppressor in other tissues, for example in the skin [17].

Acknowledgements This work was supported by VIB, a Methusalem grant from KU Leuven and the Flemish government, Janssen Pharmaceutica, and the Arthur Bax and Anna Van Luffelen foundation. References 1. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233 2. Hartmann D, de Strooper B, Serneels L et al (2002) The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts. Hum Mol Genet 11:2615–2624 3. De Strooper B, Annaert W, Cupers P et al (1999) A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398:518–522 4. Struhl G, Greenwald I (1999) Presenilin is required for activity and nuclear access of Notch in Drosophila. Nature 398:522–555

5. De Strooper B (2003) Aph-1, Pen-2, and Nicastrin with Presenilin generate an active gamma-Secretase complex. Neuron 38:9–12 6. De Strooper B, Vassar R, Golde T (2010) The secretases: enzymes with therapeutic potential in Alzheimer disease. Nat Rev Neurol 6: 99–107 7. Doody RS, Raman R, Farlow M et al (2013) A phase 3 trial of semagacestat for treatment of Alzheimer’s disease. N Engl J Med 369: 341–350 8. Wolfe MS, Xia W, Ostaszewski BL et al (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398:513–517

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9. Serneels L, Van Biervliet J, Craessaerts K et al (2009) gamma-Secretase heterogeneity in the Aph1 subunit: relevance for Alzheimer’s disease. Science 324:639–642 10. Levitan D, Greenwald I (1995) Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer’s disease gene. Nature 377:351–354 11. Shen J, Bronson RT, Chen DF et al (1997) Skeletal and CNS defects in Presenilin-1deficient mice. Cell 89:629–639 12. Wong PC, Zheng H, Chen H et al (1997) Presenilin 1 is required for Notch 1 and Dll1 expression in the paraxial mesoderm. Nature 387:288–292 13. Geling A, Steiner H, Willem M et al (2002) A gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep 3:688–694 14. Lewis HD, Pérez Revuelta BI, Nadin A et al (2003) Catalytic site-directed gamma-secretase complex inhibitors do not discriminate pharmacologically between Notch S3 and beta-APP cleavages. Biochemistry 42:7580–7586 15. van Es JH, Clevers H (2005) Notch and Wnt inhibitors as potential new drugs for intestinal neoplastic disease. Trends Mol Med 11: 496–502 16. Wong GT, Manfra D, Poulet FM et al (2004) Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J Biol Chem 279:12876–12882 17. Demehri S, Turkoz A, Kopan R (2009) Epidermal Notch1 loss promotes skin tumorigenesis by impacting the stromal microenvironment. Cancer Cell 16:55–66 18. Groth C, Fortini ME (2012) Therapeutic approaches to modulating Notch signaling: current challenges and future prospects. Semin Cell Dev Biol 23:465–472 19. Li YM, Xu M, Lai MT et al (2000) Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1. Nature 405:689–694 20. Kreft AF, Martone R, Porte A (2009) Recent advances in the identification of gammasecretase inhibitors to clinically test the Abeta oligomer hypothesis of Alzheimer’s disease. J Med Chem 52:6169–6188 21. Das C, Berezovska O, Diehl TS et al (2003) Designed helical peptides inhibit an intramembrane protease. J Am Chem Soc 125: 11794–11795 22. Bihel F, Das C, Bowman MJ et al (2004) Discovery of a subnanomolar helical

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D-tridecapeptide inhibitor of gamma-secretase. J Med Chem 47:3931–3933 Dovey HF, John V, Anderson JP et al (2001) Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J Neurochem 76:173–181 McKee TD, Loureiro RM, Dumin JA et al (2013) An improved cell-based method for determining the γ-secretase enzyme activity against both Notch and APP substrates. J Neurosci Methods 213:14–21 Borgegård T, Gustavsson S, Nilsson C et al (2012) Alzheimer’s disease: presenilin 2-sparing γ-Secretase inhibition is a tolerable Aβ peptidelowering strategy. J Neurosci 32:17297–17305 Churcher I, Ashton K, Butcher JW et al (2003) A new series of potent benzodiazepine gammasecretase inhibitors. Bioorg Med Chem Lett 13:179–183 Churcher I, Williams S, Kerrad S et al (2003) Design and synthesis of highly potent benzodiazepine gamma-secretase inhibitors: preparation of (2S,3R)-3-(3,4-difluorophenyl)-2-(4fluorophenyl)-4hydroxy-N-((3S)-1-methyl -2-oxo-5phenyl-2,3-dihydro-1H-benzo[e] [1,4]-diazepin-3-yl)butyramide by use of an asymmetric Ireland-Claisen rearrangement. J Med Chem 46:2275–2278 Yang MG, Shi JL, Modi DP et al (2007) Design and synthesis of benzoazepinonederived cyclic malonamides and aminoamides as potent gamma-secretase inhibitors. Bioorg Med Chem Lett 17:3910–3915 Quesnelle C, Kim S-H, Lee F, et al. (2012) Bisfluoroalkyl-1,4-benzodiazepinone compounds WO/2012/129353 http://clinicaltrials.gov/show/NCT01292655 http://clinicaltrials.gov/show/NCT01653470 Wei P, Walls M, Qiu M et al (2010) Evaluation of selective γ-secretase inhibitor PF-03084014 for its antitumor efficacy and gastrointestinal safety to guide optimal clinical trial design. Mol Cancer Ther 9:1618–1628 Zhang CC, Yan Z, Zong Q et al (2013) Synergistic effect of the γ-secretase inhibitor PF-03084014 and docetaxel in breast cancer models. Stem Cells Transl Med 2:233–242 Mayer SC, Kreft AF, Harrison B et al (2008) Discovery of begacestat, a Notch-1-sparing gamma-secretase inhibitor for the treatment of Alzheimer’s disease. J Med Chem 51: 7348–7351 Krop I, Demuth T, Guthrie T et al (2012) Phase I pharmacologic and pharmacodynamic study of the gamma secretase (Notch) inhibitor MK-0752 in adult patients with advanced solid tumors. J Clin Oncol 30:2307–2313

Small Molecules That Inhibit Notch Signaling 36. Chen SM, Liu JP, Zhou JX et al (2011) Suppression of the notch signaling pathway by gamma-secretase inhibitor GSI inhibits human nasopharyngeal carcinoma cell proliferation. Cancer Lett 306:76–84 37. Shi W, Harris AL (2006) Notch signaling in breast cancer and tumor angiogenesis: crosstalk and therapeutic potentials. J Mammary Gland Biol Neoplasia 11:41–52 38. Tran IT, Sandy AR, Carulli AJ et al (2013) Blockade of individual Notch ligands and receptors controls graft-versus-host disease. J Clin Invest 123:1590–1604 39. Shen Y, Lv D, Wang J et al (2012) GSI-I has a better effect in inhibiting hepatocellular carcinoma cell growth than GSI-IX, GSI-X, or GSIXXI. Anticancer Drugs 23:683–690 40. Groth C, Alvord WG, Quiñones OA et al (2010) Pharmacological analysis of Drosophila melanogaster gamma-secretase with respect to differential proteolysis of Notch and APP. Mol Pharmacol 77:567–574 41. Kamstrup MR, Biskup E, Gniadecki R (2010) Notch signalling in primary cutaneous CD30+ lymphoproliferative disorders: a new therapeutic approach? Br J Dermatol 163: 781–788 42. Gu W, Xu W, Ding T et al (2012) Fringe controls naive CD4(+)T cells differentiation through modulating notch signaling in asthmatic rat models. PLoS One 7:e47288 43. Harrison H, Simões BM, Rogerson L et al (2013) Oestrogen increases the activity of oestrogen receptor negative breast cancer stem cells through paracrine EGFR and Notch signalling. Breast Cancer Res 15:R21 44. Wang SF, Aoki M, Nakashima Y et al (2008) Development of Notch-dependent T-cell leukemia by deregulated Rap1 signaling. Blood 111:2878–2886 45. Pancewicz J, Taylor JM, Datta A et al (2010) Notch signaling contributes to proliferation and tumor formation of human T-cell leukemia virus type 1-associated adult T-cell leukemia. Proc Natl Acad Sci U S A 107:16619–16624 46. Sainson RC, Aoto J, Nakatsu MN et al (2005) Cell-autonomous notch signaling regulates endothelial cell branching and proliferation during vascular tubulogenesis. FASEB J 19:1027–1029 47. Ota H, Katsube K, Ogawa J et al (2007) Hypoxia/Notch signaling in primary culture of rat lymphatic endothelial cells. FEBS Lett 581:5220–5226 48. Nwabo Kamdje AH, Bassi G, Pacelli L et al (2012) Role of stromal cell-mediated Notch signaling in CLL resistance to chemotherapy. Blood Cancer J 2:e73

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49. Joshi I, Minter LM, Telfer J et al (2009) Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood 113:1689–1698 50. van Es JH, van Gijn ME, Riccio O et al (2005) Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435:959–963 51. van Es JH, de Geest N, van de Born M et al (2010) Intestinal stem cells lacking the Math1 tumour suppressor are refractory to Notch inhibitors. Nat Commun 1:18 52. Okamoto M, Matsuda H, Joetham A et al (2009) Jagged1 on dendritic cells and Notch on CD4+ T cells initiate lung allergic responsiveness by inducing IL-4 production. J Immunol 183:2995–3003 53. Liu S, Breit S, Danckwardt S et al (2009) Downregulation of Notch signaling by gammasecretase inhibition can abrogate chemotherapyinduced apoptosis in T-ALL cell lines. Ann Hematol 88:613–621 54. Gusscott S, Kuchenbauer F, Humphries RK et al (2012) Notch-mediated repression of miR-223 contributes to IGF1R regulation in T-ALL. Leuk Res 36:905–911 55. Mizutari K, Fujioka M, Hosoya M et al (2013) Notch inhibition induces cochlear hair cell regeneration and recovery of hearing after acoustic trauma. Neuron 77:58–69 56. Curry CL, Reed LL, Golde TE et al (2005) Gamma secretase inhibitor blocks Notch activation and induces apoptosis in Kaposi’s sarcoma tumor cells. Oncogene 24:6333–6344 57. Pandya K, Meeke K, Clementz AG et al (2011) Targeting both Notch and ErbB-2 signalling pathways is required for prevention of ErbB-2positive breast tumour recurrence. Br J Cancer 105:796–806 58. Okamoto R, Tsuchiya K, Nemoto Y et al (2009) Requirement of Notch activation during regeneration of the intestinal epithelia. Am J Physiol Gastrointest Liver Physiol 296:G23–G35 59. Luistro L, He W, Smith M et al (2009) Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties. Cancer Res 69:7672–7680 60. Debeb BG, Cohen EN, Boley K et al (2012) Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells. Breast Cancer Res Treat 134:495–510 61. Huynh C, Poliseno L, Segura MF et al (2011) The novel gamma secretase inhibitor RO4929097 reduces the tumor initiating potential of melanoma. PLoS One 6:e25264 62. Osanyingbemi-Obidi J, Dobromilskaya I, Illei PB et al (2011) Notch signaling contributes to

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lung cancer clonogenic capacity in vitro but may be circumvented in tumorigenesis in vivo. Mol Cancer Res 9:1746–1754 63. Sethi N, Dai X, Winter CG et al (2011) Tumorderived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells. Cancer Cell 19:192–205 64. Real PJ, Ferrando AA (2009) NOTCH inhibition and glucocorticoid therapy in T-cell acute lymphoblastic leukemia. Leukemia 23: 1374–1377 65. DeAngelo DJ, Stone RM, Silverman LB (2006) A phase I clinical trial of the notch inhibitor MK-0752 in patients with T-cell

acute lymphoblastic leukemia/lymphoma (T-ALL) and other leukemias. J Clin Oncol 24(18S):6585 66. Real PJ, Tosello V, Palomero T et al (2009) Gamma-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia. Nat Med 15:50–58 67. Konishi J, Kawaguchi KS, Vo H et al (2007) Gamma-secretase inhibitor prevents Notch3 activation and reduces proliferation in human lung cancers. Cancer Res 67:8051–8057 68. Purow B (2012) Notch inhibition as a promising new approach to cancer therapy. Adv Exp Med Biol 727:305–319

Chapter 24 Application and Evaluation of Anti-Notch Antibodies to Modulate Notch Signaling Wendy R. Gordon and Jon C. Aster Abstract In recent years, several groups have reported the development of antibodies that inhibit or activate Notch signaling. Modulatory antibodies are valuable experimental tools that permit specific targeting of individual Notch receptor homologs (in contrast to pan-Notch-receptor inhibitors like gamma-secretase inhibitors), and show promise as therapeutic agents. Typically, Notch responsive luciferase reporter assays are used to validate and characterize modulatory antibodies. We describe detailed methods for performing dual luciferase-based signaling assays to read out modulation of Notch activity by antibodies designed to inhibit/activate signaling. Key words Notch, Luciferase assay, Inhibitory antibody, Activating antibody, NRR

1

Introduction The Notch signaling pathway [1–3] has emerged as an attractive potential therapeutic target due to its involvement in the pathogenesis of many human diseases, such as T-cell acute lymphoblastic leukemia (T-ALL). Both Notch receptors and Notch ligands are single pass transmembrane proteins. Mammals have four Notch receptors, NOTCH1-4, and at least four functional ligands, DLL1, DLL4, JAG1, and JAG2, each of which belongs to the DSL (Delta/Serrate/LAG-2) family. Signaling initiates when the glycosylated ectodomain of a Notch receptor on the surface of one cell engages the ectodomain of a ligand expressed on a neighboring cell. In a process that involves endocytosis of Notch ligands, ligandbinding triggers exposure of a site housed in the juxtamembrane negative regulatory region (NRR) of Notch, allowing cleavage of Notch at a site just external to the transmembrane domain by ADAM (A Disintegrin And Metalloprotease) proteases. The Notch molecule then undergoes cleavage within the inner half of its transmembrane domain by gamma-secretase, which releases the Notch intracellular domain (NICD) into the cytosol. From here, NICD

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_24, © Springer Science+Business Media New York 2014

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Table 1 Modulatory notch antibodies

Target

Name of antibody

Activating or inhibitory

Source/ reference

Notch1 NRR

WC75, WC629 Anti-NRR1 N1_E6 OMP-52 M51 Hu-hN1

Inhibitory Inhibitory Inhibitory Inhibitory Inhibitory

[16] [7] [8] [9] [10]

Notch2 NRR

Anti-NRR2 N2_B6, N2_b9

Inhibitory Inhibitory

[7] [8]

Notch3 NRR

A4, A8 A13

Inhibitory Activating

[5] [5]

WC613, WC133 N1_9_b5

Inhibitory Inhibitory

[6] [8]

Notch1 ligand binding domain

Note Co-crystal structure In clinical trials Co-crystal structure

Only identified agonistic antibody

translocates to the nucleus, forms a ternary transcription activation complex with the DNA-binding factor CSL (CBF1/Su(H)/ Lag-1) and co-activators of the Mastermind family, and activates the expression of target genes. Gamma-secretase inhibitors block Notch signaling very effectively, but have detrimental “on-Notch” effects such as gut toxicity because they inhibit all mammalian Notch receptors, and also inhibit the processing of other gammasecretase substrates [4]. These inherent characteristics of gammasecretase inhibitors represent significant limitations, both in terms of experimental utility and therapeutic translation. A recent strategy to improve the specificity of Notchdirected therapeutics involves the development of specific Notch ligand (see Chapter 25) and Notch receptor-targeting antibodies [5–10] (Table 1). Isoform-specific antibodies have been generated against the ligand-binding domains and NRRs of NOTCH1, NOTCH2, and NOTCH3, mainly by pharmaceutical companies interested in exploring the therapeutic potential of targeting individual receptors. The juxtamembrane NRR is the “activation switch” of Notch and contains the site of metalloprotease cleavage, which is the rate-limiting step in Notch receptor activation. In the resting state, this site is deeply buried by inter-NRR interactions [11–14] that must be disengaged for cleavage to occur during activation by ligand. Notably, T-ALL frequently harbors gain-of-function point mutations in the NRR that lead to ligand-independent activation, underscoring the NRR’s importance and serving as the genesis for the idea that stabilization of mutant NRRs (e.g., with antibody) could be useful in the treatment of T-ALL [15].

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Several groups have developed antibodies that inhibit signaling of both normal and mutated NOTCH1 and NOTCH2 receptors, using either phage display or traditional immunization of mice with NRR fragments. Structural studies [7, 10] and epitope mapping [5, 15] show that inhibitory NRR antibodies generally bind to discontinuous epitopes that clamp the NRR in its closed conformation, preventing access to metalloproteases. These studies found that anti-NOTCH1 antibodies effectively block Notch signaling in vitro [6], in subcutaneous xenografts of tumors bearing Notch1 mutations [7], in triple negative breast cancer xenografts [10], and in T-ALL xenografts obtained from patients resistant to conventional treatment [9]. These antibodies are highly specific for NOTCH1 or NOTCH2 [7, 8] and when used alone do not lead to gastrointestinal toxicity previously associated with gamma-secretase inhibitor treatment [7]. An additional potential toxicity stems from studies showing that simultaneous blocking of NOTCH1 and NOTCH2 receptors causes neural stem cells to differentiate and adopt a neuronal cell fate [8], but the relevance of this observation to therapeutic interventions is uncertain, particularly because antibodies do not penetrate the intact blood–brain barrier. In addition, both inhibitory and activating antibodies have been generated against wild type NOTCH3 NRR [5]; since conditional Notch3 knockout mice appear healthy, inhibitory NOTCH3 antibodies are likely to be well tolerated. Less is known about the potential consequences (and applications) of NOTCH3 activating antibodies. Interestingly, NOTCH4 modulatory antibodies have yet to be reported, perhaps because of substantial divergence of the NOTCH4 NRR sequence from other mammalian Notch homologs. All of the studies characterizing Notch modulatory antibodies have used Notch-based luciferase reporter assays (see also Chapter 11). Since Notch activation ultimately leads to transcriptional activation of target genes in the nucleus, luciferase reporter assays provide very sensitive readouts of Notch activation (Fig. 1). In a basic luciferase assay, a promoter sequence that is responsive to a signaling pathway of interest (e.g., Notch) is placed upstream of a TATA box and the coding sequence for firefly luciferase, which oxidizes its substrate luciferin in the presence of ATP, oxygen, and magnesium to yield photons of light that can be quantified by a luminometer. Use of a Dual-Luciferase® assay improves accuracy by also introducing a control plasmid to normalize for cell number/health [17]. The control plasmid contains a promoter that is not affected by the signaling pathway of interest that drives constitutive expression of Renilla luciferase, which acts on a different substrate (coelenterazine) than firefly luciferase. In Notch-based luciferase assays, the most sensitive and specific response elements are artificial sequences consisting of iterated CSL-binding sites, such as a CSLx4 sequence [18] and a highly sensitive CSLx12 sequence, also known as TP-1, created by iterating a 2x CSL-binding site found in the Epstein-Barr virus TP-1 promoter six times [19, 20].

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Fig. 1 Schematic of modulatory antibody assay. In this example, a reporter cell expressing intact NOTCH1 receptor that has been transfected with a Notch firefly luciferase reporter plasmid and an internal control Renilla luciferase plasmid is being stimulated by feeder cells expressing DLL4. In the absence of modulatory antibodies, signals initiated by engagement and endocytosis of the DLL4 ligand lead to successive cleavages of NOTCH1 by ADAM metalloproteases and gamma-secretase, release of NICD1, and formation of a Notch transcription complex. The complex binds to the TP-1 promoter element to turn on transcription of firefly luciferase, which produces luminescence in the presence of its substrate D-luciferin. The binding sites for previously characterized modulatory antibodies against DLL4 (red), NOTCH1 ligand binding domain (blue), and NOTCH1 negative regulatory region (green) are also shown

Here, we discuss the details of applying anti-Notch antibodies to modulate Notch activity in cultured cells, and describe standard Notch-based dual-luciferase assays to monitor the effectiveness of anti-Notch antibody treatment. In the notes section, we discuss alternatives to the canonical protocol, such as using plated ligand instead of ligand-expressing cells. As an example, Fig. 2 shows a representative dual-luciferase assay validating the specificity of inhibitory and activating Notch3 NRR antibodies. This method should be particularly useful for groups interested in generating and characterizing their own blocking or activating antibodies.

2 2.1

Materials DNA Constructs

1. All DNA plasmids should be maxiprepped and have an OD 260/280 ratio of ~1.8. 2. Notch receptor reporters. Clone appropriate response sequence upstream of firefly luciferase into the multiple cloning region of a pGL vector (Promega). Sensitive response elements of endogenous Notch activity are 4xCSL (4xCGTGGGAA) [18]

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Fig. 2 Example of dual-luciferase reporter assay data produced using modulatory antibodies against the NOTCH3 NRR that inhibit (A4, A8) or activate (A13) Notch signaling. Reproduced from Li et al. (2008) with permission from the Journal of Biological Chemistry [12]. Isogenic U2OS T-REX cell lines stably expressing inducible NOTCH3-Gal4 chimeric receptors were transiently transfected with Gal4 UAS firefly luciferase and pR-TK Renilla luciferase plasmids and then co-cultured with OP9 cells stably expressing the ligand DLL1. (a) Specificity of the A4 and A8 antibodies (G3 is isotype control) for inhibiting NOTCH3 signaling is shown using cell lines engineered to express NOTCH1-Gal4, NOTCH2Gal4, or NOTCH3-Gal4 chimeric receptors. (b) Specific activation of NOTCH3 signaling by the A13 antibody f is also shown using the same Notch reporter cells. Antibodies were used at a concentration of 10 μg/ml. Error bars represent standard error from triplicate measurements

or TP-1 (12xCSL) [19]. If using Notch-Gal4 fusion receptors, a useful construct consists of five or nine copies of the Gal4 UAS upstream of luciferase [21]. The vectors, pGL and derivatives thereof, are commercially available from Promega. Although luciferase assays are simple, cheap, and rapid, it should noted that other readouts of Notch activation or inhibition can also be used in parallel or as a substitute for luciferase assays (see Note 1).

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3. Control reporter construct. The pRL-TK plasmid from Promega can be used as an internal control. The pRL-TK vector contains the herpes simplex virus thymidine kinase (HSV-TK) promoter and drives low to moderate levels of Renilla luciferase expression in co-transfected mammalian cells. 4. Notch expression plasmids. If not using stably expressing Notch cell lines, obtain/clone a full-length Notch or Notch-Gal4 fusion construct in a plasmid that drives expression in transiently transfected cells, such as pcDNA5 (Life Technologies). 2.2 Cell Culture Reagents/Cell Lines

1. Cell culture growth media appropriate for cell lines used in assay. For U2OS cells, MS5/MS5-Dll4 cells, 3T3/3T3Jagged2 cells, and OP9/OP9-Dll1 cells, use D10 media: DMEM supplemented with 10 % FBS, glutamine, and 1 % Penicillin–Streptomycin. 2. PBS, pH 7.4. 3. Trypsin–EDTA. 4. OptiMEM (Invitrogen). 5. Lipofectamine 2000 transfection reagent (Invitrogen). 6. Tissue culture treated 96-well plates and 6 cm dishes. 7. Optional—6-well Uplift plates (Thermo Scientific; see Note 2). 8. Notch-expressing cell line or cells for transient transfection. In particular, U2OS cells express low levels of endogenous Notch receptors and ligands and produce very clean luciferase reporter assay results compared to other cell types (like 293 T). 9. Ligand-expressing cell line (e.g., 3T3-Jagged2 [22], MS5Dll4 [23], OP9-Dll1 [24]) and parental cell line controls. Alternative option—plating recombinant ectodomain of ligand (R & D Systems) can also be used to activate full-length Notch (see Note 3).

2.3 Antibodies/ Drugs

1. Antibody that modulates Notch signaling raised against the NRR of one of the Notch receptors or against the ligandbinding repeats of Notch, or generated from phage display. 2. Appropriate IgG control for antibody. 3. Gamma-secretase inhibitor (e.g., compound E or DAPT) as a positive control for a reagent that maximally inhibits Notch signaling.

2.4 Luciferase Assay Materials and Equipment

1. Dual-Luciferase® kit from Promega, including 5× Passive Lysis Buffer, the substrate of firefly luciferase (luciferin) and the substrate of Renilla luciferase (coelenterazine). Alternative luciferase kits from other suppliers are also available (see Note 4).

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2. Dual injector plate reader (e.g., Turner Biosystems Modulus Microplate) or luminometer (e.g., Turner Biosystems TD-20). See Note 5 for protocol to perform readout of individual samples. 3. White 96-well plates.

3

Methods

3.1 Day 1: Plating of Cells for Transfection

1. If using stably expressing Notch cells, plate cells in a 6 cm dish so that they are 80–90 % confluent the next day, and proceed to Subheading 3.2.1. 2. If transiently transfecting Notch plasmids, plate U2OS cells in a 6 cm dish so that they are 80–90 % confluent the next day (~two million cells at confluency), proceed to Subheading 3.2.2. 3. Optional—plate cells instead in two wells of an Uplift dish to avoid exposing expressed Notch molecules to potentially activating/degrading trypsin and EDTA during co-culture step (see Note 2). 4. See Note 6 for reverse transfection option, which shortens the protocol by a day.

3.2 Day 2: Transfection of Cells with Reporter Plasmids and Notch DNA 3.2.1 Protocol for Stably Expressing Notch Cell Lines/Primary Cells

1. Prepare a 50:1 ratio of firefly: Renilla luciferase plasmids; 500 ng/μl of TP1 or Gal4 firefly luciferase, and 10 ng/μl of pR-TK Renilla luciferase. 2. Allow Lipofectamine 2000 to warm to room temperature and warm Opti-MEM in a 37 °C bath. 3. Add 500 μl of Opti-MEM to a 15 ml conical tube and add 20 μl of Lipofectamine 2000. Mix and let sit for 5 min. 4. While Lipofectamine 2000 is incubating, add 10 μl of reporter mix to 500 μl of Opti-MEM (5 μg firefly luciferase plasmid and 100 ng of Renilla plasmid). 5. Pipette the DNA/Opti-MEM mixture into the Lipofectamine/ Opti-MEM mixture, and mix. Let stand for 20 min. 6. Remove media from 6 cm dish, wash once with PBS. 7. Add 2 ml of Opti-MEM plus 1 ml of liposomes (DNA/OptiMEM/Lipofectamine mix). 8. Change the media back to D10 media after 3–6 h.

3.2.2 Protocol for Transient Transfection of Notch Molecules

1. Follow steps 1–3 above. 2. To Opti-MEM and reporter mix DNA (step 4 above), also add 25–250 ng of Notch plasmid. 3. Continue preparation of liposomes and transfection as above (steps 5–8).

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3.3 Day 2: Plating of Ligand-Expressing Cells

1. Determine how many conditions are to be tested, and plate cells expressing ligand as well as parental cell control for each condition in triplicate. Useful controls include a gammasecretase inhibitor positive control, and a nonspecific isotypematched immunoglobulin (Ig) control for the antibody at each concentration used. 2. Plate ligand-expressing cells in a 96-well plate so that they are 80–90 % confluent the next day. See Note 3 for alternative strategy using plated recombinant ligand.

3.4 Day 3: Co-culture with Ligand and Addition of Antibodies and Drugs

1. In this step, you will resuspend Notch-expressing cells from Subheading 3.2, add appropriate antibody or drug, and replate cells on top of the ligand-expressing cells in 96-well plates from Subheading 3.3. 2. Remove media from 6 cm dish of Notch-expressing cells transfected with reporter plasmids. 3. Wash cells with PBS. 4. Add 300 μl trypsin–EDTA and rotate plate to ensure that trypsin covers entire surface of plate. See Note 2 for an alternative strategy to using trypsin–EDTA, which may degrade/activate Notch molecules expressed on the cell surface. 5. Place at 37 °C for 3 min. 6. Tap cells against side of hood to help dislodge cells, add 3.7 ml D10 media. Resuspend cells and transfer to 15 ml conical tube. Add 2.8 ml of additional D10 media to tube to bring the volume to 6.8 ml; mix cells. The total volume of 6.8 ml is calculated based on the addition of 70 μl of cells into each well of a 96-well plate in steps 7–9 below. 7. Now the resuspended cells from step 6 will be aliquoted into separate tubes so that modulatory antibody and IgG controls can be added. Since Notch cells will be plated in triplicate on ligand-expressing cells and parental control, aliquot 6 × 70 μl = 420 μl of cells for each concentration of antibody and IgG control to be tested. A reasonable starting concentration for antibodies to be tested is 10 μg/ml, which may need to be adjusted depending on the affinity of the antibody for Notch and whether a dose response is to be performed. 8. It may also be helpful to treat an aliquot of cells with a gammasecretase inhibitor, a positive inhibitor control, to compare the potency of modulatory antibodies with a well-characterized small molecule inhibitor. Add compound E to a final concentration of 1 μM, or DAPT to a final concentration of 10 μM. 9. Finally, after removing media from 96-well plate containing ligand-expressing cells, add 70 μl of Notch cells in medium containing antibodies/drugs to appropriate wells.

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3.5 Day 4: Luciferase Assay

331

1. Remove media from 96-well plates containing co-cultured cells. 2. Wash each well with 100 μl PBS. 3. Add 20 μl of 1× passive lysis buffer to each well. 1× passive lysis buffer is prepared by diluting 5× buffer from Promega kit with doubly distilled water. 4. Place plate on a rocker for 30 min. 5. Pipette 5 μl of lysate from each well into a white 96-well luminescence plate. 6. Prepare firefly luciferase substrate solution and Renilla luciferase substrate solution in glass test tubes. 7. Use dual injection luminometer, such as Turner Biosystems Modulus Microplate (see Note 5 for protocol to sequentially measure individual samples in a luminometer such as Turner TD-20), to add 50 μl per well of firefly luciferase substrate followed by 25 μl of Renilla substrate, which also contains a firefly luciferase quenching reagent. 8. Normalize the firefly luciferase activity with Renilla luciferase activity to calculate the Notch activity (Fig. 2).

4

Notes 1. Other readouts of modulation by Notch antibodies include cell growth (e.g., by readouts such as CellTiter Blue or CellTiter Glo assays), expression of Notch target genes (e.g., by RT-PCR), and direct measurement of Notch activation using antibodies that are specific for neoepitopes at the N-terminus of NICD created by gamma-secretase cleavage. Commercial antibodies for measurement of activated Notch1, typically on western blots, are currently available through Cell Signaling Technologies. 2. If transiently transfecting Notch molecules or if a Notchexpressing cell line is not inducible, alternative strategies to trypsin-EDTA may be used to avoid activation/proteolysis of cell surface Notch molecules. Uplift plates (Thermo Scientific) have a temperature sensitive coating that allows cells to adhere at 37 °C and to detach after ~15 min at room temperature. The cells often come off in clumps, so it may be necessary to disperse the cells by passing them through a 32 G needle. Another alternative to avoid trypsin–EDTA treatment of Notch-expressing cells is to plate the ligand-expressing cells on top of the Notch-expressing cells. 3. An alternative to using ligand-expressing cells is to use plated ligand in the co-culture assay [25]. The recombinant ligands Dll4, Dll1, and others can be purchased from R & D Systems. In

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this protocol, the ligand is prepared at 10 μg/ml in PBS. 50 μl of this solution is then plated in each well of a tissue culture treated 96-well plate. Incubate plates at 4 °C overnight. Alternatively, ligand can be incubated for 2–3 h at 37 °C. Wash wells with 100 μl PBS before adding cells. As a control for plated ligand, prepare IgG control, also at 10 μg/ml in PBS. The concentration of ligand can be varied to tune Notch signaling levels. 4. Other types of luciferase kits are available from Promega and other manufacturers that do not require lysis of cells, that readout luminescence at two different wavelengths, etc. Other control reporter systems, such as β-galactosidase, can be used as internal controls (see Chapter 11). Alternatively, cell lines that stably integrate reporter plasmids may also be used. 5. If a dual-injector plate reader luminometer is not available, the luciferase assay can be performed on individual samples in a standard luminometer by sequentially adding luciferin followed by coelenterazine per the Promega protocol. 6. The protocol can be shortened by a day by using the reverse transfection method. In this method, which requires twice as many cells, cells are not plated the day before transfection. Instead, they are plated in Opti-MEM with the Opti-MEM/ DNA/Lipofectamine mix. Prepare Lipofectamine/DNA mixtures for transfection into a 6 cm dish as described in Subheading 3.2. Add the 1 ml of Opti-MEM/DNA/ Lipofectamine mix to the 6 cm plate. Next, plate the reporter cells in the 6 cm plate; when using U2OS cells, approximately two million cells should be used. The reporter cells should be collected by centrifugation at 500 × g for 5 min, resuspended in 2 ml of Opti-MEM, and added to the 6 cm plate containing 1 ml of Opti-MEM/DNA/Lipofectamine mixture.

Acknowledgements Supported in part by grants from the NIH (J.C.A.), the Leukemia and Lymphoma Society (J.C.A.), and the AHA (W.R.G.) References 1. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689 2. Gordon WR, Arnett KL, Blacklow SC (2008) The molecular logic of Notch signaling – a structural and biochemical perspective. J Cell Sci 121:3109–3119 3. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233

4. Olsauskas-Kuprys R, Zlobin A, Osipo C (2013) Gamma secretase inhibitors of Notch signaling. Onco Targets Ther 6:943–955 5. Li K, Li Y, Wu W et al (2008) Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of NOTCH3. J Biol Chem 283:8046–8054 6. Aste-Amézaga M, Zhang N, Lineberger JE et al (2010) Characterization of Notch1 antibodies that inhibit signaling of both normal

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8.

9.

10.

11.

12.

13.

14.

15.

16.

and mutated Notch1 receptors. PLoS ONE 5:e9094 Wu Y, Cain-Hom C, Choy L et al (2010) Therapeutic antibody targeting of individual Notch receptors. Nature 464:1052–1057 Falk R, Falk A, Dyson MR et al (2012) Generation of anti-Notch antibodies and their application in blocking Notch signalling in neural stem cells. Methods 58:69–78 Agnusdei V, Minuzzo S, Frasson C et al (2013) Therapeutic antibody targeting of Notch1 in T-acute lymphoblastic leukemia xenografts. Leukemia. doi:10.1038/leu.2013.183 Qiu M, Peng Q, Jiang I et al (2013) Specific inhibition of Notch1 signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells. Cancer Lett 328:261–270 Gordon WR, Vardar-Ulu D, Histen G et al (2007) Structural basis for autoinhibition of Notch. Nat Struct Mol Biol 14:295–300 Li K, Wu W, Gordon WR et al (2008) Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of NOTCH3. J Biol Chem 283: 8046–8054 Gordon WR, Roy M, Vardar-Ulu D et al (2009) Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL. Blood 113:4381–4390 Gordon WR, Vardar-Ulu D, L’Heureux S et al (2009) Effects of S1 cleavage on the structure, surface export, and signaling activity of human Notch1 and Notch2. PLoS ONE 4:e6613 Weng AP, Ferrando AA, Lee W et al (2004) Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306:269–271 Tiyanont K, Wales TE, Siebel CW et al (2013) Insights into Notch3 activation and inhibition

17.

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22.

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25.

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mediated by antibodies directed against its negative regulatory region. J Mol Biol 425: 3192–3204 Aster JC, Xu L, Karnell FG et al (2000) Essential roles for ankyrin repeat and transactivation domains in induction of T-cell leukemia by notch1. Mol Cell Biol 20:7505–7515 Hsieh JJ, Henkel T, Salmon P et al (1996) Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mol Cell Biol 16:952–959 Kato H, Taniguchi Y, Kurooka H et al (1997) Involvement of RBP-J in biological functions of mouse Notch1 and its derivatives. Development 124:4133–4141 Wallberg AE, Pedersen K, Lendahl U et al (2002) p300 and PCAF act cooperatively to mediate transcriptional activation from chromatin templates by notch intracellular domains in vitro. Mol Cell Biol 22:7812–7819 Wang Y, O’Malley BW, Tsai SY et al (1994) A regulatory system for use in gene transfer. Proc Natl Acad Sci U S A 91:8180–8184 Luo B, Aster JC, Hasserjian RP et al (1997) Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor. Mol Cell Biol 17:6057–6067 Sarmento LM, Huang H, Limon A et al (2005) Notch1 modulates timing of G1-S progression by inducing SKP2 transcription and p27 Kip1 degradation. J Exp Med 202:157–168 Schmitt TM, Zúñiga-Pflücker JC (2002) Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17:749–756 Delaney C, Varnum-Finney B, Aoyama K et al (2005) Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 106:2693–2699

Chapter 25 Application of Anti-Ligand Antibodies to Inhibit Notch Signaling Jun-ichiro Koga and Masanori Aikawa Abstract Emerging evidence suggests that Notch signaling not only regulates biological processes during development but also participates in the pathogenesis of various diseases in adults, including tumor angiogenesis, hematopoietic malignancies, and cardiometabolic syndromes. Notch signaling involves several ligands and receptors that have unique and overlapping functions. Therefore, blocking function of a ligand or receptor with a neutralizing antibody is a useful approach to examine the specific role of each Notch component. In addition, administration of Notch signaling blocking antibodies in experimental animals offers important insights into clinical translation of Notch biology. In this chapter, we describe examples of in vitro and in vivo loss-offunction experiments with blockade of Notch ligands, particularly Delta-like ligand 4 (Dll4). Key words Notch ligand, Blocking antibody, Delta-like ligand 4, Luciferase assay, Nucleofection

1

Introduction Blocking antibodies are useful tools for exploring the diverse and complex functions of Notch ligands both in vitro and in vivo. Mammalian Notch ligands consist of three Delta-like ligands (Dll1, Dll3, Dll4) and two Jagged ligands (Jagged1, Jagged2) [1, 2]. These ligands constitute the transmembrane Delta/Serrate/lag-2 (DSL) protein family, which activates canonical Notch signaling in adjacent cells in a juxtacrine fashion (trans-activation) [3]. The extracellular domain of each Notch ligand is comprised of the N-terminal (NT) domain followed by the DSL domain and epidermal growth factor (EGF)-like repeats [1]. The DSL domain is required for ligand–receptor binding [4]. After binding to ligands and undergoing subsequent conformational changes, Notch receptors are cleaved by ADAM (a disintegrin and metalloproteinase) proteases and members of the γ-secretase complex [5, 6]. The Notch intracellular domain (NICD) then enters the nucleus and forms a transcriptional activation complex composed of CSL (also known as RBP-Jκ [7]) and Mastermind-like 1 (MAML1) [3].

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4_25, © Springer Science+Business Media New York 2014

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Table 1 Blocking antibodies of Notch ligands Target

Host

Reactivity

In clinical trial

Company

Reference

Dll1

Ms

Hu

Dll4

Ms, Hu

Hu

Jagged1

Ms

Hu

R&D Systems

Jagged2

Ms

Hu

BioLegend

Dll1

Hm

Ms

Bio X Cell, BioLegend

[14]

Dll4

Hm

Ms

Bio X Cell, BioLegend

[13, 14]

Jagged1

Hm

Ms

BioLegend

[14]

Jagged2

Hm

Ms

Bio X Cell

[14]

BioLegend Phase I

BioLegend, Genentech, Regeneron, Oncomed

Hu Human, Hm Hamster, Ms mouse

The clinical significance of Notch ligands has been established in tumor angiogenesis [8–11] and T cell biology. We reported that Notch signaling, triggered by Dll4, participates in macrophage activation and the shared mechanism for cardiometabolic disorders [12, 13]. Several clinical trials on anti-Notch ligand antibodies are currently in progress. For basic research, several blocking antibodies for Notch ligands are currently available (Table 1). Moriyama et al. generated anti-Jagged1, anti-Jagged2, anti-Dll1, and antiDll4 antibodies for mouse and human ligands [14]. Ridgway et al. made a neutralizing antibody YW152F (Genentech) that targets both human and mouse Dll4 [9]. An anti-human Dll4 antibody Enoticumab (REGN421, Regeneron) is already in clinical trials for advanced malignancies. Another anti-human Dll4 antibody Demcizumab (OMP-21 M18, Oncomed) is also in phase I clinical trials in patients with advanced solid tumors. While Notch ligand targeting therapies for cancer are being translated to clinical settings, other applications remain unexplored. We will describe experimental methods in cultured cells and in mice to manipulate Notch ligand activity using specific blocking antibodies. We will mainly focus on Dll4, but similar methods can be applied to other anti-Notch ligand antibodies.

2

Materials

2.1 Cell Lines and Culture Reagents

1. 3 T3-L1 cells and human umbilical endothelial cells (HUVECs) can be obtained from public sources such as ATCC (American Type Culture Collection). Growth media for 3T3-L1 is based on Dulbecco’s Modified Eagle Medium (DMEM)

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(e.g., LONZA) and contains 10 % fetal bovine serum (FBS), 1 % pyruvate, and 1 % penicillin/streptomycin. Growth media for HUVEC is based on M199 (e.g., LONZA) and contains 20 % FBS, 10 μg/mL heparin, 100 μg/mL ECGS (Endothelial Cell Growth Supplement) (e.g., Biomedical Technologies), 1 % penicillin/streptomycin. 2. PBS, pH 7.4. 3. Trypsin–EDTA. 4. Cell culture flasks (e.g., T75 (75 cm2) flask) and plates (e.g., 6 well, 24 well, 96 well). 2.2 CSL Luciferase Assay

1. RBP-Jκ (CSL) Reporter (SABiosciences). 2. Nucleofector (LONZA), Cell Line Nucleofector Kit L (LONZA) (see Note 1). 3. Dual-Luciferase Reporter Assay System (e.g., Promega). 4. Recombinant Dll4 solution: Reconstitute 1 vial (50 μg) of mouse recombinant Dll4 (rDll4, R&D Systems) with 250 μL sterile PBS to prepare 200 μg/mL stock solution. Aliquot should be kept in −20 °C freezer. Immediately before use, dilute 5 μL stock solution with 995 μL sterile PBS to prepare 1 μg/mL rDll4 solution.

2.3 Loss-of-Function Experiments

1. Anti-Notch ligand antibodies (Table 1). Dll4; anti-mouse (HMD4-1) (Bio X cells, BioLegend), antihuman (MHD4-46) (BioLegend). Dll1; anti-mouse (Bio X cells, BioLegend), anti-human (BioLegend). Jagged1; anti-human (R&D Systems), Jagged2; anti-mouse (Bio X cells). 2. γ-secretase inhibitor; e.g., DAPT (Calbiochem).

3

Methods

3.1 Notch Activity Assay Using CSL Luciferase Assay in Cultured Cells (See Note 2)

1. Cell preparation. Prepare cells that express Notch receptor(s) or cell lines in which Notch receptor is transfected. We will use 3T3-L1 cells which endogenously express Notch1-4 receptors [15] as an example here. Plate 3T3-L1 cells in T-75 flasks with growth media optimized for 3T3-L1 cells (see Note 3). 2. Transfection of a CSL reporter. Wash cells with PBS and detach them with trypsin–EDTA. Then, transfect a CSL reporter by nucleofection according to the manufacturer’s instructions (see Note 4). After transfection, plate 2 × 106 cells/well on 6-well plates and culture 24 h.

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3. Preparation of immobilized Dll4. On the same day of transfection, aliquot 100 μL/well of mouse rDll4 solution (1 μg/mL) into wells of a 96-well culture plate and keep at 4 °C for 24 h (see Note 5). In control wells, add the same volume of PBS. 4. Notch signal activation in 3T3-L1 cells by rDll4. 24 h after plating the 3T3-L1 cells in 6-well plates, incubate cells with 5 μg/mL of anti-mouse Dll4 antibody (e.g., HMD446) for 1 h. In control group, incubate cells with the same concentration of non-immune IgG of the same isotype. Then, detach the cells with trypsin-EDTA and replate 5 × 104 cells on a 96-well plate pre-coated with rDll4. Culture the cells for further 24 h with culture medium containing 5 μg/mL antimouse Dll4 antibody or isotype IgG. 5. Luciferase assay. Detect firefly and Renilla luciferase activity with a dualluciferase assay system (e.g., Promega) according to the manufacturer’s instructions (see Note 6). 3.2 In Vitro Loss-ofFunction Experiment

1. Preparation of cells. Prepare cells which express Dll4 and Notch receptor. The following protocol is an example of loss-of-function experiments in human umbilical vein endothelial cells (HUVECs), which endogenously express Dll4 as well as Notch1 and Notch4. Trypsinize and plate 1 × 105/well of HUVECs cultured with endothelial growth medium on new 24-well culture dishes. 2. Preincubation with anti-Dll4 antibody. Culture HUVECs until cells grow to 70–80 % confluence (see Note 7) and replace the medium with the new medium containing 5 μg/mL anti-Dll4 antibody [16] (e.g., MHD446) (see Note 8). In control samples, add the same concentration of non-immune IgG of the same isotype. γ-secretase inhibitor (DAPT, 10 μM) could be used as a positive control (see Note 9). 3. Evaluation of the effect of anti-Dll4 antibody treatment. Extract RNA or protein 24 h later to examine the effect of antibody on target molecule. Figure 1 shows the effect of Dll4 blockade on expression of MCP-1 in HUVECs. 4. Evaluating the effect of Dll4 signal blockage in different stimulation conditions. Once the changes upon Dll4 antibody treatment without any stimuli are characterized, HUVECs can be further treated by various stimuli (LPS, oxLDL, flow, etc.) and the effect of Dll4 blockade examined under various conditions.

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Fig. 1 Anti-Dll4 antibody inhibited MCP-1 expression in HUVECs. HUVECs were incubated with anti-human Dll4 antibody (50 μg/mL) for 24 h. MCP-1 expression was quantified by qPCR

3.3 In Vivo Blocking of Dll4

The following procedure is an example of loss-of-function experiments in mice. 1. Preparation of antibody solution. Dilute a stock solution of anti-mouse Dll4 antibody (HMD4-2) with sterile PBS and prepare a 1.0 mg/mL working solution. The dose of injection is 250 μg (=250 μL) of the antibody per mouse [17]. Prepare an isotype control IgG solution of the same concentration for control experiments. 2. Preparation of mouse. Prepare a mouse and disinfect the injection site with 70 % alcohol. As an example, we describe an anti-Dll4 treatment on LDL receptor-deficient (Ldlr−/−) mice fed a high-fat, highcholesterol diet. This is a model system for cardiometabolic syndrome and exhibits atherosclerosis and metabolic disturbances. 3. Antibody injection. Inject 250 μL antibody intraperitoneally in the lower right quadrant of the abdomen. Injection should be performed with needles under 23 G in the Trendelenburg position. 4. Repeat injections. Repeat injection with the same amount of antibody twice a week. 5. Perform blood tests, biopsies, or in vivo imaging depending on the phenotype of interest. Alternatively, sacrifice the mice and perform autopsies for biochemical and molecular biological analyses. Figure 2 shows the effect of anit-Dll4 treatment for 12 weeks on atherogenesis in Ldlr−/− mice fed a high-fat, highcholesterol diet.

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Anti-DII4 Ab 60

*

*

50 40 30 20 10 0

Early

Late

Plaque size, x100 pixel

Stenosis of brachiocephalic artery, %

Control IgG 600

*

500 400 300 200 100 0

Early

Late

Fig. 2 Effect of Dll4 on atherogenesis. Upper panels show longitudinal section of aortic arch stained with hematoxylin and eosin. Lower graphs show atherosclerotic plaque area. Anti-mouse Dll4 antibody (250 μg/ injection) was injected twice a week from 8 weeks of age (early) or 20 weeks of age (late) for 12 weeks. * p < 0.05. (Edited from Fig. 2 of PNAS 2012 [16])

4

Notes 1. Nucleofection enables high efficacy transfection compared to conventional transfection methods including lipofection. 2. Other options to determine Notch signal activation include the detection of cleaved Notch intracellular domain (NICD) and quantification of expression levels of prototypical Notch target genes (Hes1, Hey1, Hey2). See Chapters 11, 12, 17, and 19 for details. 3. Since we are particularly interested in adipocytes, we allow 3 T3-L1 cells to differentiate into adipocytes by culturing these cells in a medium containing dexamethasone and/or insulin for 10 days. See [18] for details. 4. Antibiotics should be removed from the culture medium for transfection. Culture medium can be exchanged after 6 h incubation. Nucleofection can be replaced with transfection using a transfection reagent (e.g., Lipofectamine) after plating the cells on 6-well plates.

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5. We coat culture dishes with rDll4 (“immobilization”) and plate the Notch receptor expressing cells to activate Notch signaling. Alternatively, one can co-culture ligand expressing cells with Notch expressing cells. See Chapter 11 for details. 6. We are using reporter which is premixed with constitutively expressing Renilla luciferase, which serves as an internal control for normalizing transfection efficacies and cell viability. 7. Wait until cells reach at least 70–80 % confluence. Cell–cell contact is required for ligand–receptor interaction. Check under the microscope whether there are enough contacts between cells. Co-culture of HUVECs with other cells which express Dll4 (we have used mouse stromal cells in which human Dll4 is stably transfected (MS5-Dll4) [12]) is another method to activate Dll4/Notch signaling. 8. Keep this antibody undiluted at 4 °C in the dark. If the antibody is to be stored for more than 6 months, make aliquots and keep them at −20 °C. Avoid freeze/thaw cycles and prepare solution immediately before use. For mouse cells in general, we use anti-mouse Dll4 antibody (HMD4-2) at 50 μg/mL. 9. γ-secretase inhibitors can block all Notch activation. Since anti-Dll4 antibody treatment is specific for mDll4-mediated signaling, the effect of γ-secretase inhibitor treatment and antiDll4 antibody treatment may be different if cells can activate Notch by other ligands (Dll1, Jag1, and/or Jag2) or in a ligand-independent manner.

Acknowledgements This work was supported in part by a National Institutes of Health grant R01HL107550 to M.A. References 1. D’Souza B, Meloty-Kapella L, Weinmaster G (2010) Canonical and non-canonical Notch ligands. Curr Top Dev Biol 92:73–129 2. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233 3. Kovall RA, Blacklow SC (2010) Mechanistic insights into Notch receptor signaling from structural and biochemical studies. Curr Top Dev Biol 92:31–71 4. Cordle J, Johnson S, Tay JZ et al (2008) A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cisinhibition. Nat Struct Mol Biol 15:849–857

5. Six E, Ndiaye D, Laabi Y et al (2003) The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and gamma-secretase. Proc Natl Acad Sci U S A 100:7638–7643 6. Parr-Sturgess CA, Rushton DJ, Parkin ET (2010) Ectodomain shedding of the Notch ligand Jagged1 is mediated by ADAM17, but is not a lipid-raft-associated event. Biochem J 432:283–294 7. Jarriault S, Brou C, Logeat F et al (1995) Signalling downstream of activated mammalian Notch. Nature 377:355–358 8. Noguera-Troise I, Daly C, Papadopoulos NJ et al (2006) Blockade of Dll4 inhibits tumour

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11.

12.

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Jun-ichiro Koga and Masanori Aikawa growth by promoting non-productive angiogenesis. Nature 444:1032–1037 Ridgway J, Zhang G, Wu Y et al (2006) Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 444:1083–1087 Real C, Remédio L, Caiado F et al (2011) Bone marrow-derived endothelial progenitors expressing Delta-like 4 (Dll4) regulate tumor angiogenesis. PLoS One 6:e18323 Jenkins DW, Ross S, Veldman-Jones M et al (2012) MEDI0639: A novel therapeutic antibody targeting Dll4 modulates endothelial cell function and angiogenesis in vivo. Mol Cancer Ther 11:1650–1660 Fung E, Tung SM, Canner JP et al (2007) Delta-like 4 induces Notch signaling in macrophages: Implications for inflammation circulation 115:2948–2956 Fukuda D, Aikawa E, Swirski FK et al (2012) Notch ligand Delta-like 4 blockade attenuates atherosclerosis and metabolic disorders. Proc Natl Acad Sci U S A 109:E1868–E1877

14. Moriyama Y, Sekine C, Koyanagi A et al (2008) Delta-like 1 is essential for the maintenance of marginal zone B cells in normal mice but not in autoimmune mice. Int Immunol 20:763–773 15. Lai PY, Tsai CB, Tseng MJ (2013) Active form Notch4 promotes the proliferation and differentiation of 3 T3-L1 preadipocytes. Biochem Biophys Res Commun 430:1132–1139 16. Yamanda S, Ebihara S, Asada M et al (2009) Role of ephrinB2 in nonproductive angiogenesis induced by Delta-like 4 blockade. Blood 113: 3631–3639 17. Oishi H, Sunamura M, Egawa S et al (2010) Blockade of delta-like ligand 4 signaling inhibits both growth and angiogenesis of pancreatic cancer. Pancreas 39:897–903 18. Hemati N, Ross SE, Erickson RL et al (1997) Signaling pathways through which insulin regulates CCAAT/enhancer binding protein alpha (C/EBPalpha) phosphorylation and gene expression in 3 T3-L1 adipocytes. Correlation with GLUT4 gene expression. J Biol Chem 272:25913–25919

INDEX A Abruptex.........................................................................6, 45 Actin cytoskeleton (F-actin) ................................... 68, 69, 72 Adeno-associated virus (AAV) ................. 118, 120, 127, 128 A disintegrin and metalloproteinase (ADAM) ....................... 5, 7, 9, 87, 323, 326, 335 ADAM10 ...................................................................311 Affinity purification................................. 182, 183, 186–188, 190, 281 After Puparium Formation (APF) .....................................81 Alkaline lysis ................................................................35, 37 Alkaline phosphatase (AP) ....................................... 278–283 Allosteric inhibitor ...................................................315, 316 Alzheimer’s disease (AD) ..................... 2, 223, 312, 314–316 American Type Culture Collection (ATCC) ....................336 Amino acid .......................................56, 65, 66, 88, 194, 196, 201, 203, 205, 210, 220, 224, 232 Ammonium Persulfate (APS)................................... 235, 237 Ampicillin resistance (ApR) .................................... 32–38, 44 Amyloid precursor protein (APP) ............ 248, 312, 314, 316 Aβ40 ...........................................................................315 Aβ42 ...........................................................................315 β-APP ........................................................................312 Analytical ultracentrifugation ...........................................233 Anesthetizer .......................................................................19 Ankyrin repeat (ANK) ..........................7, 157, 231, 232, 243 Antibody activating .................................................... 324–326, 331 blocking ..............................................................335, 336 high affinity ..................................................................88 inhibitory ............................................................324–326 low affinity ..........................................................102, 110 monoclonal ........................................10, 83, 89, 104, 194 polyclonal......................................................................69 primary ...........................................63, 69, 70, 72, 75, 76, 80, 83, 97, 106–108, 110, 111, 227 secondary/fluorescent conjugated .......................63, 69, 70, 72, 74, 80, 83, 89, 97, 106, 107, 109, 111, 224, 227 uptake ...........................................................................95 uptake assay .................................... 79–85, 88–90, 95–97 Apoptosis.................................................... 18, 252, 317, 319 Arylsulfonamides ..............................................................316 Aspartyl protease ..............................................................314 Assay binding ....................................................... 193, 277–283 cell-based ............................................................225, 318

dual-luciferase......................138, 150, 325–327, 337, 338 endocytosis ...................................................................80 GSI-washout ......................................................247–252 luciferase .............................................134, 135, 138, 140, 148, 150, 151, 325–329, 331, 332, 337–338 Asymmetric cell division ....................................................79 pIIa/pIIb.................................................................79, 84

B Bacterial artificial chromosome (BAC)........................ 32, 34, 35, 55, 81, 85 Bacterial attachment site (attB) .............................. 33, 34, 38 Balancer .................................................................. 22, 24, 42 Basic Helix Loop Helix (bHLH) ..................... 102–104, 110 BDGP. See Berkeley Drosophila Genome Project (BDGP) BDSC. See Bloomington Drosophila Stock Center (BDSC) Bearded/bearded family ....................................................102 Benzoazepinone ...............................................................315 Berkeley Drosophila Genome Project (BDGP) .......................................... 182, 184, 190 βME. See β-Mercaptoethanol (βME) bHLH. See Basic Helix Loop Helix (bHLH) Binding ......................................... 6–9, 49, 54, 55, 57, 81, 85, 87, 88, 92, 98, 102, 103, 110, 111, 117, 133, 134, 144–146, 155–157, 190, 193–195, 203–206, 209, 231–235, 237, 238, 242, 248, 256, 257, 263, 269, 270, 274, 277–283, 315, 316, 323–326, 328, 335 Bioinformatics/bioinformatic analysis ................. 9, 182, 184, 188–189, 206, 266, 267, 274–275 Bioluminescence ....................................... 159–165, 170–177 Biotin/biotinylation/biotin-avidin ......................... 88–94, 98, 196, 200, 207, 233 Bi-transgenic Notch1 reporter ...........................................55 Blood–brain barrier ..........................................................325 Bloomington Drosophila Stock Center (BDSC) ........................................ 19, 23, 33, 107 Bovine serum albumin (BSA)........................ 69, 70, 90, 107, 177, 212, 220, 226, 229, 235, 280–282 BTD. See β-Trefoil domain (BTD)...........................231, 232

C Caenorhabditis elegans ............................................ 2, 3, 9, 312 Calcium/Ca2+ binding ............................................... 195, 203, 204, 206 chelation ......................................147, 149, 152, 159, 165

Hugo J. Bellen and Shinya Yamamoto (eds.), Notch Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1187, DOI 10.1007/978-1-4939-1139-4, © Springer Science+Business Media New York 2014

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NOTCH SIGNALING: METHODS AND PROTOCOLS 344 Index Calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)................................. 152, 166, 204 ethylene diamine tetraacetic acid (EDTA) .................. 131, 132, 134, 136–137, 139, 140, 152, 166, 172, 174–175, 183, 195, 196, 198, 206, 234, 236, 238, 240, 242, 259–262, 265, 329 ethylene glycol tetraacetic acid (EGTA) ............ 131, 132, 134, 136–137, 139, 140, 147, 149, 152, 159, 164–166, 197, 200, 201, 207, 228, 259–261 Carboxy-terminus (C-terminus) .......................... 7, 205, 224 Carcinogenesis ..................................................................255 cbEGF...............................................195, 200, 201, 203, 204 CBF1 ...................................................54, 115–117, 127, 324 Cell culture media CCM3 ................................................................279–281 DMEM/F12 .......................171, 172, 174–176, 225–228 Dulbecco’s modified Eagle’s medium (DMEM) ................................88, 91, 93–95, 158, 159, 171–176, 225–228, 328, 336 M3 complete medium ........................................279–281 N2 and B27 medium .................................. 172, 175, 176 Opti-MEM ......... 135, 137, 138, 140, 212, 213, 329, 332 T6E .................................................................... 249, 250 Cell death ............................................................. 3, 140, 155 Cell fate decisions .................................................................3 Cell lethal (cl) ............................................................... 19, 22 Cell lines CHO-Dll1-IRES-GFP (hamster) ..................... 147, 159 CHO-Jag1-IRES-GFP (hamster) .............................159 CHO-Notch2 (hamster) ............................................147 HEK/HEC293T/293T (human) ...................... 147, 151, 166, 224–228 human umbilical endothelial cells (human) (HUVEC) .............................................. 336, 337 Kc167 (Drosophila) .............................................. 134, 189 ligand expressing cells .......................... 88, 133, 145, 147, 149, 159, 326, 328, 330, 331, 341 murine embryonic fibroblast/primary fibroblast (mouse) ......................................... 88, 89, 96, 124 OP9 (mouse) ................................................ 88, 327, 328 OP9-delta (mouse) .....................................................252 receptor expressing cells ........................................55, 341 Schneider 2 (Drosophila) ............................................. 133 S2R + (Drosophila) .............................. 182, 184, 185, 189 3T3 (mouse) ....................................... 146, 151, 318, 328 T6E (human)...................................................... 249–251 3T3-L1 (mouse) .........................................................145 U2OS (human) ..................................... 88, 328, 329, 332 Cell surface ........................ 3, 5, 6, 8, 57, 85, 87–95, 330, 331 Cell-to-cell communication .............................................115 Central nervous system (CNS) ................................. 110, 316 Charge-coupled device (CCD)................................. 167, 170 Chemiluminescence..........................................................233 Chimeric Notch receptor..........................................146, 158

Chromatin ...........................................7, 9, 10, 248, 255–258 Chromatin immunoprecipitation (ChIP) ChIP-Seq ................................................... 257, 266–275 cross-linking ChIP ..................... 256, 257, 262–264, 272 native ChIP .........................256, 257, 264–266, 272, 273 Chromosome ........................................17–19, 22, 24, 25, 35, 39–42, 44, 51, 55, 81, 106, 107, 268 Circular dichroism ............................................................233 Cis-interactions............................................. 8, 286, 300, 303 Cistrome ............................................257, 266–272, 274, 275 Cleavage S1................................................................................5, 6 S2........................................................ 5–9, 224, 311, 312 S3......................................... 5–8, 224, 225, 248, 249, 311 Clinical trial ...................................................... 223, 315, 336 Coactivators CREB-binding protein (CBP) ...................................232 E1A binding protein p300 .........................................232 Co-culture ..........145, 149, 150, 161, 165, 327, 329–331, 341 Collier model ............................................................287–307 Collision induced dissociation (CID) ............... 211, 216–219 Complementation imaging .......................................155–168 Conditional .........................................33, 35, 47–53, 56, 325 Conditionally amplifiable origin of replication (oriV)........33 Conditional mutant mice gain-of-function .....................................................48–54 loss-of-function ......................................................48–54 Conditioned medium ................151, 164, 166, 167, 279–283 Confluens ................................... 39–41, 45, 95, 98, 137, 140, 160, 161, 165, 178, 185, 213, 226, 228, 279–281, 329, 330, 338, 341 Confocal microscopy ........................................ 68, 74, 83, 97 Conformational change ............................................5–8, 335 Context dependent ............................................. 23, 115, 247 Context specific ........................................................ 2, 9, 247 Cooperativity .................................................... 290, 292, 303 Copy control solution ............................................. 32, 35, 37 Copy number variation (CNV) ..........................................10 Corepressors CIR.............................................................................232 KyoT2/FHL1 ..................................................... 232, 256 MINT/SHARP .........................................................232 SKIP ...........................................................................232 SMRT ........................................................................232 Cosmid ...............................................................................30 Cre driver .....................................................................49–51 Cre-Lox system ......................................................48–50, 53 Cre recombinase ............................................... 48–50, 55, 56 Cre recombinase-estrogen receptor fusion protein .............53 Cre recombinase fused to mutant estrogen ligand-binding domain (CreERT2) .................................... 55, 56 CSL CBF1/suppressor of hairless/Lag-1 binding site ..........................144–146, 257, 270, 325, 326 N-terminal Rel-homology region (NTD) .......... 231, 232 C-terminal Binding Protein (CtBP) ....................................7

NOTCH SIGNALING: METHODS AND PROTOCOLS 345 Index C-terminal Rel-homology region (CTD) ................ 231, 232 Cultured cells. See Cell lines CuSO4 induction ..............................................................182 Cut .................................... 44, 66, 71, 83, 101, 102, 104, 173 Cyclin-dependent kinase-8 (CDK8) ....................................7 Cycloheximide .......................................... 249, 250, 252, 270

D Deacetylases .................................................................7, 256 Decoy ...................................................................................7 Degradation................................ 6–10, 88, 91, 92, 95–97, 99, 110, 111, 117, 207, 287, 288, 290, 291, 295 Delta/serrate/LAG-2 (DSL) delta (Dl) ....................................... 65, 80, 82, 83, 85, 282 delta-like ligand 1 (Dll1) ....................... 6, 49, 88–90, 92, 98, 147, 159, 323, 327, 328, 331, 335–337, 341 delta-like ligand 3 (Dll3) .................................... 6, 7, 335 delta-like ligand 4 (Dll4) ......................... 6, 49, 323, 326, 328, 331, 335–341 domain........................................................................335 Development ...................... 1–4, 8–10, 15, 18, 23, 41, 43, 47, 49, 52, 54, 56, 57, 59, 66, 68, 69, 71, 75, 79, 80, 89, 95, 101, 102, 104, 106, 110, 115, 156, 167, 181, 223, 238, 247, 248, 285, 312, 314, 316, 324 Developmental Studies Hybridoma Bank (DSHB) .............................65–70, 80, 89, 95, 104 4',6-Diamidino-2-phenylindole (DAPI) ............................69 1,4-Diazabicyclo[2.2.2]octane (DABCO) ............. 70, 75, 90 Diethylpyrocarbonate (DEPC) ........................................135 Differentiation ........................... 3, 18, 56, 169, 255, 285, 307 Diseases angiogenesis ........................................................318, 336 cancer..............3, 8, 47, 184, 223, 312, 314–319, 325, 336 carcinogenesis .............................................................225 cardiometabolic syndromes .........................................339 congenital disorders ........................................................1 hematopoietic malignancies ........................................335 hereditary......................................................................47 idiopathic ......................................................................47 neurological ....................................................................2 non-small cell lung carcinoma (NSCLC) ...................319 psychiatric .......................................................................2 solid tumors ................................................ 315–317, 336 stroke ..............................................................................1 T cell acute lymphoblastic leukemia (T-ALL) ...... 9, 248, 256, 316–318, 323–325 tumor ......................... 2, 56, 315, 316, 318, 319, 325, 336 Dithiothreitol (DTT) ......................... 89, 148, 151, 194, 198, 200, 206, 212, 234, 235, 238–242, 259–261, 273 DNA-binding domain..............................................144, 146 DNA labeling biotinylated DNA .......................................................233 chemiluminescent detection .......................................233 fluorescently labeled DNA .........................................233

intercalating fluorescent dyes ......................................233 radioactive labeling, isotopic DNA labeling ...............233 DNA methylation ............................................................255 Docking site ................................32, 34, 38–41, 45, 314, 315 Domain-swap ...............................................................56–57 Dominant negative ......................49, 106, 107, 117, 145, 147 Dominant negative MAML (DNMAML). See Mastermind-like (MAML) Dorsal-ventral boundary...........................................101, 105 Dosage-sensitive .............................................................4, 30 Doxycycline (Dox) ............................................ 158, 165, 167 Drosophila development adult...........................................40–43, 45, 101, 103, 104 bristle ......................................................................79, 81 embryo .............................................................. 18, 38, 42 larva .................................................................. 43, 64, 81 metamorphosis .......................................................79, 81 pupa ............................................................ 43, 45, 81–85 Drosophila melanogaster.............................. 15, 30, 63–76, 318 Drug ...................9, 10, 50, 145, 248, 249, 312, 316, 328, 330 Drug target .......................................................................2, 9 Dual-luciferase assay ........................................ 138, 326, 338

E EDTA/EGTA treatment.................. 132, 136–137, 139, 140 EEA1 .................................................................................95 EGF domain-specific O-linked N-acetylglucosamine (GlcNAc) transferase (EOGT).......................210 EGF repeats. See Epidermal growth factor like repeats (EGFr) Electrophoretic mobility shift assay (EMSA)........... 231–243 Electrospray ionisation mass spectrometry (ESI-MS)......207 E3 ligase ...............................................................................6 Embryo...................................................1, 18, 38, 42, 52, 55, 58, 104, 118–119, 121–122, 124, 125, 127, 170–178, 189 Embryogenesis ...................................................................48 Embryonic stem cell (ES cell) ...................................... 48, 57 Endocytosis/endocytic. See also Vesicle/vesicular trafficking trans-endocytosis ....................................................5, 6, 8 Endogenous Notch responsive genes................ 102, 232, 255 Endolysosomal pathway .......................................................8 Endoplasmic reticulum (ER) ............................................4–6 Endothelial Cell Growth Supplement (ECGS) ...............337 Enhancer .................................... 16, 30, 45, 48, 81, 103, 104, 131, 133, 134, 137, 140, 257, 270, 312 Enhancer of Split (E(spl))............................ 2, 102–105, 110, 132, 134, 135, 139, 248 Epidermal growth factor-like (EGF) ...................... 4, 31, 32, 34, 36, 38, 40, 69, 80, 88, 89, 95–97, 171, 172, 194, 201, 206, 210–212, 216–219, 281, 283, 304, 335 Epidermal growth factor like repeats (EGFr) ............ 4–6, 31, 32, 34, 36–38, 40, 69, 80, 95, 210–212, 216–219 Epigenetic(s) ............................................................ 255–275

NOTCH SIGNALING: METHODS AND PROTOCOLS 346 Index Escherichia coli BL21................................................................... 197, 205 EPI300 ...................................................................35, 37 SW102 ...................................................................35–37 Ethyl methanesulfonate (EMS)....................................15–25 mutagenesis ............................................................16–22 European Conditional Mouse Mutagenesis Program (EUCOMM) ....................................................49 Exocytosis/exocitic. See Vesicle/vesicular trafficking Experimental approaches..................................................233 Extracellular ....................2, 4, 6–8, 32, 57, 63, 65, 69, 80, 89, 107, 159, 209, 210, 224, 248, 278, 281, 311, 335 Extracellular domain (ECD) ................................. 4, 6–8, 32, 57, 63, 69, 80, 89, 107, 159, 209–213, 224, 248, 278, 281, 335 Extracted ion chromatograms (EIC) ................ 211, 217–219 Ex vivo ..........................................................................54, 80

F Fast protein liquid chromatography (FPLC) ....................239 Fate mapping ................................................ 55–56, 116, 117 Filopodia .......................................................... 286, 303, 307 Fine-tune .................................................................... 4, 9, 10 Fixation ............................. 68–72, 75, 80, 82, 83, 89, 96, 122 Flippase (FLP) ............................................... 17–19, 48, 106 Flippase recognition target (FRT) ....... 17–19, 48, 106, 161, 167 Flow cytometry...........................................................54, 205 Floxed, flox ...........................................................49–51, 128 FLP/FRT system ...............................................................17 Fluorescent proteins green (GFP) EGFP ...............................54, 58, 103, 105, 116–118, 121, 122, 127, 128, 145, 189 emGFP ...................................................................54 red (RFP) DsRed...................................................................121 mCherry ....................................... 103, 105, 106, 145 tomato ....................................................................56 yellow (YFP) .......................................................... 53, 58 venus ..........................................54, 58, 103, 105, 106 Fly/flies. See Drosophila melanogaster Fringe Lunatic ...........................................................................4 Manic .............................................................................4 Radical ............................................................................4 F1 screen ............................................................................18 Furin-like convertase ........................................................311 Furin-like protease............................................................5, 6

G Gain-of-function ................... 16, 29, 39, 45, 48–54, 248, 324 β-Galactosidase ..................... 54, 56, 106, 111, 150, 153, 332 Gal80ts .............................................................................. 106 GAL4/UAS binary expression system................................16 Gal4VP16 transcriptional activator (Gal4VP16)................55

Gamma secretase (γ-secretase) activity ............................................................................2 anterior pharynx 1 A/B (Aph1A/B) ...........................312 inhibitor (see γ-Secretase inhibitor (GSI)) nicastrin (NCT)..........................................................312 presenilin 1/2 (PS1/2) ................................................312 presenilin enhancer 2 (Pen2) ......................................312 Gap-repair mutagenesis ................................................34, 36 Gastrointestinal toxicity. See Intestinal/gastrointestinal/ gut toxicity Gel electrophoresis (DNA) ........................ 33, 233, 234, 272 Gel electrophoresis (protein) ......................... 93, 94, 98, 183, 199–201, 206, 207, 214, 220, 225 native (non-denaturing)..............................................233 Gene synthesis ..............................................................36–38 Genetic interactions ........................................... 39, 181, 191 Genetic screen .................................2, 9, 15–25, 31, 181, 312 Genome ............... 9, 16, 17, 20, 22–24, 31–34, 44, 48, 49, 51, 57–59, 181, 182, 184, 257, 266–269, 274, 275, 312 Genomic rescue transgene, genomic transgene.............29–45 glp-1 .....................................................................................2 Glucocorticoid ..........................................................317, 319 Glycan ...................................................... 209–211, 216–219 Glycosylation ...........................................8, 31, 194, 209, 211 Golgi complex ..................................................................4–6 Grainyhead ...............................................................102, 133 Groucho ...............................................................................7 Gut toxicity. See Intestinal/gastrointestinal/gut toxicity

H Halorhodopsin..........................................................118, 128 Hank’s balanced salt solution (HBSS) ............. 139, 147, 150, 159, 164, 165, 278, 280, 282 Haploinsufficient ......................................................3, 41–42 HERP family Hey1............................................................ 247, 251, 340 Hey2.................................................................... 247, 340 HeyL ............................................................................ 247 HES family ..............................................................102, 248 Hes genes Hes1 ................54, 117, 145, 146, 169–178, 247, 251, 340 Hes7 .............................................169, 170, 172–174, 178 Heterodimer .................................................................8, 312 Heterodimerization domain (HD domain) ...................... 6, 9 High performance liquid chromatography (HPCLC) ............................................... 195, 198 Hill-type functions ...........................................................287 Hippocampus ...........................................................124, 128 Histone .................................... 7, 54, 255–258, 269, 270, 273 Histone acetyltransferases (HAT) ................................ 7, 256 Histone deacetylase (HDAC)............................... 7, 256, 273 HDAC1......................................................................256 Histone demethylases .......................................................256 KDM5A .....................................................................256 Histone methyltransferases...............................................255

NOTCH SIGNALING: METHODS AND PROTOCOLS 347 Index Hoechst 33342 ...................................................................90 Homeostasis ....................................................... 47, 101, 317 Homolog .................................................................. 2, 4, 325 Hydrogen exchange coupled mass spectrometry ..............233

I IC50 ...........................................................................315–318 Imaginal disc eye-antennal disc ....................................................71, 73 wing disc .......................................................................73 Imaging confocal ............................................................ 74, 81, 84 live ............................... 55, 80, 83–85, 156–158, 161, 162 molecular .................................................... 155, 171, 339 real-time .....................................................................163 Immobilized ligand .................................. 149, 150, 161, 167 Immunoblot..............................................................225, 227 Immunoglobulin (Ig) ................................................ 278, 330 Immunohistochemistry ................................ 63, 72, 122, 124 Immunostaining ........................68, 70, 72, 89, 105–109, 170 IMSR. See International Mouse Strain Resource (IMSR) Inclusion bodies ................................................ 194, 241–242 Inductive signaling ...............................................................3 In silico ................................................................. 9, 220, 286 In situ ....................................................................... 106, 170 Interaction cis ..............................................................8, 286, 300, 303 protein–DNA .....................................................233, 234 protein–protein ................................9, 155, 189, 191, 233 trans ......................................................285, 288, 301, 307 Interactome ..............................................................181–191 Internalization/internalized ............................. 80, 82–85, 87, 88, 90, 92, 95–99 Internal ribosome entry site (IRES) ...................................53 International Mouse Strain Resource (IMSR) ............. 57, 58 Intestinal/gastrointestinal/gut toxicity ...... 317, 319, 324, 325 Intracellular domain........... 2, 3, 6, 7, 53, 55–57, 63, 69, 117, 132, 144, 159, 169, 209, 223, 248, 285, 311, 323, 335, 340 trafficking .........................................................79–85, 87 Intracranial viral injection......................... 118, 120, 124, 128 Intramembrane ......................................................... 7, 55, 87 Intraventricular electroporation ........ 118–119, 122, 124–128 In utero electroporation .................... 120–122, 124, 127–128 In vitro ................................ 8, 10, 54, 84, 124, 193–207, 248, 252, 281–282, 313, 318, 319, 325, 335, 338–339 In vivo.....................................................7–10, 16, 23, 30–32, 54–58, 79–85, 101–111, 120, 124, 248, 313, 318, 319, 335, 339–340 Ion-exchange column .......................................................206 IPTG. See Isopropyl β-D-thiogalactopyranoside (IPTG) IRES. See Internal ribosome entry site (IRES) Isogenization ......................................................................24 Isopropyl β-D-thiogalactopyranoside (IPTG) ........................... 195, 197, 205, 238–241 Isothermal titration calorimetry .......................................233

J Jag1 ............................................................. 49, 318, 323, 341 Jag2 ..................................................................... 49, 323, 341 Jagged1 ................................................................. 6, 335–337 Jagged2 ......................................................... 6, 328, 335–337 Juxtacrine ......................................................................3, 335

K Kinase ..................................................................... 7, 67, 328 Klumpfuss ........................................................................134 Knock-in ................................................................ 48, 53–57 Knock-out .......................................................... 49, 312, 325 conditional ....................................................................49 Knockout Mouse Project (KOMP) ....................................49

L LacZ. See β-Galactosidase LAMP-1 ............................................................................95 Lateral inhibition......................................... 3, 285, 287–300, 302–307 LCI. See Luciferase complementation imaging (LCI) Learning and memory ......................................................128 Ligand binding domain .............................................. 8, 324, 326 dependent ................ 9, 145, 147, 149, 151, 158, 163, 164 independent ................................8, 9, 133, 147, 149, 152, 158, 163–166, 324, 341 receptor complex.........................................................5–8 selectivity ....................................................................4, 6 Ligand-Fc.........................................147, 148, 151, 152, 158, 159, 161, 164, 166, 167 Lin-12 ..................................................................................2 Lineage analysis ..................................................................47 Lin-12/Notch repeat (LNR) ...................................... 6, 9, 32 Liquid chromatograph linked to tandem mass spectrometer (LC-MS/MS) .................................................210 Loss-of-function ....................................... 16–18, 20, 24, 30, 48–53, 248, 337–339 Loss of heterozygosity (LOH) ...........................................56 LoxP sites ......................................................... 48, 50, 51, 53 Luciferase assay (see Assay) click beetle green (CBG) .................... 156, 159, 161, 166 firefly ..................................................138, 144, 150, 166, 170, 325–329, 331, 338 Renilla .........................................135, 138, 139, 150, 162, 325–329, 331, 338, 341 Ub-Luc ...............................................................170, 171 Luciferase complementation imaging (LCI) ............ 155–168 Luciferase fragments ................................................155–157 Nluc, CLuc .........................................................155, 156 Luciferin coelenterazine ..................................... 162, 325, 328, 332 D-luciferin ..................................148, 159, 161, 162, 164, 166, 167, 171, 172, 174, 176, 177, 326

NOTCH SIGNALING: METHODS AND PROTOCOLS 348 Index Lysis buffer .......................................135, 148, 150–152, 183, 186, 187, 195, 197, 198, 239–241, 259–261, 263–265, 278, 280, 328, 331 Lysosomal pathway.............................................................87 Lysosome .................................................................. 8, 65, 95

M MACS analysis. See Model based Analysis for ChIP-Seq (MACS) analysis MAML. See Mastermind-like (MAML) Mastermind (Mam)........... 2, 4, 7, 9, 106, 133, 231, 256, 324 Mass spectral (MS) analysis .............................................211 Mastermind-like (MAML) .............. 116, 145, 147, 155–157 DNMAML ........................................................145, 147 MAML1 ..........................................7, 49, 231, 232, 234, 235, 237, 241, 242, 335 MAML2 ........................................................................7 MAML3 ........................................................................7 Mathematical modeling............................................286, 303 Mathematical view ...............................................................9 Matlab .............................................................. 286, 289, 290 MCS. See Multiple cloning site (MCS) β-Mercaptoethanol (βME)...............................................238 Methyltransferase .............................................................255 Microarray ........................................................................270 Micrococcal nuclease (MNase)..........257, 259, 261, 265, 273 Micro RNA (miRNA)........................................................16 Mifepristone (RU486) ........................................................48 Mindbomb (Mib) ............................................................. 5, 6 Mitotic recombination........................................................17 MNase. See Micrococcal nuclease (MNase) Model based Analysis for ChIP-Seq (MACS) analysis .................................... 267, 268, 270, 274 Model organisms ...................................2, 9, 15, 30, 181, 184 Modifications ........................... 4, 7, 8, 10, 29–32, 36, 41, 43, 45, 88, 151, 194, 209–220, 256–258, 269, 277 Modifier screen...................................................................16 Morphogen....................................................... 286, 304–307 Morphogenesis .....................................................................3 Mosaic .................................................................... 18, 65, 85 Mosaic analysis with repressible cell marker (MARCM) .....................................................106 Mosaicism ..........................................................................18 Motif search ..................................................... 268–269, 275 Mouse (mice). See Mus musculus Mouse Genome Informatics (MGI) database ..............49, 58 Multiple cloning site (MCS) ...................33, 34, 36, 104, 133 Mus musculus ......................... 3, 9, 30, 31, 47–59, 63, 69, 72, 74, 80, 83, 89, 115–128, 169–177, 210–213, 216–219, 312, 317–319, 325, 336–339, 341 Mutagenesis............... 16–22, 24, 34, 36, 38, 48, 49, 194, 203 Mutations ......................... 2, 9, 16–19, 22–25, 29–34, 36–43, 45, 48–50, 58, 143, 157, 248, 256, 317, 324, 325

N N1. See Notch1 (N1) N-acetyl-cysteine (NAC) ......................................... 172, 176 NAS. See Notch Activity Sensor (NAS) Negative regulatory region (NRR) ................... 6, 9, 323–328 Nervous system................................................... 2, 4, 23, 127 Neuralized (Neur) ...................................................... 5, 6, 79 Neural stem cell .........................169–172, 174–176, 178, 325 Neurogenic ...................................................................2, 312 Neutralizing antibodies ....................................................336 Demcizumab (OMP-21M18) ....................................336 Enoticumab (REGN421) ...........................................336 YW152F.....................................................................336 Neutral loss ....................................................... 211, 216–219 NEXT. See Notch extracellular truncation (NEXT) NICD. See Notch intracellular domain (NICD) N,N,N´,N´-tetramethylethylenediamine (TEMED) .............................................. 235, 237 Notch1 (N1) ........................... 9, 49, 55–57, 88, 90, 115, 117, 118, 159, 160, 211–213, 216–219, 224, 225, 232, 235, 239, 247, 251, 267–272, 275, 312, 315–318, 324–326, 331, 338 Notch2..................................... 49, 56, 57, 145, 159, 324, 325 Notch3......................... 49, 247, 251, 315, 316, 319, 324–327 Notch4........................................................ 56, 316, 325, 338 Notch Activity Sensor (NAS) ...................................... 54, 58 Notch extracellular truncation (NEXT) ....................... 5, 7, 8 Notch intracellular domain (NICD) ............. 3–9, 53, 63–65, 69, 101, 116, 117, 131–135, 137–141, 144, 148, 155–157, 162, 169, 223–229, 231, 248, 256, 257, 285, 311, 312, 315, 323, 331, 335, 340 Notch intracellular Green Fluorescent Protein (N-iGFP) .........................................................81 Notch/Notch receptor cleavage............................... 7, 8, 146, 156, 223, 248, 286, 300, 312, 316, 317, 323, 324 Cre fusion .........................................................55–56, 58 hNotch-111–13 ...................................... 194, 195, 201, 204 localization.................................................. 57, 64, 65, 68 NotchΔE/mNotchΔE ................................ 144, 224–228 Notch extracellular domain (NECD) .................. 6, 8, 32, 63, 69, 89, 107, 248 NotchFL..............................152, 158, 160, 161, 164, 167 RAMANK .........................................231, 232, 234, 235, 237, 239, 240, 243 signaling .......................................................1–10, 16, 18, 20, 30, 31, 41–43, 47–59, 79, 87, 88, 101–111, 115, 117, 118, 127, 128, 131–141, 143–146, 152, 155, 157, 169–178, 182, 223, 231–243, 247–249, 252, 255, 256, 258, 285–307, 311–319, 323–332, 335–341 Notch reporter TP1............................................................. 144, 147, 329 4XCSL ....................................................... 144, 146, 326

NOTCH SIGNALING: METHODS AND PROTOCOLS 349 Index Notch responsive element (NRE) ................... 102, 103, 105, 118, 127, 133, 134 Notch signaling canonical ......................................................... 3–5, 9, 335 non-canonical ....................................................... 3, 4, 49 targets .....................7, 9, 49, 247, 249, 252, 255, 286, 340 Notum dissection..........................................................71, 83 NRE. See Notch responsive element (NRE) NRR. See Negative regulatory region (NRR) N-terminal domain (NT domain) .................... 203, 232, 335 N-terminus (amino-terminus)...................................... 4, 331 Nuclear complexes ............................................ 7, 9, 231–243 Nuclear import ...............................................................7, 10 Nuclear localization sequence (NLS) ......................... 6, 7, 54 Nuclear magnetic resonance (NMR)/NMR spectroscopy .............193–195, 197, 201–205, 207 Nuclear translocation............................................ 6, 157, 249 Nucleofection ...........................................................337, 340 Nucleus ....................................... 2–5, 7, 8, 10, 101, 131, 132, 169, 231, 311, 324, 325, 335 Null, null allele ........................................... 41–43, 49–51, 56 Numb ...........................................................................79–81

O O-fucose ................................................5, 210, 211, 216–218 O-fucosylation ..............................................................4, 216 O-fucosyltransferase 1 (O-fut1) (Pofut1) .................. 4, 5, 210 O-glucose........................................................... 31, 210, 211, 216, 218, 219 O-glucosylation ........................................................ 4, 31, 32 O-glucosyltransferase 1 (Poglut1). See Rumi O-linked glucose .................................................................31 O-N-Acetylglucosamine (O-GlcNAc) ................. 4, 209, 210 Open reading frame (ORF) ...................................... 184, 190 oriV. See Conditionally amplifiable origin of replication (oriV) Oscillatory expression .......................................................170 O-xylose................................................................................4

P Paracrine ...............................................................................3 Paraformaldehyde (PFA) ..................... 69, 70, 80, 90, 97, 122 Paralog .............................................4, 15, 115, 146, 156, 194 Pattern formation ...............................................................18 PCR ...............................................................38, 39, 44, 137. See also Quantitative PCR (qPCR); Quantitative real-time PCR (RT-QPCR) P element meiotic mapping ...........................................................24 transgenesis.............................................................30, 33 Permeabilization ............................................... 68, 76, 89, 99 PEST (proline (P)/glutamic acid (E)/serine (S)/threonine (T)-rich motif ) ........................... 6, 7, 9, 105, 224 Phage attachment site (attP) ............................ 33, 39, 41, 44

Phalloidin ............................................................... 65, 69, 72 PhiC31 (ΦC31) integrase..................................................................39, 44 mediated transgenesis ....................................... 33, 38, 39 site-specific genomic integration ..................................31 Phosphate-buffered saline (PBS) .............. 69–73, 75, 76, 80, 83, 88–97, 107–109, 118, 122, 132, 134, 136, 148, 150–152, 159, 161, 162, 167, 173–177, 83, 186, 187, 190, 212, 226, 249, 250, 263, 264, 328–332, 337–339 Phosphorylation ................................................... 7, 223, 224 Poisson distribution ............................................................22 Polycomb complex ............................................................256 Post-translational modification glycosylation ...........................................................8, 209 hydroxylation ..............................................................209 phosphorylation ..............................................................7 ubiquitination .........................................................7, 209 PRC2................................................................................256 Presomitic mesoderm (PSM) ........................... 170, 172–174 Prokaryotic protein expression..........................................193 Proliferation.................................................... 3, 18, 169, 319 Proteasome ..................................................... 6, 7, 9, 10, 228 Proteasome inhibitor lactacystin ...........................................................225, 228 Protein purification ..........................195–196, 233, 234, 238–239 Protein complementation assay (PCA).............................155 Protein–protein interaction. See Interaction Protein tag alkaline phosphatase (AP) .......................... 278–281, 283 FLAG ................................................. 185, 190, 274, 281 GST.................................................................... 239, 243 hemagglutinin (HA) ........ 89, 96, 183, 185–188, 190, 191 His ............................... 200, 206, 207, 211–213, 220, 239 Myc ............................................................ 211, 212, 224 Proteolytic cleavage ...........................2, 6, 7, 55, 56, 285, 286 Proteome .......................................................... 181, 182, 184 Proteomics ........................................................ 181–191, 220 PSM. See Presomitic mesoderm (PSM) Pulling force .........................................................................6 Pulse labeling......................................................................88

Q Quality control .........................................................188, 190 Quantitative.................................. 9, 133, 146, 157, 159, 167, 210, 211, 225, 234, 277 Quantitative PCR (qPCR) .............................. 132, 135, 137, 139, 249, 251, 264, 274, 339 Quantitative real-time PCR (RT-QPCR)........ 132, 137, 249

R Rab GTPases/Rab ..............................................................85 rab5...............................................................................95 RAM (RBP-jκ Associated Molecule) ..................................7

NOTCH SIGNALING: METHODS AND PROTOCOLS 350 Index RBPj ........................................................49, 54, 55, 231, 248 RBP-jκ (Recombination signal binding protein for immunoglobulin kappa J region) ........... 115, 128, 155–157, 162, 335, 337 Real time PCR (RT-PCR) ............................... 132, 135, 257 Receptor activation .....................................4, 6, 101, 144, 156, 324 ligand binding.........................................................8, 328 notch (see Notch/Notch receptor) Recombinant ligands ........................................................ 328, 330, 331 protein ................................................ 193, 195, 197, 201 Recombineering ......................................... 31, 34–36, 38, 44 Recycling ....................................... 5, 6, 79, 87, 88, 90–94, 98 Recycling assay .......................................................91–94, 98 Refolding ..................................................................193–207 Reporter fluorescent .................................. 105, 118, 143–145, 170 Notch activity ................................23, 116, 118, 122, 124 Reverse-transcription ...............................................136, 137 RNA interference (RNAi) .......................... 16, 106, 132, 282 Room temperature (RT)........... 43, 51, 70, 72, 81, 93–97, 99, 108, 109, 136, 138, 140, 150–152, 164, 175–177, 185–187, 190, 198, 200, 213–215, 226, 228, 229, 236, 239, 242, 262, 263, 266, 279–282, 329, 331 ROSA26.......................................................................49, 53 RU486. See Mifepristone (RU486) Rumi..................................................4, 5, 31, 33, 36–38, 210

Signal activation .................................6–8, 68, 87, 131, 338, 340 receiving cell ..................................................... 3, 5, 8, 87 sending cell ....................................................... 5, 6, 8, 87 sequence...................................................... 4, 6, 212, 224 termination .................................................................6, 7 Simulation ................................................286, 287, 289, 293, 297, 298, 303–305 Single nucleotide polymorphism (SNP) .............................24 Site-directed mutagenesis ..................................... 36, 38, 203 Site-specific integration ................................................31, 32 Small interfering RNA (siRNA) .................................. 96, 98 SNP. See Single nucleotide polymorphism (SNP) Sodium 2-mercaptoethanesulfonate (MesNa) .............. 89–94 Somite .............................................................. 170, 173, 178 SOP. See Sensory organ precursor (SOP) Split ............................... 23, 71, 110, 137, 140, 248, 263, 273 Stem cell .................. 21, 54, 79, 169–172, 174–176, 178, 325 Stereomicroscope ........................................ 19, 45, 69, 80, 81 Stroke ................................................................................... 1 Structure-function analysis ........................ 10, 29–45, 47, 56, 143–145, 156, 161 Suppressor of Hairless (Su(H)) ............................... 2, 7, 102, 115, 133, 134, 324 Synaptic plasticity .................................................................3 Synthetic transcriptional reporter .............................101, 102

S

TAD. See Transactivation domain (TAD) Tamoxifen............................................................... 48–53, 56 TAN-1..................................................................................2 Target identification ......................................... 247, 248, 252 TEMED. See N,N,N´,N´-tetramethylethylenediamine (TEMED) Tetracycline (Tet) ..................................... 158, 160, 165, 167 Tetramethylrhodamine B isothiocyanate (TRITC)...... 69, 72 TEV. See Tobacco etch virus protease (TEV) Thioglycolic acid ................................................................19 Three-dimensional structure ....................................194, 204 Tobacco etch virus protease (TEV) .......................... 238–243 Trafficking. See Intracellular, trafficking Transactivation domain (TAD) ........................................ 6, 7 Transcription/transcriptional activator ........................................................ 55, 103, 146 regulation ........................................................................7 reporter ...............................................................143–153 repressor..............................................................169, 256 Transfection lipofection ...................................................................340 nucleofection ......................................................337, 340 Transgenic fly............................................................................30, 81 mouse ................................................... 47, 120, 127, 170

Sanpodo (Spdo) ............................................................ 79–81 SDS-PAGE. See Gel electrophoresis (protein) γ-Secretase inhibitor (GSI) Begacestat ...................................................................316 BMS-906024..............................................................315 compound E (CmpdE)....................................... 315, 328 DAPT ........................................116, 117, 145, 163, 249, 313, 315, 318, 328, 330, 337, 338 Dibenzazepine (DBZ) ........................................ 315–318 L-685,458........................................................... 314, 318 LY-411575..................................................................312 MCL 28170 ...............................................................312 MG132.......................................................................312 MRK-003 ....................................313, 314, 316, 318, 319 MRK-0752 .................................................................316 MW167, GSI II ......................................... 312, 314, 318 PF-03084014...................................................... 316, 317 RO4929097 ........................................................313–315 Z-IL-CHO ................................................................314 Segmentation clock ..........................................................170 Selectivity ..................................................4, 6, 207, 315, 316 Sensory organ precursor (SOP) ............................ 79, 81, 102 Serrate ............................2, 5, 6, 133, 210, 277, 278, 323, 335

T

NOTCH SIGNALING: METHODS AND PROTOCOLS 351 Index Transgenic Notch Reporter (TNR) ....................................54 Trans-interactions............................................. 288, 301, 307 Transition-state analogs....................................................314 Translation...............................................6, 57, 224, 252, 324 Transmembrane domain (TMD) ...................... 2, 6, 57, 157, 159, 312, 323 Transposable element/transposon .......................................16 β-Trefoil domain (BTD)........................................... 231, 232 TRITC. See Tetramethylrhodamine B isothiocyanate (TRITC) TritonX-100 with PBS (PBT)...................................... 69, 70 Type-I transmembrane protein.........................................4, 6

U UAS. See Upstream activating sequences (UAS) Ubiquitination mono- .............................................................................6 poly- ...............................................................................7 Ubiquitin-proteasome system...............................................9 UCSC genome browser .................................... 267, 268, 274 Ultrabithorax (Ubx) ............................................................18 Universal Proteomics Resources .......................................184 Untranslated region (UTR) 3ʹUTR ................................................................... 35, 57, 170, 171 5ʹUTR ................................................................ 170, 171 Upstream activating sequences (UAS) ................ 16, 146, 327 UTR. See Untranslated region (UTR)

V Vasculature ......................................................... 48, 123, 316 Vesicle/vesicular trafficking

endocytosis .....................................................................6 exocytosis ........................................................................6 ligand ..............................................................................6 receptor ...........................................................................6 recycling..........................................................................6

W Western blotting/blot ...........................91–94, 119, 182, 185, 186, 190, 207, 225–228, 283, 331 Whole-exome sequencing (WES)......................................10 Whole genome sequencing.................................................24 Whole-mount immunohistochemistry ...............................68 Wild-type .............................................................. 17, 18, 24, 30, 32–36, 38–42, 45, 54, 65, 121, 127, 170, 226, 235, 317 Wing notch/notching ................................................... 1, 2, 105 vein ..........................................................1, 39, 41, 42, 45 Worm. See Caenorhabditis elegans

X Xenopus .................................................................................. 6 X-gal staining ........................................... 106, 108, 109, 111 X-ray crystallography................................................193, 233 Xylosyltransferase .............................................................210

Y Yeast-two-hybrid (Y2H) ..................................................182

Z Zebrafish ..........................................................................312