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THE ANATOMICAL RECORD PART A 273A:677– 680 (2003)
Dynamic Expression of Kru ¨ ppel-Like Factor 4 (Klf4), a Target of Transcription Factor AP-2␣ During Murine Mid-embryogenesis JULIA EHLERMANN,1 PETRA PFISTERER,2 AND HUBERT SCHORLE1* Institute for Pathology, Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany 2 InDex Pharmaceuticals ABMTC, Karolinska Institutet, Stockholm, Sweden
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ABSTRACT Kru¨ppel-like factor 4 (Klf4) belongs to the family of transcription factors that are thought to be involved in the regulation of epithelial and germ cell differentiation, based on their expression in postproliferative cells of the skin, gut, and testes. Gene ablation experiments suggest that Klf4 plays a role in keratinocyte differentiation, since mice lacking Klf4 fail to establish proper barrier function and, as a consequence, die postnatally due to dehydration. Recent studies have shown that Klf4 is also expressed in postnatal male mice, in postmeiotic sperm cells undergoing terminal differentiation into sperm cells. However, prior to the current study, the expression pattern of Klf4 during early and mid-embryogenesis had not been examined. Here we demonstrate that Klf4 transcripts can be detected from embryonic day 4.5 (E4.5) on in the developing conceptus, and that Klf4 expression before E10 is restricted to extraembryonic tissues. The embryo proper displays a highly dynamic and changing Klf4 signal from E10 of murine development on. In addition to being expressed in a stripe of mesenchymal cells extending from the forelimb bud rostrally over the branchial arches to the developing eye, Klf4 is also expressed in the mesenchyme surrounding the nasal pit at day E11.5. In addition, Klf4 has been detected in the apical ectodermal ridge and adjacent mesenchymal cells in the limb buds, and in mesenchymal cells of the developing body wall in trunk areas. These findings suggest that Klf4 plays an important role in regulating cellular proliferation, which underlies the morphogenetic changes that shape the developing embryo. Anat Rec Part A 273A:677– 680, 2003. © 2003 Wiley-Liss, Inc.
Key words: Kru ¨ ppel-like factor 4 (Klf4); gut-enriched Kru ¨ ppel-like factor (GKLF); expression pattern; mesenchyme; apical ectodermal ridge; craniofacial mesenchyme; transcription factor AP-2␣; Tcfap2a
Kru¨ppel-like factor (Klf4; also known as gut-enriched Kru¨ppel-like factor (GKLF)) is a member of the family of Kru¨ppel proteins, which are named for their homology to the Drosophila Kru¨ppel gene (Anderson et al., 1995). Previous reports have shown that Klf4 is expressed in the gastrointestinal tract (Garrett-Sinha et al., 1996; Shields et al., 1996) and in the developing layers of the skin starting from embryonic day 16.5 (E16.5) of murine development (Segre et al., 1999). Early studies implicating Klf4 in regulating growth arrest (Garrett-Sinha et al., 1996; Shields et al., 1996) have been substantiated by the finding that Klf4 is a transcriptional repressor of Cyclin D1, a gene that is involved in cell cycle progression (Shie et al., 2000). Gene-targeting experiments have revealed a role for Klf4 in barrier formation in the skin (Segre et al., ©
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1999). Recently, Klf4 was described as a target gene that is repressed by transcription factor AP-2␣ (Tcfap2a, MGI
Grant sponsor: Deutsche Forschungsgemeinschaft; Grant numbers: DFG 503-3; DFG 503-6. *Correspondence to: Hubert Schorle, Institute for Pathology, Department of Developmental Pathology, University of Bonn, Sigmund-Freud Strasse 25, 53127 Bonn, Germany. Fax: ⫹49228-287-5030. E-mail:
[email protected] Received 17 March 2003; Accepted 23 April 2003 DOI 10.1002/ar.a.10089
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Accession ID: MGI:104671) during early embryogenesis. Loss of AP-2 expression has been shown to result in premature differentiation and cell cycle exit of the tissues affected (Pfisterer et al., 2002). This study prompted us to reexamine the expression of Klf4 in order to expand our current knowledge about the expression pattern of this gene.
MATERIALS AND METHODS Cloning the Probe A 1.9 kb fragment of the murine cDNA (Genbank accession number U20344) spanning bases 2–1958 was cloned from an E10.5 cDNA library (Invitrogen, Heidelberg, Germany) and ligated in the EcoRI site of pBS II (KS) as previously described (Pfisterer et al., 2002).
Whole-Mount In Situ Hybridization Whole-mount in situ hybridization was performed as previously described (Werling and Schorle, 2002). Embryos were sectioned to 30 m on a Leica Vibratome (Leica, Bensheim, Germany) and embedded in Immunomount (Thermo Shandon, Pittsburgh, PA) as described elsewhere (Maldonado-Saldivia et al., 2000). The resulting whole-mount embryos and sections were photographed with a Leica microscope (Leica) equipped with DIC optics and a Zeiss Axiocam digital camera system (Carl Zeiss, Jena, Germany). Files were processed using Adobe™ Photoshop and Illustrator software (Adobe, San Jose, CA).
RESULTS AND DISCUSSION To obtain an overview of Klf4 expression during murine development, we used a Northern blot covering stages E4.5–18.5. Furthermore, we determined the spatial expression of Klf4 during mid-embryogenesis using wholemount in situ hybridization. The embryos were processed for vibratome sectioning to further characterize the cell types and areas in which Klf4 was expressed. We detected Klf4 expression as early as E4.5 of murine development (Fig. 1). This expression persisted until birth, with a sharp drop in intensity at E10.5. In Fig. 1A, the lanes representing E4.5–9.5 contain extraembryonic as well as uterine tissues. From E10.5 on, the RNA was isolated from embryonic tissues only, and thus represents the expression of Klf4 in the embryo proper, which can be seen at stages E10.5–18.5 (Fig. 1B). Using RT-PCR analyses of mRNA from dissected embryos lacking extraembryonic material from E8.5–10.5, we were able to show that there is no Klf4 signal in the embryo proper prior to E10.5 (Pfisterer et al., 2002), indicating that extraembryonic tissues, such as trophoblast cells and giant cells, express Klf4 during early embryogenesis. Next, we performed whole-mount in situ analyses on E10.0 –13.5 embryos, which showed that Klf4 was expressed in a highly dynamic pattern in the murine embryo during these stages (Fig. 2). Klf4 expression is set up at day E10.0 in the mesenchyme surrounding the fourth arch artery (Fig. 2A, arrow) and persists until E11.5 (Fig. 2A– F). At E10.5, the next site of expression of Klf4 is the medial part of the first branchial arch (Fig. 2B). Expression in the branchial arch extends very rapidly rostrocaudally to the developing eye and the aboral (lateral) side of the second and third branchial arches until E12.0 (Fig. 2C–F). Thereafter, the intensity of expression declines and becomes more evenly distributed. The mesenchyme of
Fig. 1. Northern blot using Seegene™ (Seegene, Korea) premade blots, displaying samples from E4.5–9.5 (A) (uterine, extraembryonic, and embryonic tissues) and E10.5–18.5 (B) (embryo proper). A single band representing Klf4 mRNA can be detected (arrow). Ethidium-bromide-stained gel demonstrates equal loading of samples (A and B, lower panel)
the developing vertebrae displays a regular and repeated staining pattern from E11.5 (Fig. 2D and E, arrows) to E11.75 (Fig. 2F). In the developing limb bud, Klf4 can be detected in the apical ectodermal ridge from E11.0 to E12.5 (Fig. 2C–H). Expression in cells in the abdominal region of the ventral body can be seen from E11.5 on (Fig. 2D–I). Furthermore, Klf4 is prominently expressed in the nasal pits at E11.5 (Fig. 2D); it appears to move away from the pit itself, and can be found in cells surrounding the nasal pits (Fig. 2F, arrow). From this stage on, expression in this distinct stripe in the craniofacial region becomes less prominent and is replaced by a more diffuse expression (Fig. 2F–H). From E12.0 to E13.5 the developing vibrissae are positive for Klf4 (Fig. 2G–I). Afterwards, Klf4 expression becomes more widespread and diffuse in many areas of the developing epidermis (Fig. 2I) (GarrettSinha et al., 1996). Next, we performed vibratome sectioning of E10 –12 embryos to determine which cells were positive for Klf4 expression. Analysis of sections from the head and trunk area revealed that Klf4 is not expressed in epithelial cells, but is expressed in a compartment of adjacent mesenchymal cells (Fig. 3A–D) (Garrett-Sinha et al., 1996). However, in the limb bud (Fig. 3E) the expression appeared to be initially set up in the apical ectodermal ridge (Fig. 3F), and then extended into the surrounding mesenchymal cells (Fig. 3G). Furthermore, Klf4 signal was detected in mesenchymal cells in the thorax area of E12.5 embryos in the region of cartilaginous primordia of the developing ribs (Fig. 3H). In summary, this study revealed a highly dynamic and temporary expression pattern of Klf4 during murine mid-
DYNAMIC EXPRESSION OF Klf4
Fig. 2. Whole-mount in situ hybridizations of embryos at E10.0 (A), 10.5 (B), 11.0 (C), 11.5 (D and E) 11.75 (F), 12.0 (G), 12.5 (H), and 13.5 (I). Dark blue staining indicates sites of Klf4 expression. Arrows: (A) mesenchyme adjacent to the fourth arch artery, (B) first branchial arch, (D)
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mesenchyme of the nasal pits, (F) frontonasal mesenchyme, and (I) developing vibrissae. D: I and II refer to the sections shown in Figure 3A and B, and C and D, respectively.
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Fig. 3. Vibratome sections (A–D, F–H) and detail (E) of whole-mount in situ hybridizations from E11.5 (A–D) and E11.0 (E) embryos. Cross section through the cranial region (A–C) showing Klf4 expression in the mesenchyme (B–D), magnification of boxed area in A and C. E: Limb bud detail showing Klf4 expression in the epithelial cells of the apical ecto-
dermal ridge of an E11.0 embryo (F), and the expression of Klf4 in cells of the apical ectodermal ridge and the adjacent mesenchymal cells at E11.5 (G). H: A section through the thorax region of an E12.5 embryo, showing Klf4 expression in mesenchymal cells. Inset: Magnification of the boxed area.
embryogenesis. Based on this work and previous findings, we hypothesize that Klf4 may be involved in orchestrating differentiation processes in the limb bud and the craniofacial area. Segre et al. (1999) reported that Klf4-deficient mice not only suffer from loss of barrier function in the skin, but also do not feed correctly. Based on the expression of Klf4 in the cranial region observed in the current study, Klf4-deficient mice may also display hyperplastic cranial ganglia, leading to impaired feeding responses. Loss of Klf4 expression in the limb buds appears to be tolerated, since knockout mice do not develop any abnormalities in that region (Segre et al., 1999).
throid Kruppel-like factor: identification of a new multigene family. Mol Cell Biol 15:5957–5965. Garrett-Sinha LA, Eberspaecher H, Seldin MF, de Crombrugghe B. 1996. A gene for a novel zinc-finger protein expressed in differentiated epithelial cells and transiently in certain mesenchymal cells. J Biol Chem 271:31384 –31390. Maldonado-Saldivia J, Funke B, Pandita RK, Schuler T, Morrow BE, Schorle H. 2000. Expression of Cdcrel-1 (Pnutl1), a gene frequently deleted in velo-cardio-facial syndrome/DiGeorge syndrome. Mech Dev 96:121–124. Pfisterer P, Ehlermann J, Hegen M, Schorle H. 2002. A subtractive gene expression screen suggests a role of transcription factor AP-2 alpha in control of proliferation and differentiation. J Biol Chem 277:6637– 6644. Segre JA, Bauer C, Fuchs E. 1999. Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet 22:356 –360. Shie JL, Chen ZY, Fu M, Pestell RG, Tseng CC. 2000. Gut-enriched Kruppel-like factor represses cyclin D1 promoter activity through Sp1 motif. Nucleic Acids Res 28:2969 –2976. Shields JM, Christy RJ, Yang VW. 1996. Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol Chem 271:20009 –20017. Werling U, Schorle H. 2002. Transcription factor gene AP-2{gamma} is essential for early murine development. Mol Cell Biol 22:3149 – 3156.
ACKNOWLEDGMENTS We thank Andrea Jacob and Inge Heim for excellent technical assistance and maintenance of the animal colony. We especially thank Gerrit Klemm and his Fotolabor crew for excellent digital artwork. This study was supported by grants from Deutsche Forschungsgemeinschaft (DFG 503-3 and 503-6) to H.S.
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