A conserved function of Human DLC3 and Drosophila Cv-c in testis development

  1. Sol Sotillos
  2. Isabel von der Decken
  3. Ivan Domenech Mercadé
  4. Sriraksha Srinivasan
  5. Dmytro Sirokha
  6. Ludmila Livshits
  7. Stefano Vanni
  8. Serge Nef
  9. Anna Biason-Lauber
  10. Daniel Rodríguez Gutiérrez
  11. James Castelli-Gair Hombría

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Abstract

The identification of genes affecting gonad development is essential to understand the mechanisms causing Variations/Differences in Sex Development (DSD). Recently, a DLC3 mutation was associated with male gonadal dysgenesis in 46,XY DSD patients. We have studied the requirement of Cv-c, the Drosophila ortholog of DLC3, in Drosophila gonad development, as well as the functional capacity of DLC3 human variants to rescue cv-c gonad defects. We show that Cv-c is required to maintain testis integrity during fly development. We find that Cv-c and human DLC3 can perform the same function in fly embryos, as flies carrying wild type but not patient DLC3 variations can rescue gonadal dysgenesis, suggesting functional conservation. We also demonstrate that the StART domain mediates Cv-c's function in the male gonad independently from the GAP domain's activity. This work demonstrates a role for DLC3/Cv-c in male gonadogenesis and highlights a novel StART domain mediated function required to organize the gonadal mesoderm and maintain its interaction with the germ cells during testis development.

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    Reply to the reviewers

    Reviewer #1:

    Major comments:

    In general, the data support the conclusions. I cannot comment on the atomistic simulation experiment as it is outside of my expertise. I had some difficulties interpreting Figure 2 as the contrast in the colour panels made it difficult to assess the different staining patterns. I would recommend changing the blue to cyan for easier visibility. While I agree that there are some differences between Fig 2F and Fig 2G it is not simple for the non-expert to distinguish the gonadal mesoderm from the somatic mesoderm. I think the enlarged panels could do with also showing the overlap in staining, or at least a tracing of the different cell populations so that the gonadal mesoderm can be clearly defined. Please also add some scale bars to the figure. Figure 3 demonstrates clear differences in gonad morphology between male and female mutants but the contrast in the colour panels A-G could also be improved. Panels H-J are very clear.

    Response: As suggested by Referees 1 and 3 we have modified the colour channels in all figures. We have also enlarged the figures taking away the uninformative region and focused around the enlarged gonads and added scale bars. For Fig 2F-G, we have added a close up of the region of interest both in colour and in black and white. These changes have increased the contrast and facilitate the data interpretation to non-expert readers.

    The rescue experiment in Figure 4 is clearly presented but could the DLC3 mutants in the graph (panel b) please be named similarly to the schematic proteins shown in panel a.

    Response: We have changed the names to maintain nomenclature uniformity.

    I found the difference between the RhoGAP domain mutants and the StART domain mutants of Cv-c to be clearly defined, and correlate with DLC3 function. This is a very interesting result that indicates multiple molecular functions for the Cv-c /DLC family.

    Response: The methods are well described, statistics adequate and the data well described._

    Minor Comments:

    My only suggestion for the text is to provide a more through description of the StART domain in the introduction.

    Response: We have included the following paragraph in the introduction describing the StART domain:

    “This family of proteins share different domains: besides the Rho GTPase Activating Protein domain (GAP), they present a protein-protein interacting Sterile Alpha Motif (SAM) at the N terminal end and a Steroidogenic Acute Regulatory protein (StAR)-related lipid transfer (StART) domain at the C terminal. StART domains have been shown in other proteins to be involved in lipid interaction, protein localization and function.”

    Reviewer #2:

    My only issue with the present study is to how well the present experimental findings in Drosophila translate to humans. As far as I can tell the present studies show that inactivating mutations in Cv-c in Drosophila result in failure of germ cell enclosure by somatic cells into the testis, resulting in sterility. In humans, and in experimental mouse transgenic lines, it has been well established that absence of germ cells does not of itself lead to failure of testis differentiation and onward development, nor does it lead automatically to sex reversal or impairment of masculinization. For the latter to occur, there must be impairment/failure of fetal Leydig cell function such that insufficient androgen is produced to effect genital/bodywide masculinization. Obviously, this will happen if no testis forms as appears to be the case in the new human DLC3 mutant reported in the present manuscript (although detail on this is unfortunately lacking). This appears to be different to the previous published DLC3/STARD8 mutant sisters, in whom the phenotype appears to reflect failure of steroidogenesis. Is the proposal that DLC3/STARD8 plays a role in both testis differentiation and in Leydig cell function (steroidogenesis) or is this due to different DLC3 genes? I think the authors need to address these key issues in their discussion, if only to highlight that there are at present many gaps in our understanding.

    The reviewer says:

    “As far as I can tell the present studies show that inactivating mutations in Cv-c in Drosophila result in failure of germ cell enclosure by somatic cells into the testis, resulting in sterility.”

    Response: This sentence does not represent the spirit of our findings accurately and this probably reflects the fact that we stressed the interaction between somatic mesodermal cells and germ cells in Drosophila which probably concealed that the main defects in Cv-c mutants are caused by the abnormal interaction of the mesodermal cells with germ cells but also among themselves. Our study provides insights about a new conserved pathway required in the mesodermal cells for the maintenance of an already formed testis, and only indirectly can be considered to deal with sterility. We show that Cv-c is required in the mesodermal cells for the correct maintenance of the testis structure, that when it fails leads to the testis dysgenesis which, among other defects, releases the germ cells. We show that in the absence of Cv-c function in the testis, the mesodermal pigment cells do not form a continuous layer around the testis and the ECM surrounding the testis breaks. We also show that the interstitial gonadal cells fail to ensheath the germ cells and as a result of all these the germ cells become dispersed. These perturbations can be partially corrected by expression in the testis mesoderm of human DLC3 or Drosophila Cv-c that in both cases require a functional StART domain. Thus, our results suggest that Cv-c/DLC3 have a fundamental function on the mesodermal testis cells that has been conserved. These results indicate that, as in Drosophila, the primary cause for the gonadal dysgenesis in DLC3 human patients is due to the abnormal maintenance of the testis mesoderm cells, which include both Sertoli and Leydig cells. Thus, our proposal is that DLC3/STARD8 plays a role in testis maintenance through its function in mesodermal cells which will probably affects both Sertoli and Leydig cell function.

    To clarify the issue raised by the referee we have modified both, the introduction and the discussion to highlight that although humans and Drosophila diverged millions of years ago there are similarities regarding gonad stabilisation.

    We have modified the introduction to clarify this issue:

    “Gonadogenesis can be subdivided into three stages: specification of precursor germ cells, directional migration towards the somatic gonadal precursors and gonad compaction. In mammals, somatic cells, i.e. Sertoli cells in male and Granulosa cells in females, play a central role in sex determination with the germ cells differentiating into sperms or oocytes depending on their somatic mesoderm environment. In humans, Primordial Germ Cells (PGCs) are formed near the allantois during gastrulation around the 4th gestational week (GW) and migrate to the genital ridge where they form the anlage necessary for gonadal development (GW5-6). Somatic mesodermal cells are required for both PGCs migration and the formation of a proper gonad. Once PGCs reach their destination, the somatic gonadal cells join them (around GW 7-8 in males, GW10 in females) and provide a suitable environment for survival and self-renewal until gamete differentiation {Jemc, 2011 #413}. Thus, mutations in genes regulating somatic Sertoli and Granulosa support cell function in humans are often associated with complete or partial gonadal dysgenesis in both sexes and sex reversal in males {Zarkower, 2021 #430; Knower, 2011 #418; Brunello, 2021 #399}. Other mesodermal cells, the Leydig cells, also play an important role in the testis by being the primary source of testosterone and other androgens and maintaining secondary sexual characteristics.”

    Also we have added a paragraph in the discussion to emphasize this argument:

    “We show that in the absence of Cv-c function in the testis, the mesodermal pigment cells do not form a continuous layer around the testis and the ECM surrounding the testis breaks. We also show that the interstitial gonadal cells fail to ensheath the germ cells and as a result of all these the germ cells become dispersed from the testis. These perturbations can be partially corrected by expression in the testis mesoderm of human DLC3 or Drosophila Cv-c that in both cases require a functional StART domain. Thus, our results suggest that Cv-c/DLC3 have a fundamental function on the mesodermal testis cells that has been conserved. These results indicate that, as in Drosophila, the primary cause for the gonadal dysgenesis in DLC3 human patients is due to the abnormal maintenance of the testis mesoderm cells, which include both Sertoli and Leydig cells”.

    I would also suggest that the authors highlight another potentially more important spin-off from such studies, namely that understanding of the regulation of DLC3/STARD8 genes, and what might perturb their expression/action would appear to present a whole new area for exploration in relation to testicular dysgenesis/masculinization disorders.

    Response: We have modified the last part of the discussion to introduce referee 2’s suggestion:

    “Our work points to DLC3/Cv-c as a novel gene required specifically in testis formation. Adding DLC3 to the list of genes involved in 46X,Y complete dysgenesis opens up a new avenue to analyse the molecular and cellular mechanisms behind these disorders that could help in diagnosis and the development of future treatments”.

    Reviewer #3 :

    Major comments:

    1. This study has shown the expression pattern of cv-c and the consequence of cv-c mutation on different aspects of gonad development. However, one major comment is there is no quantification of the expression levels as well as the scoring of the mutant phenotypes.
    2. In Figure 2, for instance, I recommend that the authors display the quantification of the fluorescence intensity of the cv-c expression under all circumstances (in situ hybridization as well as protein-trap based GFP expression) to better depict the differences among the male vs female gonad.

    Response: We don’t think quantifying the stainings will add much to the results. We believe that the changes performed increasing the images’ contrast and their amplification are sufficient to illustrate our statement about cv-c being expressed in testis but not in ovaries.

    1. In Figure 3, the authors show the different gonad developmental defects associated with the cv-c mutation. Specifically, the authors show that the gonad mesoderm cells are displaced with the pigment cells failing to ensheath the germ cells. In addition, the authors also suggest that there is an increased frequency of germ cell blebbing, an indication of migratory activity. However, there is no quantification of these findings. I think the authors should display a quantitative estimation of % of the mutant gonad depicting these phenotypes vs the normal gonad to have a perspective of how penetrant the phenotypes are.

    Response: As referee suggested, we have quantified bleb phenotype. The results are presented in figure 3, panel J.

    1. In Figure 4, the authors attempt to rescue the Cv-C mutation linked gonadal defects by overexpressing different Cv-C protein variants. The rescue experiments are not very clear. The graph shows the % of normal testes under different genotypic combinations. It is not very clear what the authors mean by normal (in what context)? Since the mutation results in different defects of gonad development, I think recommend that represent the rescue in terms of these defects. It would be interesting to see for instance, what happens to the blebbing or germ cell ensheathment phenoype upon rescue. How many % of testes show the rescue as compared to cv-c mutants?

    Response: The percentages are quantified considering if the testes have any germ cell outside the gonad. We have added a line to clarify this point in the figure legend: “…quantified as encapsulated gonads with all germ cells inside the testis as assessed by Fisher-test”.

    Nevertheless, we are going to quantify the number of ECM breaks and show the results in the reviewed manuscript.

    1. Did the authors try cell-specific depletion of cv-c and examined the consequence on gonad development?

    Response: cv-c mutants are embryonic lethal because of Cv-c’s widespread requirement on various embryonic tissues during development. Induction of FRT clones in the embryonic testis mesoderm was unsuccessful because of the low number of divisions during embryogenesis. We also tried to knock down cv-c expression with 3 different RNAi lines. Unfortunately, overexpression of these RNAi with different testis Gal4 drivers did not decrease cv-c mRNA levels significantly in the mesoderm or in other tissues where cv-c is expressed. Despite these experiments unsatisfactory outcome, our finding that cv-c is expressed in the testis mesoderm cells, and the fact that we can rescue the testis phenotypes by expressing Cv-c with gonadal mesodermal specific Gal4 lines supports a testis mesoderm requirement of cv-c for its gonadal function.

    1. Another major concern is the lack of mechanistic insight of cv-c. For example, how does loss of cv-c result in gonadal dysgenesis? The authors suggested that StART domains regulate via lipid binding. The authors could examine if StART domain function is dependent on lipid-mediated interactions.

    Response: We agree with the referee that the molecular characterisation of the StART-mediated GAP-independent Cv-c function we have uncovered in this work is a very interesting finding that should be addressed by future work. However, such biochemical characterisation requires a complex approach to distinguish between the already known StART function regulating the GAP activity shown before (Sotillos Scientific Reports) and the new GAP-independent function we describe in the testes that falls beyond this work.

    The central point of this manuscript is the demonstration that both DLC3/Cv-c are involved in male gonad formation, an important conserved function for both of them that had been overlooked by previous publication. Thus, DLC3 should be considered a new gene to be analysed in the future when studying gonadal dysgenesis. A second important point raised by our work is the demonstration that DLC3/Cv-c can perform RhoGAP independent functions, something that had never been described for these proteins.

    Not withstanding this, in the revised version, we have added a new supplementary figure (1) related to the StART domain-lipid interaction analysed in-silico. The in-silico model shows that the DLC3-StART domain Ω1-loop structure displays the highest frequency of interaction with the membrane. This loop is conserved in the StART domains of several other STARD proteins and seems to modulate access to the ligand binding cavity. Ω-loops play multiple roles in protein function, often related to ligand binding, stability and folding. In this context, mutations in the proximity of the Ω1-loop, like the ones carried by the patients, may have drastic effects on overall protein stability that could affect the interaction between gonadal precursor cells.

    1. Do the cv-c mutants survive to adulthood? If yes, then it would be interesting to know how the adult testis behaves in cv-c mutants. Does it result in sterility?

    Response: Unfortunately, all studied cv-c mutants are embryonic lethal.

    1. Ensheathment is required for proper germline development and defects in ensheathment can affect soma-germline communication and germline development. Germ cell ensheathment affects the proliferation of germ cells and display defective JAK/STAT signaling. It would be interesting to know if the germ cells in cv-c mutant gonad show the proliferation defect and impaired JAK/STAT signaling.

    Response: This is an interesting suggestion. JAK/STAT signalling has a male specific function that could explain why cv-c gonadal defects are male specific. We are going to study how cv-c affects STAT signalling in the male gonad. We are currently preparing stocks combining 10XSTAT::GFP reporter with cv-c mutants and preparing samples for anti-STAT labelling. We will also analyse if embryos lacking STAT activation, activate cv-c expression in the testes.

    1. I was also wondering if the authors have examined the number of germ cells in the mutant gonads.

    Response: Yes, we have counted the number of germ cells in cv-c mutants and, if anything, there are more. We initially considered that an excess of GC proliferation could be the cause of gonad disruption. However, we have discarded this hypothesis as phospho-histone 3 stainings did not show a significant increase of GC divisions. Moreover, when we blocked cell proliferation in cv-c’ mutant gonads using UAS-p21, the testes phenotype was not rescued. We are unsure what could be responsible for the slight increase of germ cells observed.

    1. In addition, I think the quality of the images should be improved.

    Response: We have changed the colours used in the confocal images and amplified the relevant regions in all panels. We thank both referees for this suggestion as these changes have improved the figure contrast.

    Minor comments:

    1. cv-c mRNA in Figure 2 panels (Fig. 2D) should be in italics.

    Response: We have changed it.

    1. There is no scale bar in Figure panels. In addition, there is no scale bar in the zoomed images in Figure 2. Scale bars should be consistently put in the all the Figures, in particular on the first panels of the Figures.

    Response: We have added scale bars to all panels.

    1. In the line 677, the manuscript says "arrowhead". There are no arrowheads but the arrows.

    Response: Corrected

    1. Please be consistent with the labels in Figure panels: Vasa is shown in capital while Eya is not.

    Response: Corrected

    1. Please be consistent with the labeling of the Figure panels: Figure 3A vs Figure 4a.

    Response: Corrected

    1. What does the asterisk signify in Figure 2? There is no mention of asterisk in the Figure 2 legend.

    Response: The meaning of the asterisk was explained in the figure legend.

    1. There is no grey channel (sagittal view) for the panels Figure 3I and J.

    Response: We have already included sagittal views in the figure.

    1. Please be thorough in labeling the genotypes in Figures. For instance, Figure 4c depict the % of normal testis in cv-c delta StART. However, the correct genotype is twi>Cv-c StART. In addition, in Figure 4c graph, cv-c mut should be cv-cGAPmut.
    2. Please be consistent with the depiction of the "START" domain of the protein throughout the manuscript. In figure 4c for instance, it is "START" in the graph while in the figure panel 4i, it is StART.
    3. In Figure 4b, it is written DLC3-GA. Did the authors mean DLC3-S993N?
    4. In line 723, it should be anti-beta catenin.

    Response: As suggested, we have unified figure labelling.

    1. The authors have shown two images to suggest that cv-c mutant gonad depict the germ cell blebbing (Figure 3I and J). I think it would be much better to put up a graph showing the number or percentage of cv-c mutant gonads displaying the germ cell blebbing than putting two images with the same information.

    Response: We have already done the quantification and added the data as a graph in figure (3J).

    1. The previous comment is also true for Figure 6H and I. In both the panels, the authors wish to show discontinuous ECM marked by Perlecan expression in cv-c mutant gonads. I think it would be better to display a score of the number of mutant gonads depicting the discontinuous ECM.

    Response: We are repeating stainings to quantify Perlecan disruption in cv-c mutants and we will display the results as a graph in figure 6.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This manuscript uses genetics, cell biology and confocal imaging to study the loss-of-function phenotypes of Cv-c, the Drosophila ortholog of DLC3. DLC3 mutation has been recently associated with male gonad dysgenesis in DSD patients. This work showed that Cv-c acts in the somatic gonadal cells to regulate male gonad development. Interestingly, the Cv-c mutant phenotypes result in testicular dysgenesis, marked by defective germ cell ensheathment and blebbing. Using different variants of the Cv-c protein and genetics based analyses, the authors further identified that the gonad development is dependent on StART domain but is independent of the GAP domain. Overall, these results should be of interest to researchers in disorders of sexual development, germ cell biology and developmental biology fields. However, at several places in order to reach the conclusions, more rigorous experiments should be performed (see revision details). In conclusion, this work has the potential but requires statistical quantification to support the central claims.

    Major comments:

    1. This study has shown the expression pattern of cv-c and the consequence of cv-c mutation on different aspects of gonad development. However, one major comment is there is no quantification of the expression levels as well as the scoring of the mutant phenotypes.
    2. In Figure 2, for instance, I recommend that the authors display the quantification of the fluorescence intensity of the cv-c expression under all circumstances (in situ hybridization as well as protein-trap based GFP expression) to better depict the differences among the male vs female gonad.
    3. In Figure 3, the authors show the different gonad developmental defects associated with the cv-c mutation. Specifically, the authors show that the gonad mesoderm cells are displaced with the pigment cells failing to ensheath the germ cells. In addition, the authors also suggest that there is an increased frequency of germ cell blebbing, an indication of migratory activity. However, there is no quantification of these findings. I think the authors should display a quantitative estimation of % of the mutant gonad depicting these phenotypes vs the normal gonad to have a perspective of how penetrant the phenotypes are.
    4. In Figure 4, the authors attempt to rescue the Cv-C mutation linked gonadal defects by overexpressing different Cv-C protein variants. The rescue experiments are not very clear. The graph shows the % of normal testes under different genotypic combinations. It is not very clear what the authors mean by normal (in what context)? Since the mutation results in different defects of gonad development, I think recommend that represent the rescue in terms of these defects. It would be interesting to see for instance, what happens to the blebbing or germ cell ensheathment phenoype upon rescue. How many % of testes show the rescue as compared to cv-c mutants?
    5. Did the authors try cell-specific depletion of cv-c and examined the consequence on gonad development?
    6. Another major concern is the lack of mechanistic insight of cv-c. For example, how does loss of cv-c result in gonadal dysgenesis? The authors suggested that StART domains regulate via lipid binding. The authors could examine if StART domain function is dependent on lipid-mediated interactions.
    7. Do the cv-c mutants survive to adulthood? If yes, then it would be interesting to know how the adult testis behaves in cv-c mutants. Does it result in sterility?
    8. Ensheathment is required for proper germline development and defects in ensheathment can affect soma-germline communication and germline development. Germ cell ensheathment affects the proliferation of germ cells and display defective JAK/STAT signaling. It would be interesting to know if the germ cells in cv-c mutant gonad show the proliferation defect and impaired JAK/STAT signaling.
    9. I was also wondering if the authors have examined the number of germ cells in the mutant gonads.
    10. In addition, I think the quality of the images should be improved.

    Minor comments:

    1. cv-c mRNA in Figure 2 panels (Fig. 2D) should be in italics.
    2. There is no scale bar in Figure panels. In addition, there is no scale bar in the zoomed images in Figure 2. Scale bars should be consistently put in the all the Figures, in particular on the first panels of the Figures.
    3. In the line 677, the manuscript says "arrowhead". There are no arrowheads but the arrows.
    4. Please be consistent with the labels in Figure panels: Vasa is shown in capital while Eya is not.
    5. Please be consistent with the labeling of the Figure panels: Figure 3A vs Figure 4a.
    6. What does the asterisk signify in Figure 2? There is no mention of asterisk in the Figure 2 legend.
    7. There is no grey channel (sagittal view) for the panels Figure 3I and J.
    8. Please be thorough in labeling the genotypes in Figures. For instance, Figure 4c depict the % of normal testis in cv-c delta StART. However, the correct genotype is twi>Cv-c StART. In addition, in Figure 4c graph, cv-c mut should be cv-cGAPmut.
    9. Please be consistent with the depiction of the "START" domain of the protein throughout the manuscript. In figure 4c for instance, it is "START" in the graph while in the figure panel 4i, it is StART.
    10. In Figure 4b, it is written DLC3-GA. Did the authors mean DLC3-S993N?
    11. In line 723, it should be anti-beta catenin.
    12. The authors have shown two images to suggest that cv-c mutant gonad depict the germ cell blebbing (Figure 3I and J). I think it would be much better to put up a graph showing the number or percentage of cv-c mutant gonads displaying the germ cell blebbing than putting two images with the same information.
    13. The previous comment is also true for Figure 6H and I. In both the panels, the authors wish to show discontinuous ECM marked by Perlecan expression in cv-c mutant gonads. I think it would be better to display a score of the number of mutant gonads depicting the discontinuous ECM.

    Significance

    The significance of this paper is the identification of a conserved protein that has a conserved function in regulating spermatogenesis. The Drosophila embryonic testis is an ideal system to study the role of cv-c in gonadogenesis. It also sets the stage for studying the potential roles of cv-c in adult testes.

    The existing published knowledge about somatic ensheathment of germ stem cells is sufficiently covered and cv-c is a new player in this process.

    The audience for this study is developmental biologists and researchers studying disorders of sex development.

    Our expertise is somatic sex identity in adult Drosophila gonads and DSD.

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    Referee #2

    Evidence, reproducibility and clarity

    This is an interesting and generally convincing study demonstrating the potential role of DLC3 (STARD8)/Cv-c in testicular development, and in particular its role in early (fetal in human) germ cell development. The genetic manipulations and associated techniques involving Drosophila appear to be state of the art, although I would not class myself as expert enough in such techniques to be able to give a truly informed opinion.

    My only issue with the present study is to how well the present experimental findings in Drosophila translate to humans. As far as I can tell the present studies show that inactivating mutations in Cv-c in Drosophila result in failure of germ cell enclosure by somatic cells into the testis, resulting in sterility. In humans, and in experimental mouse transgenic lines, it has been well established that absence of germ cells does not of itself lead to failure of testis differentiation and onward development, nor does it lead automatically to sex reversal or impairment of masculinization. For the latter to occur, there must be impairment/failure of fetal Leydig cell function such that insufficient androgen is produced to effect genital/bodywide masculinization. Obviously, this will happen if no testis forms as appears to be the case in the new human DLC3 mutant reported in the present manuscript (although detail on this is unfortunately lacking). This appears to be different to the previous published DLC3/STARD8 mutant sisters, in whom the phenotype appears to reflect failure of steroidogenesis. Is the proposal that DLC3/STARD8 plays a role in both testis differentiation and in Leydig cell function (steroidogenesis) or is this due to different DLC3 genes? I think the authors need to address these key issues in their discussion, if only to highlight that there are at present many gaps in our understanding.

    I would also suggest that the authors highlight another potentially more important spin-off from such studies, namely that understanding of the regulation of DLC3/STARD8 genes, and what might perturb their expression/action would appear to present a whole new area for exploration in relation to testicular dysgenesis/masculinization disorders.

    Significance

    This represents a significant advance in our understanding and identifies model systems in which to gain further insight.

    I would also suggest that the authors highlight another potentially more important spin-off from such studies, namely that understanding of the regulation of DLC3/STARD8 genes, and what might perturb their expression/action would appear to present a whole new area for exploration in relation to testicular dysgenesis/masculinization disorders in humans.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors have utilised Drosophila to provide evidence that a DLC3 mutation identified in 46,XY patients with male gonadal dysgenesis is likely causative for the phenotype. They demonstrated that mutation of the Drosophila ortholog of DLC3, Cv-c, was associated with defects in embryonic testis development and that the phenotype could be rescued by expression of wildtype human DLC3 but not the patient variant. DLC3/Cv-c are members of the RhoGAP family of proteins but GAP activity was not required for in the testis while mutation of the StART domain disrupted gonad morphogenesis. Mutations in this domain were associated with human gonadal dysgenesis suggesting a conservation of function during gonad development.

    Major comments:

    In general, the data support the conclusions. I cannot comment on the atomistic simulation experiment as it is outside of my expertise. I had some difficulties interpreting Figure 2 as the contrast in the colour panels made it difficult to assess the different staining patterns. I would recommend changing the blue to cyan for easier visibility. While I agree that there are some differences between Fig 2F and Fig 2G it is not simple for the non-expert to distinguish the gonadal mesoderm from the somatic mesoderm. I think the enlarged panels could do with also showing the overlap in staining, or at least a tracing of the different cell populations so that the gonadal mesoderm can be clearly defined. Please also add some scale bars to the figure.
    Figure 3 demonstrates clear differences in gonad morphology between male and female mutants but the contrast in the colour panels A-G could also be improved. Panels H-J are very clear.
    The rescue experiment in Figure 4 is clearly presented but could the DLC3 mutants in the graph (panel b) please be named similarly to the schematic proteins shown in panel a.
    I found the difference between the RhoGAP domain mutants and the StART domain mutants of Cv-c to be clearly defined, and correlate with DLC3 function. This is a very interesting result that indicates multiple molecular functions for the Cv-c /DLC family.
    The methods are well described, statistics adequate and the data well described.

    Minor Comments:

    My only suggestion for the text is to provide a more through description of the StART domain in the introduction.

    Referees cross-commenting

    Quantification of dispersed low level punctate expression levels will be very difficult to achieve and I do not believe will alter the conclusions. I agree that quantification of the percentage of gonads that display the illustrated phenotypes would be beneficial. The manuscript nicely describes a phenotype that has been defined to be due to mutation of the StART domain. Defining the mechanism of how the StART domain regulates protein function would take the study to a new level but may not be necessary for publication of the findings.

    Significance

    The significance of this work is two-fold.

    1. A demonstration that Cv-c and DLC3 have similar functions in regulation of gonad morphogenesis, and that the patient mutations almost certainly correlate with the observed phenotypes.
    2. An intriguing demonstration that this family of proteins is not simply functioning as RhoGAPs, and that there is a specific roles for the StART domain in male gonad development.

    DLC3 has already been shown to rescue Malpighian tubule defects associated with Cv-c mutants so the functional equivalence of these proteins has already been established. The novelty of the present study relates to a demonstration that the StART domain is specifically required for male gonad morphogenesis, and the utility of experiments in Drosophila to assist in associating a human mutation with a clinical phenotype.

    This work should find a willing audience from clinical geneticists as an example of how model organism genetics can assist genetic diagnoses. It will also be of great interest to the field of reproductive biologists who wish to understand how the sex determination pathways are resolved by differential tissue development. The obvious next steps are to determine why mutations in Cv-c only affect male gonads and not females, and what is the specific role of the StART domain in this process.
    My field of study has focussed on adult gonad development and regeneration, and I found the manuscript presented in a style that was simple to follow with my suggestion that contrast of some of the colour panels could be improved.

  5. Version published to 10.7554/elife.82343 on eLife
  6. Version published to 10.1101/2022.07.28.501838v1 on bioRxiv