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.

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 self-renewal 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 and Spradling, 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.

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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, thereby promoting germ cell differentiation (Wang et al., 2011). 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, 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.

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 Drosophila stocks used in this study include: c587 (Kirilly et al., 2011), PZ1444 (Kirilly et al., 2011), UAS-SOD1 (Pan et al., 2007), UAS-axn (BL7225), UAS-sgg (BL6818), armRNAi (BL31304; BL31305), dshRNAi (BL31306; BL31307), axnRNAi (BL31705), sggRNAi (BL35364), Wnt2RNAi (BL36722; TH00483; TH00484), and Wnt4RNAi (BL29442; TH00485). TH lines for Wnt2 and Wnt4 were constructed for this study according to the published procedure (Ni et al., 2011); the coding region of Gst2 and Cat were cloned into a UASp vector to generate UAS-Gst2 and UAS-Cat using standard molecular biology techniques. Drosophila strains were maintained and crossed at room temperature on standard cornmeal/molasses/agar media unless specified. To maximize the RNAi-mediated knockdown effect, newly eclosed flies were cultured at 29°C for a week before the analysis of ovarian phenotypes.

BrdU incorporation, TUNEL labeling, and DHE staining were performed according to the published procedures (Xie and Spradling, 1998; Owusu-Ansah and Banerjee, 2009; Wang et al., 2011). Immunohistochemistry was performed according to our previously published procedures (Xie and Spradling, 1998). The following antibodies were used in this study: rabbit polyclonal anti-β-galactosidase antibody (1:100, Cappel), mouse monoclonal anti-Hts antibody (1:50, DSHB), Guinea pig polyclonal anti-Piwi antibodies (1:100; provided by H. Lin), rabbit monoclonal anti-pS423/425 Smad3 antibody (1:100, Epitomics), and rat monoclonal anti-Vasa antibody (1:50, DSHB). All images were taken with a Leica TCS SP5 confocal microscope.

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 LabChip® 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.

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Thank you for submitting your work entitled “Wnt Signaling-Mediated Redox Regulation Maintains the Germline Stem Cell Differentiation Niche” for peer review at eLife. Your submission has been favorably evaluated by Fiona Watt (Senior Editor), a Reviewing Editor, and two reviewers.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

In this manuscript, Wang et al. address the role of somatic inner germarial sheath cells (ISCs) in promoting differentiation of germline cells displaced from the stem cell niche. Using a battery of genetic tools and molecular readouts, the Wnt signaling pathway is identified as a critical regulator of germ cell differentiation that acts, at least in part, autonomously within the ISCs. Clear evidence supports the conclusion that autonomous Wnt signaling is important for the viability of ISCs and that elevated ROS in Wnt signaling mutants contributes to the reduced viability of these cells. Conditions that lead to reduced ISC number led to accumulation of less differentiated germ cells, suggesting that Wnt-mediated maintenance of ISCs is important for controlling germ cell differentiation. The work adds a new mechanism to a developing field of study focused on understanding how individual signaling pathways that mediate interactions between ISC cells and germ cells influence germ cell biology.

Essential revisions:

The reviewers are unanimous in their appraisal of the manuscript and agree that if all the conclusions in the manuscript are properly supported by experiments, then this work will generate a really interesting new model for the signaling that controls somatic cell-germ cell interactions during germ cell differentiation. There are aspects of the analysis that are not conclusive, and these need to be fully addressed in order for a resubmission to be considered for publication.

The two central messages in this paper are:

1) Wnt2 and Wnt4 work redundantly and Wnt signaling in the ISC is somehow linked in a non-autonomous fashion to an IGS to germ cell signal, that contrary to previous reports is not piwi dependent.

2) The Wnt signal controls ROS signaling in the ISC cells and this is what provides the ISC to germ cell connection.

Of these two messages, the first one is a high-point of the analysis and we suggest some changes that will make the manuscript better. However, the equally important point 2 is not convincingly demonstrated. It is critical that these are addressed.

Point 1:

a) The role of Wnt4 in the process was previously known, but the redundancy with Wnt2 was not. On this point, rather than place the emphasis on what another laboratory “got wrong”, the authors could seize this opportunity to explain the general problem with redundancy and how this manuscript managed to avoid those pitfalls.

b) One assumes that the p-value resulting from loss of Wnt4 alone (using D1), in Figure 7D is not correct, certainly looks that way from the graph, but if somehow this p value is in the e-9 range (rather that about 0.1 or so), then we have a big problem with the redundancy argument.

c) The reviewers feel that the dismissal of the published piwi data needs some substantiation. While the authors' assurance that this is due to the use of whole ovaries not accounting for ISC loss seems reasonable, the authors did not discuss an alternative signal, and they need to address this issue more directly.

Genetic experiments demonstrating that piwi expression in ISCs (or possibly terminal filament and cap cells given the broad expression of the Gal4 driver utilized) can rescue Btk29 mutant defects in germ cell differentiation clearly indicate an important role for piwi in this process (Hamada-Kawaguchi). An explanation for the ability of piwi expression to rescue the effects of Btk29 mutation on germ cell differentiation and Bam expression should be considered.

In addition, the possibility that loss of interactions between ISC projections and differentiated germ cells (described in Kirilly, et al. 2011) upon dissociation of the germarium tissue for FACS sorting might alter the in vivo effects of genetic mutations on piwi expression should be carefully considered.

Finally, the armKD image shown in Figure 5B and B' appears to have reduced piwi expression in many of the ISC cells, and the methods for quantifying Piwi protein levels are not clear. Rigorous exclusion of piwi as relevant for Wnt-mediated germ cell differentiation regulation would require an experiment in which piwi is expressed in ISCs lacking arm or dsh and assessing its ability to rescue the defects observed. The main point is that genetic evidence supports roles within ISCs for all of the above genes, and the manuscript would be enhanced by the proposal of an integrated model that considers the individual roles of each pathway in this process and how these coordinate to control germ cell differentiation.

In this context, the authors should comment on how the entire network of genes such Btk29, EGFR, Rho, Piwi, and BMP that are key factors for controlling the ISC-germ cell interactions that promote germ cell differentiation, integrates into the Wnt model. This can be addressed by textual changes only if the authors think this will enhance the paper.

Some of the Gal4 drivers used in this study are not cell type specific. It will be important to use Cap and Follicle cell drivers to confirm that the Wnt effects as stated are indeed specific to the ISC. This is an important caveat to address.

Point 2:

The ROS connection to Wnt signaling needs to be addressed with better data.

a) It is not clear, from the DHE staining shown, that knockdown of dsh affects ROS levels in ISCs. For example, the difference in DHE staining in 6B vs 6B' could be because the ISC cells are absent in a dshKD context (as suggested elsewhere in the paper). So this does not definitively establish that loss of Wnt signaling affects ROS levels in ISC cells.

b) The RNAseq data are supportive and correlative, but on its own, it is not enough to establish causality. To test whether the decrease in scavengers is the cause of the dshKD phenotype, it would be important to determine whether knockdown of scavengers also causes a loss of ISCs, as knockdown of dsh does.

c) The proliferation and apoptosis phenotypes described in Figure 3 may be due to effects during development, which would call into question whether Wnt promotes survival and proliferation during adulthood.

Here are some suggestions from the reviewers, but if the authors think of better experiments to substitute for these that will convince the reviewers, then this will be OK:

i) Controls with scavenger knockdown alone to show the DHE staining works as expected, and counter staining (e.g. LacZ in PZ1444) to identify IGS cells and show at cellular resolution the level of DHE staining in wt vs dshKD conditions. Since it seems all the ISC cells are gone by 7 days after heat shift, it may be necessary to either look earlier, before all the ISC cells have disappeared, or prevent apoptosis by some other means, such as overexpression of p35. If they find that DHE staining is actually higher in dshKD ISC cells vs wt, this would go a long way toward addressing the reviewers' concerns.

ii) Quantification of ISC number in GSTD2 + Cat double KD will determine if the ISC number changes or remains the same. The result needs to fit the model.

iii) To determine whether the proliferation and apoptosis phenotypes described in Figure 3 are due to effects during development, the authors should use tsGal80 to suppress expression until adulthood, as in Figure 2, and perform TUNEL staining of dshKD ISC cells as they did for arm*.

iv) Does germ-cell lethality revert when ROS scavengers are expressed in them?

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled “Wnt Signaling-Mediated Redox Regulation Maintains the Germline Stem Cell Differentiation Niche” for further consideration at eLife. Your revised article has been favorably evaluated by Fiona Watt (Senior Editor), a Reviewing Editor, and two reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) An integrated model of how Wnt signaling and BMP signaling might work together is confusing, more so in this version of the manuscript than the last. pMAD and Dad-lacZ are dramatically elevated in undifferentiated germ cells in germaria with reduced dsh or arm activity (Figure 4), but the final conclusion is that BMP signaling is not affected by altered Wnt because no changes in transcript levels of other dpp effectors (including dad?) were detected and because a 50% reduction of dpp ligand did not alter the observed phenotypes, except in the case of the dshKD1 background, where it appears to have rescued the phenotype quite dramatically. Given these data, the conclusion about independence of the pathways is not justified and needs to be tempered and explained better.

2) ROS induced in the germ cells acts downstream of the IGS cell Wnt signal to inhibit differentiation. Is there a rescue of normal differentiation by reducing ROS in germ cells when IGS cells still have reduced Wnt signaling? (A definitive comment on the link of Wnt signaling, ROS and differentiation will make this paper very exciting. However, if tools to test this conclusion are time consuming to put together, then adding this as a point of discussion will suffice).

3) One of our reviewers expressed concern that Wnt signaling during development might produce defective adult tissues in which proliferation and survival of ISCs is different. This can be easily addressed by repeating the proliferation and apoptosis experiments with arm*, armKD, and dshKD (Figure 3) with Gal80ts. Please pick at least one (or more) genotype to test with Gal80 to demonstrate that this is not the case. If this will involve multi generational crosses and a significant delay in revision, then please bring this up as an important caveat for the whole study.

4) Given the large effect of Wnt4 knockdown, the redundancy argument is not strong. Also, the data should include p values for should provide p-values for (Wnt2 vs Wnt2+4 and Wnt4 vs Wnt2+4.

5) c587 is described in all sections of the paper as "ISC-specific”. This is not true and needs to be changed.

6) The new data on genetic alteration of ROS levels focused on germaria with 2 cystoblasts as indicative of a differentiation phenotype. In the text associated with Figures 1 and 2, it states that most germaria have 1-2 cystoblasts, and only gemaria with more than 4 Hts/spectrosome positive germaria were scored. It is not clear how only 2 cystoblasts can be accurately scored as a "differentiation defect” if most germaria have 1 or 2 cystoblasts present. Most likely, this is due to redundancy among GST genes, but it's hard to know how to compare this data to that presented in Figure 1.

Point 1:

a) The role of Wnt4 in the process was previously known, but the redundancy with Wnt2 was not. On this point, rather than place the emphasis on what another laboratory “got wrong”, the authors could seize this opportunity to explain the general problem with redundancy and how this manuscript managed to avoid those pitfalls.

Thank the reviewers for the comment. In the revised manuscript, we have put the emphasis on the requirement of both Wnt2 and Wnt4 in ISCs for maintaining the differentiation niche and promoting germ cell differentiation.

b) One assumes that the p-value resulting from loss of Wnt4 alone (using D1), in Figure 7D is not correct, certainly looks that way from the graph, but if somehow this p value is in the e-9 range (rather that about 0.1 or so), then we have a big problem with the redundancy argument.

Indeed, knocking down wnt2 or wnt4 by one RNAi line yields significantly more CBs than the controls, indicating that both Wnt2 and Wnt4 are required for sustaining maximal Wnt signaling in ISCs to promote germ cell differentiation. However, simultaneous knockdown of both wnt2 and wnt4 in ISCs produces much severe germ cell differentiation defects, which is more than the additive effect of two single knockdowns. These results strongly argue that Wnt2 and Wnt4 function redundantly in ISCs to promote germ cell differentiation.

c) The reviewers feel that the dismissal of the published piwi data needs some substantiation. While the authors' assurance that this is due to the use of whole ovaries not accounting for ISC loss seems reasonable, the authors did not discuss an alternative signal, and they need to address this issue more directly.

Genetic experiments demonstrating that piwi expression in ISCs (or possibly terminal filament and cap cells given the broad expression of the Gal4 driver utilized) can rescue Btk29 mutant defects in germ cell differentiation clearly indicate an important role for piwi in this process (Hamada-Kawaguchi). An explanation for the ability of piwi expression to rescue the effects of Btk29 mutation on germ cell differentiation and Bam expression should be considered.

This reviewer might have misunderstood our argument. Our results on piwi mRNA and protein have clearly demonstrated that piwi expression is not downregulated in Wnt signaling-defective ISCs as suggested by Hamada-Kawaguchi et al. Others and we have confirmed the important function of Piwi in ISCs to promote germ cell differentiation (Jin et al., 2013; Ma et al. 2014). Even though Piwi overexpression can “bypass” the requirement of Wnt signaling in ISCs in promoting germ cell differentiation, it does not necessarily support the Hamada-Kawaguchi model that Wnt signaling controls the expression of Piwi to promote germ cell differentiation. However, the rescue experimental results do suggest that Piwi might function downstream (though not directly regulated by Wnt signaling) or in parallel with Wnt signaling in ISCs to promote germ cell differentiation. We have re-written the part to avoid any potential confusion.

In addition, the possibility that loss of interactions between ISC projections and differentiated germ cells (described in Kirilly, et al. 2011) upon dissociation of the germarium tissue for FACS sorting might alter the in vivo effects of genetic mutations on piwi expression should be carefully considered.

We thank the reviewers for the interesting thought. The dissociation process is rapid, and the cell-sorting process is carried out at 4^°C, which prevents transcriptional changes. Although we could not completely ruled out the suggested possibility, it is very unlikely because we have shown that Piwi protein levels are similar or upregulated in armKD/dshKD ISCs in INTACT germaria. Based on our results on piwi mRNA and protein, we are confident that defective Wnt signaling does not lead to the downregulated piwi expression in ISCs.

Finally, the armKD image shown in Figure 5B and B' appears to have reduced piwi expression in many of the ISC cells, and the methods for quantifying Piwi protein levels are not clear. Rigorous exclusion of piwi as relevant for Wnt-mediated germ cell differentiation regulation would require an experiment in which piwi is expressed in ISCs lacking arm or dsh and assessing its ability to rescue the defects observed. The main point is that genetic evidence supports roles within ISCs for all of the above genes, and the manuscript would be enhanced by the proposal of an integrated model that considers the individual roles of each pathway in this process and how these coordinate to control germ cell differentiation.

For Piwi protein expression quantification, we used the Leica quantification software to select the nucleus area and measure the total fluorescence intensity. Like any experiments, there are some degrees of variations due to expression variation, staining variation or both. After measuring 203 wild-type ISCs, 106 armKD ISCs and 139 dshKD ISCs, we could not find any evidence supporting that Piwi protein levels are down-regulated in Wnt signaling-defective ISCs. These findings on piwi mRNA and protein levels are mutually supported. In this study, we argue that Piwi is NOT the direct target of Wnt signaling in ISCs. We also believe that Piwi and Wnt signaling are relevant with each other in controlling ISC maintenance and promoting germ cell differentiation because they have similar mutant phenotypes in ISC maintenance and germ cell differentiation.

In this context, the authors should comment on how the entire network of genes such Btk29, EGFR, Rho, Piwi, and BMP that are key factors for controlling the ISC-germ cell interactions that promote germ cell differentiation, integrates into the Wnt model. This can be addressed by textual changes only if the authors think this will enhance the paper.

According to the suggestion, we have done our best to add some of our thoughts on the relationships among the pathways in the Discussion though the connections are largely unknown. Hopefully, these speculations will help stimulate future research in these areas.

Some of the Gal4 drivers used in this study are not cell type specific. It will be important to use Cap and Follicle cell drivers to confirm that the Wnt effects as stated are indeed specific to the ISC. This is an important caveat to address.

As the reviewers pointed out, c587 expresses GAL4 in not only in ISCs but also in early follicle precursor cells. In addition, the existing Gal4 drivers for cap cells, bab1-gal4 and hh-gal4, are also expressed in ISCs at low levels. There is no good GAL4 driver, which is specifically expressed in early follicle progenitors. Therefore, it is very challenging to use various GAL4 drivers to address the caveat. A recently published study by the Yu Cai group shows that wnt4 mRNAs are primarily present in ISCs, and wnt2 mRNAs are also expressed in ISCs. These results are consistent with our RNA-seq and genetic knockdown results.

Point 2:

[…] Here are some suggestions from the reviewers, but if the authors think of better experiments to substitute for these that will convince the reviewers, then this will be OK:

i) Controls with scavenger knockdown alone to show the DHE staining works as expected, and counter staining (e.g. LacZ in PZ1444) to identify IGS cells and show at cellular resolution the level of DHE staining in wt vs dshKD conditions. Since it seems all the ISC cells are gone by 7 days after heat shift, it may be necessary to either look earlier, before all the ISC cells have disappeared, or prevent apoptosis by some other means, such as overexpression of p35. If they find that DHE staining is actually higher in dshKD ISC cells vs wt, this would go a long way toward addressing the reviewers' concerns.

According to the request, we have performed DHE staining in PZ1444-labeled armKD and dshKD germaria before most of the ISCs are lost. Our results confirm that dshKD or armKD ISCs exhibit elevated levels of DHE staining. New results have been added to Figure 6—figure supplement 1B–F′.

ii) Quantification of ISC number in GSTD2 + Cat double KD will determine if the ISC number changes or remains the same. The result needs to fit the model.

According to the request, we have quantified ISC numbers in single and double knockdown germaria for GSTD2 and Cat. Indeed, the ISC number is significantly reduced in GSTD2 Cat double knockdown germaria, which correlates with the germ cell differentiation defect. The new results have been added to Figure 6P-S.

iii) To determine whether the proliferation and apoptosis phenotypes described in Figure 3 are due to effects during development, the authors should use tsGal80 to suppress expression until adulthood, as in Figure 2, and perform TUNEL staining of dshKD ISC cells as they did for arm*.

According to the request, we have performed both BrdU and TUNEL experiments on the dshKD and armKD germaria. Our results show that knocking down dsh or arm indeed leads to significantly decreased BrdU-positive ISCs and significantly increased TUNEL-positive ISCs. Now new results have been provided in Figure 3H–Q.

iv) Does germ-cell lethality revert when ROS scavengers are expressed in them?

When arm and dsh are knocked down in ISCs, the severe germ cell differentiation defect appears, but the GSC loss is rarely observed. When ROS scavengers, including Cat, SOD and GST2, are expressed in ISCs, the germ cell differentiation defect caused by arm and dsh knockdown is drastically rescued, but GSC numbers remain largely unchanged. We have added the new results in Figure 6—figure supplement 2G.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

1) An integrated model of how Wnt signaling and BMP signaling might work together is confusing, more so in this version of the manuscript than the last. pMAD and Dad-lacZ are dramatically elevated in undifferentiated germ cells in germaria with reduced dsh or arm activity (Figure 4), but the final conclusion is that BMP signaling is not affected by altered Wnt because no changes in transcript levels of other dpp effectors (including dad?) were detected and because a 50% reduction of dpp ligand did not alter the observed phenotypes, except in the case of the dshKD1 background, where it appears to have rescued the phenotype quite dramatically. Given these data, the conclusion about independence of the pathways is not justified and needs to be tempered and explained better.

The reviewers must have misread our manuscript. We show that some accumulated CB-like cells are positive for pMad and Dad-lacZ expression, indicative of upregulated BMP signaling activities. Our quantitative results in Figure 4H–H′ show that the heterozygous dpp mutations, dpp^hr4 and dpp^hr56, can partially rescue the germ cell differentiation defect caused by dshKD and dshKD2, indicating “Wnt signaling in ISCs regulates germ cell differentiation partly by preventing BMP signaling in the differentiation niche.” However, we show that Wnt signaling does not regulate mRNA levels for BMP pathway components in ISCs, which might give the reviewers the impression “BMP signaling is not affected by altered Wnt (signaling)”. In the revised manuscript, we have made it clear that Wnt signaling in ISCs is required to prevent BMP signaling, and add the information to the model in Figure 7I.

2) ROS induced in the germ cells acts downstream of the IGS cell Wnt signal to inhibit differentiation. Is there a rescue of normal differentiation by reducing ROS in germ cells when IGS cells still have reduced Wnt signaling? (A definitive comment on the link of Wnt signaling, ROS and differentiation will make this paper very exciting. However, if tools to test this conclusion are time consuming to put together, then adding this as a point of discussion will suffice).

That is a great question. All the existing tools cannot answer the question satisfactorily. We are in the process of generating new transgenic fly strains, allowing us to conditionally remove ROS in germ cells while Wnt signaling are still defective in IGS cells. It will take a long time to generate new transgenic fly strains and bring all the genetic elements together to accomplish this goal. We will leave this question for the future studies. In the Discussion, we have added the sentence “In the future, it will be important to determine if increased ROS levels in early germ cells contribute to their differentiation defects caused by defective Wnt signaling in ISCs”.

3) One of our reviewers expressed concern that Wnt signaling during development might produce defective adult tissues in which proliferation and survival of ISCs is different. This can be easily addressed by repeating the proliferation and apoptosis experiments with arm*, armKD, and dshKD (Figure 3) with Gal80ts. Please pick at least one (or more) genotype to test with Gal80 to demonstrate that this is not the case. If this will involve multi generational crosses and a significant delay in revision, then please bring this up as an important caveat for the whole study.

In order to address this question, we would need at least a few months to bring five transgenes together, which also requires chromosomal recombination. Based on our existing experimental results, this is NOT a big issue. Our experimental results have shown that Wnt signaling is required in adult ISCs to promote germ cell differentiation and maintain themselves. In addition, we have gain-of-function and loss-of-function experiments to demonstrate that Wnt signaling is required to maintain ISCs by regulating proliferation and survival. Since we are not able to provide the experimental results excluding the possibility, we have added the statement “However, we could not completely rule out the possibility that defective Wnt signaling leads to the loss of adult ISCs due to their developmental defects.”

4) Given the large effect of Wnt4 knockdown, the redundancy argument is not strong. Also, the data should include p values for should provide p-values for (Wnt2 vs Wnt2+4 and Wnt4 vs Wnt2+4.

As suggested, we have added the P values for Wnt2 versus Wnt2+4 and Wnt4 versus Wnt2+4 in Figure 7D. Our results have indicated that Wnt2 and 4 double knockdowns have significantly severe germ cell differentiation defects than Wnt2 and Wnt4 single knockdowns, which support the redundant roles of Wnt2 and Wnt4.

5) c587 is described in all sections of the paper as “ISC-specific”. This is not true and needs to be changed.

As suggested, we have modified the “ISC-specific” into “c587-mediated”.

6) The new data on genetic alteration of ROS levels focused on germaria with 2 cystoblasts as indicative of a differentiation phenotype. In the text associated with Figures 1 and 2, it states that most germaria have 1-2 cystoblasts, and only gemaria with more than 4 Hts/spectrosome positive germaria were scored. It is not clear how only 2 cystoblasts can be accurately scored as a “differentiation defect” if most germaria have 1 or 2 cystoblasts present. Most likely, this is due to redundancy among GST genes, but it's hard to know how to compare this data to that presented in Figure 1.

A normal germarium contains 0 to 2 CBs with an average of one CB. Due to the weak differentiation defect of GstD2KD+CatKD, we have quantified the exact CB number to determine if there is any germ cell differentiation defect. Our statistic analysis results allow us to conclude that there is any weak and yet significant differentiation defect. In Figure 4H–H′, dshKD1 and dshKD2 germaria contain 15 and 13 CBs, respectively. Using the germaria carrying 4 or more CBs as the differentiation defect is a more efficient quantification method in Figures 1 and 2.

Author details

1. Su Wang

   1. Stowers Institute for Medical Research, Kansas City, United States
   2. Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, United States

   Contribution

   SW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Yuan Gao

   Center for Life Sciences, College of Life Sciences, School of Medical Sciences, Tsinghua University, Beijing, China

   Contribution

   YG, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Xiaoqing Song

   Stowers Institute for Medical Research, Kansas City, United States

   Contribution

   XS, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

4. Xing Ma

   1. Stowers Institute for Medical Research, Kansas City, United States
   2. Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, United States

   Contribution

   XM, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

5. Xiujuan Zhu

   Stowers Institute for Medical Research, Kansas City, United States

   Contribution

   XZ, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

6. Ying Mao

   Center for Life Sciences, College of Life Sciences, School of Medical Sciences, Tsinghua University, Beijing, China

   Contribution

   YM, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

7. Zhihao Yang

   Center for Life Sciences, College of Life Sciences, School of Medical Sciences, Tsinghua University, Beijing, China

   Contribution

   ZY, Contributed unpublished essential data or reagent

   Competing interests

   The authors declare that no competing interests exist.

8. Jianquan Ni

   Center for Life Sciences, College of Life Sciences, School of Medical Sciences, Tsinghua University, Beijing, China

   Contribution

   JN, Contributed unpublished essential data or reagent

   Competing interests

   The authors declare that no competing interests exist.

9. Hua Li

   Stowers Institute for Medical Research, Kansas City, United States

   Contribution

   HL, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

10. Kathryn E Malanowski

    Stowers Institute for Medical Research, Kansas City, United States

    Contribution

    KEM, Acquisition of data

    Competing interests

    The authors declare that no competing interests exist.

11. Perera Anoja

    Stowers Institute for Medical Research, Kansas City, United States

    Contribution

    PA, Acquisition of data

    Competing interests

    The authors declare that no competing interests exist.

12. Jungeun Park

    Stowers Institute for Medical Research, Kansas City, United States

    Contribution

    JP, Acquisition of data

    Competing interests

    The authors declare that no competing interests exist.

13. Jeff Haug

    Stowers Institute for Medical Research, Kansas City, United States

    Contribution

    JH, Acquisition of data

    Competing interests

    The authors declare that no competing interests exist.

14. Ting Xie

    1. Stowers Institute for Medical Research, Kansas City, United States
    2. Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, United States

    Contribution

    TX, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    tgx@stowers.org

    Competing interests

    The authors declare that no competing interests exist.

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