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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: Dev Biol. 2018 Mar 21;438(1):1–9. doi: 10.1016/j.ydbio.2018.03.018

JAK/STAT signaling prevents excessive apoptosis to ensure maintenance of the interfollicular stalk critical for Drosophila oogenesis

Antoine Borensztejn 1,1,2, Alexandra Mascaro 1,2, Kristi A Wharton 1,*
PMCID: PMC5951713  NIHMSID: NIHMS955397  PMID: 29571611

Abstract

Apoptosis not only eliminates cells that are damaged or dangerous but also cells whose function during development in patterning or organogenesis is complete. The successful formation of germ cells is essential for the perpetuation of a species. The production of an oocyte often depends on signaling between germline and somatic cells, but also between specialized types of somatic cells. In Drosophila, each developing egg chamber is separated from the next by a single file of interfollicular somatic cells. Little is known about the function of the interfollicular stalk, although its presumed role in separating egg chambers is to ensure that patterning cues from one egg chamber do not impact or disrupt the development of adjacent egg chambers. We found that cells comprising the stalk undergo a progressive decrease in number during oogenesis through an apoptotic-dependent loss. The extent of programmed cell death is restricted by JAK/STAT signaling in a cell-autonomous manner to ensure that the stalk is maintained. Both a failure to undergo the normal reduction in stalk cell number, or to prevent excessive stalk cell apoptosis results in a decrease in fecundity. Thus, activation of JAK/STAT signaling in the Drosophila interfollicular stalk emerges as a model to study the tight regulation of signaling-dependent apoptosis.

Keywords: Apoptosis, JAK/STAT, Interfollicular stalk, Polar cells, Upd, Drosophila oogenesis

1. Introduction

Cell death by apoptosis is an essential process in both development and homoeostasis to eliminate cells that are abnormal or no longer needed (Baehrecke, 2002; Fuchs and Steller, 2011). Elimination of cells via apoptosis is critical to metazoan development. In both the developing mammalian and Drosophila nervous systems, up to 50% of cells undergo apoptosis, and in the human ovary more than 80% of developing oocytes undergo cell death prior to birth (Hidalgo and ffrench-Constant, 2003; Reynaud and Driancourt, 2000; White et al., 1994; Zhou et al., 1997). More specifically, apoptosis has also been shown to regulate the effectiveness of developmental organizing centers by controlling the number of cells producing critical signals (Borensztejn et al., 2013; Nonomura et al., 2013; Sanz-Ezquerro and Tickle, 2000).

While the molecular machinery leading to cell destruction is well understood, involving the activation of caspase proteases that cleave cellular proteins to induce death (Chowdhury et al., 2008; Feinstein-Rotkopf and Arama, 2009), much less is known about the developmental signals that act upstream to initiate or repress apoptosis. The ability to mark, genetically manipulate, and image individual cells in Drosophila makes it an excellent system to elucidate the molecular signals that regulate precise developmental apoptosis, such as studies showing a specific reduction from 10 to 3 midline glial cells during Drosophila embryogenesis (Zhou et al., 1997; Bergmann et al., 2002) and the shaping of the retina in the adult eye by apoptosis (Monserrate and Baker Brachmann, 2006). In Drosophila developing egg chambers, JAK/STAT signaling is required to ensure the apoptotic reduction of polar cells from 3 to 6 at each pole to exactly two by stage 5 (Besse and Pret, 2003; Borensztejn et al., 2013; Khammari et al., 2010; Vachias et al., 2010).

During Drosophila oogenesis, each oocyte develops from an egg chamber that becomes increasingly mature, as it descends from the germarium to the uterus, akin to an assembly line. Sequential division of germline cells at the anterior tip of the germarium gives rise to a 16-cell cyst. The cyst is enveloped by somatic follicle cells to form the egg chamber as it buds off into the vitellarium, where strings of progressively maturing egg chambers advance through 14 stages of oogenesis to produce a mature egg (Fig. 1A) (Bastock and St Johnston, 2008; King, 1970). Roughly 20 strings of maturing egg chambers, ovarioles, make up each of two oovaries in the adult fly. In addition to specialized somatic cells surrounding each germ cell cyst, such as the polar cells that provide patterning signals from the two ends of the egg chamber, a small number of cells arranged in single file comprise the interfollicular stalk, which separates each egg chamber from the next. The separation of egg chambers appears critical for the proper execution of patterning events necessary for the production of a mature oocyte and its eggshell (Adam, 2004; Bai and Montell, 2002; Chen et al., 2011; Grammont and Irvine, 2001; Nystul and Spradling, 2010). Developmental cues produced by somatic follicle cells are also crucial for establishing the future animal's anterior/posterior and dorsal/ventral axes (Andreu et al., 2012; Goff et al., 2001; Gonzalez-Reyes and St Johnston, 1998; Moussian and Roth, 2005; Nilson and Schupbach, 1999; Ruohola et al., 1991).

Fig. 1.

Fig. 1

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Stalk cells are produced in excess and their number is restricted during oogenesis. (A) Each ovarian ovariole is a chain of egg chambers at progressively more advanced stages of development (stages (st) 2–9 of 14 shown) generated in the germarium. Individual egg chambers composed of a sixteen germline cell (blue) cyst encased in somatic follicle cells are separated by interfollicular stalks (red) derived from progenitors (yellow) in region 2b/3 of the germarium that also give rise to the polar cells (green) (Horne-Badovinac and Bilder, 2005; Margolis and Spradling, 1995; Nystul and Spradling, 2010). During stage 9, the border cells (orange) begin to migrate to reach the anterior tip of the oocyte, bringing the anterior polar cells with them (Montell et al., 1992; Silver, 2005). (B–C') Lamin C (red) marks stalk cell nuclei (and muscle sheath cell nuclei (x)). Stalks posterior to st2 (B) and st7 (C) egg chambers are shown, nuclei are stained with Hoescht (blue), scale bar is 25 µ. (D) Number of cells comprising stalks following st2, st3 and 4 (st3/4), st5 and 6 (st5/6) and st7 and 8 (st7/8) egg chambers in wild type ovaries (OrR). Percentage of stalks composed of 0–5 cells (black bars), 6–10 cells (gray), and greater than 10 cells (white). Mean number (x̄) of cells per stalk as oogenesis proceeds. Number (n) of stalks analyzed. Statistical significance of the difference in number of cells at each stage compared with the previous stage is indicated by an unpaired Wilcoxon rank sum test with tie correction (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001).

All somatic follicle cells arise from the two follicle stem cells (FSCs) located between regions 2a and 2b of the germarium (Fig. 1A) (de Cuevas and Matunis, 2011). Within region 3 of the germarium, future stalk and polar cells are distinguished from main body epithelial follicle cells (Chang et al., 2013). The stalk cells form a linear connection between each consecutively developing egg chamber within an ovariole but little else is known about their function. As the sole source of the JAK/STAT signaling pathway ligand, Unpaired (Upd), (Borensztejn et al., 2013; Harrison et al., 1998; Hombría and Brown, 2002; Hombría et al., 2005; McGregor et al., 2002), the polar cells act as an organizing center to impart pattern information to adjacent follicle cells at different stages of oogenesis (McGregor et al., 2002; Xi et al., 2003). A need for tight regulation of Upd levels is thought to underlie the precise reduction of polar cells to two via apoptosis. The elimination of supernumerary polar cells depends on activation of JAK/STAT signaling in the polar cells themselves or surrounding follicle cells (Borensztejn et al., 2013).

wCuriously, while loss of JAK/STAT activity in stalk cells results in a failure to promote apoptosis in polar cells, a disruption in JAK/STAT signaling is reported to result in shorter interfollicular stalks, presumably due to fewer stalk cells (Baksa et al., 2002; Borensztejn et al., 2013; Chang et al., 2013; Hayashi et al., 2012; McGregor et al., 2002). Previously, specification of stalk cell identity was thought to require JAK/STAT signaling (Baksa et al., 2002; McGregor et al., 2002), however, subsequent findings indicated that neither loss of the downstream transducer of JAK/STAT, Stat92E, function in the germarium, nor loss of heparan sulphate proteogylcans (HSPGs), which impact Upd signaling, block specification of stalk cell identity or formation of the early stalk (Chang et al., 2013; Hayashi et al., 2012). While stalks are shorter when JAK/STAT signaling is compromised, the stalk still forms, therefore, we considered the possibility that JAK/STAT signaling is required to maintain stalk cells during oogenesis and not for specification of stalk cell identity. Such a failure in stalk cell maintenance would be consistent with the shorter stalks or merged egg chambers observed in hop or stat92E mutants (Chang et al., 2013; McGregor et al., 2002).

Here, we test the hypothesis that JAK/STAT signaling plays a role in stalk cell maintenance. Our studies indicate that in wild type ovarioles, the number of cells comprising the stalk reduces progressively during oogenesis due to apoptosis. We find that abnormal interfollicular stalk length influences egg production, whereby blocking apoptosis, which produces longer than normal stalks, results in lower fecundity, and inducing excessive apoptosis to produce shorter stalks also reduces egg production. Thus, the apoptotic reduction of stalk cells appears to be tightly regulated to ensure maximal fecundity. We also found that in the absence of JAK/STAT signaling, excessive apoptosis of stalk cells occurs, eliminating more stalk cells than normal, indicating that JAK/STAT signaling has an anti-apoptotic function with regard to stalk cell survival, in contrast to its known pro-apoptotic function in eliminating supernumerary polar cells (Borensztejn et al., 2013). Our findings presented here, coupled with previous findings, indicate that JAK/STAT activity in the stalk has both anti-apoptotic and pro-apoptotic functions.

2. Results

2.1. Stalk cells are produced in excess and their number is restricted through apoptosis

We determined the exact number of cells comprising individual interfollicular stalks at different stages of oogenesis (Fig. 1). Ovarioles were dissected from multiple ovaries and stalks following stage 2 through stage 8 egg chambers were considered in this analysis, with counts from consecutive stages 3 and 4, 5 and 6, and 7 and 8 grouped. Egg chambers stained with Hoechst to visualize all nuclei were staged according to King (King, 1970). Stalk cell identity was based on the presence of Lamin C, which is highly expressed in stalk cell nuclei (Pearson et al., 2016). Stage 2 stalks showed an average of 9.0 cells/stalk with a reduction to 7.4 cells/stalk by stage 3 and 4 (stage 3/4) (p = 1.4 × 10−4; Fig. 1B–D), and a further decrease in cell number, 6.9–6.2 cells/stalk (p = 0.0091) for stage 5/6 and stage 7/8 stalks, respectively (Fig. 1D).

We tested for the possibility that apoptosis could account for this progressive loss of stalk cells with stage. Dying cells were directly visualized by TUNEL staining in wild type ovaries. We found at least one dying cell in 6.4% (n = 125) of stalks between stages 2 and 8 (Fig. 2A). An antibody against death caspase 1 (Dcp-1) as an alternative method also identified apoptotic stalk cells in wild type ovarioles (Fig. 2B). To further test the involvement of apoptosis on stalk cell restriction, we expressed the baculoviral p35 gene which encodes a caspase inhibitor that is known to block apoptosis when expressed in Drosophila cells by binding to the active sites of effector caspases (Bump et al., 1995; Zhou et al., 1998). p35 expression was targeted by the 109-53-Gal4 driver whose expression is limited within the vitellarium to stalk cells, after their specification and once the interfollicular stalk is formed, with no expression in the polar/stalk cell precursors in region 2b and 3 of the germarium (Fig. 2C). To have greater temporal control over 109-53-Gal4-induced expression, we made use of a ubiquitously expressed (under tubulin control) temperature sensitive transcriptional repressor GAL80ts that prevents 109-53-Gal4-induced transcription at the permissive temperature (25 °C), and allows GAL4-directed expression of UAS-p35, for example, at the restrictive temperature (29 °C). The normal decrease in stalk cell number that we observed in wild type was unaltered in the control line, 109-53-Gal4 tubGal80ts > UAS-eGFP (Fig. 2D). However, when p35 was expressed in 109-53-Gal4 tubGal80ts > UAS-p35 females, stage 5/6 and 7/8 stalks were found to have more cells (x̄ = 7.0 versus 6.2, p = 0.030 at stage 5/6 and x̄= 7.4 vs 6.1, p = 0.0020 at stage 7/8, Fig. 2E), indicating that blocking apoptosis prevents the normal loss of stalk cells as oogenesis proceeds. We also found that knocking down the effector caspase Drice (109-53 Gal4 tubGal80ts > UAS-drice-RNAi) resulted in longer stalks when compared to a UAS-GFP-RNAi control at stage 7/8 (109-53 Gal4 tubGal80ts > UAS-drice-RNAi x̄ = 8.0 cells vs x̄ = 5.9 in control, p = 9.0 × 10−6). We made use of a different stalk cell Gal4, c306-Gal4, which is also expressed in some somatic cells of the germarium, as well as in polar cells and neighboring cells at the termini of egg chambers, likely to become border cells in later stage egg chambers (Fig. 2H–J”) to drive p35 expression and found a persistence of stalk cells in c306-Gal4 tubGal80ts > p35 stage 7/8 stalks compared to controls (x̄ = 6.9 versus 6.1, p = 0.0086, Fig. 2K–L). Together, these data indicate that during the normal progression of oogenesis the interfollicular stalk undergoes a reduction in cell number via apoptosis.

Fig. 2.

Fig. 2

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Supernumerary stalk cells are eliminated by apoptosis during normal development. (A) A TUNEL positive cell (red) in stalk linking stage 6 and stage 7 egg chambers (109-53-Gal4/+). (B) A Death caspase 1 (Dcp-1) positive cell (green) in stalk linking stage 6 and stage 7 egg chambers (tubP-Gal80ts/+;109-53-Gal4/UAS-GFP-RNAi) with insets showing Lamin C and Dcp-1 staining or Hoechst staining. (C) UAS-mCD8GFP/tubP-Gal80ts; 109-53-Gal4/+ ovarioles with GFP expression shown in green, nuclei stained with Hoescht (blue), and stalk cell nuclei stained with anti-Lamin C (red). 109-53-Gal4 is expressed in the terminal filament, epithelial muscle sheath, and all of the stalk cells but no other cell type in the vitellarium or the germarium. (D–G, K–L) Percentage of stalks composed of 0–5 cells (black bars), 6–10 cells (gray) and greater than 10 cells (white), based on both Lamin C and Hoechst staining. Mean number (x̄) of cells per stalk as oogenesis proceeds. Number (n) of stalks analyzed. (D) tubP-Gal80ts/+ ;109-53-Gal4/UAS-eGFP. (E) tubP-Gal80ts/UAS-p35; 109–53-Gal4/+, with statistical significance of a difference in E compared to D at each stage indicated. (F) tubP-Gal80ts/+ ;109-53-Gal4/UAS-GFP-RNAi. (G) tubP-Gal80ts/UAS-drice-RNAi;109–53-Gal4/+ with statistical significance of a difference in F compared to G at each stage. (K) c306-Gal4/+ ;;tubP-Gal80ts/+. (L) c306-Gal4/+ ;UAS-p35/+ ;tubP-Gal80ts/+ , with statistical significance of a difference in L compared to K at each stage. Statistics for D–G, K–L were computed via an unpaired Wilcoxon rank sum test with tie correction. Adjusted p-values are reported with a Holm-Bonferonni multiple comparison correction. (H–J”) c306-Gal4/+ ;mCD8GFP/+ ;tub-Gal80ts/+ showing that c306-Gal4 is variably expressed in somatic cells of the germarium, including a germarium with low c306-Gal4 expression in main panel and one with greater expression in insert (H), and in all stalk cells, as well as variable polar and terminal follicle cells presented in (I) from stage 3 to stage 4. After stage 8, c306-Gal4 expression is lost in the polar cells but appears strongly in the border cells in st10 (J-J”). Boxed area in J is magnified in J′ and J″. Scale bar is 25µ in A–C and H–J, 5µ in insets in B, and 10 µ in J′–J″.

2.2. JAK/STAT signaling inhibits stalk cell apoptosis

JAK/STAT signaling has been shown to play a role in the maintenance of stalk cell identity as well as in promoting polar cell apoptosis (Borensztejn et al., 2013; Chang et al., 2013). We considered the possibility that JAK/STAT signaling could influence the apoptotic loss of stalk cells. To test this, JAK/STAT signaling was knocked down in stalk cells by expressing RNAi that targets the gene encoding the JAK/STAT receptor, Dome. We tested the expression of two different UAS-dome-RNAi lines (#1682 and #647) driven by c306-Gal4 or 109-53-Gal4. (The ability of each dome-RNAi line to compromise Dome function was validated by its ability to disrupt border cell migration, a process known to require JAK/STAT signaling, when driven in border cells by c306-Gal4 (Fig. 3H)). We found that even though c306-Gal4 is expressed in the stalk/polar cell progenitors in region 2b of the germarium, driving dome-RNAi under c306-Gal4 control did not affect newly formed stage 2 stalks compared to controls (x̄ = 8.8 in control versus 9.5, p = 0.55 for c306-Gal4 tub-Gal80ts > dome-RNAi#647 and 8.7, p = 0.98 for c306-Gal4 tubGal80ts > dome-RNAi#1682, Fig. 3A compared to 3B,C). These results are consistent with those of Chang et al., 2013 who showed that loss of function clones for the downstream JAK/STAT transducer, stat92E, had no effect on proliferation of stalk cell progenitors, stalk cell fate specification, or formation of the stalk (Chang et al., 2013). While knocking down JAK/STAT signaling did not affect stalk formation at stage 2, we observed a significant reduction in stalk cell number in later stage stalks compared to controls (c306-Gal4 tub-Gal80ts > dome-RNAi#647 x̄ = 6.0 versus 7.0 in control, p = 0.0081 at stage 5/6, x̄ = 5.0 versus 6.4, p = 0.0036 at stage 7/8; c306-Gal4 tub-Gal80ts > dome-RNAi#1682 x̄ = 6.3 versus 7.2, p = 0.0021 at stage 3/4, x̄ = 4.6 versus 7.0, p = 2.8 × 10−11 at stage 5/6, and x̄ = 4.0 versus 6.4, p = 4.9 × 10−6 at stage 7/8. Fig. 3A–C). This more pronounced loss of stalk cells, observed when JAK/STAT signaling is compromised, indicates that JAK/STAT signaling is required to maintain stalk cells and suggests that it normally acts to inhibit or restrict excessive stalk cell loss through apoptosis.

Fig. 3.

Fig. 3

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JAK/STAT signaling inhibits stalk cell apoptosis. (A–F; J–K) Percentage of stalks composed of 0–5 cells (black bars), 6–10 cells, (gray) and greater than 10 cells (white), reflecting counting of stalk cells based on both Lamin C and Hoechst staining. Mean number (x̄) of cells per stalk as oogenesis proceeds, and number (n) of stalks analyzed. (A) c306-Gal4/+ ;;tubP-Gal80ts/+. (B) c306-Gal4/+ ;;UAS-dome-RNAi#647/tubP-Gal80ts, with statistical significance of the difference in number of cells at each stage compared to compared to A. (C) c306-Gal4/+ ;;UAS-dome-RNAi#1682/tubP-Gal80ts, with statistical significance of the difference in number of cells at each stage compared to A. (D) tubP-Gal80ts/+ ;109-53-Gal4/UAS-dome-RNAi#1682, with statistical significance of the difference in number of cells at each stage compared to tubP-Gal80ts/+ ;109-53-Gal4/eGFP from 2D. (E) tubP-Gal80ts/UAS-p35;109-53-Gal4/UAS-dome-RNAi#1682 with statistical significance of the difference in number of cells at each stage compared to D. (F) tubP-Gal80ts/UAS-p35;109-53-Gal4,UAS-eGFP/UAS-dome-RNAi#1682 with statistical significance of the difference in number of cells at each stage compared to D (J) yw1118, (K) hopTUM, with statistical significance of the difference in number of cells at each stage compared to J. Significance is indicated by the same code as in Fig. 1 and is based on unpaired Wilcoxon signed-rank test with tie correction. Adjusted p-values are reported with a Holm-Bonferonni multiple comparison correction (G) Percentage of stalks (from stages 2–8) exhibiting at least one TUNEL positive cell for 109-53-Gal4/+ and 109-53-Gal4/UAS-dome-RNAi#1682. Statistically significant difference according to a Chi-square test is indicated with a bar according to the same code as in Fig. 1. (H, left) Schematic of stage 10 egg chamber (anterior to left). Final position of the polar cell (red)/border cell (green) cluster shown adjacent to the oocyte after migration during stage 9. Migration distance of polar cell/border cell clusters is determined within four domains of stage 10 egg chambers, from anterior pole (0%) to oocyte (100%). (H, right) Percentage of stage 10 egg chambers exhibiting a polar cell/border cell cluster within each quartile domain from c306-Gal4/+ ;;tubP-Gal80ts/+ , c306-Gal4/+ ;;UAS-dome-RNAi#1682/tubP-Gal80ts or c306-Gal4/+ ;;UAS-dome-RNAi#647/tubP-Gal80ts ovarioles. n = number of egg chambers analyzed for each genotype. (I) Confocal image of a stalk between stages 8 and 9 of hopTum ovaries, scale bar is 25µ. Hoescht (in blue) stain the nuclei, Lamin C (in red) stains the stalk cell nuclei. Note that expression of a constitutively active form of Hop is associated with an elongated stalk composed here of 8 cells.

We obtained similar results driving dome-RNAi expression with the 109-53-Gal4 driver, whose expression is limited to stalk cells after stalk formation (109-53-Gal4 tubGal80ts > UAS-dome-RNAi#1682, x̄ = 3.8 versus 6.1, p = 2.09 × 10−6 at stage 7/8, Fig. 3D compared to 2D). In this case the knock down of JAK/STAT signaling in cells of the stalk indicates that the disruption of signal reception in these cells is sufficient to allow an excessive loss of stalk cells as oogenesis proceeds. (The number of cells at stage 2 in 109-53-Gal4 tub-Gal80ts > dome-RNAi#1682 was higher in the experiment presented in Fig. 3D (x̄ = 9.8 compared to x̄ = 8.6 in control from Fig. 2D, p = 0.010) but other repeats of this experiment did not show a significant difference).

To test if apoptosis is responsible for stalk cell loss associated with the knock down of JAK/STAT signaling, we co-expressed p35 with dome-RNAi and found the loss of stalk cells in stage 5/6 and 7/8 stalks was suppressed (109-53-Gal4 tub-Gal80ts > UAS-p35 UAS-dome-RNAi, x̄ = 7.0 versus 5.5, p = 0.016 at stage 5/6, and x̄ = 6.6 versus 3.7, p = 3.1 × 10−6 at stage 7/8; Fig. 3D,E). To control for the possibility that co-expression of two UAS constructs could influence the effectiveness of UAS-dome-RNAi expression, we co-expressed UAS-dome-RNAi and UAS-eGFP (109-53-Gal4 tub-Gal80ts > UAS-eGFP UAS-dome-RNAi#1682) and compared the number of stalk cells to those observed when UAS-dome-RNAi was expressed alone (109-53-Gal4 tub-Gal80ts > dome-RNAi#1682; Fig. 3D and F). The number of stalk cells at each stage was comparable, indicating that the presence of two UAS constructs did not dilute the level of UAS-dome-RNAi expression and its effect on stalk cell loss. Thus, we can conclude that the expression of UAS-p35 prevents stalk cell loss associated with the knock down of dome (Fig. 3E,F). Further evidence supporting our hypothesis that JAK/STAT signaling in stalk cells acts to limit the extent of apoptosis, we found that more than 25% of stalks expressing dome-RNAi (109-53-Gal4 > dome-RNAi#1682; n = 109) exhibit at least one TUNEL positive cell compared to 6.4% of control stalks (109–53-Gal4; n = 125, p = 0.00016) (Fig. 3G).

Based on this these findings we next tested whether an increase in JAK/STAT activity could inhibit apoptosis, and protect stalk cells from loss. We counted the number of cells per stalk in ovarioles from females homozygous for hopTum, a constitutively active allele of Hop, the JAK kinase (Harrison et al., 1995). In hopTum ovaries, we observed distinctly longer interfollicular stalks at stage 5/6 and 7/8 composed of more cells than the control (x̄ = 8.3 versus 6.8, p = 0.0054 at stage 5/6, and x̄ = 8.1 versus 6.3, p = 7.6 × 10−4 at stage 7/8, Fig. 3I–K). Thus, elevated JAK/STAT signaling is able to prevent the normal loss of stalk cells. Together, our results indicate that during oogenesis, a portion of interfollicular stalk cells normally undergo apoptosis between stage 2 and stage 8, and that JAK/STAT activity in these cells serves to limit apoptosis, ensuring that an excessive loss of stalk cells does not occur and the stalk is maintained.

2.3. Stalk cell maintenance is required throughout oogenesis to prevent egg chamber fusion

The loss of the interfollicular stalk has been associated with the merging of at least two adjacent egg chambers, such that a disorganized structure composed of multiple germline cell cysts results. Such fused egg chambers remain surrounded by somatic follicle cells but fail to undergo normal oocyte and eggshell patterning (Adam, 2004; Assa-Kunik et al., 2007; Bai and Montell, 2002; Chang et al., 2013; Grammont and Irvine, 2001; McGregor et al., 2002). It has been reported that females, or ovaries, that are mutant for components of the JAK/STAT signaling pathway exhibit egg chamber fusions presumably due to defects in stalk formation (Besse et al., 2002). However, our results, aligned with those of Chang et al., 2013, indicate that JAK/STAT signaling is not required for formation of the stalk per se. Thus, we investigated the possibility that a loss in JAK/STAT signaling could result in the merging of adjacent egg chambers due to the complete or nearly complete loss of stalk cells. Indeed, in ovarioles from 109-53-Gal4 > dome-RNAi #1682 females, we found large, elongated egg chambers with more than 16 germline cells surrounded by somatic follicle cells, and no Lamin C-positive cells that would be indicative of stalk cells (Fig. 4A). Stage 2 stalks were always present in these ovarioles, with fusions of adjacent egg chambers occurring at stage 3 or later, well after stalks are formed. These results are consistent with an excessive loss of stalk cells after their normal specification and formation into the linear interfollicular stalk. In some cases where a definitive linear stalk is absent, egg chambers abut one another without the fusion of adjacent germline cysts and are separated by disorganized follicle cells (Fig. 4B). The intervening cells are often uniformly positive for Eyes absent (Eya) (Fig. 4D) indicating a follicle cell identity consistent with the loss of stalk cells, as stalk cells do not express Eya (Fig. 4C).

Fig. 4.

Fig. 4

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Stalk cell number regulation is required for oogenesis. (A–F) Confocal images of ovarioles labeling nucleic acids with Hoechst (blue) and either stalk and muscle sheath cell nuclei with Lamin C (red) in A–B, E–F or Eya (in green) in C–D. (A) 109-53-Gal4/UAS-dome-RNAi#1682 ovarioles show fused egg chambers with no epithelium in between them. A, A’, and A′’ show different focal planes within the same ovariole. Note that though the egg chambers have fused, there are constricted areas where the borders of egg chambers would be expected. (B) tubP-Gal80ts/+ ;109-53-Gal4/UAS-dome-RNAi ovarioles sometimes have fused egg chambers that are completely adjacent and adhered but epithelial cells remain between them. 22.9% (n = 48) of ovarioles from counts in Fig. 3D contained at least one fused egg chamber versus 0% (n = 46) in control in Fig. 2D. (C–D) Confocal images with Eya in green and Hoechst in blue of control (tubP-Gal80ts/+;109-53-Gal4/UAS-eGFP, C) where the stalk cells are unlabeled by Eya (indicated with an arrow) and dome-RNAi (tubP-Gal80ts/+;109-53-Gal4/UAS-dome-RNAi, D) where an earlier stage stalk partially remains and is unlabeled (arrow) but fused egg chamber shows Eya positive cells in follicle cells connecting the abutting egg chambers ovarioles stained with Hoescht (in blue) and Eya (in green). (E–F) Examples of UAS-hid/+ ;tub-PGal80ts/+ ;109-53-Gal4/+ ovarioles that are either completely fused (E) or fused with epithelium in between (F). Scale bars for A–F are 25µ. (G) Percentage of ovarioles with egg chambers fused beginning at the indicated stage from flies of genotypes tub-PGal80ts/+ ;109-53-Gal4/UAS-GFP-RNAi and UAS-hid/+ ;tub-PGal80ts/+ ;109–53-Gal4/+ . (H) Number of eggs laid in 24 h by 5 three day old females of the respective genotypes: w1118;tubP-Gal80ts/+ ;109–53-Gal4/+ , w1118/+ ;tubP-Gal80ts/+ ;109-53-Gal4/GFP-RNAi, w1118;tubP-Gal80ts/p35;109-53-Gal4/+ , w1118/yw;tubP-Gal80ts/+ ;109-53-Gal4/dome-RNAi#1682 (dark gray), and UAS controls w1118, w1118/+ ;;UAS-GFP-RNAi/+ , w1118;UAS-p35/+ , w1118/yw;;UAS-dome-RNAi#1682/+ (light gray), with 10–11 replicates for each. UAS-only controls for each UAS construct tested were not statistically significant from the w1118 control. Statistical significance of the difference in eggs laid was compared within each group using a type II ANOVA with a 95% confidence interval with Dunnett's post-hoc test to compare each other genotype to w1118;tubP-Gal80ts/+ ;109–53-Gal4/+ or w1118, respectively and indicated by adjusted p-value where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

Egg chamber fusions associated with a complete loss of stalk cells occurred in 66% (n = 101) of ovarioles where the pro-apoptotic protein Hid is expressed in the stalk to promote apoptosis (Fig. 4E–G). In this case tubGal80ts was used to escape hid-induced lethality during earlier development by only switching adult females to the restrictive temperature 3 days after emergence and aging them 3 additional days, thus, allowing hid to be expressed in stalk cells. As with the expression of dome-RNAi, stage 2 stalks were always maintained in 109-53-Gal4 tubGal80ts > hid ovarioles. As a control, 109-53-Gal4 tubGal80ts > GFP-RNAi animals were raised in the same conditions in parallel and did not exhibit any egg chamber fusions (n = 74). Thus, we conclude that the interfollicular stalk is not only critical to separate egg chambers during their formation but that the stalk is also critical after egg chamber formation to maintain their separation. In the absence of stalks, the main body epithelial follicle cells appear to become disorganized and are unable to provide a suitable boundary to separate individual germline cysts for proper oogenesis.

2.4. Elimination of excessive stalk cells is required for efficient egg production

Our results indicate that stalk cell number is tightly regulated during oogenesis. We next asked if altered stalk length had an effect on egg production. The number of eggs laid by females in which stalk cell apoptosis was prevented (109-53-Gal4 tubGal80ts > UAS p35) or in which excessive stalk cell apoptosis was allowed, but under conditions that resulted in complete egg chamber fusions (109-53-Gal4 tubGal80ts > UAS dome-RNAi #1682), was compared to control females (Gal80ts;109-53 > ) (Graphical abstract). The fecundity of five 3 day old females over 24 h was determined in 10–11 different replicates. When stalk cell apoptosis was blocked (109-53-Gal4 tubGal80ts > p35), females laid significantly fewer eggs than the controls (x̄ = 600 when driving UAS-p35 versus x̄ = 708 in 109-53-Gal4 > , p = 0.0024; Fig. 4H). When JAK/STAT signaling was knocked down in stalk cells as a result of dome-RNAi expression, a reduction in the number of eggs laid was also observed (x̄ = 404 when driving UAS-dome-RNAi#1682 versus x̄ = 708 in control, p = 1.4 × 10−11). Thus, genotypes that result in either longer (109-53 Gal4 tubGal80ts > UAS p35) or shorter (109–53 Gal4 tubGal80ts > UAS dome-RNAi#1682) stalks than wild-type, result in a decrease in fecundity.

3. Discussion

Previous studies have shown that JAK/STAT signaling is required for the presence of interfollicular stalks (Baksa et al., 2002; Chang et al., 2013; McGregor et al., 2002), although its specific role and the mechanistic basis of this requirement was not known. Did cells lacking JAK/STAT signaling fail to become fated as stalk cells? Or did they fail to be maintained and were they eliminated, perhaps undergoing cell death? Here, we provide evidence that stalk cells are produced in excess during normal oogenesis and their number is reduced by apoptosis. We show that JAK/STAT signaling is required to ensure that excessive stalk cell clearance is prevented, thus, maintaining the interfollicular stalks. Furthermore, we show that maintenance of the stalk after its formation is required to prevent egg chamber fusions, and importantly, that variations in stalk length that deviate from normal affect fecundity.

3.1. Stalk cells are produced in excess and their numbers decrease through apoptosis during oogenesis

A detailed quantification of cells per interfollicular stalk during different stages of normal oogenesis indicates that the number of stalk cells comprising the stalk as it forms at the end of the germarium continually decreases as egg chamber maturation progresses. Apoptosis contributes to stalk cell loss as indicated by the presence of TUNEL and Dcp-1-positive stalk cells in wild type ovarioles. Furthermore, the normal reduction in stalk cells is blocked by the expression of the caspase inhibitor p35 or knockdown of the caspase Drice. Thus, the interfollicular stalk in Drosophila ovarioles emerges as a new model tissue whose cell number is tightly regulated by apoptosis during development.

3.2. JAK/STAT signaling ensures maintenance of somatic interfollicular stalk cells through a pro-survival function

Here, we show that JAK/STAT signaling exhibits an anti-apoptotic or pro-survival function in maintaining interfollicular stalk cells. Interestingly, this finding is distinct from the pro-apoptotic role of JAK/STAT signaling shown to be important in the reduction of polar cell number within the egg chamber (Borensztejn et al., 2013). We find that cell-autonomous manipulation of JAK/STAT signaling, by RNAi mediated inhibition of the JAK/STAT receptor Dome, results in an increase in apoptotic cells and an increase in stalk cell loss. In contrast, overactivation of JAK/STAT signaling inhibited normal developmental apoptosis, and led to longer than normal stage 3–8 stalks.

Knockdown of dome in somatic precursors in the germarium did not affect the specification of stalk cells, the formation of the stalk, or the number of cells comprising the earliest stalk at stage 2 but led to a greater decrease in the number of cells in stalks after stage 2 than in wild type females. In this case, we did not observe Lamin C positive cells within the follicular epithelium of egg chambers, which could reflect that in the absence of JAK/STAT signaling, cells leave the stalk to become incorporated into the follicular epithelium, but rather our data support the loss of stalk cells by cell death. It is possible that the loss of Lamin C could reflect a change in cell fate, but our results and studies by Chang et al. (2013) indicate that stalk cell fate is not influenced by JAK/STAT signaling.

The JAK/STAT signaling pathway has previously been described as an anti-apoptotic signaling pathway in various contexts in both mammals and Drosophila. stat5A/B−/− mice show a strong increase of apoptosis in bone marrow (Shelburne et al., 2003). In the Drosophila wing and eye disc, Stat92E, the downstream component of JAK/STAT signaling, has been shown to be a positive regulator of the apoptosis inhibitor Diap1 (Betz et al., 2008). However, in other contexts, JAK/STAT activity has been reported to have a pro-apoptotic function. In mice, the mammary glands are reduced in size after weaning through stat-3 dependent apoptosis (Chapman et al., 1999). And previous work in Drosophila shows that JAK/STAT activity in any one of multiple cell types (stalk cells, terminal follicle cells, or polar cells) is required for supernumerary polar cells to undergo apoptosis (Borensztejn et al., 2013). The polar cells are the sole source of the JAK/STAT ligand Upd in the ovariole and knocking down this source results in a decrease in stalk cells and in some cases an increase in polar cells. Interestingly, preventing the reception of the Upd signal in stalk cells by expressing dome-RNAi results in an increase in polar cells, indicating that JAK/STAT signaling in the stalk cells is required for promoting apoptosis of polar cells. Thus, coupled with our findings presented here, JAK/STAT signaling in the stalk cells acts in a cell non-autonomous pro-apoptotic manner to eliminate supernumerary polar cells (Borensztejn et al., 2013) and in a cell-autonomous anti-apoptotic/pro-survival manner to maintain cells of the stalk. It is possible that stalk cells respond to the Upd signal by down regulating the cell death machinery and producing a secreted factor that signals back to polar cells to promote apoptosis. It is also clear that polar cells respond to the Upd signal differently than the stalk cells, as blocking reception of the signal by expressing dome-RNAi in polar cells leads to an increase in their number (pro-apoptotic) (Borensztejn et al., 2013). While the molecular underpinnings of these different responses remain to be elucidated, our findings highlight a model with which to investigate the factors regulating JAK/STAT-dependent control of apoptosis as inhibitory versus promoting.

3.3. Stalk cell number needs to be tightly regulated

We found that females whose interfollicular stalks are either longer or shorter than wild type lay fewer eggs. These findings indicate that the length of the stalk is critical, wherein variations by only a few cells have a detrimental effect on fecundity. Stalks are certainly important for separating each cyst of 16 germ cells with its associated somatic cells to define a single egg chamber as it buds off the germarium. However, once established must the separation of egg chambers be maintained? Results from the exogenous expression of the proapoptotic gene hid in stalks after their formation indicates that stalk cell maintenance is critical for the continued integrity of egg chambers as oogenesis proceeds. Even after egg chambers have been properly formed and separated, complete or even partial elimination of the interfollicular stalk results in disorganized and/or merged egg chambers that are have been reported incapable of producing a functional oocyte (Adam, 2004; Assa-Kunik et al., 2007; Bai and Montell, 2002; Chang et al., 2013; Chen et al., 2011; Grammont and Irvine, 2001; McGregor et al., 2002). The most severe fusion of egg chambers was apparent when dome was knocked down in genotypes lacking Gal80ts (109-53-Gal4 > UAS-dome-RNAi, Fig. 4A). Abutting egg chambers with intervening follicle cells were apparent in ovarioles from females with Gal80ts (109-53-Gal4 tubGal80ts > UAS dome-RNAi). These likely resulted from the perdurance of the transcriptional repressor GAL80 despite shifting females to the restrictive temperature. Alternatively, stalk maintenance could be required up to a certain stage in order to prevent complete fusion, although we did not have any evidence of a bias in egg chamber staging with complete versus partial fusions.

To summarize, we find during normal Drosophila oogenesis a structure critical for proper egg production undergoes apoptosis in a highly regulated manner. Activation of JAK/STAT signaling in these interfollicular stalk cells acts as a pro-survival pathway to ensure maintenance of this link between developing egg chambers, preventing the loss of stalk cells through apoptosis. These results coupled with previous findings identify that activation of JAK/STAT signaling in stalk cells can inhibit apoptosis of stalk cells but promote apoptosis of adjacent polar cells. Thus, it appears that specific mechanisms must be in place such that the output of JAK/STAT signaling distinguishes between a pro-versus anti-apoptotic function. Further studies to investigate the factors responsible for this difference will advance our understanding of how specific apoptotic machineries are regulated by JAK/STAT signaling.

4. Material and methods

4.1. Drosophila lines

109-53-Gal4 is expressed specifically in the stalk cells of the vitellarium, in the terminal filament of the germarium, and the epithelial muscle sheath surrounding the ovarioles (Fig. 2A). c306-Gal4 is expressed in somatic cells of germarium, stalk, polar and terminal follicle cells of vitellarium (Fig. 2D–F; (Manseau et al., 1997)). Lines from BDSC: 109-53-Gal4 (p{GawB}109-53, #7025), UAS-p35 (w*;P{UAS-p35.H}BH1, #5072), hopTUM/FM7c (#8492), UAS-dome-RNAi#1682 (P{TRiP.JF01682}attP2, #28983), UAS-dome-RNAi #647 (P{TRiP.HMS00647}attP2, #32860), UAS-GFP-RNAi (y[1]sc[*] v[1];P{y[+t7.7]v[+t1.8] = VALIUM20-EGFP.shRNA.4}attP2, #41553), UAS-mCD8::GFP, drice-RNAi (y1 sc* v1; P{TRiP.HMS00398}attP2, #32403), UAS-eGFP(y[*] w[*]; P{w[+mC] = UAS-2xEGFP}AH3, #6658), Gal80ts (w[*]; P{w[+mC] = tubP-GAL80[ ts]}20; TM2/TM6B, Tb[1], #7019). UAS-hid flies were generated by Zhou et al. (1997).

A temperature sensitive Gal80 gene was expressed under the control of a tubulin promoter to block transcriptional activation by GAL4 at the permissive temperature (25 °C), and only allowing GAL4-induced UAS-target gene expression when flies were shifted to the restrictive temperature (29 °C). All crosses were performed at 25 °C except where noted. Progeny from tubGal80ts;109-53-Gal4 or c306-Gal4;;tubGal80ts crossed to UAS-GFP-RNAi or UAS-eGFP(as a control), dome UAS-RNAi lines, or UAS-p35 were raised through development at 25 °C. The newly emerged females (0–1 day) were shifted and maintained at 29 °C for 3 days (for c306-Gal4 crosses) or 4 days (for 109-53-Gal4 crosses) prior to ovary dissection to allow expression of UAS constructs. Based on King (1970) we expect egg chambers to progress from stage 1 to stage 8 within 60–65 h.

The ability of each RNAi line to disrupt JAK/STAT signaling in ovaries was verified (Fig. 3G). For the UAS-hid experiment, flies were crossed at 18 °C until 3 days after emergence, then maintained for 3 additional days at 29 °C prior to ovary dissection. For the UAS-drice-RNAi experiment, flies were crossed at 18 °C until emergence, then maintained for 3 days at 29 °C prior to ovary dissection.

4.2. Immunohistochemistry

Ovaries from 3 to 4 day old females were dissected in PBS and fixed for 20 min at room temperature (RT) in 4% paraformaldehyde in PBS, pre-absorbed in 2% BSA, 0.3% Tween-20 or Triton X-100 in PBS for 1 h at RT, incubated with primary antibodies overnight 4 °C, secondary antibodies for 2 h at RT, stained with 1 µg/ml Hoechst 33342 (Molecular Probes) for 10 min at RT, and ovarioles were separated and mounted in 80% glycerol 0.5% N-propylgalate mounting media. Antibody dilutions: mouse monoclonal anti-Lamin C (DSHB, LC28.26) 1:200, mouse monoclonal anti-Fasciclin 3 (DSHB, 7G10) 1:40, mouse monoclonal Eyes Absent (DSHB, eya10H6) 1:200, rabbit polyclonal anti-GFP (Life Technologies), anti-mouse-Cy3 (Life Technologies) 1:200, and anti-rabbit-Alexa Fluor 488 1:200 (Cell Signaling). To detect apoptosis, ovaries stained with Lamin C were fixed a second time (4% paraformaldehyde, PBS) prior to ApopTag® Red In situ Apoptosis Detection Kit (Milipore). Ovaries were incubated in equilibration buffer 10 s RT, 1 h 37 °C in Tdt enzyme solution, 10 min RT in stop/wash buffer, washed 0.3% Tween-20, PBS, and Rhodamine antibody solution for 30 min at RT. Confocal images were taken using a Zeiss 510Meta LSM or Zeiss 800 with multiple consecutive Z-sections (0.2–0.3 mm per slice), processed with ImageJ (1.46 R) and annotated using Adobe Photoshop CS5.1 and Adobe Illustrator CC 2015.

4.3. Cell counting

The number of cells per stalk were counted in stalks through stage 8 of oogenesis in ovarioles of 3–4 day old females. Stalk cells, marked with Lamin C, were counted directly using manual focusing on a Nikon FXA or Zeiss Axio Imager. M1 to ensure all cells were counted. The stage of a stalk was assigned according to the stage of the egg chamber just anterior to it. Stalk cells (Lamin C-positive) associated with a fusion of adjacent egg chambers were not included in cell number counts due to an inability to accurately stage them. Results presented in Figs. 1D and 2A–C are based on ovaries from 3 females per genotype. Results in Figs. 1D, 2A–C, and 2D–E and 3D–F (2D–E and 3D–F were all done as one experiment) are based on ovaries from 6 females per genotype. The experiment in Fig. 3J–K was completed with 4 females per genotype.

4.4. Egg laying experiments

Flies were crossed at 25 °C to obtain the listed genotypes. Within 24 h of emergence, 5 females were moved to 29 °C and aged for 2.5 days and were maintained in cages with 3 males. Females laid eggs for 24 h on apple juice plates, with plates changed after 12 h and counted before hatching.

Acknowledgments

We are grateful to Denise Montell, Developmental Studies Hybridoma Bank (DSHB), Bloomington Drosophila Stock Center (BDSC, NIH P40 OD018537), Vienna Drosophila RNAi Center, and NIG-Fly for providing us with fly stocks. We warmly thank Amanda Howard for MATLAB advice, and Kristin White, Anne-Marie Pret, Francois Agnès, Trudi Schupbach, Eric Wieschaus, and members of the Wharton lab for discussions

Funding

Research, publication, A.B and A.M. were supported by an award from the National Institute of General Medical Sciences [RO1GM068118] to K.A.W. A.M. was also supported by an NRSA training grant [T32 GM007601].

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