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Germline self-renewal requires cyst stem cells, while stat regulates niche adhesion in Drosophila testes

Adults maintain tissue-specific stem cells through niche signals. A model for niche function is the Drosophila melanogaster testis, where a small cluster of cells called the hub produce locally available signals that allow only adjacent cells to self-renew. We show here that the principal signalling pathway implicated in this niche, chemokine activation of STAT, does not primarily regulate self-renewal of germline stem cells (GSCs), but rather governs GSC adhesion to hub cells. In fact, GSC renewal does not require hub cell contact, as GSCs can be renewed solely by contact with the second resident stem cell population, somatic cyst stem cells (CySCs), and this involves BMP signalling. These data suggest a modified paradigm whereby the hub cells function as architects of the stem cell environment, drawing into proximity cellular components necessary for niche function. Self-renewal functions are shared by the hub cells and the CySCs. This work also reconciles key differences in GSC renewal between Drosophila testis and ovary niches, and highlights how a niche can coordinate the production of distinct lineages by having one stem cell type rely on a second.

inactivation of stat ts , but rescued the CySCs by restoring somatic expression of stat (Figure S1B, D-E; Table 1, Tj Gal4, c587 Gal4 or EyaA3 Gal4 entries). As expected, CySCs persisted because stat had been restored to this lineage specifically ( Figure 1A, Zfh-1, green, marks CySCs), but now stat-depleted germline cells were maintained indefinitely in most testes (57/64 or 89% still had spermatogonia eight days after stat depletion; Figure 1C; see Table 1 for quantitation) 4 . In particular, amplifying spermatogonia and spermatocytes were observed at all stages of differentiation in their normal organization emanating from the testis apex near the hub, shown by normal Bam accumulation, branching fusomes, continual cycling through S phase, and spermatocyte Ptb accumulation ( Figure 1D-G). These testes appeared to harbor as many (or even more) differentiating germ cells than wild-type testes. This is in striking contrast to the unrescued situation, where most testes had lost all germ cells to differentiation by eight days, and 0/37 had spermatogonia present (Figure 1B, C; Table 1).
The continual production of normally differentiating germ cells indicated that stat-depleted germ cells must still be functioning as GSCs. In normal testes, GSCs are closely apposed to the hub. Surprisingly, when germline stat was depleted, germ cells lost contact with the hub (Figure 2A vs. B), and the nearest germ cells were now located several cell tiers away from the hub. The first few tiers of these displaced germ cells remained in contact with CySCs as judged by Zfh-1 expression, and these germ cells retained the hallmarks of GSCs, including expression of M5-4, appearance of dot spectrosomes, and cycling as individual cells ( Figure  2C-E). While these markers are not unique to GSCs, but shared by gonialblasts (the differentiating daughters of GSCs), the appearance of these characteristics along with the continual, robust production of normally differentiating germ cells demonstrates that the displaced, stat-depleted germ cells still self-renew as GSCs.
The only hallmark of GSCs that was disrupted under conditions of stat-depletion was the reliably oriented plane of cell division. Normally, GSCs divide perpendicular to the hub surface 5 , providing for a mechanism of asymmetric stem cell division, with one daughter cell invariantly pushed away from renewing influences, allowing its differentiation program to initiate. Such oriented divisions are directly dependent on tethering of one centrosome in the GSC to its interface with the hub 6 , 7 . While centrosome staining of wild-type mitotic GSCs shows invariant orientation 6 , the division plane of stat-depleted, displaced GSCs became randomized ( Figure 2F shows one oriented mitotic GSC (arrow) and one nonoriented mitotic GSC (arrowhead); ratio of oriented:non-oriented mitotic GSCs was 1:1.7, n=16 GSCs; see Methods). Thus, this alternate niche configuration likely does not precisely parse the stem cell daughters into one renewing and one differentiating cell, demonstrating the niche's capacity to function in a less regimented, population mode for self-renewal, where the production of differentiating daughter cells is stochastic 8 . We do not know whether GSCs in this alternate niche configuration completely bypass the other characteristics of asymmetric division, including stem cell inheritance of the mother centrosome and the mitotic checkpoint associated with centrosome attachment 7 , 9 . In testes with stat-depleted GSCs, the CySCs were rescued due to lineage-specific restoration of stat expression, and these Zfh-1-positive cells now surrounded the hub (which is the source of the STAT-activating ligand). We noted that the stat-depleted, displaced GSCs were interspersed among Zfh-1-expressing cells ( Figure 2G). These Zfh-1-expressing cells did not adopt hub character ( Figure S2 and Figure 2C), suggesting that it was their CySC fate and not any influence of hub cells that was integral to the maintenance of GSCs under these conditions. In fact, stat-depleted GSCs could not be rescued by restoration of stat solely to the hub cells (Table 1, Upd Gal4 entry). The requirement for CySCs was further confirmed because stat-deficient GSCs were rescued, although to a lesser degree, by cyst lineage restoration of zfh-1, a target of STAT in CySCs (Table S1). This is consistent with previous findings that Zfh-1 controls a CySC signal that can support GSC renewal 3 .
If CySCs are required for GSC renewal, loss of the CySC population should lead to GSC loss, even if the GSCs retain stat. To test this, we specifically depleted stat in the CySC lineage by temperature shift of the stat ts allele accompanied by Gal4 stat rescue in germline cells ( Table 1, Nanos Gal4 VP16 entry). Unshifted controls averaged 36 Zfh-1-positive cells/testis (n=10), while testes depleted for stat in CySCs for eight and sixteen days averaged only 14.2 (n=25) and 9.5 (n=56) Zfh-1-positive cells/testis respectively. Over the same period, we observed a gradual loss of GSCs. While unshifted controls had 10 GSCs/ testis on average (n=10), testes eight and sixteen days after stat loss in CySCs harbored only 3.1 (n=90) and 0.7 (n=56) GSCs/testis on average, respectively. This result is consistent with a requirement for CySCs in GSC renewal. We had expected that CySC stat-depletion would cause all CySCs to differentiate. The retention of some CySCs may be due to the hypomorphic nature of the stat ts allele, or alternatively, a partially redundant mechanism for CySC maintenance.
Next, we investigated why stat-depleted GSCs became displaced from the hub. It is known that an intrinsic increase in STAT activation in CySCs causes these cells to outcompete GSCs for niche access 10 . Since our strategy to examine stat-depleted GSCs utilized Gal4driven rescue of stat in CySCs, we considered whether higher than normal STAT levels in CySCs led to excess STAT activation and hence could account for the observed GSC displacement from the hub. However, this did not explain the displacement of GSCs in our experiment. As expected, testes expressing excess somatic stat but otherwise stat/+ indeed led to some displacement of GSCs (Table 1: Tj Gal4/UAS stat; stat 85c9 /+). However, even after eight days, most such testes still retained GSCs at the hub (90% of testes had GSCs, averaging 2.4 GSCs/testis; Figure 2H, Table 1). Only upon depletion of stat in the germline were all GSCs lost from the hub, and this occurred within two days (Table 1: Tj Gal4/UAS stat; stat ts /stat 85c9 ). To confirm that intrinsic reduction of STAT in the germline led to GSC displacement, we knocked down stat by RNAi in the germline. This experiment involved no extra stat expression in CySCs, yet also led to detachment of most GSCs from the hub ( Figure 2I). These testes similarly exhibited continual, robust production of normally differentiating germ cells, and displaced GSCs clustered next to CySCs, which now surrounded the hub (Table 1). Thus, STAT is required intrinsically in GSCs to promote attachment to the hub. We note that under the conditions of stat depletion that must be used with the stat ts allele, there could be some residual STAT function in germ cells. Thus, we cannot eliminate the possibility that STAT plays some direct role in self-renewal of GSCs. However, a principal role in adhesion is sufficient to explain earlier mosaic experiments 1 , 2 . We suggest that stat null GSCs were lost, not because stat was essential for self-renewal, but rather, because an attachment defect led to their displacement from the niche, and nearby wild-type germ cells de-differentiated 11 to take their place.
If STAT primarily regulates attachment, then adhesion defects should precede other signs of differentiation in GSCs. To ensure reliably oriented divisions, an elaborate cortical complex is postulated to be established in GSCs at the hub interface, linking one centrosome to the adherens junction ( Figure 3A and C, respectively) 6 , 7 . Thus, in testes with global stat depletion, we examined these components in GSCs prior to overt signs of differentiation, scoring only GSCs that were still located next to the hub and that did not yet express the differentiation marker Bam ( Figure 3A′, B′). At sixteen hours after stat depletion, there were on average 4.5 such undifferentiated GSCs per testis (n=26 testes) compared to 7.0 for controls (n=12). We found that 47% of such GSCs (n=86) exhibited defective centrosome placement ( Figure 3B, arrows), compared to only 8% (n=64) in controls ( Figure 3A). In addition, we expressed E-cadherin-GFP in germline cells, which selectively labeled the adherens junctions at the GSC-hub interface ( Figure 3C′). In stat-depleted testes, Ecadherin-GFP became significantly delocalized in most GSCs, with 44% of GSCs (n=81) completely lacking enrichment at the hub interface compared to only 16% (n=102) in controls ( Figure 3C, D; arrowheads and arrows show reduced or absent E-cadherin enrichment, respectively). We observed similar dispersal of Par3-GFP from the interface ( Figure 3E, F; quantified in Table S2); although Par3 has not been postulated to be part of the cortical complex in GSCs, it fortuitously localized in prominent puncta at the hub-GSC interface, adjacent to E-cadherin. Thus, stat-depleted GSCs exhibit severe disruption of the hub-GSC interface, and this precedes overt differentiation. We also found that increasing germline E-cadherin expression moderately suppressed the germ cell loss phenotype ( Figure  3G), suggesting that regulation of adhesion is a key role for STAT in the germline.
If cell-intrinsic Jak/STAT pathway activation does not primarily regulate GSC self-renewal, what does? The BMP pathway has been implicated in GSC renewal 12 -14 . The ligands, gbb and dpp, are each expressed in CySCs (and in hub cells), and pathway activation has been observed in GSCs 12 -14 . In testes with germline stat depletion, we observed strong BMP pathway activation in the displaced GSCs, shown by accumulation of phosphoSmad and the BMP target gene dad ( Figure 4A, B). Interestingly, we noted that the levels of pSmad and Dad-lacZ were much higher in testes with stat-deficient displaced GSCs than in wild-type GSCs (wild-type not shown), possibly because higher levels of STAT in CySCs led to higher BMP ligand levels. Sustained zfh-1 expression in the cyst lineage, which generates ectopic GSCs far from the hub 3 , also led to strong BMP pathway activation in the ectopic GSCs ( Figure 4C). Thus, Jak/STAT activation in CySCs leads to BMP ligand expression 15 , likely using zfh-1 as an intermediary.
In testes with germline stat depletion, we also found that dampening the BMP pathway by expression of the extracellular BMP antagonists sog CR1 or kekkon5 suppressed rescue of the germline ( Figure 4D). Thus, BMPs are functionally important in CySC-supported GSC selfrenewal. A primary role for BMPs in male GSCs makes the testis much more similar to the Drosophila ovary, where BMP pathway activation regulates GSC renewal via repression of the differentiation gene, bam 16 , 17 , as also occurs in the male germline 18 . A remaining difference, however, is that, in the male, BMP pathway activation is insufficient for induction of ectopic GSCs. Thus, there may be another assisting signal that also emanates from the CySCs. The Jak/STAT pathway is also similar between the two niches, with pathway activation primarily required in the somatic cell populations 3 , 19 , 20 . However, while the testis GSCs use Jak/STAT-regulated centrosome tethering to attach and orient the division plane, the ovary uses Jak/STAT-independent spectrosome localization to ensure an asymmetric outcome to stem cell divisions 21 .
One can formulate a revised model for this paradigmatic niche taking our data along with recent work ( Figure 4E). The hub cells are architects of the stem cell environment, establishing close proximity of the three cell populations because STAT influences adhesion in nearby germline and somatic cells, probably through adherens junctions (this work) 22 and integrin linkages 10 , respectively. STAT activation also governs self-renewal in the somatic lineage via zfh-1 and chinmo induction 3 , 23 . These CySCs then activate BMP, which is used to renew neighboring, hub-adherent germ cells. Thus, self-renewal activity relies not just on the hub, but also on one of the resident stem cell lineages. BMPs are expressed from both hub and CySCs 12 , 13 , possibly to generate sufficient quantity of BMP for renewal; alternatively, the ligand composition from hub versus CySCs may be qualitatively different. In either case, the parsing of different roles such as adhesion and self-renewal among multiple niche components is a departure from previous niche paradigms, and is likely to be utilized in the much more complex niche environments in mammals.

Fly Stocks and Crosses
The temperature-sensitive stat allele (stat F , FBal0060556) was used in trans with a strong stat allele, either stat 85c9 , stat 06346 , or stat 397 (FBal0130583, FBal0009559, FBal0130586, Erika Bach). Germ cell stages present in adult testes were tabulated before and at various time points after stat was inactivated by a shift from 18° (the permissive temperature) to 29° (the non-permissive temperature). Spermatogonia were identified by their smaller cell size and the presence of bright Hoechst staining. Included in this category were GSCs attached to the hub and individual or paired germ cells away from the hub. Spermatocytes were identified by increased cell size and much reduced Hoechst staining. Gal4 drivers were Traffic Jam Gal4 (Kyoto Stock Center, DGRC #104055), c587 Gal4 (Erika Matunis), or EyaA3 Gal4 3 for CySC expression, Nos Gal4 VP16 (Ruth Lehmann and Erica Selva) for germline expression, and Upd Gal4 (Doug Harrison) for hub expression. Rescue transgenes were UAS Stat-HA (FBtp0036673, Erika Bach, NYU, NY) and UAS zfh-1 (FBtp0016144, Bloomington Stock Center). Bam expression was visualized using a Bam-GFP reporter transgene (Dennis McKearin, UT Southwestern, Tx) and staining for GFP. Germline depletion of stat was also accomplished using an RNAi transgene (#106980; Vienna Drosophila Resource Center) crossed to Nos Gal4 VP16. We analyzed cortical components of the GSC at the interface with hub cells by crossing Nos Gal VP16 with UAS E-cadherin-GFP (also called UAS DEFL, Kyoto Stock Center) or UAS Par3GFP (Daniel St Johnston, MRC, UK) in stat mutant testes. Dad expression was visualized using the enhancer trap insertion P{lacZ}Dad P1883 (FBal0094852), followed by lacZ immunostain. The BMP pathway was dampened by coexpressing either UAS sog CR1 (activated form of sog, FBtp0018246, Ethan Bier) 24 or UAS kekkon5 (FBtp0018362, Joe Duffy) 25 in the CySC lineage along with UAS stat in stat ts flies. These are both extracellular BMP ligand antagonists. Various time points after stat temperature inactivation, the types of germ cells present were scored, and compared to controls (the same genotype, lacking BMP antagonist expression). Significance was determined by the Chi-square test of independence (http:// faculty.vassar.edu/lowry/odds2x2.html).

Immunostaining
Testes were dissected and fixed in 4% paraformaldehyde in Buffer B (16.7 mM KH2PO4/ K2HPO4, 75 mM KCl, 25 mM NaCl, 3.3 mM MgCl2, pH 6.8) with 0.1% Triton X-100 for 20 minutes. Samples were blocked for one hour in 4% normal donkey serum in PBS with 0.1% Triton. Primary antibodies were incubated overnight in block, and were goat anti-Vasa

S Phase Labeling
To label cells in S phase, freshly-dissected testes were incubated for 30 minutes in 10μM Edu 26 then fixed. Testes were stained as above with other antibodies, followed by visualization of the Edu label with the Click-iT EdU Alexa Fluor 647 Kit (Invitrogen) according to the manufacturer's instructions.

Spindle orientation and centrosome attachment
In order to examine spindle orientation in stat-depleted GSCs, we examined the first tier of germ cells nearest to the hub eight days after stat inactivation. Mitotic germ cells were identified by centrosomin enrichment 27 , DNA compaction, and nuclear breakdown (revealed by loss of vasa exclusion from the nucleus). At this stage of the cell cycle, spindle orientation is reflected by centrosome position. Germ cells were scored as oriented if one centrosome was located within an arc inscribing approximately one quarter of the germ cell contour nearest the hub.
Centrosome attachment to the hub interface in GSCs sixteen hours after stat depletion was scored by γtubulin stain, which reveals centrosomes in all stages of the cell cycle. GSCs were scored as having an attached centrosome if one centrosome was visible at the GSC-hub interface (in some cells, centrosome separation might not yet have occurred). GSCs were scored as having unattached centrosomes if both centrosomes were visible and neither was anchored at the hub-GSC interface.

Quantitation of germline Par3-GFP mislocalization
Germline Par3-GFP localization was quantified by measuring the average intensity along an arc, drawn either along the hub-GSC interface or along each lateral edge of the GSC. In all cases, Z slices with the highest intensity were chosen. The two lateral edge measurements were averaged, and this value was used as the denominator in a ratio with the hub interface value (hub/lateral edges). The resulting ratios from all scored GSCs were averaged, and a p value calculated by a two tailed Students t test.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. Testes with global stat depletion exhibited germ cell loss (lack of magenta cells) and CySC loss (Zfh-1, green; note, a few CySCs always persisted near the hub). (C) Global stat depletion led to progressive germ cell loss, first the more undifferentiated spermatogonia (blue; this category includes GSCs/single germ cells) followed by spermatocytes (orange), while most testes with germline stat depletion maintained spermatogonia (blue) indefinitely. Similar results were obtained with three CySC lineage Gal4 drivers and three strong stat alleles with the stat ts allele (Table 1). (D-G) Stat-depleted germ cells continually progressed through differentiation, shown by normal expression of the spermatogonial differentiation marker Bam (D, green), the appearance of branching fusomes through germ cell cysts (E, αspectrin, green), synchronously cycling germ cell cysts (F, Edu pulse-labeled cells, green), and expression of the spermatocyte marker Ptb (G, green). Bar:10μm.   Table S2. (G) Germline UAS E-cadherin expression in stat ts testes delayed germ cell loss compared to stat ts alone or with UAS Par3 expression. Asterisk denotes hub. Bar:10μm.  1 Expanded Fas3-positive somatic cell population in many testes made identification of hub and the number of GSCs attached to it impossible. GSCs/testis count includes only those testes without an expanded Fas3-positive population.
2 Testes with germline stat rescue maintained some CySCs, thus germ cell loss was modest (see text).