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. Author manuscript; available in PMC: 2014 May 28.
Published in final edited form as: Dev Cell. 2013 May 16;25(4):350–363. doi: 10.1016/j.devcel.2013.04.013

Tissue repair through cell competition and compensatory cellular hypertrophy in postmitotic epithelia

Yoichiro Tamori 1, Wu-Min Deng 1,*
PMCID: PMC3891806  NIHMSID: NIHMS472954  PMID: 23685249

Summary

In multicellular organisms, tissue integrity and organ size are maintained through removal of aberrant or damaged cells and compensatory proliferation. Little is known, however, about this homeostasis system in postmitotic tissues, where tissue-intrinsic genetic programs constrain cell division and new cells no longer arise from stem cells. Here we show that, in postmitotic Drosophila follicular epithelia, aberrant but viable cells are eliminated through cell competition, and the resulting loss of local tissue volume triggers sporadic cellular hypertrophy to repair the tissue. This “compensatory cellular hypertrophy” (CCH) is implemented by acceleration of the endocycle, a variant cell cycle composed of DNA synthesis and gap phases without mitosis, dependent on activation of the insulin/IGF-like signaling pathway. These results reveal a remarkable homeostatic mechanism in postmitotic epithelia that ensures not only elimination of aberrant cells through cell competition but also proper organ-size control that involves compensatory cellular hypertrophy induced by physical parameters.

Introduction

In multicellular organisms, the cellular communities continually experience various stresses and damages from exogenous and endogenous sources. When the insults cause an emergence of aberrant cells or abrupt cell death, the cellular community is threatened by a risk of cancer, organ dysfunction or developmental anomaly, which may lead to organismal mortality. Maintenance of tissue integrity requires elimination of these aberrant or damaged cells and subsequent additional divisions of the surrounding normal cells, which are induced by mitogenic signals from the dying cells (Huh et al., 2004; Pérez-Garijo et al., 2004; Ryoo et al., 2004). This tissue homeostasis process, termed apoptosis-induced compensatory proliferation is crucial for the maintenance of tissue integrity in proliferating tissues (Fan and Bergmann, 2008). In Drosophila imaginal discs, the Caspase-9-like initiator Caspase, DRONC, has been shown to be upregulated in apoptotic cells to coordinate apoptosis and compensatory proliferation through activation of c-Jun N-terminal kinase (JNK) pathway (Kondo et al., 2006). JNK activation then leads to ectopic upregulation of mitogenic morphogens such as Wingless (Wg) and Decapentaplegic (Dpp), Drosophila homologs of Wnt and BMP/TGF-ß, respectively, to induce the proliferation of surrounding cells (Pérez-Garijo et al., 2004; Ryoo et al., 2004).

The sensing and removal of aberrant cells by their neighbors involve cell competition, a remarkable homeostatic process at the cellular level (Johnston, 2009; Tamori and Deng, 2011). Cell competition was first experimentally confirmed in Drosophila by Morata and Ripoll (1975) in the study of growth parameters of Minute (M) mutations. Minutes are a group of dominant mutations that are defective in producing ribosomal proteins (Lambertsson, 1998) and are lethal to cells when homozygous because the mutant cells lack functional ribosomes and cannot synthesize proteins. Flies heterozygous for Minute (M/+), on the other hand, are viable and of normal size, although they require a few days more than wild-type flies to complete embryonic development. In mosaic imaginal discs containing both M/+ and wild-type cells, the M/+ cells are disproportionately eliminated and do not contribute to the adult animal (Morata and Ripoll, 1975). Meanwhile, growth of the wild-type cells is correspondingly increased, sometimes leading the entire compartment to be constructed from just these cells (Simpson, 1979; Simpson and Morata, 1981; Moreno et al., 2002). Cell competition has also been reported in imaginal discs containing mutations of genes involved in the regulation of cell proliferation (Prober and Edgar, 2000; de la Cova et al., 2004; Moreno and Basler, 2004; Neto-Silva et al. 2010; Tamori et al. 2010; Ziosi et al. 2010; Vincent et al., 2011; Rodrigues et al. 2012) or maintenance of epithelial apical-basal polarity (Brumby and Richardson, 2003; Grzeschik et al., 2007; Igaki et al., 2009; Menéndez et al., 2010; Tamori et al., 2010; Hafezi et al., 2012).

Studies on Minute and mahjong (mahj) mutations, which cause cell competition both in flies and mammals (Morata and Ripoll, 1975; Moreno et al., 2002; Oliver et al., 2004; Tamori et al., 2010), suggest that cell competition is an evolutionarily conserved cellular homeostatic process. Mahjong is the Drosophila homolog of mammalian VprBP (HIV protein Vpr binding protein)/DCAF1 (DDB1-and Cul4-associated factor 1) and a binding protein of Lethal giant larvae (Lgl), a neoplastic tumor suppressor gene (Tamori et al. 2010). Depletion of mahjong induces cell competition both in Drosophila imaginal epithelia and mammalian MDCK (Madin-Darby canine kidney) cells (Tamori et al. 2010). Mahjong has also been shown to interact with the Merlin/NF2 tumor suppressor in mammalian systems (Li et al. 2010). The unphosphorylated form of Merlin, presumably stabilized in a closed conformation, is able to mediate growth inhibition. The unphosphorylated Merlin translocates into the nucleus and binds to DCAF1, the substrate receptor subunit of CRL4DCAF1 and mammalian Mahjong homolog, and inhibits CRL4DCAF1-mediated ubiquitylation of target proteins. Gene-expression profiling analysis suggests that Merlin, through inhibition of CRL4DCAF1, regulates the expression of genes involved in cell-cycle progression, growth arrest and apoptosis (Li et al., 2010). Collectively, the cellular competitive ability regulated by Mahjong can be considered as a consolidated output of diverse gene expression involved in cell proliferation and apoptosis.

Most previous reports have shown that slowly proliferating cells undergo apoptosis when they are surrounded by rapidly proliferating cells. Activation of Cyclin D/Cdk4 or the insulin/IGF (insulin-like growth factor)-like signaling (IIS) pathway to accelerate cell division or cellular growth, respectively, however, does not cause cell competition (de la Cova et al., 2004). A difference in cell growth or proliferation rate thus does not always trigger cell competition, and it remains to be elucidated how cells determine winners and losers in cell competition.

During the process of cell competition, optimal winner cells eliminate neighboring suboptimal loser cells and subsequently undergo compensatory proliferation to replace the region previously occupied by the loser cells, with the result that tissue integrity and organ size are finely normalized (de la Cova et al., 2004; Simpson et al., 1981). The process therefore not only assures elimination of potentially deleterious cells but is also tightly connected to organ-size control (Baker, 2011; de Beco et al., 2012; Tamori and Deng, 2011). In postmitotic tissues, although removal of damaged cells has been observed (Campisi, 2003), little is known about the role of the neighboring normal cells in these processes. Here, we show that cell competition can occur in a postmitotic epithelium, eliminating aberrant but viable cells and how the space previously occupied by the lost cells is filled in postmitotic tissues, where tissue-intrinsic genetic programs constrain cell division and new cells no longer arise from stem cells.

Results

Cell Competition Occurs in Postmitotic Epithelia

To examine whether cell competition can occur between postmitotic cells we used the Drosophila follicular epithelium as a model system. Drosophila follicle cells undergo mitotic divisions up to oogenesis stage 6, after which they switch into three rounds of endoreplication during stages 7–10A. At stage 10B, they leave the endocycle, and the main-body follicle cells (a single layer of columnar epithelium surrounding the oocyte) undergo synchronized amplification of genomic loci encoding eggshell proteins (Calvi et al., 1998). Therefore, the follicular epithelium (FE) after stage 7 is a non-proliferating postmitotic tissue (Figure 2A). We focused on Minute and mahjong first because of their conserved role in cell competition between flies and mammals (Morata and Ripoll, 1975; Moreno et al., 2002; Oliver et al., 2004; Tamori et al., 2010). In Minute (M(3R)w or M(2)92) mosaic FE, heterozygous (M/+) cells adjacent to wild-type cells frequently underwent apoptosis during both the mitotic and postmitotic stages (Figures 1A, 1D and 1G). In contrast, when the entire FE consisted of M/+ cells, no apoptosis was detected (Figure 1C), suggesting that the apoptosis of M/+ cells in mosaic FE is cell-competition dependent. In mahj mosaic FE, competition-dependent apoptosis of mahj homozygous mutant (mahj−/−) cells adjacent to wild-type or mahj heterozygous (mahj+/−) cells was also observed during postmitotic stages (Figure 1B and 1F), whereas no apoptosis was detected when all follicle cells were mahj−/− (Figure 1E). The surviving mahj−/− cells had a defect in producing VM32E, a structural constituent of vitelline membrane (Figure S1A), suggesting that mahj−/−cells cannot develop into appropriate eggshells. These M/+ or mahj−/− clones completely disappeared from most of the mosaic FE during late stages of oogenesis (Figures S1B and S1C). The competition-dependent apoptosis of mahj−/− or M/+ cells was suppressed when these two types of cells were adjacent (Figure 1H). The survival of mahj−/− and M/+ cells in the less competitive environment further suggests that cell competition can occur in postmitotic epithelia.

Figure 2.

Figure 2

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Whole Process of Cell competition Occur in Postmitotic Stages. (A) A schematic representation of cell cycles in Drosophila FE and its time scale and Ay-Gal4:PR system to control the timing of UAS-gene expression during postmitotic stages. (B) No apoptosis in the stage-10B FE entirely covered by the mahj-RNAi–expressing clones (expressing GFP). (C) Quantification of apoptotic cells in the mahj-RNAi mosaic FE. n = 184 apoptotic cells from 22 egg chambers, means ± s.d. of more than three independent experiments, *P < 0.001. (D) 24 hours after induction of mahj-RNAi–expressing clones (expressing GFP) in a stage-11 FE. Apoptotic cells were stained with anti-cleaved Caspase-3, red in (B) and (D). Nuclei were labeled with DAPI, blue in (B) and (D). Scale bars, 10 μm. See also Figure S2.

Figure 1.

Figure 1

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Cell Competition Can Occur in Postmitotic Follicular Epithelia. (A) Quantification of apoptotic cells in the M/+ mosaic FE. n = 224 apoptotic cells from 29 egg chambers, means ± s.d. of more than three independent experiments, *P < 0.001. (B) Quantification of apoptotic cells in the mahj mosaic FE. n = 258 apoptotic cells from 44 egg chambers, means ± s.d. of more than three independent experiments, *P < 0.001. (C) No apoptosis in the stage-10B FE of M(3R)w, ubi-GFP heterozygous (M/+) fly. (D) Competition-dependent apoptosis in stage-10B mosaic FE with M(3R)w/+− (expressing GFP) and wild-type (lacking GFP) clones. (E) No apoptosis in the stage-10B FE entirely covered by the mahj−/− clones. (F) Competition-dependent apoptosis in stage-10B mosaic FE with mahj−/− (lacking GFP) and wild-type (expressing GFP) clones. (G) Competition-dependent apoptosis in stage-10B mosaic FE with M(2)92/+− (expressing GFP moderately) and wild-type (expressing GFP strongly) clones. (H) Stage-10A mosaic FE with mahj−/− (expressing RFP strongly) and M/+ (expressing RFP moderately) clones. Apoptotic cells were stained with anti-cleaved Caspase-3, red in (C–G) and green in (H). Nuclei were labeled with 4′, 6-diamidino-2-phenylindole (DAPI), blue in (C–H). Scale bars, 10 μm. See also Figure S1.

These experiments did not, however, reveal whether the win-loss determination was made during the postmitotic stages, because the FLP-FRT mitotic recombination system generates mutant clones when cells still proliferate (before stage 7). We therefore generated Mahj-knockdown cells during the postmitotic stages using a UAS-mahj-RNAi construct driven by a progesterone inducible Gal4, AyGal4:PR (an actin promoter flip-out cassette fused to a Gal4:Progesterone Receptor) (Rogulja and Irvine, 2005). This system allowed us to control the timing of mahj-RNAi expression by administration of the progesterone analog RU486. We confirmed that no Gal4 expression was detected in follicle cells prior to RU486 administration (Figure S2A, S2B and S2C). Because the total time of follicle-cell endocycle stages is about 24 hours at 25°C (Mahowald and Kambysellis, 1980) (Figure 2A), we dissected egg chambers after 24 hours of RU486 administration and examined those older than stage 10A, which contained Mahj-knockdown cells induced during postmitotic stages. Indeed, these Mahj-knockdown cells underwent competition-dependent apoptosis after stage 10B (Figure 2C and 2D); many of these cells died and were eliminated in stage-14 egg chambers (Figure S2D). In contrast, when the entire FE expressed mahj-RNAi, no apoptosis was observed (Figure 2B). Collectively, these results led us to conclude that the entire process of cell competition, including the win-loss determination, can be implemented in the postmitotic epithelium.

dMyc, Yki and nTSGs Are Not Involved in Postmitotic Cell Competition in the FE

There are several other types of genes whose mutations have been described to induce cell competition in proliferating Drosophila imaginal epithelia. dMyc, a Drosophila homolog of the c-myc protooncogene, has been suggested to be a major player to determine winners and losers in cell competition, because the relative expression level of dMyc has a decisive influence on competitiveness. dMyc mutant cells are outcompeted by neighboring wild-type cells, whereas cells expressing higher level of dMyc outcompete neighboring wild-type cells (de la Cova et al., 2004; Moreno and Basler, 2004). Previously, it has been shown, however, that dMyc mutant follicle cells are not outcompeted by surrounding wild-type cells in the FE (Maines et al., 2004) but are smaller because of a defect in endoreplication (Maines et al., 2004). We observed the same phenotypes in dMyc-knockdown follicle cells induced by dMyc-RNAi expression. The mosaic clones expressing dMyc-RNAi did not show apoptosis but were smaller during postmitotic stages (Figures 3A; Table S1). On the other hand, dMyc overexpressing follicle cells were larger than wild-type cells but did not induce apoptosis of neighboring wild-type cells (Figure 3B). These results suggest that differences in expression levels of dMyc do not induce cell competition in postmitotic FE.

Figure 3.

Figure 3

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Defect in dMyc, Yki or nTSGs Does Not Cause Cell Competition in Postmitotic FE. (A) Stage-11 mosaic FE with dMyc-RNAi–expressing clones (expressing GFP). (B) Stage-10B mosaic FE with dMyc–expressing clones (expressing GFP). (C) Stage-10B mosaic FE with ykiB5 homozygous mutant (lacking GFP) and wild-type (expressing GFP) clones. (D) Stage-10A mosaic FE with a constitutively active form of Yki (YkiM123)–expressing clones (expressing GFP). (E) Stage-10A mosaic FE with lgl4 homozygous mutant (lacking GFP) and wild-type (expressing GFP) clones. (F) Stage-10B mosaic FE with scrib1 homozygous mutant (expressing GFP) and wild-type (lacking GFP) clones. Apoptotic cells were stained with anti-cleaved Caspase-3, red. Nuclei were labeled with DAPI, blue. Scale bars, 10 μm.

The Hippo pathway is a hyperplastic tumor-suppressor pathway controlling organ size through regulation of cell growth, proliferation, and apoptosis (Halder and Johnson, 2011). Relative difference in the level of Hippo pathway activity between neighboring cells has also been shown to lead to cell competition in Drosophila imaginal discs (Tyler et al., 2007), as is the case with dMyc; mutant clones of yorkie (yki), the Drosophila homolog of the Hippo pathway transducer, Yap, were outcompeted by neighboring wild-type cells and, conversely, Yki overexpressing clones outcompeted neighboring wild-type cells (Neto-Silva et al., 2010; Ziosi et al., 2010). We observed, however, that mosaic clones of yki homozygous mutant follicle cells did not undergo apoptosis in the FE (Figure 3C). Similarly, mosaic clones overexpressing a constitutively active form of Yki (YkiM123) (Zhang et al., 2008), which was sufficient to induce hyperplastic overgrowth in developing imaginal epithelia, did not induce apoptosis of neighboring wild-type cells (Figure 3D).

Neoplastic tumor-suppressor genes (nTSGs) such as lethal giant larvae (lgl) and scribble (scrib) are also involved in cell competition in imaginal discs. The mutant clones of these genes made in a heterozygous imaginal disc through mitotic recombination are slower in growth than their wild-type neighbors and are ultimately eliminated by cell competition (Brumby and Richardson, 2003; Grzeschik et al., 2007; Igaki et al., 2009; Menéndez et al., 2010; Tamori et al., 2010). lgl or scrib homozygous mutant follicle cell clones, however, were not out-competed during postmitotic stages in mosaic FE (Figures 3E and 3F). Together, our results suggest that Minute and mahjong are the only mutations described so far that induce cell competition in both the proliferating epithelium and the postmitotic FE.

“Compensatory Cellular Hypertrophy” of Winner Cells Is Implemented by Acceleration of the Endocycle

Cell competition in proliferating tissues such as developing imaginal epithelia is normally accompanied by compensatory proliferation of winner cells, which fill the space left by the apoptotic loser cells (Simpson and Morata, 1981; de la Cova et al., 2004). In mahj mosaic FE, however, no dividing cells were detected after stage 7 (Figure 4A), indicating that neighboring wild-type cells in the postmitotic epithelium did not undergo compensatory proliferation. Instead, we found that winner cells underwent “compensatory cellular hypertrophy” (CCH)–some wild-type cells appeared larger than other cells in confocal sectional images (Figure 4B). The hypertrophic cells were very rare in control mosaic egg chambers with GFP-expressing follicle cell clones (Figure 4C), but were obvious in M/+ mosaic FE (Figure S3). Vertical sections of the z-stack confocal images revealed that the apical-basal height of the hypertrophic cells was about the same as that of neighboring “normal-sized” cells (Figure 4B, right panels), indicating that these hypertrophic cells had greater cellular volume. Moreover, their nuclear volumes were approximately two to four times as large (Figures 4D and 4E; Table S1). DNA staining revealed that the average signal intensities of the larger and normal nuclei were similar (Figures 4E and 4F) indicating that the DAPI intensities in nuclei of hypertrophic cells was proportional to the change of nuclear volume, so the size change was not due to relaxed chromosomal packaging. The increase in nuclear and cellular volume was unlikely to be the result of cell fusion, because multi-nucleated cells were never observed in mahj mosaic FE. Together, these results suggest that hypertrophic cells may have undergone extra rounds of endocycling to reach the larger nuclear and cellular sizes.

Figure 4.

Figure 4

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Winner Cells Undergo Compensatory Cellular Hypertrophy Implemented by Acceleration of Endocycle. (A) Stage-10B mahj mosaic FE stained for phospho-histone H3 (PH3) (red). (B) Compensatory cell hypertrophy (CCH) in some wild-type clones in a stage-10B mahj mosaic FE stained for Discs large (Dlg) (red). Right panels, vertical sections of two different sites marked with arrows in the left panel. Black line drawings trace the apical and basal sides of the cells. Black box, hypertrophic cells. (C) Quantification of CCH in the control GFP or mahj mosaic FE. n = 100 egg chambers for each genotype, means ± s.d. of more than three independent experiments, *P < 0.001. (D) Quantification of nuclear volumes in the wild-type or mahj mosaic FE. n = 20 cells for each of the four different cell types, means ± s.d. of more than three independent experiments, *P < 0.001. (E) Three different-sized nuclei of wild-type cells in mahj mosaic FE. (F) DAPI signal intensities of the nuclei shown in (E). (G) FACS analysis of DNA contents in non-mosaic mahj heterozygous follicle cells without heat shock. (H) FACS analysis of DNA contents in mahj mosaic follicle cells after heat shock. DNA contents of GFP-negative mahj−/− clones or GFP-positive winner clones (mahj+/− and +/+) are shown in magenta or green, respectively. The merged area where the values of mahj−/− (magenta) and winner clones (green) overlap is shown in black. Increase of the fifth peak indicating a 32C DNA content and emergence of the sixth peak indicating a 64C DNA content were observed in GFP-positive winner clones (H, arrow). (I) BrdU incorporation detected by anti-BrdU (green) in the mahj mosaic stage-10B FE. mahj−/− clones, lacking RFP. White circles, hypertrophic winner cells. Nuclei were stained with DAPI, blue in (A) and (I) and white in (B) and (E). Scale bars, 10 μm. See also Figure S3.

To confirm that the CCH is a result of extra rounds of endocycling in the hypertrophic cells we isolated follicle cells from mahj mosaic egg chambers (the winner wild-type and mahj heterozygous mutant cells marked with GFP) and analyzed the genomic DNA content by fluorescence-activated cell sorting (FACS) analysis. The follicle cells isolated from the mahj/GFP heterozygous flies (FRT42D mahj1/FRT42D ubi-GFP) without mitotic-clone induction showed four major cell populations, with 2C, 4C, 8C, and 16C of nuclear DNA content, suggesting these cells undergo up to three normal rounds of endocycling (Figure 4G). A minor fifth peak indicating 32C DNA content (1.2% of the whole cell population) was also detected in the mahj/GFP heterozygous follicle cells, similar to what has been reported in wild-type egg chambers (Calvi et al., 1998; Cayirlioglu et al., 2001) (Figure 4G). When mitotic clones were induced in these mahj/GFP egg chambers, in contrast, the fifth peak indicating 32C DNA content became larger (4.2% of the whole cell population) and a small sixth peak indicating 64C DNA content (1.4% of the whole cell population) was detected in the GFP-positive cell population (green in Figure 4H). The GFP-negative cell populations (magenta in Figure 4H) indicating mahj homozygous mutant cells showed the first three peaks (2C, 4C and 8C), but the fourth peak (16C) was barely detectable (Figure 4H). These results further confirmed that the hypertrophic cells underwent one or two extra rounds of endocycling and that mahj−/− cells were smaller than wild-type neighboring cells. Based on these observations, hereafter, we defined CCH as hypertryophic cells with larger nuclear volumes (2 or more fold change) caused by extra endocycles.

Analyses of 5-bromo-2-deoxyuridine (BrdU) incorporation in mahj mosaics revealed that the hypertrophic cells did not prolong the endocycle stage but instead accelerated endocycles within the normal time window to achieve their increased size. Follicle cells normally show oscillating genomic BrdU incorporation patterns in mitotic and endocycle stages and a punctate pattern after transitioning into the gene-amplification stages (Calvi et al., 1998). In mahj mosaic FE at stage 10B, all main-body follicle cells, including hypertrophic cells, showed synchronized punctate BrdU incorporation patterns (Figure 4I) and must therefore have left endocycle on schedule.

CCH Implemented by Endocycle Acceleration Is Dependent on IIS Activation

The insulin/IGF (insulin-like growth factor)-like signaling (IIS) pathway has been shown to regulate cellular growth and endoreplication rates (Cavaliere et al., 2005; Hietakangas and Cohen, 2009; Zielke et al., 2011). To determine whether IIS activation is required for the CCH, we blocked activation of the IIS pathway with expression of an RNAi construct for Drosophila Akt (dAkt-RNAi), a key regulator of IIS pathway (Hietakangas and Cohen, 2009), or a dominant-negative form of the insulin receptor (InRDN) (Wu et al., 2005) in the winner (wild-type and mahj+/−) cells but not in the mahj−/− cells in postmitotic mahj mosaic FE using the FRT ptc-Gal4 mosaic system (see Experimental Procedures for detail). The blockade of IIS did not suppress competition-dependent apoptosis of mahj−/− cells (Figure 5B) but did significantly suppress CCH of the winner cells (Figures 5A, 5C and 5D) indicating that IIS activity is required for CCH. To assess the activity of PI3K, a target of InR, in mahj mosaics, we used a tubulin promotor-GFP-pleckstrin homology domain fusion (tGPH) as a marker (Britton et al. 2002). In mahj mosaic FE, weak cytoplasmic tGPH was observed in the normal wild-type cells. In the hypertrophic cells, however, cytoplasmic tGPH was decreased, and membrane localization increased (68.53% s.d. ± 13.38 of the hypertrophic cells, n = 100) (Figures 5E and 5F), indicating that insulin-PI3K signaling is upregulated in the hypertrophic cells.

Figure 5.

Figure 5

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CCH Implemented by Endocycle Acceleration is Dependent on IIS Activation. (A) Stage-10B mahj mosaic FE with overexpression of dAkt-RNAi in the winner (wild-type and mahj+/−) cells (expressing GFP). (B) Stage-10B mahj mosaic FE with overexpression of InRDN in the winner (wild-type and mahj+/−) cells (expressing GFP). (C) Quantification of CCH in the control GFP, mahj, or mahj−/− with IIS pathway activation-blocked-winner cells mosaic FE. n = 100 egg chambers for each genotype, means ± s.d. of more than three independent experiments, *P < 0.001. (D) Quantification of nuclear volumes of wild-type or wild-type expressing dAkt-RNAi or InRDN with mahj−/− clones. n = 20 cells for each of three different cell types, means ± s.d. of more than three independent experiments. (E) A stage-10A mahj mosaic FE with mahj−/− (lacking RFP in their nuclei) and wild-type cells (expressing RFP in their nuclei). The tGPH signals (green) observed in the mahj−/− or nomal size wild-type cells were increased in the membranes of hypertrophic wild-type cells (arrowhead). Right panel shows signal intensities of tGPH plotted using Interactive 3D Surface Plot, an ImageJ plugin. (F) Quantification of tGPH signal intensities and their local differences between membrane and cytoplasm in the normal and hypertrophic wild-type cells in mahj−/− mosaic FE. n = 20 cells for each of two different cell types, means ± s.d. of more than three independent experiments. The values of pixel intensity were measured using ImageJ Plot Profile. (G) Quantification of nuclear volumes of wild-type or InRCA–overexpressing clones. n = 20 cells for each of two different cell types, means ± s.d. of more than three independent experiments, *P < 0.001. (H) 24 hours after induction of InRCA expressing clones (expressing GFP) in a stage-10B FE stained for Dlg (red). (I) BrdU incorporation detected by anti-BrdU antibody (red) in the stage-10B mosaic FE with InRCA–overexpressing clones (expressing GFP). Nuclei were labeled with DAPI, white in (A), (B), (E) and (H) and blue in (I). Scale bars, 10 μm. See also Figure S4.

To determine whether upregulation of IIS accelerates the endocycle in follicle cells, we used overexpression of a constitutively active form of the insulin receptor (InRCA) (Wu et al., 2005), which was sufficient to induce hypertrophy in follicle cells (Figure S4B). To control the timing of transgene overexpression, we again used the AyGal4:PR system. In stage-10B egg chambers dissected 24 hours after RU486 administration, overgrowth of InRCA-expressing cells was readily observed (Figure 5H). Their nuclear volumes were approximately 2- to 4-fold that of their wild-type neighbors (Figure 5G and Table S1). This increase in nuclear volume was not caused by prolonged endocycling after stage 10A, because the overgrown InRCA-expressing cells entered the gene amplification stage normally (Figure 5I). Together, these results suggest that CCH implemented by endocycle acceleration within the normal window of endocycle stages is dependent on IIS activation.

CCH Does Not Require JNK Signaling Activation

In proliferating epithelia, an induction of sporadic apoptosis has been shown to trigger compensatory proliferation of neighboring tissues (Huh et al., 2004; Pérez-Garijo et al., 2004; Ryoo et al., 2004). The stress-responsive c-Jun N-terminal kinase (JNK) pathway is activated in the apoptotic cells to upregulate mitogenic signaling in the neighboring normal cells (Pérez-Garijo et al., 2004; Ryoo et al., 2004; Sun and Irvine, 2011). When the apoptotic cells are kept alive by the expression of baculovirus caspase inhibitor, p35, hyperplastic overproliferation is induced because of persistent JNK activity in the “undead” cells (Pérez-Garijo et al., 2009). In postmitotic FE, CCH instead of compensatory proliferation was observed in some wild-type follicle cells when sporadic apoptosis was induced by overexpression of the proapoptotic gene Reaper (Rpr) (Figures 6A and 6B). The Rpr-overexpressing apoptotic cells were extruded from the apical side of the epithelial layer (Figures 6D and 6E). To determine whether JNK signaling mediates CCH in the postmitotic FE, we monitored the expression of a JNK activation reporter, puckered (puc)-lacZ (puc encodes a JNK phosphotase that negatively regulates JNK activity) (Martín-Blanco et al., 1998; Dobens et al., 2001). JNK activation, however, was not detected in the mosaic FE where sporadic apoptosis was induced by overexpression of Rpr (Figure 6C). In contrast, in mahj mosaic FE, we found that JNK was highly activated in the winner cells abutting the mahj−/− cells during postmitotic stages (Figure 6F).

Figure 6.

Figure 6

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JNK Signaling Activation Is Not Required For CCH. (A) 18 hours after induction of Rpr-expressing clones (expressing GFP) in a stage-10B mosaic FE. Mitotic cells were labeled with anti-PH3 (red). (B) Quantification of nuclear volumes in the mosaic FE with Rpr- or Rpr- and p35-overexpressing clones. n = 20 cells for each of the four different cell types, means ± s.d. of more than three independent experiments, *P < 0.001. (C) JNK is not activated in a stage-10B mosaic FE with Rpr-expressing clones (expressing GFP). (D) 12 hours after induction of Rpr-expressing clones (expressing GFP) in a stage-10B mosaic FE. (E) A vertical section of the site marked with a white line in (D) showing apical extrusion of apoptotic Rpr-expressing clones (expressing GFP). (F) JNK activation patterns in a stage-10B mahj mosaic FE. (G) Stage-10B mosaic FE with mahj−/− (lacking GFP) and wild-type (expressing GFP) clones. (H) and (I) Vertical sections of two different sites marked with white lines in (G) showing apical protrusions (arrows) of wild-type clones (expressing GFP) to the neighboring mahj−/− clones (lacking GFP) and an apoptotic mahj−/− cell engulfed by the wild-type neighbors (star). (J) Stage-10B mahj mosaic FE with overexpression of Puc in the winner (wild-type and mahj+/−) cells (expressing GFP). (K) Quantification of CCH in the wild-type, mahj or mahj−/− with JNK activation-blocked-winner cells mosaic FE. n = 100 egg chambers for each genotype, means ± s.d. of more than three independent experiments. (L) Quantification of apoptotic cells in the mahj mosaic FE with or without JNK activation-blocked-winner cells. n = 1483 (mahj−/− cells with WT), 1824 (mahj−/− cells with WT + Puc), or 1719 (mahj−/− cells with WT + BskDN) apoptotic cells from 50 different egg chambers for each mosaic situation, means ± s.d. of more than three independent experiments, *P < 0.001. The expression of puc-lacZB48, an enhancer-trap reporter line for puckered (Martín-Blanco et al., 1998; Dobens et al., 2001), was stained with anti-β-galactosidase antibodies, red in (C), (F) and (J). Plasma membranes were stained with anti-Dlg, red in (D–E) and (G–I). Nuclei were stained with DAPI, white in (A) and (J) and blue in (C–I). Arrowheads, CCH. Scale bars, 10 μm. See also Figure S5.

Recently, non-autonomous activation of JNK signaling in wild-type cells surrounding mutant clones of neoplastic tumor-suppressor genes such as scrib has been shown to induce genes involved in the phagocytic pathway to promote engulfment of the mutant cells (Ohsawa et al. 2011). In the postmitotic mahj mosaic FE, we found that some mahj−/− cells were engulfed by neighboring wild-type cells (Figure 6F). This engulfment by the neighboring winner cells was prominent in the vertical sections of the z-stack confocal images of the mosaic epithelial layer (Figures 6G, 6H and 6I); the winner cells protruded their apical side to engulf neighboring mahj−/− cells. The competition-dependent apoptosis of mahj−/− cells, however, was not suppressed in the homozygous mutant background for genes involved in engulfment such as draper (drpr), wasp or phosphatidylserine receptor (psr), in the postmitotic FE (Figure S5D, S5E and S5F), suggesting that the engulfment is a passive response to the dying loser cells to remove the apoptotic corpses. Interestingly, when JNK signaling was blocked with overexpression of Puc (Drosophila JNK phosphatase) or a dominant negative (DN) form of Basket (Adachi-Yamada et al., 1999a) (Drosophila JNK, Bsk) in the winner (wild-type and mahj heterozygous) cells but not in the mahj−/− cells, both the engulfment and competition-dependent apoptosis of mahj−/− cells were significantly suppressed (Figures 6J, 6L, S5A and S5B). These JNK blockades, however, did not suppress the CCH of the winner cells (Figures 6J, 6K, S5A and S5B). Also, mosaic clones overexpressing an active form of Drosophila JNKK, Hemipterous, (HepCA) (Adachi-Yamada et al. 1999b) did not induce CCH in the postmitotic FE (Figure S5C). These results indicate that CCH does not require JNK activation, unlike the mechanism of compensatory proliferation in proliferating epithelia. In support of this conclusion, in the mahj mosaic FE or in the mosaic FE with Rpr-expressing clones, CCH was observed not only in the cells neighboring the mahj−/− or Rpr-expressing apoptotic cells, but also several cells away, where JNK activity was low (Figures 6C and 6J).

CCH Is Induced by Loss of Local Tissue Volume in Postmitotic Epithelia

When Rpr-expressing apoptotic cells were kept alive by p35 co-expression in the postmitotic FE, the undead cells induced neither overproliferation nor CCH of the surrounding wild-type cells (Figure 7A). These undead cells were normal in their morphology and size (Figures 6B and 7A; Table S1). In contrast, some winner cells in mahj mosaics exhibited CCH even when the apoptosis of mahj−/− cells was inhibited by p35 expression (Figures 7B and 7D). The undead mahj−/− cells were smaller than normalsized wild-type cells (Figures 7B; Table S1). Furthermore, winner cells in M/+ mosaics did not exhibit significant CCH when apoptosis of M/+ cells was inhibited by p35 expression (Figures 7C and S6B). The undead M/+ cells were normal in their morphology and size (Figures 7C and S6A). Because the single common event entailing CCH among these different situations (apoptosis of M/+ or mahj−/− loser cells, sporadic apoptosis induced by Rpr-expressing clones, or smaller-sized undead mahj−/− cells) is loss of local tissue volume, we hypothesized that CCH is sporadically induced by loss of local tissue volume resulting from cellular growth or viability defects of some cells in the epithelium and that apoptosis is not necessary to induce CCH. To test this hypothesis we generated small and non-mitotic but viable dMyc knockdown cells (Maines et al., 2004) in the postmitotic FE using AyGal4:PR system. As shown in Figure 3, dMyc-knockdown follicle cells induced by dMyc-RNAi expression did not show apoptosis but were significantly smaller than wild-type cells during postmitotic stages (Figures 3A, 7E and 7F; Table S1). In these mosaic FE, sporadic CCH was observed not only in the neighboring wild-type cells but also several cells away from the dMyc-knockdown cells (Figures 7F and 7G) suggesting that the CCH is induced by the loss of local tissue volume in the postmitotic epithelium.

Figure 7.

Figure 7

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The CCH is sporadically induced by loss of local tissue volume of epithelial tissues. (A) 48 hours after induction of Rpr- and p35-expressing clones (expressing GFP) in a stage-10B mosaic FE. Mitotic cells were labeled with anti-PH3 (red). (B) Stage-10B mosaic FE with mahj−/− MARCM clones expressing p35 (marked with GFP). (C) Stage-10B M/+ mosaic FE with overexpression of p35 both in the wild-type (expressing GFP strongly) and M/+ clones (expressing GFP moderately). (D) Quantification of CCH in the wild-type or p35–expressing mahj mosaic FE. n = 100 egg chambers for each genotype, means ± s.d. of more than three independent experiments, *P < 0.001. (E) Quantification of nuclear volumes in the mosaic FE with wild-type or dMyc-RNAi–expressing clones. n = 20 cells for each of the three different cell types, means ± s.d. of more than three independent experiments, *P < 0.001. (F) 24 hours after induction of dMyc-RNAi–expressing clones (expressing GFP) in a stage-10B mosaic FE. (G) Quantification of CCH localization in the mosaic FE with undead mahj−/− clones (overexpressing p35) or dMyc-RNAi expressing clones. n = 306 (WT with mahj−/− + p35, white bars) or 346 (WT with dMyc-RNAi, hatched bars) hypertrophic cells from 50 different egg chambers for each mosaic situation, means ± s.d. of more than three independent experiments. No distances from the mutant clones or between two different mosaic mutant genotypes differed significantly (P > 0.01). Data of the CCH localization was analyzed by one-way analysis of variance (ANOVA). Average of CCH rates between two different genotypes for each distance was analyzed with two-tailed unpaired t-tests assuming equal variances (P > 0.01). (H) A model for tissue repair through CCH in postmitotic epithelial tissues. In cell competition, aberrant but viable loser cells are eliminated by neighboring winner cells through JNK-dependent engulfment (upper left). Damaged apoptotic cells are extruded from the apical side of epithelial layer (lower left). In either case, local tissue volume is lost, and it induces IIS activation-dependent sporadic cellular hypertrophy. Plasma membranes were labeled with anti-Dlg, red in (B), (C) and (F). Nuclei were stained with DAPI (white). Arrowheads, CCH. Scale bars, 10 μm. See also Figure S6.

Discussion

Cell Competition in Postmitotic FE

Cell competition displays three major phenotypic hallmarks: (1) competition-dependent apoptosis; loser cells at the clone boundary undergo apoptosis, (2) survival of loser cells in homogeneous situation; loser cells remain viable in a homogeneous field where they come into contact only with the same loser cells, and (3) survival of loser cell when competitive pressure is lowered; loser cells survive when they confront M/+ cells (Johnston, 2009; Tamori and Deng, 2011). In postmitotic FE, M/+ and mahj−/− cells show all of these three hallmarks; this phenomenon can therefore be regarded as cell competition, even though cell competition has only been previously described between proliferating cells.

Most of the mutant cells that have been shown to be eliminated in cell competition are slower-growing cells in proliferating tissues. In these tissues, cell growth is normally used non-discriminately to refer to both increases in cell number and cell size. Based on our study, we would like to separate these two processes, and use “cell proliferation” to refer to the increase of cell number, and use “cell growth” to refer only to the increase of cell size. The postmitotic FE provides an excellent opportunity to examine whether a difference in proliferation is generally involved in determining winners and losers. In this tissue, our data show that the entire process of cell competition, including the win-loss determination, can be implemented in the postmitotic epithelium, where cells no longer proliferate. These findings indicate that cell proliferation rate is not necessarily the determining factor for winners and losers in cell competition. Although not dividing, these follicle cells nevertheless undergo endoreplication to increase nuclear and cellular volume, raising the question whether different cellular growth rates between neighboring cells can trigger cell competition. In postmitotic FE, our studies indicate that the cellular growth rate does not seem to have a prominent role in determining winners and losers, either. First, the M/+ follicle cells showed no obvious growth defect but were outcompeted by wild-type cells in the postmitotic FE. Second, neither knockdown of dMyc, which causes slower cellular growth, nor overexpression of dMyc, which accelerates cellular growth, results in cell competition in these postmitotic follicle cells.

Cell competition is not just a struggle for existence of cells but a tissue intrinsic homeostasis system to increase organismal fitness via maximization of the quality of somatic cells (Baker, 2011). The primary role of the somatic follicular epithelial cells is to cover the germ-line cells and keep their development appropriate. Consistent with this notion, mahj mutant cells have a defect in producing VM32E, a structural constituent of vitelline membrane. Although the criterion for comparison of cellular fitness might be dependent on tissue types, the important concept shown in this study is that not only proliferating tissues but also postmitotic tissues can eliminate aberrant cells through cell competition to maintain tissue integrity. Further studies designed to identify molecules that play a pivotal role in cell competition and to elucidate the mechanisms in different types of mutant cells and tissues will eventually reveal the whole picture of the phenomenon.

JNK and Elimination of Loser Cells in Postmitotic FE

It has been shown that, in proliferating tissues, ectopic activation of JNK signaling in apoptotic cells plays a key role in compensatory proliferation to upregulate mitogenic signaling in surrounding normal cells (Pérez-Garijo et al., 2004; Ryoo et al., 2004; Sun and Irvine, 2011). Our data, however, indicate that JNK signaling is not involved in the induction of CCH in the postmitotic FE. The non-cell-autonomous JNK activation observed in the wild-type cells adjacent to mahj−/− cells seems to be involved in engulfment to eliminate the loser cells, as the blockade of JNK activation strongly suppressed the engulfment and apoptosis of mahj−/− cells. This JNK-induced engulfment may be specific in cell competition, since JNK activation was not observed in the mosaic FE with Rpr-expressing apoptotic clones which were apically extruded (Figure 7F). An important question is whether or not engulfment plays an active role in killing neighboring loser cells.

Li and Baker (2007) reported apoptosis-independent engulfment in cell competition. Their results suggest that the corpse engulfment by winner cells is not simply a passive response to the presence of dying loser (M/+) cells but is required to kill and eliminate neighboring loser cells (Li and Baker, 2007). Ohsawa et al. (2011) revealed that non-cell-autonomous JNK activation in normal cells surrounding scrib, dlg, or vps25 mutant clones induced upregulation of genes involved in the phagocytic pathway, which caused them to engulf the nTSGs mutant cells (Ohsawa et al., 2011). More recently, Lolo et al. (2012), however, showed that most of the apoptotic loser cells are not engulfed by neighboring winner cells but by recruited hemocytes, which are required for the removal of the apoptotic corpses. They, therefore, concluded that engulfment is a consequence but not a cause of loser cells’ death (Lolo et al., 2012).

In postmitotic FE, engulfment does not seem to have an active role in inducing apoptosis, as mahj−/− cells still underwent competition-dependent apoptosis when genes involved in engulfment were disrupted. In addition, undead mahj−/− cells expressing p35 were not engulfed by neighboring wild-type cells. Together, these observations suggest that engulfment is more likely a passive response to the dying loser cells to remove the apoptotic corpses in the FE. Since blocking of JNK activation in winner cells strongly suppressed both the engulfment and the apoptosis of mahj−/− cells, an unknown signal downstream of JNK activation may have been produced by the winner cells to signal the death of the abutting loser cells, and JNK dependent engulfment is involved in clearing the dying cells.

Mechanisms of Compensatory Cellular Hypertrophy

Several lines of evidence shown in this study indicate that the CCH is triggered by a loss of local tissue volume resulting from defects in cell growth or viability in the postmitotic epithelium (Figure 7F). The next important question is how the loss of local tissue volume leads to upregulation of IIS signaling sporadically in the postmitotic tissue. A key fact in this process is that CCH is induced sporadically not only in the neighboring cells but also in cells located several cells away from the location where local tissue volume is lost. This interspersion of CCH suggests that IIS upregulation is probably not triggered by a signaling molecule from the apoptotic cells or small-sized cells, but by a global physical alteration resulting from the loss of local tissue volume. One aspect of this physical stress is probably tensile force, because postmitotic tissues are comprised of a predetermined number of cells. A loss of cell number or reduction in size of some cells would increase the overall stretched state of the postmitotic tissue, and the increase of the tensile force is probably not evenly distributed among all cells. Future studies on how tensile force induces IIS activation would address the molecular basis of this mechanism.

The fact that hypertension induces cellular hypertrophy has been corroborated by some experimental studies showing that cyclic mechanical stretch enhances the hypertrophic growth of mammalian cardiomyocytes (Blaauw et al., 2010; Leychenko et al., 2011) or chick skeletal muscle cells (Sasai et al., 2010). The involvement of the IIS pathway in mechanical stretch-induced hypertrophy has been indicated in some previous reports (Hu et al., 2007; Sasai et al., 2010). Tensile force-induced CCH may therefore be a conserved tissue homeostasis system in postmitotic tissues. Indeed, cellular hypertrophy related tissue homeostasis has been shown in several other systems. In Drosophila brains, increased cell size of subperineurial glia (SPG) resulting from polyploidization is required for maintaining the SPG envelope surrounding the growing brain (Unhavaithaya and Orr-Weaver, 2012). Cellular hypertrophy of hepatocytes through an increase in ploidy, without proliferation, makes the first contribution to liver regeneration after partial hepatectomy in mice (Miyaoka et al., 2012). These indicate that the CCH through endoreplication may have a conserved role across cell types and phyla. Recently, overcrowding-induced live-cell delamination has been shown to be a conserved process that buffers growing cell numbers in epithelial tissues (Eisenhoffer et al., 2012; Marinari et al., 2012). Although this mode of epithelial homeostasis uses a mechanism different from “loss of local tissue volume”-induced cellular hypertrophy in postmitotic epithelia, both systems rely on plastic cellular behaviors. Fine control of tissue integrity and organ size is therefore ensured by these plastic cellular behaviors even under the constraints of tissue-intrinsic genetic programs.

Experimental Procedures

Fly Stocks and Genetics

Drosophila stocks were maintained by standard methods at 25°C. For generation of mosaic mutant clones, third-instar larvae or pupae were heat-shocked for 2 hours at 37°C on two consecutive days. For generation of UAS-transgene overexpression clones, adult flies were heat-shocked for 15–30 min at 37°C. To control the timing of UAS-transgene overexpression with the AyGal4:PR system, we heat-shocked adult female flies for 15–30 min at 37°C to generate flip-out mosaic clones, and 1 day after the heat shock, we transferred them to vials with yeast paste containing 20 μg/ml RU486 (mifepristone, Sigma). To overexpress UAS-transgenes only in the winner cells (wild-type and mahj heterozygous cells) in the mahj mosaic follicular epithelia during postmitotic stages we used recombined chromosomes, FRT42D ptc-Gal4 UAS-GFP and FRT42D hRFP mahj1 with temperature-sensitive Gal80 (Gal80ts). ptc-Gal4 drove UAS-transgene expression in all main-body follicle cells after stage 7. Clones homozygous for ptc-Gal4 UAS-GFP do not compete with wild-type cells (Figure S4A). To overexpress p35 in mahj−/− cells, we used mosaic analysis with a repressible cell marker (MARCM) system. Fly strains and all genotypes of flies used in each experiment are described in Supplemental Information.

Immunohistochemistry and Image Analysis

Immunofluorescent stainings of follicular epithelia were performed according to standard procedures for confocal microscopy as described previously (Deng et al., 2001). The following antibodies were used: rabbit anti-β-galactosidase (1:2000, MP Biomedicals), mouse anti-BrdU (1:60, BD Biosciences), rabbit anti-cleaved Caspase-3 (1:100, Cell Signaling), mouse anti-Dlg (1:40, Developmental Studies Hybridoma Bank), rabbit anti-phosphohistone H3 (1:200, BD Biosciences), rabbit anti-VM32E (1:100; a gift from V. Cavaliere, University of Bologna, Italy) (Andrenacci et al., 2001). Alexa Fluor 488, 546 and 633 (1:400, Invitrogen) were used for secondary antibodies. Images were acquired with a Zeiss LSM 510 confocal microscope. Nucleus volumes were measured with Volumest, an ImageJ plugin for volume estimation by stereological methods (Merzin, 2008). Signal intensity of DAPI was plotted with Interactive 3D Surface Plot, an ImageJ plugin.

Fluorescence-activated cell-sorting analysis

Follicle cell isolation was conducted as described previously (Bryant et al., 1999). Ovaries of 250–300 females per experiment were dissected in Grace’s insect medium supplemented with 10% fetal calf serum and 1× antibiotic antimycotic. Ovaries were washed three times in calcium-free phosphate-buffered saline and incubated in 0.7 ml of 0.25% trypsin with intermittent vortexing at room temperature for 15 min. Supernatant was passed through a 40-μm nylon filter into 1 ml of Grace’s medium and pelleted at 4,000 rpm for 7 min in an Eppendorf Minispin. The filtration step was repeated two to three additional times until the supernatant became clear. Follicle cells were then resuspended in 0.5 ml of Grace’s medium containing 1 μl of 5 mM Vybrant DyeCycle Violet Stain (Life Technologies), incubated at room temperature for 30 min, washed once in calcium-free phosphate-buffered saline and stored on ice. A flow cytometer (FACSAria; Becton Dickinson) determined follicle cell ploidy by fluorescence-activated cell-sorting analysis of Vybrant DyeCycle–stained cell preparations with excitation at 407 nm for Vybrant DyeCycle stain and at 488 nm for GFP. We used BD CS&T Beads (BD Biosciences) and SPHERO Rainbow Fluorescent Particles (Sperotech) as a calibration standard.

Quantification of CCH

We defined CCH as hypertrophic cells with larger nuclear volumes (2 or more fold change) than normal wild-type cells (139.62 μm3 ± s.d. 13.4) caused by extra endocycles, and counted the number of egg chambers bearing CCH cells when a CCH inducing condition existed in that egg chamber. The average percentages of CCH were quantified from 100 egg chambers of more than three independent experiments for each genotype.

Quantification of Nuclear Volumes in CCH

The nuclear volumes of randomly selected clones for each genotype in mosaic FE were measured. If the measured nuclear volume was more than double that of normal cells (139.62 μm3 ± s.d. 13.4), we counted the cells as CCH. The average measured value of cells counted as CCH in each mosaic FE was shown in each graph for quantification of nuclear volumes.

Statistical Analysis

Two-tailed unpaired t-tests assuming equal variances were performed for all statistical analyses. P < 0.01 was considered statistically significant for all analyses.

Supplementary Material

01

Highlight.

Cell competition occurs in the postmitotic epithelium.

Compensatory cellular hypertrophy (CCH) as a tissue repair mechanism.

CCH is implemented by acceleration of endoreplication cycle.

Loss of local tissue volume triggers sporadic epithelial CCH over a long range.

Acknowledgments

We thank N. Baker, D. Bilder, L.L. Dobens, L.A. Johnston, J. Jiang, D. Pan, the Vienna Drosophila RNAi Center and the Bloomington Drosophila Stock Center for providing fly stocks; and V. Cavaliere for providing anti-VM32E antibody. We also thank J.S. Acuna, Y. Fujita, J.S. Poulton and A.B. Thistle for critical reading of the manuscript; J.S. Acuna, Y.C. Huang, D. Jia, B. Palmer, Z. Shu, S.-A. Yang and G. Xie for help in dissection of ovaries for the FACS analysis; and R. Didier from the Flow Cytometry Lab of FSU College of Medicine and T.J. Fellers from the Biological Science Imaging Resource of FSU for their technical support. This work was supported by National Science Foundation grant (IOS-1052333) and National Institutes of Health grant (R01 GM072562) to W.-M.D.

Footnotes

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