Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche

Adult stem cells continuously undergo self-renewal and generate differentiated cells. In the Drosophila ovary, two separate niches control germ line stem cell (GSC) self-renewal and differentiation processes. Compared to the self-renewing niche, relatively little is known about the maintenance and function of the differentiation niche. In this study, we show that the cellular redox state regulated by Wnt signaling is critical for the maintenance and function of the differentiation niche to promote GSC progeny differentiation. Defective Wnt signaling causes the loss of the differentiation niche and the upregulated BMP signaling in differentiated GSC progeny, thereby disrupting germ cell differentiation. Mechanistically, Wnt signaling controls the expression of multiple glutathione-S-transferase family genes and the cellular redox state. Finally, Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in the differentiation niche. Therefore, this study has revealed a novel strategy for Wnt signaling in regulating the cellular redox state and maintaining the differentiation niche. DOI: http://dx.doi.org/10.7554/eLife.08174.001


Introduction
Stem cells have two important properties, self-renewal and differentiation, which are critical for continuously generating new functional cells to maintain tissue homeostasis. The self-renewal property is controlled in various stem cell systems by interplays between signals from the niche and intrinsic factors (Li and Xie, 2005;Morrison and Spradling, 2008;Losick et al., 2011). Germ line stem cells (GSCs) in the Drosophila ovary and testis are attractive systems for studying stem cell selfrenewal at the molecular and cellular level (Fuller and Spradling, 2007;Xie, 2013). Although stem cell differentiation was widely thought to be a developmentally default state, we have recently proposed that GSC lineage differentiation is also controlled extrinsically by a differentiation niche formed by inner germarial sheath cells (ISCs, also known as escort cells). However, it remains unclear how the maintenance and function of the differentiation niche are regulated at the molecular level. In this study, we show that autocrine Wnt2/4 signaling maintains the differentiation niche by regulating ISC proliferation and survival via redox regulation.
In the Drosophila ovary, two or three GSCs at the tip of the germarium, the most anterior region of the Drosophila ovary, continuously self-renew and generate differentiated GSC daughters, cystoblasts (CBs). The CBs further divide four times synchronously with incomplete cytokinesis to form 2-cell, 4-cell, 8-cell, or 16-cell cysts (de Cuevas et al., 1997). GSCs and their differentiated progeny can be reliably identified by their unique morphology of germ line-specific intracellular organelles known as fusomes: GSCs and CBs contain a spherical fusome known as the spectrosome, whereas differentiated germ cell cysts contain a branched fusome (Lin et al., 1994). GSCs can be reliably distinguished from CBs by their direct contact with cap cells ( Figure 1A). Cap cells function as the self-renewing niche to maintain GSCs by activating BMP signaling and maintaining E-cadherin-mediated cell adhesion (Song et al., 2002;Xie andSpradling, 1998, 2000). In addition, various classes of intrinsic factors work with BMP signaling and E-cadherin to control GSC self-renewal (Xie, 2013). Therefore, GSC self-renewal is controlled by coordinated functions of niche-initiated signaling pathways and intrinsic factors.
Following GSC division, differentiating GSC daughters, CBs, are always positioned away from the self-renewal niche. ISCs sit on the surface of the germarium to send their cellular processes to wrap up underneath CBs, mitotic cysts, and early 16-cell cysts, which move posteriorly (Decotto and Spradling, 2005;Kirilly et al., 2011;Morris and Spradling, 2011). Our recent study suggests ISCs and their associate long cellular processes act as the differentiation niche to promote GSC progeny differentiation in the Drosophila ovary because disrupting long ISC processes leads to an accumulation of CB-like cells, indicative of a germ cell differentiation defect (Kirilly et al., 2011). A series of genetic studies have further supported the existence of the differentiation niche.
The epidermal growth factor (EGF) signaling pathway is active in ISCs to promote GSC lineage differentiation partly by repressing dally expression (Schultz et al., 2002;Liu et al., 2010). In addition, Rho signaling is also required in ISCs to promote GSC differentiation partly by repressing dally and dpp expression. dally encodes a proteoglycan protein, which is capable of promoting Dpp/BMP diffusion to the differentiation niche (Guo and Wang, 2009;Hayashi et al., 2009). Ecdysteroid signaling also operates in ISCs to promote germ cell differentiation because inactivating ecdysteroid receptors EcR and Usp in ISCs disrupts cyst formation (Morris and Spradling, 2012). One potential mechanism is that ecdysteroid signaling controls the formation of ISC cellular processes, thereby promoting the interaction between ISCs and germ cells (Konig and Shcherbata, 2015). Gap junction protein Inx2 functions in ISCs to promote germ cell differentiation, but its transmitted substances between ISCs and germ cells remain identified (Mukai et al., 2011). The importance of gap junctions between ISCs and germ cells could also explain why ISC cellular processes are important for germ cell differentiation. Therefore, physical interactions and signaling-mediated communications between ISC cellular processes and GSC progeny likely contribute to GSC progeny differentiation collectively.
In addition, chromatin regulators are also important in ISCs to promote GSC differentiation. Eggless, a Drosophila H3K9 trimethyltransferase, maintains ISCs and represses dally and dpp expression in ISCs, eLife digest An animal or plant has many different types of cells that have specific roles in the life of the organism. These cells are organized into tissues. In most tissues in adult animals, small groups of cells called stem cells are responsible for replacing the other cells that have been lost due to disease, injury, or as part of normal body maintenance.
The 'germ line' stem cells of female fruit flies-which produce female sex cells (or eggs)-are an effective system for studying how stem cells are regulated. These cells live in an area of the ovary called a stem cell niche. Each time a stem cell divides, it produces one stem cell and one other daughter cell. This daughter cell then moves into another niche called the 'differentiation' niche and undergoes a series of divisions that produce the egg cells. The differentiation niche is formed by escort cells and is crucial for producing the egg cells, but it is not clear how the escort cells promote this process, or how the niche is maintained.
Wang et al. have now studied the differentiation niche in more detail. The experiments show that a cell communication system called Wnt signaling maintains the differentiation niche by controlling the ability of the escort cells to grow and divide. If Wnt signaling is defective, the differentiation niche is lost, which disrupts the formation of egg cells.
Further experiments show that two proteins called Wnt2 and Wnt4 in the differentiation niche-which activate Wnt signaling-act as signals to regulate the niche, mainly by controlling the expression of four particular genes. These four genes encode enzymes that remove 'reactive oxygen species' from cells. Wang et al.'s findings have revealed an important role for Wnt signaling in maintaining the differentiation niche. The next step is to figure out the details of how this works.
thereby promoting germ cell differentiation . Similarly, Piwi functions in ISCs likely as a chromatin regulator to control germ cell differentiation partly by repressing dpp expression (Jin et al., 2013;Ma et al., 2014). dBre1 (a E3 ubiquitin ligase) and dSet1 (a H3K4 trimethylase) together control H3K4 trimethylation in ISCs and promote germ cell differentiation partly by limiting BMP signaling from the differentiation niche (Xuan et al., 2013). The potential chromatin factor without children (Woc) maintains the ISC-germ cell physical interaction via regulation of Stat-Zfh1 (Maimon et al., 2014). The histone demethylase Lsd1 is required in ISCs to promote germ cell differentiation by maintaining ISC survival, maintaining ISC morphology, and preventing BMP signaling from the differentiation niche (Eliazer et al., 2011(Eliazer et al., , 2014. Therefore, ISCs function as the differentiation niche by preventing BMP signaling and direct communication. A recent study showed that tyrosine kinase Btk29A maintains Wnt signaling in ISCs by phosphorylating Drosophila β-catenin homolog Armadillo (Arm) (Hamada-Kawaguchi et al., 2014).  It also argues that Wnt4 activates Wnt signaling to maintain Piwi expression and repress E-cadherin expression in ISCs, thereby promoting germ cell differentiation. In contrast, this study has demonstrated that both ISC-expressing Wnt2 and Wnt4, but not Wnt4 alone, act through known Wnt pathway components to maintain active Wnt signaling, promoting germ cell differentiation. More importantly, we show that Wnt signaling is required to maintain ISCs by promoting ISC survival and proliferation. Surprisingly, Wnt signaling is dispensable for Piwi expression. Instead, we demonstrate that Wnt signaling controls the expression of four Gst genes to maintain the reduced redox, thereby promoting ISC maintenance and germ cell differentiation. Finally, knocking down one of the Gst gene, GstD2, in ISCs leads to the germ cell differentiation defect. Therefore, our study has revealed a novel mechanism which autocrine Wnt signaling utilizes to maintain ISCs and promote germ cell differentiation.

Results
Canonical Wnt signaling is required in the differentiation niche to promote germ cell differentiation To identify the genes that function in ISCs to promote germ cell differentiation, we used c587-driven UAS-RNAi expression to knockdown individual genes in ISCs. c587 is a GAL4 line that is specifically expressed in ISCs and early follicle cell progenitors based on UAS-GFP expression (Song et al., 2004) ( Figure 1B). To facilitate the identification of GSCs and differentiated germ cells, spectrosomes and fusomes are labeled by Hts staining (Lin et al., 1994), and germ cells are visualized by Vasa staining (Lasko and Ashburner, 1988). In contrast to the control germarium containing 0 to 2 CBs, knocking down Wnt downstream genes armadillo (arm) and disheveled (dsh) in ISCs leads to an accumulation of many spectrosome-containing CBs (collectively referred to single germ cells at least one cell diameter away from cap cells) ( Figure 1C,D). Based on the fact that control germaria rarely contain three CBs, the germaria containing four or more CBs are considered to exhibit a germ cell differentiation defect. Over 90% of arm and dsh knockdown germaria (armKD1 and dshKD1) exhibit the germ cell differentiation defect ( Figure 1E). Similarly, c587-driven expression of the RNAi lines against different arm or dsh sequences (armKD2 and dshKD2) also generates the similar germ cell differentiation defect ( Figure 1E). In Drosophila, Wnt ligands bind the receptor complex composed of Frizzled (Fz), Frizzled 2 (Fz2) and Arrow to activate Dsh and stabilize Arm, which forms a protein complex with a TCF (T Cell Factor)-like Pangolin in the nucleus to activate target gene expression, whereas Axin (Axn) and Shaggy (Sgg) negatively modulate Wnt signaling by promoting Arm degradation (Logan and Nusse, 2004). Simultaneous knockdown down of both fz and fz2 (fzKD+fz2KD) in ISCs recapitulate the germ cell differentiation defect caused by either armKD or dshKD, although either fzKD or fz2KD yields a much weaker germ cell differentiation defect ( Figure  Consistently, overexpression of axn and sgg in ISCs also leads to the germ cell differentiation defect similar to that caused by armKD and dshKD ( Figure 1E,G,H). These results indicate that Wnt signaling is required in ISCs to promote germ cell differentiation.
The expression of a constitutively active mutant arm S10 (arm*) leads to hyperactive Wnt signaling independently of Wnt ligands (Pai et al., 1997). c587-driven arm* expression alone does not cause any obvious GSC maintenance and germ cell differentiation defects, but results in severe egg chamber budding defects likely due to defective follicle cell development ( Figure 1I). Interestingly, c587-mediated arm* expression can fully rescue the germ cell differentiation defect caused by the two independent dshRNAi knockdowns, indicating that Arm functions downstream of Dsh in ISCs to promote germ cell differentiation ( Figure 1J-L). These results further suggest that the two dshRNAi lines are highly specific. Taken together, our findings demonstrate that canonical Wnt signaling works in ISCs to promote GSC lineage differentiation.

Wnt signaling is required in adult ISCs to promote GSC lineage differentiation
The c587-gal4 driver is known to be expressed in somatic precursor cells in the developing female gonad, which give rise to terminal filament, cap cells, adult ISCs, and follicular stem cells (Zhu and Xie, 2003). To determine if adult ISCs require active Wnt signaling for GSC lineage differentiation, we took advantage of c587-deriven expression of a temperature-sensitive gal4 repressor gal80 (c587>>UAS-gal80 ts ) to allow the expression of UAS-RNAi lines against dsh and arm only in adult ISCs using temperature shift. At 25˚C, gal80 ts is functional to prevent gal4-driven expression of a UAS transgene, but at 29˚C, gal80 ts is inactivated to allow gal4-driven gene expression (McGuire et al., 2003). After the c587>>gal80 ts control and c587>>gal80ts UAS-dshRNAi or UAS-armRNAi females eclosed at 25˚C (RNAi expression is extremely low or not expressed), they either continued to be kept at 25˚C for one week (keeping RNAi expression repressed) or were moved to 29˚C for one week (turning on the RNAi expression due to gal80 inactivation). For the c587>>gal80 ts control females, which were cultured at 25˚C or 29˚C as adults for one week, their germaria contain the normal number of CBs (Figure 2A,B). Interestingly, for the c587>>gal80 ts UAS-dshRNAi or UAS-armRNAi females, which were cultured at constant 25˚C, their germaria contain the normal or close to normal numbers of CBs ( Figure 2B-F). In contrast, for the c587>>gal80 ts UAS-dshRNAi or UAS-armRNAi females, which were shifted from 25˚C to 29˚C for one week, their germaria contain excess CBs, indicative of the germ cell differentiation defect ( Figure 2B,C′-F′). These results demonstrate that Wnt signaling is required in adult ISCs to promote germ cell differentiation.

Wnt signaling maintains ISCs by promoting proliferation and survival
Our previous studies have shown that a severe ISC loss also causes the similar germ cell differentiation defect (Kirilly et al., 2011;Wang et al., 2011). Then, we determined if Wnt signaling controls ISC maintenance by using the PZ1444 reporter to quantify ISC numbers in control and Wnt signalingdefective germaria. PZ1444 expresses nuclear LacZ in ISCs and cap cells, which can be distinguished based on nucleus size and location (Xie and Spradling, 2000). In the one-week-old control germaria, PZ1444 labels 30-35 ISCs in addition to cap cells ( Figure 3A,C). In the one-week-old arm and dsh knockdown germaria, only fewer than 5 ISCs remain ( Figure 3B-D). Some knockdown germaria have completely lost ISCs ( Figure 3B), whereas others retain one or a few ISCs ( Figure 3D). Consistently, overexpression of axn and sgg also leads to a severe ISC loss ( Figure 3E,F). These results demonstrate that Wnt signaling is required to maintain ISCs.
Wnt signaling maintains ISCs possibly by promoting cell proliferation, survival, or both. Interestingly, arm*-expressing germaria contain significantly more ISCs ( Figure 3G). In contrast with the one-week-old control germaria containing 32 ISCs, the one-week-old arm*-expressing germaria carry 130 ISCs ( Figure 3C). Consistently, c587-mediated axn and sgg knockdown, which increases Wnt-signaling activity (Heslip et al., 1997;Willert et al., 1999), also leads to more ISCs ( To further investigate how Wnt signaling maintains the ISC population, we examined ISC proliferation and apoptosis in the control, armKD, dshKD, and arm*-expressing germaria using BrdU incorporation and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling)-labeling assays, respectively. In order to avoid severe ISC loss in these experiments, we purposely cultured the control, armKD, dshKD, and arm*-expressing females at 29˚C for shorter time than earlier experiments. BrdU incorporation labels replicating ISCs in the S-phase of the cell cycle, whereas TUNEL labeling detects fragmented DNA in dying cells. Based on BrdU-labeling results, defective Wnt signaling significantly decreases ISC proliferation, whereas hyperactive Wnt signaling significantly increases ISC proliferation ( Figure 3H-L). Based on TUNEL-labeling results, defective Wnt signaling significantly increases ISC apoptosis, whereas hyperactive Wnt signaling significantly decreases ISC apoptosis ( Figure 3M-Q). These results suggest that Wnt signaling maintains the ISC population by promoting proliferation and increasing survival.
Wnt signaling is required in ISCs to prevent BMP signaling in differentiated GSC progeny and maintain long ISC cellular processes Our previous studies have also shown that ISC loss disrupts germ cell differentiation by increasing BMP signaling (Kirilly et al., 2011;Wang et al., 2011;Ma et al., 2014). In the Drosophila germarium, BMP signaling leads to production of phosphorylated Mad (pMad) and activation of Dad-lacZ reporter expression in GSCs (Chen and McKearin, 2003;Kai and Spradling, 2003;Casanueva and Ferguson, 2004;Song et al., 2004). In the control germaria, pMad and Dad-lacZ expression is restricted to GSCs ( Figure 4A,A′,D,D′). In the armKD and dshKD germaria, pMad and Dad-lacZ expression is activated in the accumulated CBs locating one to a few cells away from cap cells in addition to GSCs ( Figure 4B,B′,C,C′,E,E′,F,F′). These results indicate that BMP signaling is spread to the differentiation zone in the Wnt signaling-defective germarium.
To investigate how Wnt signaling regulates germ cell differentiation at the molecular level, we compared the gene expression changes in fluorescence-activated cell sorting (FACS)-purified GFP-labeled control, Wnt signaling-defective dsh knockdown, and axn-overexpressing (AxnOE) ISCs using deep RNA sequencing (RNA-seq). Our RNA-seq results show that known Drosophila BMP-signaling components and regulators, including dally and dpp, which are often upregulated in defective ISCs (Liu et al., 2010;Kirilly et al., 2011;Wang et al., 2011;Jin et al., 2013;Ma et al., 2014), remain unchanged in the dshKD and AxnOE ISCs in comparison with the control ISCs ( Figure 4G). To further determine if upregulated BMP signaling is responsible for the germ cell differentiation defect resulted from defective Wnt signaling, we determined the effect of heterozygous dpp mutations on the differentiation defect caused by dshKD because they have been shown to suppress the differentiation defect caused by BMP-signaling upregulation (Kirilly et al., 2011;Wang et al., 2011). Our results indicate that the dpp heterozygous mutation dpp hr4 significantly rescues the germ cell differentiation defect caused by dshKD1 and dshKD2, whereas the dpp heterozygous mutation dpp hr56 significantly rescues that caused by dshKD1 but not dshKD2 ( Figure 4H). This rescue effect by the dpp heterozygous mutations is unlikely caused by decreasing GSCs (Figure 4H′). These results suggest that Wnt signaling in ISCs regulates germ cell differentiation partly by preventing BMP signaling in the differentiation niche.
Long ISC cellular processes are also required to promote germ cell differentiation (Kirilly et al., 2011). They can be easily visualized by c587-driven expression of membrane-tethered GFP, CD8GFP. In the control germaria, ISC cellular processes wrap up differentiated germ cells ( Figure 4I,I′). In contrast, cellular processes in the remaining ISCs of the armKD and dshKD germaria fail to extend into the accumulated CBs ( Figure 4J-K′). These results indicate that Wnt signaling is required to maintain ISC cellular processes.

Wnt signaling is dispensable for maintaining Piwi protein expression in ISCs
A recent study proposes that Wnt signaling upregulates piwi expression in ISCs, thereby promoting germ cell differentiation (Hamada-Kawaguchi et al., 2014). To verify if Piwi protein is indeed downregulated in Wnt signaling-defective ISCs, we quantified the expression of Piwi protein in the The control germarium shows that GSCs are positive for Dad-lacZ expression, but one CB is negative. (E-F′) In the armKD (E, E′) and dshKD (F, F′) germaria, GSCs are Dad-lacZ-positive as in the control. Some of the accumulated CBs are negative for Dad-lacZ, but the other ones exhibit low Dad-lacZ expression (arrows). (G) RNA sequencing (RNA-seq) results on the purified ISCs show that mRNA levels for the known BMP pathway components, including dpp, gbb (also encoding a BMP ligand) and dally, remain largely unchanged in axn-overexpressing ISCs or dskKD ISCs in comparison with the control (FKPM = reads per kilobase per million mapped reads). (H, H′) Our CB and GSC quantification results indicate that the heterozygous dpp mutations have partial suppression on the germ cell differentiation defects caused by dshKD, and the suppression is not due to the changes in GSC numbers (50 germaria examined for each genotype). (I-K′) armKD (J, J′) and dshKD (K, K′) ISCs lack their CD8GFP-positive cellular processes extending into the accumulated CBs (arrowheads) in contrast with the control ISCs extending the cellular processes wrapping up underneath germ cells (arrowheads, I′). (I′-K′) A higher magnification of highlighted areas in (I-K). DOI: 10.7554/eLife.08174.008 PZ1444-labeled dshKD or armKD ISCs. Piwi protein shows comparable expression levels among the examined 203 control ISCs, 48 armKD1 ISCs, 58 armKD2 ISCs, and 46 dshKD2 ISCs ( Figure 5A-D). Surprisingly, Piwi protein levels are significantly elevated in the dshKD1 ISCs (93 examined) in comparison with the control ISCs ( Figure 5D). In addition, we also used FACS to purify the GFPlabeled control ISCs, axnOE ISCs, and dshKD1 ISCs for RNA-seq. The RNA-seq results show that the piwi mRNA levels are slightly upregulated in the axnOE ISCs and are significantly upregulated in the dshKD ISCs in comparison to the control ISCs ( Figure 5E). Therefore, our results indicate that Wnt signaling promotes germ cell differentiation unlikely by maintaining Piwi expression in ISCs. However, Wnt signaling possibly works with Piwi in an unknown means to promote germ cell differentiation because the loss of their functions in ISCs leads to similar germ cell differentiation defects (Jin et al., 2013;Hamada-Kawaguchi et al., 2014;Ma et al., 2014).

Wnt signaling maintains the cellular redox state in ISCs, GSCs, and early differentiated germ cells by maintaining glutathione-S-transferase (Gst) gene expression
Our RNA-seq results also show that four Gst genes, GstD2, GstD4, GstD10, and GstE3, decrease their mRNA expression levels significantly in both dshKDand axn-overexpressing ISCs in comparison with the control ( Figure 6A). GST proteins comprise a family of eukaryotic metabolic enzymes for eliminating hydrogen peroxide (H 2 O 2 ) and catalyzing the conjugation of the reduced form of glutathione (GSH) to oxidized substrates for the purpose of detoxification. Thus, downregulated expression of the Gst genes in the Wnt signaling-defective ISCs might cause the increase in cellular reactive oxygen species (ROS), which can be detected by dihydroethidium (DHE) fluorescence. In the  anterior half of control germaria, ISCs and underneath differentiating germ cells show low DHE fluorescence, indicating that these cells have low cellular ROS levels, including ISCs ( Figure 6B; Figure 6-figure supplement 1A,B′). In contrast, dshKD and armKD ISCs elevate DHE fluorescence, and germ cells underneath also increase DHE fluorescence, further supporting the idea that defective Wnt signaling results in the increased cellular ROS in ISCs and their interacting germ cells ( Figure 6B′; Figure 6-figure supplement 1C-G). As a distinct GST family member from the GST-D family members, GST2 has similar function in regulating the cellular redox state (Singh et al., 2001). Consistently, GST2 overexpression in the dshKD ISCs can also restore low DHE fluorescence in the anterior half of the germaria, suggesting that Wnt signaling controls the cellular redox state in ISCs, GSCs, and early GSC progeny by regulating Gst gene expression ( Figure 6B″; Figure  . Taken together, these results demonstrate that Wnt signaling in ISCs maintains the reduced redox state, which is partly responsible for ISC maintenance and germ cell differentiation. GstD2 works with Cat in ISCs to control germ cell differentiation Our RNA-seq results indicate that GstD2 is the most abundantly expressed Gst genes in ISCs. In addition, it is also the most severely downregulated Gst gene in the Wnt signaling-defective ISCs, which prompted us to further investigate its function in promoting germ cell differentiation ( Figure 6A). We generated two independent RNAi lines against GstD2, among which Line 1 (GstD2KD1) efficiently knocks down GstD2 mRNA but Line 2 (GstD2KD2) does not based on quantitative RT-PCR results on the purified ISCs ( Figure 6K). In contrast with the control germaria containing one CB on average, the GstD2KD1 germaria contain two CBs, which are significantly more than the control ( Figure 6L,O). As expected, the GstD2KD2 germaria behave like the control, containing one CB on average ( Figure 6O). Interestingly, c587-mediated expression of Gst2 and Cat can also rescue the moderate germ cell differentiation defect caused by GstD2KD1, indicating that the differentiation defect is caused by GstD2 knockdown in ISCs ( Figure 6O). Considering the fact that additional three Gst genes are downregulated in the Wnt signaling-defective ISCs, these results indicate that Gst genes are required in ISCs to promote germ cell differentiation.
Both Cat and Gst genes work together to remove cellular hydrogen peroxide (H 2 O 2 ). The c587mediated Cat knockdown (CatKD) germaria behave like the control germaria, containing one CB on average ( Figure 6M,O). Interestingly, c587-mediated CatKD significantly enhances the germ cell differentiation defect caused by GstD2KD1, and consequently the double knockdown germaria accumulate significantly more CBs than the single knockdown germaria ( Figure 6N,O). In addition, the CatKD germaria carry a normal number of ISCs, whereas the GstD2KD germaria carry slightly fewer ISCs ( Figure 6P,Q,S). Consistently, the GstD2KD1 CatKD germaria carry significantly fewer ISCs than the GstD1KD1 or CatKD germaria ( Figure 6R,S). These results indicate that Cat and Gst genes work together in ISCs to maintain ISCs and promote germ cell differentiation, and further suggest that redox control is important for ISC maintenance.

Autocrine Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in ISCs
Our RNA-seq results indicate that four Wnt-like genes are expressed in the purified ISCs, but wingless shows little expression ( Figure 7A). Wnt2 and Wnt4 are expressed at high levels, whereas Wnt5 and Wnt6 are present in much lower levels ( Figure 7A). A recent study has shown that wnt4 mRNAs are indeed restricted to ISCs, whereas wnt2 mRNAs are also present in ISCs (Luo et al., 2015). Knocking down Wnt2 or Wnt4 alone in ISCs by two or three independent RNAi lines results in no or slight germ cell differentiation defect based on CB numbers ( Figure 7B-D). This is in contrast with the recent study claiming that Wnt4 alone in ISCs is responsible for germ cell differentiation (Hamada-Kawaguchi et al., 2014). Consistent with the idea that Wnt2 and Wnt4 function redundantly in ISCs, simultaneous knockdown of Wnt2 and Wnt4 via different combinations of RNAi lines leads to a severe germ cell differentiation defect, which is similar to that caused by dshKD or armKD ( Figure 7D-H). These results demonstrate that Wnt2 and Wnt4 in ISCs serve as redundant autocrine signals for promoting germ cell differentiation.

Discussion
Although the differentiation niche is critical for promoting GSC progeny differentiation, little is known about its regulation. Here, we have identified Wnt2 and Wnt4 as autocrine signals to maintain the GSC differentiation niche partly through redox regulation ( Figure 7I). First, Wnt signaling is required in ISCs for their maintenance by promoting cell proliferation and survival. Defective Wnt signaling causes a severe ISC loss, thereby preventing germ cell differentiation. Second, Wnt signaling is required to maintain the reduced redox state by sustaining the expression of Gst genes. This represents a novel connection between Wnt signaling and redox control. In addition, this study has also revealed that the reduced redox state is critical for ISC survival and thus for promoting germ cell differentiation. Third, Wnt signaling in ECs promotes GSC progeny differentiation partly by repressing BMP signaling in differentiated GSC progeny. Fourth, Wnt2 and Wnt4 represent redundant autocrine signals for maintaining Wnt signaling in the germ cell differentiation niche. Wnt signaling has been shown to control stem cell self-renewal directly (Holland et al., 2013), whereas ROS has shown to prime hematopoietic progenitor differentiation in Drosophila (Owusu-Ansah and Banerjee, 2009). This study has demonstrated the importance of Wnt signaling in maintaining the GSC differentiation niche by reducing ROS and thus promoting GSC lineage differentiation. Therefore, this study not only has identified critical signals for maintaining the GSC differentiation niche but also has revealed a novel function of Wnt signaling in the regulation of cellular redox.
This study has demonstrated that autocrine Wnt signaling controls ISC maintenance, thereby promoting germ cell differentiation. First, canonical Wnt signaling is required in ISCs to promote germ cell differentiation. c587-mediated knockdown of Wnt signal transducers Fz/fz2, dsh, and arm as well as c587-directed overexpression of Wnt signaling inhibitors sgg and axn leads to similar germ cell differentiation defects. Moreover, c587-directed overexpression of a constitutively active form of arm* can fully rescue the germ cell differentiation defect caused by dsh knockdown. Second, Wnt signaling maintains ISCs by promoting proliferation and survival. c587-mediated knockdown of dsh and arm leads to a severe ISC loss, whereas c587-mediated knockdown of sgg and axn or c587-directed arm* overexpression expands the ISC population. In addition, hyperactive Wnt signaling increases ISC proliferation and decreases apoptosis, whereas Wnt signaling downregulation increases ISC apoptosis and decreases proliferation. Thus, canonical Wnt signaling maintains the differentiation niche by promoting ISC proliferation and survival. However, we could not completely rule out the possibility that defective Wnt signaling leads to the loss of adult ISCs due to early developmental defects. Third, ISC-expressing Wnt2 and Wnt4 function redundantly in the differentiation niche to promote germ cell differentiation. Our RNA-seq results show that wnt2 and wnt4 mRNAs are present in the purified ISCs at high levels, while other wnt genes are expressed at much lower levels. c587-mediated wnt2 and wnt4 double knockdown results in more severe germ cell differentiation defects than wnt2 or wnt4 single knockdown.
Piwi has recently been shown to be required in the differentiation niche for promoting germ cell differentiation partly by repressing dpp expression (Jin et al., 2013;Ma et al., 2014). Although a recent study proposes that Wnt signaling controls germ cell differentiation by regulating piwi expression (Hamada-Kawaguchi et al., 2014), this study shows that Piwi protein and mRNAs are not downregulated in Wnt signaling-defective ISCs, suggesting that Wnt signaling does not sustain piwi expression in the differentiation niche to promote GSC progeny differentiation. Instead, this study has further revealed that Wnt signaling maintains the differentiation niche by controlling the cellular redox. First, our RNA-seq results show that GstD2, GstD4, GstD10, and GstE3 mRNA expression levels are significantly downregulated in the purified Wnt signaling-defective ISCs in comparison with the control ISCs. Second, defective Wnt signaling in ISCs elevates ROS levels in themselves and underneath germ cells, indicating that Wnt signaling is required in the differentiation niche to maintain low ROS levels. It remains unclear if increased ROS levels in early germ cells contribute to their differentiation defects. Third, c587-directed GST2, SOD1, and CAT overexpression can dramatically suppress the ROS elevation, the germ cell differentiation retardation, and the ISC loss caused by defective Wnt signaling, indicating that ROS elevation is responsible for the germ cell differentiation defect and the ISC loss. Finally, c587-mediated knockdown of GstD2 and Cat results in the ISC loss and the germ cell differentiation defect, indicating that ROS elevation in ISCs is sufficient to cause ISC loss and retard germ cell differentiation. This study has, for the first time, demonstrated that autocrine Wnt signaling controls cellular redox state in the differentiation niche and thus promotes germ cell differentiation.
So far, various studies have revealed a number of important signaling pathways and factors in ISCs to promote germ cell differentiation by maintaining ISC cellular process-mediated germ cell-soma interaction and preventing BMP signaling (Xie, 2013). This study shows that Wnt signaling is required for preventing BMP signaling in differentiated germ cells and for maintaining long ISC cellular processes. Rho, Eggless, Woc, Lsd1, Piwi, EGFR signaling, and ecdysone signaling have been shown to be required in ISCs to maintain long ISC cellular processes encasing germ cells (Schultz et al., 2002;Kirilly et al., 2011;Wang et al., 2011;Eliazer et al., 2014;Ma et al., 2014;Maimon et al., 2014;Konig and Shcherbata, 2015). Since properly differentiated germ cells are also required to maintain long ISC cellular processes (Kirilly et al., 2011), it is difficult to distinguish the cause and effect of the germ cell differentiation defect and the ISC cellular process loss. In contrast, three known strategies operate in the differentiation niche to prevent BMP signaling, thereby producing a permissive environment for germ cell differentiation. First, Lsd1, Rho, and Piwi are required in ISCs to repress dpp mRNA expression, thereby directly preventing BMP signaling in the differentiation niche (Eliazer et al., 2011;Kirilly et al., 2011;Jin et al., 2013;Ma et al., 2014). dpp encodes a BMP ligand, which activates BMP signaling important for GSC self-renewal (Xie and Spradling, 1998). Second, Rho, Eggless, and EGFR signaling function in ISCs to repress the expression of dally, preventing BMP diffusion from the self-renewal niche to the differentiation niche (Liu et al., 2010;Kirilly et al., 2011;Wang et al., 2011). Dally, which is a proteoglycan protein facilitating BMP differentiation and signaling, is expressed in cap cells, the GSC self-renewal niche, to restrict BMP signaling to GSCs (Akiyama et al., 2008;Guo and Wang, 2009). Third, a recent study has proposed that cap cell-initiated Wnt signaling maintains the expression of tkv encoding a type I BMP receptor in ISCs, thereby preventing BMP signaling in differentiated germ cells (Luo et al., 2015). Our findings from this study have also supported the idea that autocrine Wnt signaling in ISCs promotes GSC progeny differentiation partly by repressing BMP signaling. However, our findings have also argued that inactivating Wnt signaling does not regulate the mRNA expression levels of tkv and other BMP pathway components in ISCs. Therefore, future research will be needed to investigate the molecular mechanisms for Wnt signaling in the differentiation niche to prevent BMP signaling and maintain long ISC cellular processes.
The GFP-positive dshKD or axn-overexpressing ISCs were sorted by FACS, and mRNA isolation was carried out according to the published procedure (Ma et al., 2014). Following manufacturer's directions and using the Illumina TruSeq Stranded mRNA LT Kit (Illumina, MA, United States; Cat. No. RS-122-2101/2), short fragment libraries were constructed. The resulting libraries were purified using Agencourt AMPure XP system (Backman Coulter, CA, United States; Cat. No. A63880) and were then quantified using a LabChipÒ GX (PerkinElmer, MA, United States) and a Qubit Fluorometer (Life Technologies, CA, United States). All libraries were pooled, re-quantified, and run as 50 bp single-end lanes on an Illumina HiSeq 2500 instrument, using HiSeq Control Software 2.0.5 and Real-Time Analysis (RTA) version 1.17.20.0. Secondary Analysis version CASAVA-1.8.2 was run to demultiplex reads and generate FASTQ files.