Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
In Figure 1, it is very difficult to identify where CySCs end and GSCs begin without using a cell surface marker for these different cell types. In addition, the methods for quantifying the mitochondrial distribution in GSCs vs. CySCs are very much unclear and appear to rely on colocalization with molecular markers that are not in the same cellular compartment (Tj-nuclear vs. Vasa-perinuclear and cytoplasmic) the reader has no way to determine the validity of the mitochondrial distribution. Similarly, the labelling with gstD1-GFP is also very much unclear - I see little to no GFP signal in either GSCs or CySCs in panels 1GK. Lastly, while the expression o SOD in CySCs does increase the gstD1-GFP signal in CySCs, the effects on GSCs claimed by the authors are not apparent.
We appreciate the reviewer’s detailed feedback on Figure 1 and the concerns raised regarding identifying CySCs and GSCs, as well as the methods used for quantifying mitochondrial distribution and gstD1-GFP labeling. Below, we address each point and describe the revisions made to improve clarity and rigor
Distinguishing CySCs and GSCs and Mitochondrial Distribution in GSCs vs. CySCs in Figure1
We acknowledge the difficulty in distinguishing CySCs from GSCs without the use of additional cell surface markers. To improve clarity, we have now included a membrane marker discslarge (Dlg) in our revised Figure 1 and S1 to delineate cell boundaries more clearly. Additionally, we provide higher-magnification images to indicate the mitochondria in CySCs and GSCs. We also agree that ing on mitochondrial distribution might be far-fetched. In the revised manuscript, we have limited our analysis to mitochondrial shape, which was found to be different in GSC and CySC (Fig. 1, D, F, G, and S1B). We have clarified our quantification methods in the revised Methods section, providing details on the image processing and analysis pipeline used to assess mitochondrial distribution.
Clarity of gstD1-GFP Labelling:
We recognize the reviewer’s concern regarding the weak GFP signal in these panels. To improve visualization, we have included fresh set of images by optimizing the contrast and presenting additional monochrome images with higher exposure settings to better illustrate gstD1-GFP expression (Figure 1L,1Q, and S1C’’’-D’’’). Additionally, we have demarcated the cell boundaries using Dlg along with individual labelling of Vasa+ and Tj+ cells. Due to technical difficulty associated with acquisition of images, we could not co-stain Vasa, Tj and Dlg together. Therefore, quantified the gstD-GFP intensity separately for GSCs and CySCs under similar acquisition conditions (Figure 1R).
Effects of SOD depletion on GSCs:
While our initial analysis suggested changes in gstD1-GFP expression in GSCs upon Sod1 depletion in CySCs, we acknowledge that the effects may not be as apparent in the provided images. In response, we have expanded our quantification, included a statistical analysis of gstD1-GFP intensity specifically in GSCs and CySCs (Figure 1S), and added more representative images in the revised figure panels (Figure S1C-D’’’) to support our claims.
In Figure 2, while the cell composition of the niche region does appear to be different from controls when SOD1 is knocked down in the CySCs, at least in the example images shown in Figures 2A and B, how cell type is quantified in figures 2E-G is very much unclear in the figure and methods. Are these counts of cells contacting the niche? If so, how was that defined? Or were additional regions away from the niche also counted and, if so, how were these regions defined?
Thank you for your regarding the quantification of cell types in Figures 2E-G. We counted all cells that were Tj-positive and Zfh1-positive in individual testis, while for GSCs, only those in direct contact with the hub were included. This clarification has been incorporated into the revised figure legend and methods (line no.400-407). We have now provided a clearer description in the text to improve transparency in our analysis.
In Figure 3, it is quite interesting that there is an increase in Eya+, differentiating cyst cells in SOD1 knockdown animals, and that these Eya+ cells appear closer to the niche than in controls. However, this seems at odds with the proliferation data presented in Figure 2, since Eya+ somatic cells do not normally divide at all. Are they suggesting that now differentiating cyst cells are proliferative? In addition, it is important for them to show example images of the changes in Socs36E and ptp61F expression.
Thank you for your insightful observations. We acknowledge the apparent contradiction and appreciate the opportunity to clarify our interpretation.
Regarding the increase in Eya+ differentiating cyst cells in Sod1RNAi individuals and their proximity to the niche, we do not suggest that these differentiating cells are proliferative. Instead, we propose that the knockdown of Sod1 may alter the timing or regulation of cyst cell differentiation, leading to an accumulation of Eya+ cells near the niche. To clarify this point, we have revised the manuscript (line no. 186-189) to emphasize that our proliferation data specifically refers to early-stage somatic cells, not Eya+ differentiating cyst cells.
We also appreciate the reviewer's request for example images illustrating the changes in Socs36E and Ptp61F expression. We could not access the antibodies specific to Socs36E and Ptp61F. Hence, we had to rely on the measurements were obtained using real-time PCR from the tip region of testis. We have clarified the same in the figure legends (line 700).
Overall, the various changes in signaling are quite puzzling-while Jak/Stat signaling from the niche is reduced, hh signaling appears to be increased. Similarly, while the authors conclude that premature differentiation occurs close to the niche, EGF signaling, which occurs from germ cells to cyst cells during differentiation, is decreased. Many times these, changes are contradictory, and the authors do not provide a suitable explanation to resolve these contradictions.
We appreciate the reviewer’s thoughtful feedback on the signaling changes described in our study. We acknowledge that the observed alterations in Jak/Stat, Hedgehog (Hh), and EGF signaling may appear contradictory at first glance. However, our data suggest that these changes reflect a complex interplay between different signaling pathways that regulate cyst cell behavior in response to specific genetic perturbation.
Regarding Jak/Stat and Hh signaling, while Jak/Stat activity is reduced in the niche, the increase in Hh signaling may reflect a compensatory mechanism or a context-dependent response of cyst cells to reduced Jak/Stat input. Prior studies have suggested that Hh signaling can function in parallel and independently of Jak/Stat signaling (PMID: 23175633) and our findings align with this possibility.
The reduction in EGFR signaling in this context appears contradictory to existing literature. One possible explanation is that, the altered GSC -CySC balance and loss of contact in Tj>Sod1i testes, leads to insufficient ligand response, thereby failing to activate EGFR signaling. (line no.222-224, 313-318).
Reviewer #2 (Public review):
We sincerely appreciate the reviewer’s detailed feedback, which has helped refine our manuscript. In this study we have focussed on the role of ROS generated due to manipulation of Sod1 in the interplay between GSC and CySCs. In this regard, we have conducted additional experiments and incorporated quantitative data into the revised manuscript. Additionally, we have refined the text and provided further context to enhance the clarity. Key revisions include:
(1) Clarification of Quantification Methods – We have refined intensity measurements by incorporating a membrane marker (Dlg) to better delineate cell boundaries and have normalized Ptc and Ci expression per cell to improve clarity.
(2) Cell-Specific ROS Measurement – We separately measured ROS in germ cells and cyst cells and performed independent Sod1 depletion in GSCs to determine its direct effects.
(3) Mitochondrial Analysis – We revised our approach, focusing on mitochondrial shape rather than asymmetric distribution, and removed overreaching claims.
(4) Proliferation Analysis – We reanalyzed FUCCI data by normalizing to total cell count, supporting the conclusion that increased proliferation, rather than differentiation delay, underlies the observed phenotype.
(5) E-Cad Quantification – We specifically analyzed E-Cad levels at the GSC-hub interface to strengthen conclusions on GSC attachment.
(6) JAK/STAT Signaling – While we could not obtain a STAT92E antibody, we clarified the spatial limitations of our current analysis and revised the text accordingly.
(7) Rescue Experiments and Gal4 Titration Control – We performed additional control experiments to confirm that observed effects are not due to Gal4 dilution.
(8) Image Quality and Terminology Corrections – We enhanced figure resolution, corrected terminology (e.g., "cystic" to "cyst"), and revised ambiguous phrasing for clarity and accuracy.
As suggested, we have also changed the manuscript title to better align with our results:
Previous Manuscript Title: Non-autonomous cell redox-pairs dictate niche homeostasis in multi-lineage stem populations
Updated Manuscript Title: Superoxide Dismutases maintain niche homeostasis in stem cell populations
Specific responses to the reviewer’s:
While the decrease in pERK in CySCs is clear from the image and matched in the quantification, the increase in cyst cells is not apparent from the fire LUT used. The change in fluorescence intensity therefore may be that more cells have active ERK, rather than an increase per cell (similar arguments apply to the quantifications for p4E-BP or Ptc). Therefore, it is hard to know whether Sod1 knockdownresults in increased or decreased signaling in individual cells.
Thank you for your insightful . To clarify, in the Fire LUT images, only pERK intensity is shown, not the cyst cell number. In our context, while there are more cells, the overall pERK intensity is lower, eliminating any ambiguity about whether the change is occurring per cell or due to an increased number of circulating cells. Moreover, for Ptc and Ci levels, we have normalized Ptc and Ci expression intensity per cell to enhance clarity and ensure an accurate interpretation of signaling changes.
There are several places in which the authors could strengthen their manuscript by explaining the methods more clearly. For example, it is unclear how the intensity graphs in Figure 1Q are obtained. The curves appear smoothed and therefore unlikely to be from individual samples, but this is not clearly explained. However, this quantification method is clearly not helpful, as it shows the overlap between somatic and germline markers, suggesting it cannot accurately distinguish between the two cell types. Additionally, using a nuclear marker (Tj) for the cyst cells and cytoplasmic marker (Vasa) for the germ cells risks being misleading, as one would not expect much overlap between cytoplasmic gstD1-GFP and nuclear Tj. Also related to the methods, it is unclear how Vasa+ cells at the hub were counted. The methods suggest this was from a single plane, but this runs the risk of being arbitrary since GSCs can be distributed around the hub in 3D. (As a note, the label on the graph "Vasa+ cells" is misleading, as there are many more cells that are Vasa-positive than the ones counted.)
We appreciate the reviewer’s careful evaluation of our manuscript and their insightful suggestions for improving the clarity of our methods. Below, we address each concern raised and describe the revisions made accordingly.
Clarification of Intensity Graphs in Figure 1Q
We have removed this graph, as we recognize that the markers previously used were not appropriate for distinguishing the different cell types. To address this concern, we have revised the text and now included a membrane marker discs-large (Dlg) in our revised Figure 1 and S1 to more clearly delineate cell boundaries. Due to technical difficulty associated with acquisition of images, we could not co-stain Vasa, Tj and Dlg together. Therefore, quantified the gstD-GFP intensity separately for GSCs and CySCs under similar acquisition conditions (Figure 1R).
Counting of Vasa+ Cells at the Hub
We appreciate the reviewer’s concern regarding our method for counting Vasa+ cells. In our original analysis, we included GSCs as the Vasa-positive cells that were in direct contact with the hub. To account for the three-dimensional arrangement of GSCs, we used the Cell counter plugin of Fiji and performed counting across different focal planes to ensure all hub-associated cells were considered. For better clarity on cell distribution around the hub, we have presented a single focal place image sliced through mid of the hub zone. To enhance transparency, we have now provided a more detailed explanation of our counting approach in the Methods section (line no 400- 403).
We agree that the label "Vasa+ cells" may be misleading, as many cells express Vasa beyond the specific subset being counted. To address this, we have changed the label to " GSCs" to reflect the subset analyzed more accurately.
The crucial experiment for this manuscript is presented in Figures 1 G-S, arguing that Sod1 knockdown with Tj-Gal4 increases gstD1-GFP expression in germ cells. This needs strengthening as the current quantifications are not convincing and appear to show an overlap between Tj (a nuclear cyst cell marker) and Vasa (a cytoplasmic germ cell marker). Labeling cell outlines would help, or alternatively, labeling different cell types genetically can be used to determine whether the expression is increased specifically within that cell type. Similarly, the measurement of ROS shown in the supplemental data should be conducted in a cell-specific manner. To clearly make the case that Sod1 knockdown in cyst cells is impacting ROS in the germline, it would be important to manipulate germ cell ROS independently. Without this, it will be difficult to prove that any effects observed are a result of increased ROS in the germline rather than indirect effects on the germline of altered cyst cell behaviour.
We appreciate the reviewer’s insightful feedback regarding the specificity of Sod1 knockdown effects in germ cells and the need for clearer quantification in Figures 1G–S. Below, we address each concern and outline the modifications made:
Clarification of Cell Type-Specific Expression:
We acknowledge the overlap observed between Tj (nuclear cyst cell marker) and Vasa (cytoplasmic germ cell marker) in the presented images. To strengthen our claim that gstD1GFP expression increases specifically in germ cells upon Sod1 knockdown, we have now labelled cell outlines using membrane marker discs-large (Dlg) to better distinguish cell boundaries, along with individual labelling of Vasa+ and Tj+ cells. Due to technical difficulty associated with acquisition of images, we could not co-stain Vasa, Tj and Dlg together.
Cell-Specific Measurement of ROS:
We agree that a cell-type-specific ROS measurement is critical to establishing a direct effect on germ cells. To address this, we have now performed ROS measurements separately in germ cells and cyst cells under similar acquisition conditions. These data are now included in the revised (Figure 1R). Similarly, upon CySC-specific Sod1 depletion, we performed measurement of gstD1-GFP intensity which was found to be enhanced in GSCs, along with expected increase in CySCs (Fig 1S). We have independently manipulated ROS levels in GSCs (Nos Gal4> Sod1i) and observed that elevated ROS negatively impacts GSCs, leading to a reduction in their number, while having an insignificant effect on adjacent CySCs.(Fig S2 E, F).
Quantifications of mitochondrial localization in Figure 1 should include some adequate statistical method to evaluate whether the distribution is random or oriented towards the GSC/CySC interface. From the image provided (Figure 1B), it would appear that there are two clusters of mitochondria, on either side of a CySC nucleus, one cluster towards a GSC and one cluster away. Therefore evaluating bias would be important. Additional experiments will be necessary to support the statement that "Redox state of GSC is maintained by asymmetric distribution of CySC mitochondria". This would require manipulating mitochondrial distribution in CySCs.
We appreciate the reviewer’s suggestion regarding the quantification of mitochondrial localization. We agree that ing on mitochondrial distribution might be far-fetched. In revised manuscript, we have demarcated the cell boundary and limited our analysis to mitochondrial shape which was found to be different in GSC and CySC (Fig. 1, D, F, G and S1B). Mitochondrial shape was quantified based on the mitochondrial area and circularity (Figure 1F and G). To prevent any misinterpretation, we have removed the statement, "Redox state of GSC is maintained by asymmetric distribution of CySC mitochondria."
One point raised by the authors is that the increase of somatic cell numbers is driven by accelerated proliferation, based on an increased number of cells in various stages of the cell cycle as assessed by the FUCCI reporter. However, there are more somatic cells in this genetic background, so it could be argued that the observed increase in different phases of the cell cycle is due to an increased number of cells. In order to argue for an increased proliferation rate, the number of cells in each phase should be divided by the total number of cells, expecting to see an increase in S and G2/M phases along with a decrease in G1. Otherwise, the simplest explanation is a block or delay in differentiation, meaning that more cells remain in the cell cycle.
We appreciate the regarding the interpretation of our FUCCI reporter data. We acknowledge that the observed increase in the number of cells in various phases of the cell cycle could be influenced by the overall higher number of somatic cells in this genetic background.
To address this concern, we have now re-analyzed our FUCCI data by normalizing the number of cells in each phase to the total number of cells and we did not observe a significant shift in the proportion of cells in S and G2/M phases relative to G1. This suggests presence of more proliferative cells, that is less cells in Go phase, rather than alterations in the timing of cell cycle progression stages. We are not sure about a block in differentiation because we see an enhanced accumulation of Eya+ cells near the niche. We have also supported our FUCCI data with pH3 staining where we have found more pH3+ spots under SOD1 depleted background. We have revised our manuscript accordingly (Figure 2I, K and S2U) to reflect this interpretation and appreciate the constructive feedback.
In Figure 3, the authors claim that knockdown of Sod1 in the soma decreases the attachment of GSCs to the hub-based on lower E-Cad levels compared to controls. Previous work has shown that in GSCs, E-Cad localizes to the Hub-GSC interface (PMID: 20622868). Therefore, the authors should quantify E-Cad staining at the interphase between the germ cells and the niche.
We appreciate the reviewer’s . As suggested, we have now quantified ECad staining specifically at the interface between the germ cells and the niche. Our analysis confirms that E-Cad levels are significantly reduced at this interphase upon Sod1 knockdown in the soma compared to controls, supporting our conclusion that Sod1 depletion affects GSC attachment to the hub as well as the whole niche. The revised Figure 3M now includes these quantifications, and we have updated the figure legend and results section accordingly.
The authors show decreased expression of the JAK/STAT targets socs36E and ptp61F, arguing that this could be a reason for decreased GSC adhesion to the hub. However, these data were obtained from whole testes and lacked spatial resolution, whereas a STAT92E staining in control and tj>Sod1 RNAi testes could easily prove this point. Indeed, previous work has shown that socs36E is expressed in the CySCs, not GSCs (PMID: 19797664), suggesting that any decrease in JAK/STAT may be autonomous to the CySCs.
We appreciate the reviewer’s observation regarding the spatial resolution of our JAK/STAT target expression analysis. To improve accuracy, we have attempted to collect only the tip of the testes while excluding the rest; however, we acknowledge that this approach may still obscure cell-specific changes. We had attempted to procure the STAT92E antibody but, despite multiple inquiries, we did not receive a positive response. While we agree that STAT92E staining would have strengthen our findings, we are currently unable to perform this experiment. Nevertheless, our observations align with prior work indicating that socs36E is predominantly expressed in CySCs (PMID: 19797664). We have revised the manuscript text accordingly to clarify this limitation.
Additional considerations should be taken regarding the rescue experiments where PI3KDN and Hh RNAi are expressed in a Tj>Sod1 RNAi background. To rule out that any rescue can be attributed to titration of the Gal4 protein when an additional UAS sequence is present, a titration control would be useful. These pathways are not described accurately since Insulin signaling is necessary for the differentiation of somatic cells (not maintenance as written in the text), and its inhibition has been shown to increase the number of undifferentiated somatic cells (PMID:27633989). As far as Hh is concerned, the expression of this molecule is restricted to the niche. It would be important to establish whether the expression is altered in this case, especially as the authors rescue the Sod1 knockdown by also knocking down Hh. One possibility that the authors need to rule out is that some of the effects they observe are due to the knockdown of Sod1 (and/or Hh) in the hub as Tj-Gal4 is expressed in the hub as well as the CySCs (PMID:27546574).
We appreciate the reviewer’s insightful s and suggestions. Below, we address each concern and describe the steps we have taken to incorporate the necessary modifications in our revised manuscript.
Titration Control for Rescue Experiments
We acknowledge the reviewer’s concern regarding potential Gal4 titration effects when introducing additional UAS constructs. To address this, we conducted a control experiment quantifying SOD1 levels in control, Tj > Sod1 RNAi, and Tj > Sod1 RNAi, UAS hhRNAi backgrounds using real-time PCR (Figure S4 M). The Sod1 levels in single and double UAS copy conditions were comparable, indicating that Gal4 titration does not significantly affect the results.
Clarification of Insulin Signaling Role
We appreciate the reviewer’s insight regarding the involvement of insulin signaling in this context. Initially, we included data on PI3K/TOR as we found it intriguing. However, as the data didn’t add much to the overall observations, we have removed them to ensure clarity and prevent any potential confusion.
Hh Expression and Niche Consideration
We recognize the importance of evaluating whether Hedgehog (Hh) expression is altered in the Sod1 RNAi background. We have already quantified hh in qRT-PCR (Figure S4C).
Potential Effects of Sod1 and Hh Knockdown in the Hub
We acknowledge the concern that Tj-Gal4 is expressed in both the hub and CySCs, potentially affecting hub function upon Sod1 and Hh knockdown. To address this, we have included additional data using the CySC-specific driver C-587 Gal4 to distinguish CySC-intrinsic effects from potential hub contributions. Our results show that while the phenotypic changes are consistent across both drivers, the effects are significantly stronger with Tj-Gal4, suggesting a role of the hub in this process. These findings have been incorporated into the revised manuscript (Fig S1G-H, M-N).
In general, the GSCs (and other aspects) are difficult to see in the images; enlargements or higher-resolution images should be provided. Additionally, the manuscript contains several mistakes or inaccuracies (examples include referring to ROS having "evolved" in the abstract when it is cells that have evolved to use ROS, or the references to "cystic" cells when they are usually referred to as "cyst" cells, or that "CySCs also repress GSC differentiation by suppressing transcription of bag-of-marbles" when CySCs produce BMPs that lead to suppression of bam expression in the germline). These would need editing for both clarity and accuracy.
We appreciate the reviewer’s insightful feedback and have made the necessary revisions to address the concerns raised.
Image Clarity and Resolution:
We have provided higher-resolution images in some of the revised images for better understanding. The revised figures now offer better clarity for key observations.
Clarification of Terminology and Accuracy:
The phrase regarding ROS in the abstract has been revised to reflect that cells have evolved to utilize ROS, rather than ROS itself evolving (line no. 27).
References to "cystic" cells have been corrected to "cyst" cells for consistency with standard terminology.
The statement about CySCs repressing GSC differentiation has been revised for accuracy, clarifying that CySCs produce BMPs, which lead to the suppression of bam expression in the germline (line no. 84).
We have carefully reviewed the manuscript for any additional inaccuracies or ambiguities to ensure clarity and precision. We appreciate the reviewer’s constructive s, which have helped improve the manuscript.
Reviewer #3 (Public review):
In response to Reviewer 3’s comments, we would like to highlight the point that in the present study we have focussed on the interplay between CySC and GSC and have accordingly conducted our experiments. We did observe some changes in the hub and do not rule out the effect of hub cells in exacerbating some of our phenotypes. We have included additional controls to highlight the effect of CySC ROS. These points have been appropriately discussed in the manuscript. Key revisions include:
(1) Data Clarity & Visualization: To improve mitochondrial lineage association, we incorporated a membrane marker (Dlg) in Figure 1, enhancing the distinction between CySCs and GSCs. Additionally, we refined gstD-GFP quantifications in individual cell types and provided high-resolution images.
(2) ROS Transfer & Measurement: We revised our discussion to acknowledge indirect ROS transfer mechanisms and added separate ROS quantifications in GSCs and CySCs, confirming higher ROS levels in CySCs (Figure 1R).
(3) Tj-Gal4 Specificity & Niche Characterization: Recognizing Tj-Gal4 expression in hub cells, we included C587-Gal4 as a CySC-specific driver, demonstrating that hub cells contribute partially to the phenotype (Figure S1G,H,M,N).
(4) Signaling Pathway Validation: We optimized dpERK staining, included controls (Tj>EGFRi), and clarified limitations regarding MAPK signaling. Due to lethality, we could not perform an EGFR gain-of-function rescue. We also validated increased Hh signaling via qPCR and a Tj>UAS Ci control (Figure S4).
(5) Conceptual & Terminological Refinements: We revised our discussion of BMP signaling, ROS gradients, and testis-specific terminology. All figures and labels now accurately represent GSC scoring (single Vasa⁺ cells in contact with the niche).
(6) Figure & Methods Improvements: We enhanced image resolution, provided grayscale versions where needed,and expanded Materials & Methods to clarify experimental conditions.
These revisions strengthen our conclusions and address the reviewer’s concerns, ensuring a more precise and transparent presentation of our findings. To align with the reviewer’s s we have changed the title of the manuscript to “Superoxide Dismutases maintain niche homeostasis in stem cell populations”.
Specific responses to the reviewer’s comments:
(1) Data
a. Problems proving which mitochondria are associated with which lineage.
We acknowledge the challenge of distinguishing CySCs from GSCs without additional cell surface markers. To enhance clarity, we have incorporated the membrane marker Discs-large (Dlg) in our revised Figure 1 to better delineate cell boundaries, providing a clearer depiction of mitochondrial distribution in GSCs and CySCs.
b.There is no evidence that ROS diffuses from CySCs into GSCs.
We acknowledge the reviewer’s concern. There are reports which talks about diffusion of ROS across cells on which we have included a few lines in the discussion (line no. 274-276). We do understand that our previous quantifications showed ROS diffusion from CySC to GSC rather indirectly. Therefore, in revised manuscript we have measured ROS separately in the two cell populations. We found that the CySCs show higher ROS profile than GSCs (Fig 1R).
c.The changes in GST-GFP (redox readout) are possibly seen in differentiating germ cells (i.e., spermatogonia) but not in GSCs. This weakens their model that ROS in CySC is transferred to GSCs.
Thank you for your observation. We acknowledge that the changes in gstD-GFP (redox readout) are more prominent in differentiating germ cells. It is known that differentiating cells show higher ROS profile than the stem cells. Hence, expectedly the intensity of gstDGFP was lesser in stem cell zone compared to the differentiating zone. In our manuscript we are focussed on the redox state among stem cell populations. Therefore, we have included better quality images and measured the gstD1-GFP intensity individually in GSCs and CySCs (Figure 1R) by demarcating the cell boundaries (Figure 1M, S1C-D’’’). We found that CySCs show higher ROS profile than GSCs and enhancement of ROS in CySC by Sod1 depletion resulted in a consequent increase in ROS in GSCs. We believe this revision strengthens our model by addressing the potential discrepancy and providing a more comprehensive understanding of ROS dynamics within the GSC niche.
d.Most of the paper examines the effect of SOD depletion (which should increase ROS) on the CySC lineage and GSC lineage. One big caveat is that Tj-Gal4 is expressed in hub cells (Fairchild, 2016), so the loss of SOD from hub cells may also contribute to the phenotype. In fact, the niche in Figure 2D looks larger than the niche in the control in Figure 2C, arguing that the expression of Tj in niche cells may be contributing to the phenotype. The authors need to better characterize the niche in tj>SOD-RNAi testes.
We appreciate the reviewer’s insightful regarding the potential contribution of hub cell to the observed phenotype. We acknowledge that Tj-Gal4 is expressed in hub cells and this could influence the niche size and overall phenotype.
To address this concern, we have included an additional control using C587-Gal4, a CySC specific driver, to distinguish CySC-specific effects from potential hub contributions. All the effects on cell number observed in Tj>Sod1i was replicated in C587>Sod1i testis, except that the observed phenotypes were comparatively weaker. These indicate partial contribution of hub cells to the observed phenotype, exacerbating its severity. However, the effect of Sod1 depletion in CySC on GSC lineages remains significant. These findings have been incorporated into Figure S1- G,H,M and N) and incorporated in the discussion (line no.308311).
e. The Tj>SOD1-RNAi phenotype is an expansion of the Zfh1<sup+ CySC pool, expansion of the Tj+ Zfh1- cyst cells (both due to increased somatic proliferation) and a non-autonomous disruption of the germline.
We appreciate the reviewer’s observation. Our data confirm that Tj>SOD-RNAi leads to an expansion of both Zfh1<sup+ CySCs and Tj+ Zfh1- cyst cells, which we attribute to increased somatic proliferation. Additionally, we observe a non-autonomous disruption of the germline, likely due to dysregulated signaling from the altered somatic niche.
f. I am not convinced that MAPK signaling is decreased in tj>SOD-i testes. Not only is this antibody finicky, but the authors don't have any follow-up experiments to see if they can restore SOD-depleted CySCs by expressing an EGFR gain of function. Additionally, reduced EGFR activity causes fewer somatic cells (not more) (Amoyel, 2016) and also inhibits abscission between GSCs and gonial blasts (Lenhart 2015), which causes interconnected cysts of 8- to 16 germ cells with one GSC emanating from the hub.
We acknowledge that the dpERK antibody can be challenging. We took necessary precautions, including optimizing staining conditions and using positive control (Tj>EGFRi) (Figure: S4B). Our results consistently showed a decrease in dpERK levels in Tj>Sod1i testes, supporting our conclusion.
We agree that inclusion of an experiment using EGFR gain-of-function to rescue the effects of CySC-Sod1 depletion would have strengthened our findings. We had attempted this experiment; however, the progenies constitutively expressing EGFR under Sod1RNAi background were lethal, preventing us from completing the analysis.
We agree that our observations do not align with the reported effects of EGFR signaling on somatic cell numbers and abscission and we appreciate the references provided. Based on our observations, we feel that modulation of MAPK signaling in the niche probably, happens in a context-dependent manner. One possible explanation is that, the altered GSC -CySC balance and loss of contact in Tj>Sod1i testes, leads to insufficient ligand response, thereby failing to activate EGFR signaling. While it is well established that ROS can enhance EGFR signaling to promote cellular proliferation and early differentiation, our results indicate a more nuanced regulation in this context. However, further detailed analysis is required to completely understand the regulatory controls. We have clarified this point in the manuscript (line no.
313-320).
g. The increase in Hh signaling in SOD-depleted CySCs would increase their competitiveness against GSCs and GSCs would be lost (Amoyel 2014). The authors need to validate that Hh protein expression is indeed increased in SOD-depleted CySCs/cyst cells and which cells are producing this Hh. Normally, only hub cells produce Hh (Michel,2012; Amoyel 2013) to promote self-renewal in CySCs.
We appreciate the reviewer’s suggestion regarding the validation of Hh protein expression and its source. Since Tj-Gal4 is expressed in the hub, it is likely activating the Hh pathway and promoting CySC proliferation. Unfortunately, we could not procure Hh antibody to directly assess its protein levels. However, to address this, we performed real-time PCR from RNA derived from the tip region and found a significant increase in hh mRNA levels in SOD-depleted cyst cells. These findings support our hypothesis that elevated Hh signaling enhances CySC competitiveness, leading to GSC loss. To support this idea, we have included a Tj>Ci positive control which caused abnormal proliferation of Tj+ cells resulted in ablation of GSCs. We have incorporated these results in the revised manuscript (Results section, Figure S-4).
h.The increase in p4E-BP is an indication that Tor signaling is increased, but an increase in Tor in the CySC lineage does not significantly affect the number of CySCs or cyst cells (Chen, 2021). So again I am not sure how increased Tor factors into their phenotype.
We acknowledge the reviewer’s concern regarding the role of increased Tor signaling in our phenotype. The observed increase in Tor could indeed be a downstream effect of elevated ROS levels. However, establishing a direct causal relationship between Sod1 and Tor would require additional experiments, which we feel might be a good study in its own merit. To maintain clarity and focus in the revised manuscript, we have opted not to include this preliminary data at this stage.
I.The over-expression of SOD in CySCs part is incomplete. The authors would need to monitor ROS in these testes. They would also need to examine with tj>SOD affects the size of the hub.
We value the reviewer's . To address this, we have now monitored ROS levels in the testes upon SOD overexpression in CySCs using DHE (Figure S5 I). Our results indicate a significant reduction in ROS levels compared to controls.
Additionally, we examined hub size upon Sod1 overexpression and observed a slight, but statistically insignificant, reduction. As our study primarily focuses on ROS-mediated GSCCySC interactions, we did not include a detailed investigation on hub size regulation.
(2) Concept
Why would it be important to have a redox gradient across adjacent cells? The authors mention that ROS can be passed between cells, but it would be helpful for them to provide more details about where this has been documented to occur and what biological functions ROS transfer regulates.
We thank the reviewer for this insightful . We acknowledge that the concept of a redox gradient was not adequately conveyed, as the cell boundary was not clearly defined. To address this, we have revised our interpretation to propose that high ROS levels in one cell may influence the ROS levels in an adjacent cell through either direct transfer or as a secondary effect of altered niche maintenance signaling, rather than through the establishment of a gradient.
Regarding ROS transfer between cells, it has been documented in several biological contexts. For instance, hydrogen peroxide (H2O2) can diffuse through aquaporins, influencing signaling pathways in neighbouring cells (PMID: 17105724). We have incorporated these details and relevant references into the revised manuscript to enhance the conceptual understanding of ROS transfer.
(3) Issues with the scholarship of the testis
a. Line 82 - There is no mention of BMPs, which are the only GSC-self-renewal signal. Upd/Jak/STAT is required for the adhesion of GSCs to the niche but not self-renewal (Leatherman and Dinardo, 2008, 2010). The author should read a review about the testis. I suggest Greenspan et al 2015. The scholarship of the testis should be improved.
We appreciate the reviewer’s feedback regarding the role of BMPs in GSC selfrenewal, we have added this in the revised manuscript (line no. 83) We have now incorporated a discussion on BMP signaling as the primary self-renewal signal for GSCs, distinguishing it from the role of Upd/JAK/STAT in niche adhesion, as highlighted in Leatherman and Dinardo (2010). Additionally, we have cited and reviewed the work by Greenspan et al. (2015) and ensure a more comprehensive discussion of GSC regulation. These revisions can be found in the line no. 285-289 of the revised manuscript.
b. Line 82-84 - BMPs are produced by both hub cells and CySCs. BMP signaling in GSCs represses bam. So it is not technically correct to say the CySCs repress bam expression in GSCs.
We acknowledge the reviewer’s clarification regarding BMP signaling and its role in repressing bam expression in GSCs. We have revised the relevant section (line no.83-85).
c.Throughout the figures the authors score Vasa+ cells for GSCs. This is technically not correct. What they are counting is single, Vasa+ cells in contact with the niche. All graphs should be updated with the label "GSCs" on the Y-axis.
We appreciate the reviewer’s careful assessment of our methodology. We acknowledge that scoring Vasa⁺ cells alone does not definitively identify GSCs. Our quantification specifically considers single Vasa⁺ cells in direct contact with the niche. To ensure clarity and accuracy, we have updated all figure legends and Y-axis labels in the relevant graphs to explicitly state "GSCs" instead of "Vasa⁺ cells."
(4) Issues with the text
a. Line 1: multi-lineage is not correct. Multi-lineage refers to stem cells that produce multiple types of daughter cells. GSCs produce only one type of offspring and CySCs produce only one type of offspring. So both are uni-lineage. Please change accordingly.
We acknowledge the incorrect usage of "multi-lineage" and agree that both GSCs and CySCs are uni-lineage, as they each produce only one type of offspring. We have revised Line 1 accordingly and also updated the title.
b. Lines 62-75 - Intestinal stem cells have constitutively high ROS (Jaspar lab paper), so low ROS in stem cell cells is not an absolute.
We appreciate the clarification. We have revised Lines 62–75 to acknowledge that low ROS is not universal in stem cells, citing the Jaspar lab study on intestinal stem cells (Line 70). Thank you for the valuable insight.
c. Line 79: The term cystic is not used in the Drosophila testis. There are cyst stem cells (CySCs) that produce cyst cells. Please revise.
We have revised the text to replace "cystic" with the correct terminology, referring to cyst stem cells (CySCs) in the manuscript.
d. Line 90 - perfectly balanced is an overstatement and should be toned down.
Thank you for the suggestion. We have revised it to “balanced” instead of "perfectly balanced."
e. Line 98 - division of labour is not supported by the data and should be rephrased.
Thank you for the feedback. We have rephrased it (line no. 98-101) to avoid the term "division of labor".
f. Line 200 - the authors provide no data on BMPs - the GSC self-renewal cue - so they should avoid discussing an absence of self-renewal cues.
We appreciate the reviewer’s point. We have revised it to avoid discussing the absence of self-renewal cues, given that we do not present data on BMP signaling. This ensures that our conclusions remain within the scope of the provided data.
(5) Issues with the figures
a The images are too small to appreciate the location of mitochondria in GSCs and CySCs.
b. Figure 1
c. cell membranes are not marked, reducing the precision of assigning mitochondria to GSC or CySCs. It would be very helpful if the authors depleted ATP5A from GSCs and showed that the puncta are reduced in these cells, and did a similar set of experiments for the Tj-Gal4 lineage. It would also be very helpful if the authors expressed membrane markers (like myrGFP) in the GSC and then in the CySC lineage and then stained with ATP5A. This would pinpoint in which cells ATP5A immunoreactivity is occurring.
d. The presumed changes in gst-GFP (redox readout) are possibly seen in differentiating germ cells (i.e.,spermatogonia) but not in GSC. iii. Panels F, Q, and S are not explained and currently are irrelevant.
e. Figure 3K - The evidence to support less Ecad in GSCs in tj>SOD-i testes is not compelling as the figure is too small and the insets show changes in Ecad in somatic cells, not GSC. d. Figure 4:
f. Panel A, B The apparent decline (not quantified) may not contribute to the phenotype.
ii.dpERK is a finicky antibody and the authors are showing a single example of each genotype. This is an important experiment because the authors are going to use it to conclude that MAPK is decreased in the tj>SOD-i samples. However, the authors don't have any positive (dominantactive EGFR) or negative (tj>mapk-i). As is standing, the data is not compelling. The graph in F does not convey any useful information.
g. Figure S1D - cannot discern green on black. It is critical for the authors to show monochromes (grayscale) for thereabouts that they want to emphasize. I cannot see the green on black in Figure S1D.
h. Figure S4 - there is no quantification of the number of Tj cells in K-N.
We appreciate your detailed feedback regarding the figures in our manuscript. Below, we address each concern and outline the revisions we have made.
(a) Image Size and Mitochondrial Localization in GSCs and CySCs
We acknowledge the need for larger images to better visualize mitochondrial localization. We have now increased the resolution and size of the images in Figure 1. Additionally, we have included high-magnification insets to enhance clarity (Figure 1 B#)
(b) Figure 1 B,B#,C
(i) We have now marked cell membranes using Dlg to improve the precision of mitochondrial assignment to GSCs and CySCs and then stained for ATP5A, which clearly demarcates ATP5A immunoreactivity in specific cell types.
(ii) We have revisited the gstD-GFP (redox readout) data and now provide revised images (Figure S1C-D’’’) and quantification (Figure 1 R,S) to better illustrate changes in the redox state. It is indeed intense in differentiating germ cells as expected but also present in the stem cell zone.
(iii) Panels F, Q, and S have now been removed in the revised figure legend.
(C) Figure 3K: We have digitally magnified the figure size and improved contrast to better visualize E-cadherin levels. The insets have been revised to ensure they focus specifically on GSCs rather than somatic cells. Earlier, we quantified the E-cadherin intensity changes in the GSC-hub interface and provided statistical analysis to support our findings (Figure 3M).
(d) Figure 4: (i) Panels A and B have now been quantified, and we provide statistical comparisons to support our observations. (ii) We acknowledge the variability of dpERK staining. To strengthen our conclusions, we have provided negative (Tj>MAPK-i) controls (Figure S4 B). Additionally, we have removed panel F (MAPK area cover) to avoid confusion.
(e) We appreciate the suggestion regarding grayscale images and have provided the monochrome images for mitochondria and gstD-GFP image representation. We have now removed Figure S1D as it was no longer required.
(f) Figure S4: The quantification of the number of Tj-positive cells was actually included in the main figure along with statistical analysis.
(g) We sincerely appreciate the reviewer’s insightful s, which have significantly improved the quality and clarity of our manuscript. We hope that our revisions adequately address the concerns raised.
(6) Issues with Methods
a. Materials and Methods are not described in sufficient depth - please revise.
b. Note that Tj-Gal4 has real-time expression in hub cells and this is not considered by the authors. The ideal genotype for targeting CySCs is Tj-Gal4, Gal80TS, hh-Gal80. Additionally, the authors do not mention whether they are depleting throughout development into adulthood or only in adults. If the latter, then they must have used a temperature shift, growing the flies at 18C and then upshifting to 25C or 29C during adult stages.
c. The authors need to show data points in all of the graphs. Some graphs do this but others do not.
d. The authors state that all data points are from three biological replicates. This is not sufficient for GSC and CySC counts. Most labs count GSCs and CySCs from at least 10 testes of the correct genotype.
We appreciate the reviewer’s valuable feedback and have made the necessary revisions to improve the clarity and rigor of our study. Below, we address each concern in detail:
Materials and Methods
We have revised the Materials and Methods section to provide a more detailed description of the experimental procedures, including genotypes, sample preparation, and quantification methods.
Tj-Gal4 Expression and Experimental Design
We acknowledge the reviewer’s point regarding Tj-Gal4 expression in hub cells. While Tj-Gal4 is active in hub cells, our focus was on CySCs, and we have now included a discussion of this caveat in the revised manuscript (line no. 308-311)
Thank you for your suggestion on the ideal genotype for targeting CySCs. While we attempted to procure hh-Gal80, we couldn’t manage to get it, so we opted for another well-established Gal4 driver, C-587 Gal4, to target CySCs. Our results indicate that although the phenotypic changes are consistent across both drivers, the effects are significantly stronger with Tj-Gal4, highlighting the role of CySCs in this process with partial contributions from the hub. These findings have been incorporated into the revised manuscript (lines 309–311).
We now clarify whether gene depletion was conducted throughout development or restricted to adulthood. For adult-specific depletion using the UAS-Gal4 system, crosses were set up at 25°C, and after two days, progenies were shifted to 29°C and aged for 3–5 days at 29°C. This process is now explicitly detailed in the revised Methods section (line no. 345-348).
Data Presentation in Graphs
We have updated all graphs to ensure that individual data points are shown consistently across all figures.
Sample Size for GSC and CySC Counts
We acknowledge the reviewer’s concern regarding biological replicates. Our initial study was based on 10 biological replicates, each set consisting of at least 7-8 testes per genotype, in line with standard practice in the field. This change is reflected in the revised Results and Methods sections.
