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Novel intrinsic factor Yun maintains female germline stem cell fate through Thickveins

Summary Germline stem cells (GSCs) are critical for the reproduction of an organism. The self-renewal and differentiation of GSCs must be tightly controlled to avoid uncontrolled stem cell proliferation or premature stem cell differentiation. However, how the self-renewal and differentiation of GSCs are properly controlled is not fully understood. Here, we find that the novel intrinsic factor Yun is required for female GSC maintenance in Drosophila. GSCs undergo precocious differentiation due to de-repression of differentiation factor Bam by defective BMP/Dpp signaling in the absence of yun. Mechanistically, Yun associates with and stabilizes Thickveins (Tkv), the type I receptor of Dpp/BMP signaling. Finally, ectopic expression of a constitutively active Tkv (TkvQD) completely suppresses GSC loss caused by yun depletion. Collectively, these data demonstrate that Yun functions through Tkv to maintain GSC fate. Our results provide new insight into the regulatory mechanisms of how stem cell maintenance is properly controlled.


INTRODUCTION
The self-renewal (maintenance) and differentiation of adult stem cells are critical for proper tissue homeostasis, so the balance of self-renewal and differentiation of adult stem cells must be tightly controlled. Disruption of this balanced control leads to either excessive stem cell proliferation or precocious stem cell depletion, eventually resulting in various diseases, including cancer and aging (Lin, 2008;Morrison and Spradling, 2008). Therefore, understanding of the underlying mechanisms controlling stem cell self-renewal and differentiation is important for the development of potential therapeutics for human diseases.
The adult Drosophila ovary is an excellent system to study the regulation of stem cell self-renewal and differentiation in vivo (Hsu et al., 2019;Ishibashi et al., 2020;Zhang and Cai, 2020). A structure, termed germarium, locates at the anterior end of a Drosophila ovariole, which contains the GSC niche, GSCs, and developing cysts ( Figure 1A). Two or three GSCs are located in the niche at the anterior tip of the germarium, which is composed of several types of somatic stromal cells including terminal filaments (TFs), cap cells (CPCs), and escort cells (ECs). GSCs can be easily identified by their location and the intracellular organelle known as the spectrosome: they physically contact with cap cells and contain an anteriorly anchored sphericalshaped spectrosome ( Figure 1A). GSCs normally undergo asymmetric self-renewing divisions, producing a self-renewed GSC daughter and a differentiating daughter, called a cystoblast (CB). CB is displaced from the niche (one cell diameter away from cap cells and containing a randomly localized spectrosome) to initiate differentiation. CB undergoes four rounds of synchronized divisions with incom-plete cytokinesis, producing a 2-to 16-cell developing cyst. The developing cyst contains a branched organelle (fusome) that interconnects individual cystocytes (Lin et al., 1994). The niche utilizes several extrinsic signals, such as bone morphogenetic protein (BMP) (Dpp and Gbb), Upd cytokine, and Wnt, to maintain GSC self-renewal (Luo et al., 2015;Wang et al., 2008a;Spradling, 1998, 2000). The short-range BMP/Dpp signaling is the major niche signal, which functions in one cell diameter in the niche to maintain GSC self-renewal and repress the transcription of the differentiation factor Bam (Bag-of-marbles) (Chen and McKearin, 2003;Losick et al., 2011;Tabata and Takei, 2004;Tu et al., 2020). Upon binding of BMP/Dpp ligands to the hetero-dimeric receptor composed of type I receptor (like Tkv) and type II receptor (like Punt), Tkv is activated and results in the phosphorylation of MAD (Mothers against dpp, co-SMAD in Drosophila, pMAD), pMAD forms a complex with Med (Medea, R-SMAD in Drosophila), and they translocate in the nucleus of GSCs to regulate the expression of their target genes such as Dad (Daughters against dpp) (Araujo et al., 2011;Chen and McKearin, 2003;Decotto and Spradling, 2005;Losick et al., 2011;Tabata and Takei, 2004;Spradling, 1998, 2000). Additionally, niche-expressing E-cadherin is required to anchor GSCs in the niche for long-term maintenance (Song and Xie, 2002). Somatic and germline cells deploy several mechanisms acting in concert to spatially restrict BMP/ Dpp signaling within the niche (Chen et al., 2010;Guo and Wang, 2009;Hayashi et al., 2009;Liu et al., 2010;Losick et al., 2011;Luo et al., 2015;Ting, 2013;Xia et al., 2010Xia et al., , 2012. As BMP/Dpp signaling is inactivated in multiple ways in CBs, bam expression is de-repressed, which further drives germline cell differentiation through multiple independent mechanisms (Casanueva and Ferguson, 2004;Chen and McKearin, 2003;Chen et al., 2011;Fu et al., 2015;Li et al., 2009Li et al., , 2013McKearin and Ohlstein, 1995;McKearin and Spradling, 1990;Ohlstein and McKearin, 1997;Pan et al., 2014;Shen et al., 2009;Song et al., 2004). However, how BMP/Dpp signal is precisely controlled in GSCs for GSC maintenance is not fully understood. (legend on next page)

RESULTS AND DISCUSSION
We identified a novel factor that we named Yun (''luck'' in Chinese) in a large-scale RNAi screen for regulators of adult intestinal stem cell maintenance, proliferation, and differentiation (Liu et al., 2022;Ren et al., 2022;Zhao et al., 2022aZhao et al., , 2022b. yun (CG7705) encodes a novel protein of 592 aa without any known domain and motif (Figures 1B and S1A). During the course of our study, another group showed that it is implicated in cell proliferation in larval brain and spermatogenesis and named it diamond (dind) (Graziadio et al., 2018), but its mechanism in brain cell proliferation and spermatogenesis remained unexplored, and it is unclear whether Yun functions in GSCs regulation. As Yun could be detected in female germline cells, including GSCs, we asked whether Yun plays any role in GSC regulation ( Figure S1B). Depleting yun in GSCs using a functional yun shmiR by nosGal4 in germline resulted in almost complete elimination of germline cells and GSCs, indicating that Yun may be required for GSC maintenance (Figures 1B, S1C, and S1D) (Zhao et al., 2022b). Detailed examinations showed that most of the nos > yun shmiR germaria was devoid of germline cells compared with control germaria ( Figures 1C-1E). And no GSCs were observed in those nos > yun shmiR germaria containing a few germline cells, indicating that these germline cells are developing cysts (Figures 1F and S1E-S1I). GSC loss could be due to defective GSC self-renewal or apoptosis. However, no increased apoptosis was observed in yun-depleted germline cells, excluding the possibility that yun-defective GSCs are lost due to apoptosis (Figures S1J-S1L).
To further confirm that Yun intrinsically controls GSC maintenance, we performed mosaic clonal analysis of yun null mutants using Flp/FRT system ( Figure 1B) (Zhao et al., 2022b). We first examined the mosaic clones that were induced at larval stages in adult animals. Control GSC clones underwent constant self-renewal divisions and produced developing egg chambers; although no GSCs were observed in yun mutant clones, many empty germaria were followed by differentiating egg chambers containing yun mutant germline cells, indicating that yun-defective GSCs could not be maintained and underwent differentiation ( Figures 1G-1I and S1M-S1P). We then induced mosaic clones in adult flies. Marked control GSCs were constantly observed, while yun mutant GSCs could not maintain their stem cell fate and differentiated ( Figures 1J-1L and S1Q-S1S). We further investigated GSC maintenance by examining the percentage of marked GSCs at different time points after clone induction (ACI). Compared with the marked control GSCs that still remained in the niche 2 and 3 weeks ACI, respectively, yun mutant GSCs were rapidly lost, and no marked yun mutant GSCs could be observed 3 weeks ACI ( Figure 1M). Collectively, these data indicate that Yun is intrinsically required for GSC maintenance.
The differentiation factor Bam plays major role in GSC self-renewal and differentiation (McKearin and Spradling, 1990). We then examined the expression of Bam in the absence of yun using Bam antibody and a bam-GFP reporter, respectively (Chen and McKearin, 2003). Bam protein could not be detected in control GSCs, while Bam protein was detected within the nos > yun shmiR niche, indicating that elevated Bam protein may cause the loss of yun-defective GSCs (Figures 2A-2D). Furthermore, Bam protein was detected within the niche of nos > stop > Gal4>yun shmiR germaria, which bypassed the possible side effects of early-on depletion of yun using nosGal4 ( Figures 2E-2H) (Ma et al., 2014). Moreover, Bam protein was detected in yun mutant GSCs within the niche (Figures 2I-2L). bam-GFP is repressed in control GSCs, while it was detected in putative GSCs in the nos > yun shmiR niche ( Figures 2MÀ2P and S2A-S2D). Altogether, these data show that upregulation of Bam in yun-defective GSCs is likely the cause of GSC loss. Consistently, simultaneous depletion of Bam in nos > yun shmiR germaria completely rescued GSC loss observed in nos > yun shmiR germaria, and nos > Bam RNAi , yun shmiR germaria were identical to nos > Bam RNAi germaria ( Figure S2E). Collectively, these data show that Yun is intrinsically required for GSC maintenance by repressing Bam expression.
Niche-derived BMP/Dpp signaling is necessary and sufficient for GSC self-renewal and Bam suppression (Casanueva and Ferguson, 2004; Chen and McKearin, 2003; Kai and Spradling, 2003;Song et al., 2004;Spradling, 1998, 2000). Next, we determined the activation of Dpp signaling in yun-defective GSCs. The levels of pMAD were strongly diminished in nos > yun shmiR and nos > stop > yun shmiR germaria compared with those in control GSCs (Figures 2MÀ2O, 2Q-2T, and S3A-S3C). The levels of pMAD were also dramatically reduced in marked yun mutant GSCs compared with those in control GSCs ( Figures 2U-2X). The expression of Dad-lacZ, the downstream target of Dpp signaling, was almost undetectable in nos > yun shmiR germline cells compared with that of control ( Figures S3D-S3F). The expression of Dad-lacZ in yun mutant GSCs was also dramatically diminished ( Figures S3G-S3H 0 ). Previous study showed that adhesion of GSCs to cap cells by E-cadherin is important for GSC maintenance (Song and Xie, 2002), so we asked whether the levels of E-cadherin were affected upon Yun depletion.  The results showed that no reductions in the levels of E-cadherin were observed in the nos > yun shmiR niche compared with those in the control, indicating that the loss of yun-defective GSCs is not caused by reduced adhesion of GSCs to cap cells ( Figures S3I and S3J 0 ). Altogether, these data show Yun is intrinsically required for Dpp signaling activation to suppress Bam expression for GSC maintenance.
How does Yun regulate GSC self-renewal through Dpp signaling? Since Dpp signaling is defective in the absence of Yun, we asked whether Yun functions through the key components of the Dpp signaling pathway. Interestingly, we found the subcellular localization of Yun in GSCs is similar to that of Tkv, and Yun often co-localizes with Tkv ( Figure 3A and S4A). Similar phenomena were observed between endogenous Yun and GFP-tagged Tkv under its endogenous promoter in the germarium ( Figure S4A). These data suggest that Yun may function through Tkv to maintain GSC fate. We found that Yun and Tkv could not be detected in the recycling endosomes at the same time, indicating that the receptor recycling pathway may not account for the GSC maintenance defects observed in the absence of yun ( Figure S4B). Furthermore, the coimmunoprecipitation results showed that both overexpressed and endogenous Yun associate with endogenous Tkv (Figure 3B). We then examined whether Yun affects Tkv levels. Interestingly, the levels of Tkv in the niche of nos > yun shmiR germaria were dramatically reduced compared with those in control GSCs, indicating that Yun affects Tkv protein levels ( Figures 3C and 3D 0 ) (Luo et al., 2015). We also examined whether Tkv affects the levels of Yun. However, the levels of Yun proteins were largely unaffected in the absence of tkv, indicating that Tkv is not likely to affect the levels of Yun proteins ( Figure S4C). Collectively, these data indicate that Yun functions through Tkv to regulate Dpp signaling, thereby maintaining GSC fate.
To further confirm this conclusion, we carried out rescue experiments. Expression of a constitutively active Tkv (tkv QD ) resulted in strong Dpp signaling activation in the germ cells ( Figures 4A and 4B). Compared with diminished Dpp signaling in the nos > yun shmiR germaria, Dpp signaling was ectopically activated in all nos > tkv QD , yun shmiR ovarioles, identical to that of nos > tkv QD ovarioles ( Figures 4A-4E). The levels of Yun proteins were significantly eliminated in germline cells of nos > tkv QD , yun shmiR ovarioles ( Figure S4D). These data support the notion that Yun functions through Tkv to regulate GSC self-renewal.
Consistently, the germaria and developing follicles in nos > tkv QD , yun shmiR ovarioles were filled with GSCs and GSC-like cells, identical to those of nos > tkv QD ovarioles ( Figures 4F-4I). Moreover, precocious bam expression observed in nos > yun shmiR GSCs was completely suppressed in nos > tkv QD , yun shmiR ovarioles, identical to nos > tkv QD ovarioles ( Figures 4J-4N). Altogether, these data show that Yun acts through Tkv to ensure Dpp signaling in GSCs, thereby maintaining GSC fate within the niche.
As the major niche-derived extrinsic signal, Dpp is spatially restricted within the niche to ensure GSC maintenance and proper GSC lineage development. To achieve such a tight spatial control of Dpp activity, multiple strategies are deployed (Chen et al., 2011). Extrinsically, cap cellexpressed glypican Dally limits Dpp diffusion to confine high Dpp concentrations within the niche (Guo and Wang, 2009;Hayashi et al., 2009;Liu et al., 2010). Viking, the type IV collagen, sequesters Dpp around GSCs to limit the functional range of Dpp (Wang et al., 2008b). Escort cell-expressed Tkv functions as a receptor sink/trap to remove excess diffusible Dpp to restrict Dpp activity locally within the niche (Luo et al., 2015;Xu et al., 2018). Intrinsically, different mechanisms were identified to promote Dpp signal activation in GSCs but rapidly dampen it in CBs (Chen et al., 2011). These mechanisms involved the post-translational regulation of Dpp signaling components in CBs: (1) the degradation of activated Tkv by the Fused-Smurf complex (Xia et al., 2010), (2) the translation repression of MAD by the Brat-Pumilo complex , and (3) the regulation of Sax levels by miR-184 (Iovino et al., 2009). It is also reported that Bam acts redundantly with Smurf to turn down Dpp signaling through an unknown mechanism (Casanueva and Ferguson, 2004). Inside GSCs, a variety of mechanisms are deployed to ensure proper Dpp signal activation for GSC selfrenewal. Many intrinsic factors have been identified to regulate Dpp signaling. Petola and TSC1/2 (tuberous sclerosis complex 1 / 2 ) are required to maintain Dpp signaling but are dispensable for bam repression (Sun et al., 2010;Xi et al., 2005). Lissencephaly-1 (Lis-1) directly binds to MAD to stabilize it and facilitate its phosphorylation, thereby regulating Dpp signaling (Chen et al., 2010).
Here we provide evidence that Yun associates with and stabilizes Tkv to ensure Dpp signal activation for GSC maintenance. These data suggest that in GSCs the Dpp signaling pathway is regulated at multiple levels to ensure GSC maintenance.
Dpp signaling also acts as a short-range signal to maintain self-renewal of male GSCs, another well-established system to stem cell biology Shivdasani and Ingham, 2003). We are interested to examine whether Yun is also required for male GSC maintenance. Interestingly, Yun is not expressed in the testis niche, and no obvious defects were observed upon yun depletion in male GSCs (not shown). These data suggest that although Dpp signaling is required in both niches, these two niches utilize different mechanisms to ensure GSC self-renewal.

Fly lines and cultures
Flies were maintained on standard media at 25 C. Crosses were raised at 18 C in humidity-controlled incubators or as noted. Information for alleles and transgenes used can be found either in FlyBase or as noted.