Cell death during Drosophila melanogaster early oogenesis is ...

[Autophagy 5:3, 298-302; 1 April 2009]; ©2009 Landes Bioscience

Brief Report

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Cell death during Drosophila melanogaster early oogenesis is mediated through autophagy

Ioannis P. Nezis,1,2 Trond Lamark,3,† Athanassios D. Velentzas,2,† Tor Erik Rusten,1 Geir Bjørkøy,3 Terje Johansen,3 Issidora S. Papassideri,2 Dimitrios J. Stravopodis,2 Lukas H. Margaritis,2 Harald Stenmark1,* and Andreas Brech1,* 1Centre

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for Cancer Biomedicine; University of Oslo and Institute for Cancer Research; Department of Biochemistry; Rikshospitalet-Radiumhospitalet HF; Montebello, Oslo Norway; of Biology; Department of Cell Biology and Biophysics; University of Athens; Athens, Greece; 3Biochemistry Department; Institute of Medical Biology; University of Tromsø; Tromsø, Norway 2Faculty

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death, the accumulation of autophagosomes and autolysosomes in the cytoplasm represents the most distinct morphological features of this type of cell death. Caspases might also function in dying cells with an autophagic morphology.3,5 During necrotic cell death the plasma membrane of the cell breaks down causing inflammation.6,7 It is currently unclear to what extent these types of cell death are distinct or interconnected, what molecular mechanisms control them, and how they are physiologically regulated in vivo. The structural and functional unit of an insect’s ovary is the egg chamber. During oogenesis in Drosophila melanogaster and other higher Dipteran, the egg chambers are formed in the anterior of the ovary, a region called germarium. As they gradually develop, they move posteriorly towards the exit of the ovariole, forming an array of developmentally ordered egg chambers.8,9 Stem cells for both the germline and soma reside in the germarium. At the anterior of the germarium, overlying somatic cells create a niche to maintain the germline stem cells.9,10 One of the two germline stem cells undergoes an asymmetric division to produce another stem cell and a cystoblast. The cystoblast undergoes four rounds of mitotic division with incomplete cytokinesis to produce a germline cyst, containing one oocyte and fifteen interconnected nurse cells, within region 2a of the germarium. Somatic stem cells reside in region 2 of the germarium and give rise to mesenchymal somatic cells that surround each germline cyst and compress it into the characteristic lens shape of region 2b of the germarium. As the somatic cells continue to envelop the cyst, they differentiate into an epithelial monolayer of follicle cells to form the egg chamber.9,11 Consequently, the formed egg chamber develops through 14 distinct developmental stages until it reaches maturity at the final stage 14.8,9 As previously demonstrated, programmed cell death during Drosophila and higher Dipteran oogenesis occurs in the germarium, as well as during mid-oogenesis and late oogenesis12-15 (reviewed in refs. 16 and 17). Cell death during mid-oogenesis, known as follicular atresia, has been sporadically observed during midoogenesis,13-15,18,19 but also as a response to nutritional deprivation, ecdysone signaling inhibition, treatment with chemotherapeutic drugs, and ectopic death of follicle cells in Drosophila.20-24 The atretic egg chambers were found to contain degenerated nurse

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Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. Herein, we report that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila melanogaster. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atg1 or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development.

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Key words: apoptosis, autophagy, Drosophila, nurse cells, programmed cell death

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Introduction

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Programmed cell death constitutes a highly conserved, genetically regulated process, implicated in a wide variety of different biological systems, including insects’ oogenesis, that leads to the self-destruction of superfluous cells through the activation of a cell death program.1 Programmed cell death has traditionally been grouped into three major subtypes based on morphological criteria.2-4 Apoptotic cell death is mainly characterized by caspase activation, chromatin condensation and DNA fragmentation. In autophagy-mediated cell

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*Correspondence to: Harald Stenmark; Centre for Cancer Biomedicine; Institute for Cancer Research; University of Oslo and Department of Biochemistry; Rikshospitalet-Radiumhospitalet HF; Montebello, N-0310; Oslo, Norway; Tel.: +47.22934951/4937; Fax: +47.22508692; Email: [email protected]/ Andreas Brech; Centre for Cancer Biomedicine; Institute for Cancer Research; University of Oslo and Department of Biochemistry; Rikshospitalet-Radiumhospitalet HF; Montebello, N-0310; Oslo, Norway; Tel.: +47.22934951/4937; Fax: +47.22508692; Email: [email protected] Submitted: 09/29/08; Revised: 11/17/08; Accepted: 11/19/08 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/article/7454

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Cell death through autophagy during Drosophila early oogenesis

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cells, mainly characterized by the presence of active caspases, fragmented DNA and condensed chromatin.13-15,21-23 Programmed cell death of nurse and follicle cells is also required for the normal maturation of the egg chambers during the late stages of D. melanogaster, Dacus oleae and Ceratitis capitata oogenesis.13,14,25-30 In the present study, we demonstrate that autophagy occurs during developmentally-induced cell death observed both in the germarium and during midoogenesis in Drosophila melanogaster. Importantly, we demonstrate that genetic inhibition of autophagy results in significant reduction of DNA fragmentation, suggesting that autophagy acts upstream of caspase activation.

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To measure cell death during Drosophila melanogaster oogenesis we used the TUNEL assay. Such analysis revealed that cell death in the germarium occurred in 26% of the ovarioles, in young, well-fed flies (Fig. 1A, Table 1A). In contrast, the percentage of cell death during middle stages of oogenesis was 9.7% under the same conditions (Fig. 2A, Table 1B). In the germarium the degenerated germline cells are usually located in region 2 and, during middle stages of oogenesis, in egg chambers of developmental stages 7, 8 and 9 (Figs. 1 and 2). In order to detect caspase activity, we performed immunolabeling with the anti-active caspase-3 (cleaved caspase-3) antibody. The degenerated germaria revealed an intense staining in region 2, indicating the presence of activated caspase-3 proteases and therefore caspase-dependent cell death (Fig. 1B). A similar staining pattern is observed in the degenerating mid-stage egg chambers (Fig. 2B). We next investigated whether autophagy occurs during cell death in the germarium and mid-stage egg chambers of Drosophila melanogaster. GFP-tagged Atg8a or its vertebrate homologue LC3, has been extensively used to monitor autophagy as it labels the autophagic membranes.31-33 However, the pH sensitivity of GFP makes it impossible to follow GFP-Atg8a after the short-lived autophagosomes fuse with lysosomes.

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Results and Discussion

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Figure 1. Developmentally-induced cell death within Drosophila melanogaster germaria exhibits autophagic features. (A) TUNEL staining demonstrating the occurrence of DNA fragmentation in the region 2 of germarium (arrow). (B) High magnification confocal micrographs of D. melanogaster germarium stained for active caspase-3 (B) and DNA (B’). Caspase activity is evident mainly in region 2 (arrow). Note the condensed nuclei in the cells exhibiting caspase activity (arrow). (C) Confocal micrographs of egg chambers of flies expressing the UAS-mCherry-DrAtg8a transgene exclusively in the germline (genotype: UAS-mCherryDrAtg8a/+; nanos-VP-16 Gal4/+). Note the punctate staining in region 2 of germaria (arrows). (D–G) Transmission electron micrographs of normal (D) and degenerating (E–G) germline cysts. In the normal germ line cysts the nuclei exhibit many invaginations and protrusions. Note the regular pattern of the ring canals (arrows, D) compared to the one observed in degenerating germline cysts (small arrows, E). In contrast, the nuclei in the degenerated cysts have rather a round shape, areas with condensed chromatin and extensive nuclear membrane dilation (E and F). The degenerating germline cyst contains many autophagosomes and autolysosomes (arrows in E–G). r1: germarium region 1, r2: germarium region 2, r3: germarium region 3, GCN: germline cell nucleus, NE: nuclear envelope. Scale bars: (A–C): 5 μm, (D and E): 1 μm, (G): 100 nm, (F): 0.5 μm.

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Atg1Δ3D/TM6B

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Atg7d4 GLC

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(B) Cell death in stage 7–9 degenerating egg chambers Genotype TUNEL positive number w1118 Atg1Δ3D Atg7d4

195 (n = 30)

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141 (n = 22)

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153 (n = 24)

Materials and Methods

Drosophila culturing conditions. Drosophila melanogaster insects were kept at 25°C and fed on standard diet (3.5% mashed potato, 0.85% agar, 4.15% syrup, 1.5% yeast, 0.2% nipagin, 0.4% propionic acid). Fifteen to 20 adult insects (equal numbers of males and females) were kept in single vials for four to five days before dissection. Dissections were carried out in cold Ringer’s solution or PBS and ovaries were separated into single ovarioles. Fly strains and crosses. The w1118 strain was used as wild-type control. The atg1Δ3D FRT80B and the atg7d4FTR42D fly lines were a gift from Thomas P. Neufeld. Germline mutant cells were generated by using the FLP recombinase-dominant female sterile technique43 using the following ovoD1 strains: (1) yw,hsflp;ovoD1FRT80B/TM6B/MKRS and (2) yw,hsflp,slbolacZ; FRT42DovoD1/CyO (gifts from Pernille Rørth). Females of the genotype yw,hsflp;Atg1Δ3DFRT80B/TM6B and yw,hsflp;FRT42Datg7d4 were crossed to yw,hsflp;ovoD1FRT80B/ TM6B and yw,hsflp;ovoD1FRT42D/CyO males respectively and were allowed to lay eggs for one day. Larval progeny were heat-shocked on day 4 and day 5 (L2 and L3 of larvae development) for 1 h and 30 min at 37°C in a circulating water-bath, to induce the generation of mitotic clones in the developing ovary. Adult females carrying germline mutants were dissected and analyzed as described below. Construction of the mCherry-DrAtg8a transgene. The pUASp vector for the mCherry Drosophila Atg8a (CG32672) fusion, pPWmCherry-Atg8a, was constructed in three steps. A PCR fragment of mCherry deleted for its stop codon was amplified from pmCherryC1,44 using the oligos 5'-TCT ACC GGT CGC CAC CAT GGT GAG CAA GGG CGA GGAG-3' (forward) and 5'-TTT GGA TCC CTT GTA CAG CTC GTC CAT GCC-3' (reverse). The PCR product was cut with AgeI, treated with T4 DNA polymerase (to make blunt ends), cut with BamHI, and subcloned into DraI- and BamHI-cut pENTR1A (initially deleted for a 423 bp EcoRI fragment containing the toxic ccdB gene) to produce pENTR-mCherry. Next Atg8a was subcloned as a BglII-XbaI fragment from pUASpGFP-Atg8a45 into pENTR-mCherry cut with BamHI and XbaI to give pENTR-mCherry-Atg8a. Finally, pPW-mCherry-Atg8a was made by a Gateway LR reaction between pENTR-mCherry-Atg8a and the destination vector pPW (obtained from the Drosophila Gateway Vector Collection; www.ciwemb.edu/labs/murphy/ Gateway%20vectors.html). The resulting construct was verified by sequencing and introduced into w1118 flies with a standard P element transformation.

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Fusion with late endosomes to create amphisomes may also lead to an environment where the fluorescence from GFP is quenched due to low pH. We therefore made a transgenic fly in which DrAtg8a is fused to the acid-insensitive fluorescent protein mCherry that can be easily monitored into amphisomes and autolysosomes. Upon expression of mCherry-DrAtg8a in the germline using the germline-specific driver, nanos-VP16-Gal4,34,35 several mCherry-DrAtg8a puncta were detected in region 2 of the germarium and in the degenerating mid-stage egg chambers, indicating autophagic activity (Figs. 1C and 2C). When mCherry-DrAtg8a was coexpressed with GFP-LC3, we observed that the number of mCherry-positive structures in degenerating mid-stage egg chambers was significantly higher compared to the number of GFP-positive structures (Fig. 2D and E). This observation reveals that mCherry-DrAtg8a is a very useful marker for monitoring autophagy in Drosophila, since it can be used for detecting both autophagosomes and autolysosomes. Examination of the ultrastructural morphology of degenerated germaria revealed that the cytoplasm of the germline cells indeed contained a variety of autophagosomes and autolysosomes. Autophagic compartments were filled with cytoplasm, numerous vesicles, dense masses and multi-lamellar membranes of various sizes (Fig. 1E–G). Additionally, while the nuclei in the normal non-degenerating germ line cysts were very irregular in shape with many invaginations and protrusions, the nuclei in the degenerated cysts had a round shape and condensed chromatin (Fig. 1D–F). Surprisingly, all these nuclei had irregular and extensive nuclear membrane dilation (Fig. 1E and F). Together, the above data demonstrate that autophagy occurs in caspase-dependent cell death during early oogenesis in Drosophila melanogaster. To explore the potential role of autophagy in the cell death process of the germline cells in the germarium and in mid-stage egg chambers, we generated germline mutant cells for the core Drosophila autophagy genes Dratg1 and Dratg7,36-38 and scored for apoptotic cell death using the TUNEL assay in order to detect DNA fragmentation. Interestingly, cell death in the germarium of Dratg1 germline mutants was reduced to 8.4% (compared to 26% of the control, Table 1A) and to 3.5% for mid-stage egg chambers (compared to

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(A) Cell death at region 2 of germarium Genotype TUNEL positive

9.7% of the control, Table 1B). atg7 germline mutants exhibited a similar reduction in apoptotic cell death. The percentage of germaria being positive for TUNEL was 13.8% and the percentage of midstage egg chambers being positive for TUNEL was 4.8% (Table 1A and B). The above data indicate that inhibition of autophagy reduces DNA fragmentation, suggesting that during Drosophila early oogenesis autophagy acts upstream of caspase activation and DNA fragmentation. How can autophagy promote cell death by acting upstream of caspase activation? One possible explanation is that proteins crucial for cell survival and organelles are degraded by autophagy, thus promoting cell death. Moreover, autophagy could also act in parallel and cooperatively with caspases for the most efficient degradation of the tissue, as previously suggested.39-42 In future studies we will try to identify molecular mechanisms that illuminate the contribution of autophagy in the cell death process.

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Table 1 Quantification of TUNEL staining in region 2 of germaria and stage 7–9 degenerating egg chambers

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Figure 2. Developmentally-induced cell death in the middle developmental stages of oogenesis in Drosophila melanogaster. (A) Confocal micrographs of D. melanogaster middle-stage egg chambers stained for TUNEL. The abnormal atretic egg chamber contains fragmented DNA (arrow). (B) Caspase-3 activity is evident in the nurse cells of an abnormal mid-stage egg chamber (arrows). (C) Confocal micrographs of egg chambers of flies expressing the UAS-mCherry-DrAtg8a transgene exclusively in the germline (genotype: UAS-Cherry-DrAtg8a/+; nanosVP-16 Gal4/+). Note the punctate staining in the abnormal degenerating egg chambers (arrows). (D) Confocal micrographs of egg chambers of flies expressing both UAS-mCherry-DrAtg8a and UAS-GFPLC3 transgenes (genotype: UAS-Cherry-DrAtg8a/ UAS-GFP-LC3; nanos-VP-16 Gal4/+). The number of red structures (mCherry-Atg8a expression-D’) is significantly higher compared to the number of green structures (GFP-LC3 expression-D). Note that it is easy to distinguish between autophagosomes (yellow arrow) exhibiting both green and red colour, and autolysosomes (red arrow) exhibiting only red colour. The white arrow points to an autolysosome which contains a very limited portion of GFP-positive content, suggesting that the GFP signal was quenched after acidification. (E) Graphic presentation of the average number of GFP-positive or mCherry-positive structures per degenerating mid-stage egg chamber. The number of fluorescent structures was counted in randomly obtained middle confocal sections from 20 mid-stage degenerating egg chambers which were dissected out from five different animals. Scale bars: 5 μm.

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Immunofluorescence labeling and confocal microscopy. Drosophila egg chambers were processed for TUNEL staining and immunofluorescence labeling as previously described.29,41 The primary antibody used in the present study was an anti-rabbit antibody against cleaved caspase-3 (Cell Signalling, USA) used at a concentration 1:500. The secondary antibodies, conjugated with either Cy2 or Cy3, were purchased from Jackson Immunoresearch. Draq5 (Biostatus, USA) or Hoechst (Merck, Germany) was used to stain DNA at a dilution of 1:1000. Finally, the egg chambers were mounted in antifading mounting medium (Prolong Antifade, Molecular Probes) and observed under a Zeiss LSM510 confocal laser scanning microscope. Conventional transmission electron microscopy (TEM). Dissected egg chambers were processed for transmission electron microscopy as follows: ovaries were dissected from female insects, separated into individual follicles in Ringer’s solution and fixed in 2% glutaraldehyde in PBS for 1 hour and 30 min at room temperature. After being rinsed two times with PBS, for 20 min each at room temperature, the follicles were stained en block overnight in PBS containing 0.5% uranyl acetate in the dark. They were then washed two times in PBS for 15 min each at 4°C and post-fixed in PBS containing 2% osmium tetroxide for 1 hour at 4°C. After two washes in PBS for 20 min each at 4°C, the specimens were dehydrated through a graded series of ethanol www.landesbioscience.com

concentrations, infiltrated in propylene oxide and embedded in Epon-Araldite (Fullam, Inc., New York, USA). Ultrathin sections were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate and observed under a Philips EM300 transmission electron microscope.

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We are very grateful to Thomas P. Neufeld, Pernille Rørth and Ruth Lehmann for fly lines. We are also very grateful to Sharon Gorski and Kim McCall for sharing results before publication. This work was supported by grants from the Hartmann Family foundation (H.S.), and the Functional Genomics Programme of the Norwegian Research Council (I.P.N., T.E.R., H.S. and A.B.) and co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to L.H.M.

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