The polyhomeotic (ph) gene of Drosophila is a member of the Polycomb group (Pc-G) genes, which are required for maintenance of a repressed state of homeotic gene transcription, which stabilizes cell identity throughout development. The ph gene was recovered in the course of a gain-of-function screen aimed at identifying genes with a role during ovarian follicle formation in Drosophila, a process that involves coordinated proliferation and differentiation of two cell lineages, somatic and germline. Subsequent analysis revealed that ph loss-of-function mutations lead to production of follicles with greater or fewer than the normal number of germ cells associated with reduced proliferation of somatic prefollicular cells, abnormal prefollicular cell encapsulation of germline cysts and an excess of both interfollicular stalk cells and polar cells. Clonal analysis showed that ph function for follicle formation resides specifically in somatic cells and not in the germline. This is thus the first time that a role has been shown for a Pc-G gene during Drosophila folliculogenesis. In addition,we tested mutations in a number of other Pc-G genes, and two of them, Sex combs extra (Sce) and Sex comb on midleg(Scm), also displayed ovarian defects similar to those observed for ph. Our results provide a new model system, the Drosophilaovary, in which the function of Pc-G genes, distinct from that of control of homeotic gene expression, can be explored.

Drosophila melanogaster oogenesis provides an outstanding opportunity for studying several fundamental aspects of developmental biology(for reviews, see King, 1970; Spradling, 1993; Spradling et al., 1997). Indeed, ovarian follicle formation in Drosophila, involving coordinated regulation of proliferation, differentiation and morphogenesis of two distinct cell lineages, somatic and germline, occurs throughout adult life through continuous activity of somatic and germline stem cells, and is thus readily accessible for observation and analysis. Each ovary is composed of 12 to 16 ovarioles, which represent linear strings of maturing follicles, also called egg chambers. Each follicle contains a single oocyte developing independently of its neighbors within a somatic follicular epithelium. Germline and somatic stem cells reside in the germarium, at the anterior end of each ovariole (Fig. 1). At the most anterior part of the germarium, overlying somatic cells (terminal filament and cap cells) create a niche that maintains the germline stem cell population via secretion of several signaling molecules(Spradling et al., 2001; Lin, 2002). Oogenesis starts with the asymmetric division of a germline stem cell, which generates both a daughter stem cell and a differentiated daughter cell, the cystoblast. Within region 1 of the germarium (Fig. 1), each cystoblast undergoes exactly four synchronous divisions with incomplete cytokinesis to give rise to a germline cyst comprised of 16 cystocytes connected to each other by cytoplasmic bridges called ring canals. One of the two cells with four ring canals becomes an oocyte and proceeds through meiosis, while the remaining 15 cystocytes develop as nurse cells and begin endoreplication of their DNA. The population of germline cysts during these stages is surrounded by somatic inner sheath cells(Margolis and Spradling,1995). Morphologically unidentifiable somatic stem cells reside in the region 2a/2b boundary of the germarium and give rise to mesenchymal somatic cells (or prefollicular cells) invaginating from the germarium wall to individuate each germline cyst (Fig. 1). This migration is associated with a characteristic transformation of the germline cyst, consisting of both flattening and lengthening of the cyst so that it spans the width of the germarium. Part of this population of prefollicular cells continues to divide and differentiates into a monolayer of polarized epithelial follicle cells(Margolis and Spradling,1995). The most mature cyst in the germarium and its surrounding follicular cells form a stage 1 follicle in region 3(Fig. 1). Anteriorly to this chamber, other prefollicular cells interdigitate to form an interfollicular stalk of five to seven cells that separates the newly born follicle from the rest of the germarium and persists between all the maturing follicles of the ovariole. Prefollicular cells give rise to a third population of specialized somatic cells, the polar cells, which represent pairs of cells defining the anterior and posterior poles of each follicle(Fig. 1). Lineage studies have previously demonstrated that precursors for stalk and polar cells separate from the general follicle cell lineage within germarial region 2b, although specific markers distinguishing these cell types can be visualized only once follicle budding is complete (Tworoger et al., 1999).

Ovarian follicle formation thus requires the coordinated control of proliferation and differentiation of both germline and somatic cells. Regulation of somatic cell proliferation and patterning has been shown to involve both soma-soma and germline-soma intercellular signaling. For instance, the apically located somatic terminal filament and cap cells are a source of Hedgehog (Hh) and Wingless (Wg) signals, both regulating somatic stem cell proliferation and/or maintenance(Forbes et al., 1996; Zhang and Kalderon, 2000; Zhang and Kalderon, 2001; Song and Xie, 2003). Hh has been shown to play an additional role in prefollicular cell differentiation and morphogenesis (Besse et al.,2002). Mature 16-cell germline cysts also instruct prefollicular cell morphogenesis during cyst encapsulation, since, in the absence of germline cells, prefollicular cells do not undergo any cell shape changes(Spradling et al., 1997). Indeed, both secreted proteins (Brainiac, Egghead, Gurken/TGF-α) encoded by genes with germline-specific function and transmembrane proteins expressed in prefollicular cells (Torpedo/EGFR) are thought to act as components of germline-to-soma signaling pathways required for correct encapsulation of mature 16-cell cysts by prefollicular cells(Goode et al., 1992; Goode et al., 1996a; Goode et al., 1996b; Rubsam et al., 1998). In addition, specification of polar cells and interfollicular stalk cells involves germline-to-soma Delta/Notch signaling. If Notch function is removed in somatic cells, or that of Delta in germline cells, no polar cells are specified (Grammont and Irvine, 2001; Lopez-Schier and St Johnston, 2001). In the absence of polar cells, the separation of germline cysts by somatic cells is defective, and this leads to accumulation of several cysts in the same follicle(Grammont and Irvine, 2001). Polar cells also seem to be a source of signaling molecules necessary for specification of interfollicular stalk cell fate. Delta/Notch signaling may be involved, since induction of mutant Delta clones in the somatic cells leads to an absence of interfollicular stalks(Lopez-Schier and St Johnston,2001). In addition, JAK/STAT signaling is required. Lack of Stat92E or Hopscotch (the Drosophila JAK homolog) in somatic cells leads to an absence of interfollicular stalk cells and to an increase in the number of polar cells, whereas overexpression of Unpaired leads to an increase in the number of stalk cells and to an absence of polar cells(Baksa et al., 2002; McGregor et al., 2002).

In the course of a gain-of-function screen aimed at identifying genes with somatic function during oogenesis, we found that overexpression of polyhomeotic (ph) leads to a significant reduction in female fecundity. The ph gene is a member of the Polycomb group(Pc-G) genes. Genes of the Pc-G and the trithorax-group (trx-G) have been shown to control early embryonic development, as well as imaginal disc morphogenesis, through maintenance, but not initiation, of the spatial pattern of homeotic and segment polarity gene expression during embryonic and imaginal disc development (Dura and Ingham,1988; Ingham and Martinez-Arias, 1992; Simon et al., 1992; Paro,1993; Randsholt et al.,2000; Francis and Kingston,2001). Several lines of evidence indicate that Pc-G proteins associate in multimeric complexes associated with chromatin, thereby ensuring gene silencing throughout development(Fauvarque and Dura, 1993; Chan et al., 1994), while trx-G proteins counteract the silencing effect of Pc-G proteins(Poux et al., 2002). Mutations in Pc-G genes exhibit synergistic and dose-dependent effects,suggesting cooperative interactions between proteins encoded by these genes. In addition, each Pc-G protein recognizes approximately 100 sites on polytene chromosomes from Drosophila salivary glands, but these sites do not fully overlap between these proteins(Zink and Paro, 1989; DeCamillis et al., 1992; Lonie et al., 1994). Two-hybrid and immunoprecipitation studies have shown that the Pc-G proteins form at least two non-overlapping protein complexes. One complex,named PRC1, includes Polycomb (Pc), Posterior Sex Combs (Psc), Polyhomeotic(PH), dRing/Sex Combs Extra (Sce) and Sex Comb on Midleg (Scm)(Shao et al., 1999; Saurin et al., 2001; Fritsch et al., 2003), while the other contains Extra Sex combs (Esc) and Enhancer of zeste (E(z))(Ng et al., 2000; Tie et al., 2001). Nevertheless, it has been shown that Pc, Psc and PH are differentially distributed on regulatory sequences of the engrailed-related gene invected, suggesting that there may be multiple Pc-G protein complexes with different compositions that function at different target sites(Strutt and Paro, 1997). Since some Pc-G proteins are not part of these complexes, for example Polycomb-like(Pcl), it is supposed that there are other as yet unidentified complexes of Pc-G proteins (for a review, see Otte and Kwaks, 2003).

The ph locus is located on the X chromosome at 2D2-3 position and consists of two different transcription units, termed polyhomeotic-distal (ph-d) and polyhomeotic-proximal (ph-p), which correspond to a tandem duplication of DNA (Dura et al.,1987; Deatrick et al.,1991). Mutants homo- or hemizygous for lesions in either one of the two units display the same phenotypes during embryonic development,suggesting that these two molecular units encode redundant functions(Dura et al., 1987). Furthermore, inactivation of one unit is transcriptionally compensated by the other, thereby maintaining a nearly wild-type level of ph product(Fauvarque et al., 1995).

We show here that overexpression of the polyhomeotic gene leads to specific defects in follicle formation. Complementary analysis revealed that ph function is required within germarial somatic cells for their differentiation and proliferation. Removal of ph function leads to production of follicles with more or less than 16 germ cells, abnormal accumulation of cysts in the germarium, expansion of the interfollicular stalks between adjacent follicles, and polar cell differentiation defects. In addition, loss-of-function mutations reduce prefollicular and follicular cell proliferation in the germarium. Finally, we show that two other Pc-G members, Sce and Scm, are also implicated in early oogenesis.

Somatic gain-of-function screen

The mutagenesis was performed using standard methodology, by mobilizing a P{y+}UAS transposon[http://flybase.bio.indiana.edu/.bin/fbidq.html?FBrf0093303(J. Merriam)] located on the X chromosome in a yw genetic background. This insert has UAS sequences and the hsp70 basal promoter oriented to transcribe leftward out of the 5′ P end. In total, 2500 independent insertions were generated (French Consortium, a part of the lines was published in Monnier et al.,2002). Males from each resulting line were crossed to w;da-GAL4 females (Wodarz et al.,1995), which provide the GAL4 protein under the control of the ubiquitous daughterless promoter. Flies were raised at 25°C on standard media. The fecundity of the female progeny was first analyzed by counting the number of eggs laid per female and per day. Ovaries from selected lines were then dissected, stained with a nuclear marker (DAPI), and tested for their morphology. Since the vector used to generate P{y+}UAS lines was a pUAST construct, this GAL4/UAS system allows overexpression of genes only in the somatic line (Rorth,1996).

We isolated by reverse PCR the genomic sequences flanking the selected P{y+}UAS insertions. Genomic DNA was extracted, digested by MspI and then circularized by ligation. The PCRs were performed under the following regime:(1) denaturation at 92°C for 5 minutes; (2) 40 cycles at 89°C for 5 seconds, 92°C for 10 seconds, 51°C for 45 seconds and 72°C for 2 minutes; and (3) a final 2-min extension at 72°C. The primer sequences correspond to the 5′ P end. Primer sequences used to amplify the fragment were: OUY311 (5′-TTGATTCACTTTAACTTGCAC-3′) and OUY521(5′-ACACAACCTTTCCTCTCAACAA-3′). The PCR products were used directly in a nested PCR with the following primer sequences: OUY521 and OUY32(5′-GCTTTCGCTTAGCGACGTG-3′). The cycle conditions were: (1)92°C for 2 minutes; (2) 20 cycles at 89°C for 5 seconds, 92°C for 10 seconds, 56°C for 45 seconds and 72°C for 2 minutes; and (3)72°C for 2 minutes. Amplification products were then sequenced directly by using the primer sequence OUY53(5′-ATACTTCGGTAAGCTTCGGCTATCGACG-3′).

Drosophila stocks

The control fly strains used were: wc and w1118. The ph504 (noted ph0) amorphic allele carries lesions in both ph-pand ph-d units (Dura et al.,1987). All other ph alleles are hypomorphic alleles. The phlac strain was induced by PlacW-element mutagenesis and corresponds to an insertion into the proximal unit, close to the first intron-splicing site (Fauvarque et al., 1995). The A101 enhancer-trap insertion(Ruohola et al., 1991) was used for tissue-specific β-galactosidase staining of polar cells. For testing ph overexpression, patched-GAL4 (Bloomington Stock number 2017), da-GAL4 (Wodarz et al., 1995), e22c-GAL4 (Bloomington Stock number 1973) and hs-GAL4 (Bloomington Stock number 3738) lines were used in combination with PH-C7 (gift from F. Maschat), m20 and m35 (Netter et al.,2001) P[UAS:cDNA ph] lines. Flies were raised on standard media at 25°C.

Clonal analysis

Induction of Flipase expression by heat shock was done using either an X-chromosomal or a II chromosomal hs-flp construct by heat shocking females just after the eclosion at 38°C for 1 hour, once. Somatic overexpression of ph was performed by generating Flip-out/GAL4 clones in females hs-flp/+; UAS-phx/Act>CD2>Gal4 UAS-GFP(Pignoni and Zipursky, 1997; UAS-phx denotes either PH-C7, m20 or m35). Flies were dissected 8 or 9 days after eclosion. Clones were detected by the presence of GFP expression. Mutant clones for ph were generated by mitotic recombination using the FLP/FRT system(Xu and Rubin, 1993) in females hs-flp tub-lacZ FRT101/ph504 w FRT101 or ubi-nls-GFP FRT101/ph504 w FRT101; hsflp38/+. hs-flp tub-lacZ FRT101 and ubi-nls-GFP FRT101; hs-flp38 lines were gifts from S. Goode (unpublished) and A. Guichet, respectively; the ph504 w FRT101 was previously described (Beuchle et al.,2001). Flies were dissected 2, 4 or 8 days after eclosion. Clones were detected by the loss of lacZ or GFP expression. FRT-mediated recombination events were induced specifically in the germline using an ovo-flp transgene and revealed by loss of nuclear GFP expression (I. Brun and C. Desplan, unpublished results) in ubi-nls-GFP FRT101/ ph504 w FRT101; ovo-flp/+ females. Flies were dissected 4 or 8 days after eclosion. Mutant clones for other Pc-G genes were also generated and the following strains were used: w; FRT82B ScmD1/TM6B, w; FRT82B Sce1/TM6B, yw;FRT42D Su(z)2I.b8/SM6b, yw FRT42D PclD5/CyO, yw; FRT42B sca AsxXT129/CyO, yw hs-flp; FRT42B hs-nGFP(Beuchle et al., 2001), yw hs-flp; FRT42D ubi-nls-GFP/CyO and yw hs-flp; FRT82B ubi-nls-GFP(gift from A. Guichet). AsxXT129 and PclD5 have been reported to be null mutations, Su(z)2I.b8 is a deficiency that removes Psc and Su(z)2 (Soto et al.,1995), Sce1 has been reported to be a null mutation (Breen and Duncan,1986) and ScmD1 is a frameshift mutation that genetically behaves as a null allele(Bornemann et al., 1998). Flies were dissected 8 or 9 days afer eclosion. In all cases, flies were kept at 25°C on standard media.

Staining ovaries

For antibody staining, ovaries were dissected in PBS and fixed in 3:1 heptane:4% formaldehyde in PBT (0.1% Tween-20 in PBS) or in 4%paraformaldehyde in PBT for 25 minutes. The ovaries were washed three times in PBT, blocked in 2% BSA for 1 hour, then incubated with primary antibody for 3 hours at room temperature or at 4°C overnight. The primary antibodies used in this study were rabbit polyclonal anti-β-galactosidase (1:200,Boehringer), mouse monoclonal anti-β-galactosidase 40-1a [1:200,Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal anti-Orb 6H4(1:30, DSHB), mouse monoclonal anti-Fasciclin III 7G10 (1:10, DSHB), rabbit polyclonal anti-α-Spectrin (1:1000)(Byers et al., 1987), rabbit polyclonal anti-phospho-histone H3 (1:1000, Upstate Biotechnology), mouse monoclonal anti-Hts 1B1 (1:5, DSHB), mouse monoclonal anti-Armadillo N27A1(1:100, DSHB) and mouse monoclonal anti-Eyes Absent eya10H6 (1:200, DSHB). The fluorescence-conjugated secondary antibodies were purchased either from Molecular Probes or Jackson Immunoresearch and used at a 1:200 dilution. All samples were mounted in Cytifluor (Kent). For DNA labeling, the ovaries were additionally incubated either for 20 minutes with 0.4 mg/ml RNase A and were subsequently stained with 50 μg/ml propidium iodide (Molecular Probes) or with 1 μg/ml of DAPI (Sigma).

For DAPI staining, tissues were fixed in 4% formaldehyde in PBS for 25 minutes and rinsed twice, first in PBT then in PBS. Ovaries were placed for one night in PBS:glycerol (1:1), with 1 μg/ml of DAPI (Sigma). Samples were examined either with a Leica DMR microscope or by confocal microscopy using a Leica DMR-BE microscope.

A gain-of-function screen reveals that somatic overexpression of ph results in follicle formation defects

We performed a genetic screen to identify genes that when overexpressed in the ovarian somatic line would affect early phases of oogenesis (GAL4/UAS system; see Materials and methods and Fig. 1). Female progeny from the cross between the P{y+}UAS insertion line 4061 and the da-GAL4 line exhibited reduced fecundity compared with control w; da-GAL4/+ and 4061/w females (data not shown). Twenty-two percent of 4061/w; da-GAL4/+ ovarioles(n=202) exhibited defects in oogenesis(Fig. 2B,C), not present in control ovarioles (Fig. 2A),which concerned both germarial and vitellarial regions. We observed both compound follicles with more than 16 germline cells(Fig. 2B, arrow) and apposed follicles separated by two follicular epithelia but presenting no intervening interfollicular stalk (Fig. 2C,arrow). In these same ovarioles, germarial region 2b/3 was often abnormally swollen and long and contained advanced cysts normally corresponding to stage 5 follicles of the vitellarium (Fig. 2C, arrowhead), indicating that follicle budding is defective.

The P{y+}UAS insertion in the 4061 line was found to be located in the first exon of ph-p, 99 bp upstream of the ATG(Fig. 2D), in the sense orientation. This configuration, along with the fact that induction of ovarian phenotypes depends on the presence of the da-Gal4 driver, suggests that the observed phenotypes are very likely due to an upregulation of this gene. This was substantiated by the fact that 4061/w; da-GAL4/+females showed a partial homeotic haltere-to-wing transformation and abnormal expression of the vestigialQE-lacZ transgene in haltere imaginal discs (data not shown) (Kim et al.,1996; Weatherbee et al.,1998). Indeed, the opposite homeotic transformation, from wing to haltere, is obtained in flies carrying homozygous/hemizygous loss-of-function ph alleles, which corresponds to the phenotype associated with dominant gain-of-function mutations in genes of the Antennapedia and bithorax complexes (Dura et al.,1985).

In order to confirm that somatic overexpression of ph perturbs oogenesis, we took advantage of available P[UAST:cDNA-ph] lines(PH-C7, m20 and m35; see Materials and methods). All the da-, ptc-, e22c- and hs-GAL4/UAS-ph combinations we tested were lethal before adult eclosion. We thus used the heat-shock inducible flip-out/GAL4 system to generate mosaic follicles containing overexpressing ph somatic cell clones. As previously observed in 4061/w; da-GAL4/+ females, we found both compound(Fig. 2E) and apposed follicles(data not shown), which were associated with the presence of large clones marked by GFP protein expression (Fig. 2E′). As visualized after anti-Orb staining (Orb protein accumulates specifically in the oocyte; Lantz et al., 1992), compound follicles contained two germline cysts developing together within a single follicle (Fig. 2E″,arrowheads). We next looked earlier for anomalies during germline cyst encapsulation in the germarium. In a wild-type germarium, prefollicular cells in region 2b (see Fig. 1)extend long and thin processes, which separate individual linearly arranged germline cysts. These cell extensions accumulate several cytoskeletal proteins, including Fasciclin III (Fas III)(Fig. 2F′), which is a homophilic cell adhesion molecule (Ruohola et al., 1991). When ph was overexpressed, germaria appeared long with multiple mature germline cysts in an extended region 2b/3(Fig. 2G-G″, abnormal germarium in which most of the germarial cells overexpressed ph; Fig. 2G, same scale as Fig. 2F). These nascent follicles were only partially individualized by somatic cells, as visualized by anti-Fas III staining (Fig. 2G″, asterisks) and budding off from the germarium was severely delayed or deficient. This partial individualization of the germline cysts by prefollicular cells could explain the `curved' aspect of the follicular epithelium systematically observed in multicyst follicles(Fig. 2B,E). As it has been shown that the absence of polar cell specification leads to formation of multicyst follicles (Grammont and Irvine,2001), we next looked for the distribution of polar cells in multicyst follicles associated with overexpression of ph. We always found four groups of polar cells when two germline cysts were encapsulated together (Fig. 2H′,H″,H‴ are three different focal planes of the same multicyst follicle). In this example, two pairs of polar cells are present at each pole of the follicle (Fig. 2H′,H‴, solid arrowheads), and two groups of polar cells in the middle of the follicular epithelium(Fig. 2H″,H‴,empty arrowheads). Thus, ph overexpression affects the behavior of somatic germarial cells, leading to encapsulation of multiple germline cysts and delayed budding, but polar cell specification is not altered.

ph is required specifically in somatic cells for follicle formation

In order to assess the endogenous requirement of ph, we next looked for ovarian defects associated with induction of mitotic cell clones using the ph504 (noted ph0) amorphic allele, which carries lesions in both ph-p and ph-d units(Dura et al., 1987). Since it has been previously reported that ph is transcribed and translated in both somatic and germline cells, in the germarium and in the vitellarium until stage 10, except in the oocyte (DeCamillis and Brock, 1994), we aimed at determining whether phfunction is required in both cellular compartments, or restricted to one of them. Therefore, we first generated ph mutant germline clones using the Flp/FRT system and the ovo promoter to drive flipaseexpression specifically in the germline. Among more than 150 ovarioles with ph0 germline clones observed, we never detected any ovarian defects (data not shown), showing that ph is not required in the germline for early oogenesis.

We next generated amorphic ph0 somatic clones using a heat-shock promoter to drive flipase expression. flipaseexpression was induced at eclosion and 4-, 6- and 8-day-old females were dissected in order to recover clones in the follicular epithelium of different stage follicles. Surprisingly, no or few ph0 cells,detected by lack of -lacZ or -GFP reporter expression, were observed in the great majority of dissected ovarioles, while large twin clones and wild-type clones generated in parallel were readily observed (data not shown). Indeed, ph0 cell clones being expulsed from the somatic epithelium were frequently observed (data not shown), which probably explains the low number of clones recovered. Similar observations were reported for ph null clones induced in wing imaginal discs(Santamaria et al., 1989). In addition, it is possible that the division rate of ph0cells may be affected.

Despite the presence of very few ph0 cells(Fig. 3A′), follicles with more than 16 germline cells were observed(Fig. 3A). These follicles result from the encapsulation of several germline cysts together as a constant 15:1 ratio between nurse cells and oocyte was always observed using anti-Orb staining (Fig. 3A″). Apposed follicles were also observed (data not shown). In order to explain these encapsulation defects, we examined prefollicular cell behavior in the germarium. In the germaria recovered bearing both ph+ and ph0 cells in region 2b/3(Fig. 3B, ph0 cells marked by an asterisk in inset), staining with Hu-li tai shao (Hts) antibodies (Hts is present on lateral membranes of prefollicular cells; Lin et al.,1994) showed that while ph+ prefollicular cells had encapsulated germline cysts by extending long, thin cell processes centripetally (Fig. 3B′,arrow), ph0 cells remained at the periphery of the germarium with a relatively round morphology in comparison(Fig. 3B′, asterisks in inset). These results suggest that ph function is necessary in prefollicular cells for these cells to be able to acquire their specific adhesive and/or migratory properties necessary for proper encapsulation of individual germline cysts.

As early aspects of the prefollicular cell developmental program are affected in ph0 cells, we next asked whether their subsequent differentiation into interfollicular, polar and follicular cells was also affected. Whereas wild-type interfollicular stalks contain five to seven cells organized in a single line, we noted the presence of abnormal stalks, with more than a dozen cells organized in two or three lines upon induction of ph0 mutant cell clones(Fig. 3C-C″). In some cases, these abnormal stalks were mosaic, containing both ph0 and ph+ cells(Fig. 3C′), although the majority recovered were made up exclusively of ph+cells.

We next examined expression of polar cell markers. In wild-type follicles,polar cells are arranged as two groups of somatic cells at each follicle extremity, as detected by various markers including Eyes Absent(Fig. 3E″, arrowheads),Fas III (see Fig. 5A″,arrow) and several enhancer trap lines (such as A101, see Fig. 5F′, inset). It has been previously described that in wild-type females the number of cells expressing these markers is greater than two at the extremities of young follicles (in particular stage 2 and 3 follicles), then progressively restricted to two by stage 5 (Besse and Pret, 2003). The Eyes Absent (Eya) protein is normally expressed in prefollicular cells then specifically turned off in polar cells, while maintained in follicular cells (Bai and Montell, 2002; Torres et al.,2003) (Fig. 3E″, arrowheads). When ph0 clones were induced, two groups of anterior and posterior polar cells were invariantly observed both in normal and multicyst follicles (data not shown). However, the presence of more than two polar cells at the extremities of stage 5 or older follicles was observed in rare ph0 polar cell clusters,marked by the absence of Eya expression, indicating an excess compared to wild-type follicles at these stages (Fig. 3D,D′) (five polar cells do not express Eya). Importantly, ph0 follicular cells correctly express Eya protein(Fig. 3E-E″, bracket),like neighboring ph+ follicular cells(Fig. 3E-E″).

Finally, we observed that ph0 follicular cells expressed Fas III at a higher level than neighboring ph0/ph+ or ph+/ph+ follicular cells(Fig. 3F′, insert, and F″). A high level of Fas III is characteristic of both polar and undifferentiated prefollicular cells. Since the level of Fas III expressed by ph0 cells is lower than that expressed by neighboring polar cells (Fig. 3F″,arrowhead), it is possible to conclude that these ph0cells represent undifferentiated follicular cells. Thus, induction of ph0 somatic cell clones in the ovary perturbs germline cyst encapsulation and stalk morphogenesis, generates an excess of stalk and polar cells and maintains follicular epithelial cells in a precursor state.

ph mutant germaria exhibit abnormal encapsulation by prefollicular cells and abnormal budding of germline cysts

We next examined ovaries associated with a whole-fly ph partial loss-of-function context. We found ovarian defects associated with one of the previously described homozygous viable phlac mutations(Dura et al., 1987)(Fig. 2D) and showed that these defects are specifically due to this mutation, since they are maintained after outcrossing over six generations (data not shown). As visualized after DAPI staining, phlac ovarioles exhibited follicles with either more (Fig. 4A,B; compare to wild-type ovariole in Fig. 2A)or fewer (Fig. 4C, arrow) than 16 germ cells. When a follicle contained more than 16 germ cells (23%ovarioles contained at least one compound follicle over 248 ovarioles observed), we found groups of nurse cells with either equivalent(Fig. 4A, arrow) or different(Fig. 4B, arrows) degrees of polyploidy. All compound follicles were multicyst, since they contained several oocytes (anti-Orb staining, data not shown). In addition, apposed chambers were also observed in phlac females (data not shown). When a follicle contained fewer than 15 nurse cells (5.6%, n=248), one of the adjacent chambers contained the complementary number of nurse cells or degenerating nurse cells(Fig. 4C; empty arrowhead points to a pycnotic nucleus). Thus, perturbing ph function in the phlac mutant leads to defects in follicle formation.

In the phlac mutant, encapsulation defects were correlated to abnormal behavior of prefollicular cells. In 14.5%(n=248) of the germaria, delayed budding of the emerging follicles was found (Fig. 4D-G). In these germaria, germline cyst encapsulation was defective in different ways. Whereas germline cysts arrange themselves in a linear fashion in region 2b of wild-type germaria (Fig. 2F′), numerous cysts were found encapsulated in an anarchic fashion in phlac mutant germaria(Fig. 4D′, arrowheads). Associated with this, groups of Fas III+ prefollicular cells were found aggregated at the periphery of the germarium not participating in the encapsulation process (Fig. 4D′,G′, asterisks). In other germaria, abnormal prefollicular cell encapsulation leads to splitting of germline cysts and envelopment of fewer than 16 germline cells(Fig. 4E″). In more strongly affected ovarioles, an absence of prefollicular cell migration between adjacent germline cysts was associated with an accumulation of several mature cysts in region 3 of the germarium(Fig. 4F′,F″,arrows). In these cases, prefollicular cell invagination was observed anteriorly (Fig. 4F′,F″, arrowheads) which will therefore probably lead to budding of a multicyst follicle. In other abnormal ovarioles, no clear limit between germarium and vitellarium could be found; in these cases, Fas III expression remained very strong all along the ovariole(Fig. 4G′). Thus, as described above for ph0 cells, we also observed an alteration in the differentiation of phlac prefollicular cells impaired in their adhesive and migratory properties during germline cyst encapsulation.

Differentiation of interfollicular and polar cells is affected in phlac mutants

As differentiation of specialized germarial somatic cells are affected in ph0 clones, we next examined expression of stalk and polar cell differentiation markers in phlac females. In wild-type ovarioles, interfollicular stalk cells express high levels ofα-Spectrin (Fig. 5A′, inset), and low levels of Fas III(Fig. 5A″, inset). We found that phlac mutant stalks contained an excess number of cells (10 to 50) frequently organized into two or three lines (32%, n=231) (Fig. 5B,C). Such structures were comprised of cells with at least some characteristics of interfollicular stalk cells because they strongly expressed α-Spectrin(Fig. 5B′). However,since they also strongly express Fas III(Fig. 5C′), their differentiation state was ambiguous.

Second, we examined polar cell differentiation. In wild-type follicles,polar cells are detected using either anti-Fas III antibody or the enhancer trap line A101 (neuralized-lacZ), which are both markers for mature polar cells from stage 2 onward (data not shown and Fig. 5A″, arrow). In phlac ovarioles, whether follicles contained more or fewer than the normal number of germline cells, there were always two groups of polar cells, one at each extremity, anterior and posterior(Fig. 5D,D′, follicle with fewer than 15 nurse cells; Fig. 5E,E′, multicyst follicle). However, these groups often contained more than two polar cells (Fig. 5F,F′, insets; 11 polar cells can be seen at the posterior pole of a stage 4 multicyst follicle). Although it has been shown previously that early stage 2 and 3 follicles in wild-type ovarioles also frequently contain more than two polar-cell marker expressing cells, a maximum of five of these cells was observed (Besse and Pret,2003) (Fig. 5G). In addition, by stage 4, in wild-type ovarioles, the restriction to two polar cells at each pole was almost complete and from stage 5 onwards there were never more than two observed (Besse and Pret, 2003) (Fig. 5G). In contrast, phlac mutant ovarioles exhibited a significant proportion of stage 4, as well as some stage 5 and up to stage 9, follicles with more than two polar cells(Fig. 5G and data not shown). The presence of groups with more than two polar cells after stage 4 was not strictly correlated with an abnormal number of germline cells within the follicle but was always associated with an abnormal interfollicular stalk. However, abnormal interfollicular stalks associated with two polar cells at each extremity were also observed. In addition, ph mutant ovarioles exhibited a shift in the dynamics of A101 staining: the appearance of this staining is delayed since 100% (n=112) of stage 2 phlac follicles exhibited no marked cells compared to 34%(n=78) of stage 2 wild-type follicles. In conclusion, phmutations perturb interfollicular stalk cell differentiation and stalk morphogenesis, delay the polar cell differentiation program and generate an excess of both of these cell types. Since stalk and polar cells arise from the same precursors (Tworoger et al.,1999), the differentiation of these precursors seems specifically affected in phlac mutants.

Proliferation of germarial somatic cells is reduced in phlac mutant females

We next wanted to know whether the excess in number of polar and interfollicular stalk cells was associated with an alteration in proliferation of precursor cells. We investigated the proliferative activity of phlac germarial somatic cells using antibodies against phospho-histone 3 (PH3), which specifically stain cells undergoing mitosis(Hendzel et al., 1997), and anti-Fas III antibodies to identify the different regions of the germarium(e.g. Zhang and Kalderon,2001) (Fig. 2F′). As no positive marker for ovarian somatic stem cells has been described so far, and as inner germarial sheath cells in 2a/2b junction have been recently shown to be mitotically active(Song and Xie, 2002; Song and Xie, 2003), any PH3+ somatic cells found just anteriorly to Fas III staining were considered to be somatic stem cells and inner sheath cells. Control phlac/+ and mutant phlac ovarioles were double-stained with antibodies against Fas III and PH3, and the number of proliferating stem and inner sheath cells (between region 2a and 2b),prefollicular cells (region 2b), and follicular cells (region 3) was scored for each genetic background. Very abnormal ovarioles, as those shown in Fig. 4G′, were not included in this study because the different germarial regions could not be delimited easily. As shown in Table 1, the number of mitotic somatic cells is significantly lower for phlac females compared to phlac/+control sisters for prefollicular and follicular cells in regions 2b and 3 of the germarium. Although there is also a difference between these two genetic contexts for mitotic activity in the 2a/2b region, the low frequency of this type of event and the presence of inner sheath cell mitotic activity may explain the fact that it is not possible to demonstrate the statistical significance of this difference. We also verified that, as in the wild-type context, there is no proliferation of stalk and polar cells after stage 1 in phlac ovarioles (data not shown). Therefore, the excess number of stalk and polar cells observed in ph mutant ovarioles cannot be explained by excess proliferation of precursors in the germarium. Nonetheless, the reduced proliferation of prefollicular and follicular cells in the germaria of ph mutant ovarioles probably contributes to the germline cyst encapsulation and budding defects observed.

Other mutations in Pc-G genes lead to ovarian defects

As this is the first time that a study reveals a role for a Pc-G member in ovarian folliculogenesis, we next analyzed the role of other members of the Pc-G for early oogenesis defects by clonal analysis using amorphic mutations. We first analyzed mutant Sce1 somatic mitotic clones. The viability of Sce1 cells does not seem to be affected since Sce1 cell clones are produced at a high frequency and since these clones have the same size as twin clones(Fig. 6A′ and data not shown). Associated with induction of Sce1 cell clones, we observed several defects, including multicyst follicles with several oocytes(Fig. 6A; two oocytes are indicated by arrows) and disorganized encapsulation in germarial region 3(Fig. 6B,B′). In addition, abnormally long interfollicular stalks containing from 10 to over 50 cells were found between wild-type (Fig. 6C) or abnormal follicles (Fig. 6B, arrowhead). In some cases, a continuous cable of somatic cells can be observed lying along one side of the follicles(Fig. 6D). These defects were always found associated with the presence of Sce1 mutant cells, although few Sce1 cells suffice to create a very abnormal stalk (Fig. 6C, empty arrowhead). All interfollicular cells from an abnormal mosaic stalk strongly express α-Spectrin as in wild-type stalks(Fig. 6D,D′); however,abnormal perdurance of Fas III was also observed(Fig. 6C,C′). Therefore,as described for ph mutations, interfollicular stalk cells display an intermediate differentiation state. This was also the case for follicular cells since Sce1 mutant epithelial follicular cells also exhibited strong expression of the Fas III protein compared with neighboring Sce+ cells (Fig. 6E,E′, insets). Finally, no excess of polar cells was observed when Sce1 clones were induced in these cells(data not shown).

Almost indistinguishable defects (multicyst follicles and giant stalks)were also obtained with the ScmD1 mutation when mutant clones were induced under the same conditions (data not shown). In contrast,no ovarian defects were provoked upon induction of somatic mitotic clones using amorphic mutant alleles for four other Pc-G genes under the same conditions, namely Psc, Su(z)2, Asx and Pcl.

Gain-of-function and loss-of-function studies reveal a new role for the ph gene in early oogenesis

During Drosophila oogenesis, a new role for ph was first revealed by the reduced fecundity and associated ovarian anomalies observed upon analysis of a P{y+}UAS insertion in the first exon of the ph-plocus (4061 line). That the ovarian phenotypes characterized for this line are due to overexpression of ph is supported by three lines of evidence: (1) the ovarian phenotypes produced depend on the presence of a GAL4 driver (da-GAL4); (2) the 4061/w; da-GAL4/+flies also present a ph gain-of-function haltere-to-wing transformation; and (3) flip-out clone overexpression in ovarian somatic cells of several UAS-ph cDNA transgenes gave very similar ovarian phenotypes. In particular, overexpression of ph is associated with production of multicyst follicles in which several (two to four) germline cysts develop within a single follicular epithelium. Importantly, each multicyst follicle contains several pairs of polar cells corresponding to the number of cysts present in the follicle. Therefore, unlike for other mutants[Notch (Grammont and Irvine,2001) and Hedgehog (Forbes et al., 1996)] for which inclusion of several cysts in one follicle has been attributed to a problem in polar cell specification, this does not seem to be the case here. Interestingly, multicyst follicles produced by ph overexpression are covered by a follicular epithelium that is not completely regular, showing indentations that appear to mark boundaries between cysts as evidenced by the presence of polar cells at the level of the indentations. This suggests that, earlier, cyst individualization may have begun and been subsequently aborted. In support of this, analysis of the associated germaria showed an abnormally long region 3, with adjacent mature germline cysts between which prefollicular cells fail to complete centripetal migration (visualized by specific anti-Fas III antibody staining of prefollicular cells). Overexpression of ph may thus specifically affect the expression of proteins necessary for recognition and/or adhesion between prefollicular cells and germline cysts for encapsulation. These effects seem to be specific to this stage since later interactions between these two cell lineages, for instance between follicular epithelial cells and the nurse cells and oocyte, are not perturbed by overexpression of ph.

Surprisingly, a similar phenotype to that observed in the phoverexpression study, multicyst follicles (several cysts within one follicle),was observed with loss-of-function ph alleles (discussed further below). Importantly, in contrast to ph overexpression, multicyst follicles in ph loss-of-function mutant ovaries always have only two groups of polar cells, one at each pole. Therefore, it seems that, unlike for overexpression of ph, delayed or deficient polar cell specification in ph mutants contributes to inclusion of several cysts within a single follicle. Thus, ph overexpression and loss-of-function phenotypes are distinguishable, indicating that the origin of the phenotypes is probably different.

ph function is necessary in somatic cells of the germarium for both their proliferation and differentiation

The implication of the ph gene in ovarian somatic cells was also studied using two different loss-of-function mutations: the hypomorphic phlac mutation, which consists of a PlacWtransposon inserted in the first intron of ph-p(Fauvarque et al., 1995); and via clonal analysis of the amorphic ph504 (noted ph0) allele, which eliminates the functions of both ph-p and phd (Dura et al., 1987). The origin of the multicyst phenotype caused by ph loss-of-function mutations was characterized more precisely by analysis of the process of follicle formation in the germarium. This study showed that several early aspects of the somatic cell developmental program(including proliferation, morphogenesis and differentiation) are perturbed by these ph mutations.

On the one hand, the rate of division of germarial somatic cells is reduced in a ph hypomorphic mutant background as assayed by immunohistochemical analysis of the mitosis-specific PH3. This probably contributes to delayed follicle encapsulation and budding, evidenced by the accumulation of mature germline cysts in germarial region 3 of phmutant ovarioles, and, consequently, by the formation of multicyst follicles. Although the same type of analysis was not possible upon induction of clones of the ph0 amorphic mutation in somatic ovarian cells, the fact that these clones are very small or absent compared with control clones suggests that a proliferation defect may also be associated with this ph mutation.

On the other hand, the morphogenetic properties of prefollicular cells and their differentiation into polar cells, interfollicular stalks and follicular epithelia are also specifically perturbed in ph mutants, which also probably contributes to formation of multicyst follicles. Our ph0 clonal analysis showed that ph function is necessary specifically in somatic cells, and not in the germline, for proper follicle formation. Almost all the phenotypes observed in ph mutant ovaries were reproduced upon induction of ph0 clones in prefollicular cells and their descendants.

First, prefollicular cell individualization of germline cysts is compromised in germarial regions 2a and 2b of ph mutant ovarioles. Fas III, which is specifically upregulated in prefollicular cells in wild-type germaria, is expressed normally in ph mutant prefollicular cells(phlac and ph0), but these cells remain at the periphery of the germarium and fail to undergo normal morphological changes necessary for germline cyst encapsulation, allowing multiple cysts, not individualized by somatic cells, to accumulate in region 3. In other ph mutant germaria in which prefollicular cells have migrated between germline cysts, encapsulation is disorganized, and germline cysts can be split and follicle budding significantly delayed. Therefore, in the germarium, interaction between prefollicular cells and the germline is defective.

Second, prefollicular cell differentiation into polar cells,interfollicular stalk cells and follicular epithelial cells is delayed and/or incomplete in the presence of ph mutations. Using a polar cell specific marker (A101/neu-lacZ), we showed that specification of polar cells, normally appearing by stage 2 in wild-type follicles, was delayed in ph mutant ovarioles until stage 3. In addition, multicyst follicles produced in ph mutant ovarioles contained two pairs of polar cells, one at each extremity of the anterior/posterior axis. Therefore,polar cell specification, which is necessary for individualization of germline cysts, is perturbed by ph mutations. Interfollicular stalk cell differentiation was assayed by expression of the stalk-specific marker,α-Spectrin, and by loss of Fas III, which is normally expressed at high levels in precursor prefollicular cells. In stalks of both ph mutant and ph0/ph+ mosaic ovarioles, althoughα-Spectrin expression was normally upregulated, Fas III was also present at high levels, indicating an ambiguous state of differentiation. Poor differentiation of stalk cells was further substantiated by the abnormally long and disorganized interfollicular stalks that showed intercalation defects. Abnormal perdurance of the early prefollicular cell marker Fas III was also observed at the level of the follicular epithelium in very affected ph mutant ovarioles, as well as in ph0 epithelial cell clones. Taken together, these results suggest that ph mutations result in the prolongation of a precursor state for polar, stalk and follicular epithelial cells.

Third, ph mutant ovarioles exhibit an excess of polar cells (up to 11) at both the anterior and posterior poles of follicles, which persists beyond stage 9, accompanied by an excess of adjacent interfollicular stalk cells (from 10 to 50). ph0 clones induced in the polar cell lineage also produced an excess of polar cells after stage 5, and the presence of ph0 cells was also associated with abnormally long stalks. Overproliferation of the pool of precursor cells common to both polar and stalk cells would result in an excess of these cells. However, the mitotic activity of germarial somatic cells was assayed by PH3 staining and a reduction compared to wild-type was observed in regions 2a, 2b and 3. It has recently been shown that an excess of polar cells is normally present in early stage follicles in wild-type females, and that the final pair of polar cells is selected from this group via apoptosis-induced cell death(Besse and Pret, 2003). However, in this study, a maximum of six such pre-polar cells was observed when apoptosis was specifically blocked. Since ph mutant ovarioles exhibit up to 11 polar cells, it would seem that apoptosis of pre-polar cells is probably not the only aspect of polar cell development affected. Finally,it is possible that the process by which the pool of polar and stalk cell precursors, distinct from the progenitors of the epithelial follicle cells, is set aside may be affected by ph mutations, leading to both problems in their number and differentiation(Tworoger et al., 1999). Determination of this pool probably involves several cell-cell signaling pathways in region 2b of the germarium, implicating Delta/Notch and EGFR signaling initiating from the germline and Hedgehog signaling from anterior terminal filament somatic cells (Goode et al., 1996b; Zhang and Kalderon, 2000; Grammont and Irvine, 2001; Lopez-Schier and St Johnston, 2001; Besse et al., 2002). ph may participate, in parallel or within one(or several) of these signaling pathways, to the regulation of the somatic cell differentiation program in the germarium. So far, attempts to uncover genetic or molecular interactions between ph and genes of these signaling pathways have proven unfruitful (K.N., unpublished results).

Requirements for the Polycomb group in oogenesis

The ph gene was first characterized as one of the Drosophila Pc-G genes, which encode transcriptional repressors required for maintaining the spatial pattern of homeotic gene expression during embryonic and larval development (Christen and Bienz, 1994; McKeon et al.,1994; Soto et al.,1995). ph has additional functions during development,since it has also been implicated in restriction of anterior compartment expression of engrailed and hedgehog in the wing imaginal disc (Maschat et al., 1998; Maurange and Paro, 2002). Our present results, which implicate ph function and that of two other Pc-G genes (Sce and Scm) in somatic cell development during early oogenesis, thus suggest that Pc-G function may be more generalized than previously thought. One other study reported ovarian defects associated with two temperature-sensitive alleles(pcoox736hs and pcomy939hs) of the E(z) gene, but the defects observed do not resemble those of ph (degeneration of nurse cells and little growth in the size of the follicle beyond stage 3 or 4) (Philips and Shearn, 1990).

We also examined ovarian phenotypes associated with mutations in several other Pc-G genes. The product of the Scm gene interacts directly with PH (Peterson et al.,1997), and, with PH, forms part of the same complex, PRC1, in Drosophila embryos (Shao et al.,1999). We found that the presence of large somatic cell clones of ScmD1 (amorphic mutation) leads to similar, though not completely overlapping, phenotypes than those observed for mutations in the ph gene. In particular, our results suggest that somatic cells are poorly differentiated and this leads to formation of multicyst follicles and abnormal interfollicular stalks with an excess number of cells. We also tested other Pc-G members, some which also belong to the PRC1 complex(Sce and Psc), while others do not (Asx, Su(z)2 and Pcl). Ovarian defects were observed only with a mutation in the Sce gene, and these defects closely resembled those obtained with ScmD1. Since follicle cell clones mutant for ScmD1 and Sce1 covered large areas of the follicular epithelium, like wild-type clones, we can conclude that, unlike for ph0, ScmD1 and Sce1 somatic cells are not affected in their proliferative property and/or viability. In addition, ScmD1 and Sce1 mutations did not affect polar cell number or differentiation. These results indicate that these anomalies were specific to mutations in the ph gene. Thus, we show that several components of the PRC1 complex, but not all, seem to be implicated in follicle formation and their functions do not seem to overlap fully. In addition, none of the genetic interactions between Pc-G genes known to exist for embryonic segment identity were reproduced in the ovary system (data not shown). We can make two hypotheses concerning the role of these Pc-G genes in ovarian folliculogenesis: (1) either each Pc-G gene acts specifically on its own specific subset of target genes in somatic cells of the ovary, possibly regulating the transcriptional machinery directly rather than forming particular Pc-G complexes that alter chromatin structure; or (2) repression of target genes in somatic cells of the ovary occurs via Pc-G complexes in a chromatin-dependent manner, but the complexes involved differ markedly in composition from those identified for embryonic cell identity. Further experiments will be needed to distinguish between these two possibilities.

We thank the laboratories that made their fly strains and reagents available, as well as the Developmental Studies Hybridoma Bank at the University of Iowa(http://www.uiowa.edu/~dshbwww/)for antibodies. We also thank J. Deutsch, F. Perronet, L. Théodore, N. Randsholt, S. Ronsseray, A. Guichet and F. Bernard for helpful discussions and/or comments on the manuscript. This work was supported, in part, by a French doctoral fellowship (MENRT) and laboratory grants from the Centre National de la Recherche Scientifique and the Association pour la Recherche sur le Cancer (5523, 7511 and 4501).

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