Author Response
The following is the authors’ response to the original reviews.
Response to reviews
We would like to extend our thanks to the reviewers who took the time to carefully read our paper and provide thoughtful insights and suggestions on how to strengthen our conclusions. All reviewers agreed that our study presented strong data supporting a role for triglyceride lipase brummer (bmm) in regulating testis lipid droplets and spermatogenesis in Drosophila, and that our findings advance our understanding of lipid biology during sperm development. Reviewers made several helpful suggestions on how to strengthen our manuscript even further. Below, we outline how we revised our manuscript in response to reviewer comments to ensure we clearly communicate our data and conclusions with readers, and properly contextualize our findings.
REVIEWER 1
In this study, the authors investigate the role of triglycerides in spermatogenesis. This work is based on their previous study (PMID: 31961851) on triglyceride sex differences in which they showed that somatic testicular cells play a role in whole body triglyceride homeostasis. In the current study, they show that lipid droplets (LDs) are significantly higher in the stem and progenitor cell (pre-meiotic) zone of the adult testis than in the meiotic spermatocyte stages. The distribution of LDs anti-correlates with the expression of the triglyceride lipase Brummer (Bmm), which has higher expression in spermatocytes than early germline stages. Analysis of a bmm mutant (bmm[1]) - a P-element insertion that is likely a hypomorphic - and its revertant (bmm[rev]) as a control shows that bmm acts autonomously in the germline to regulate LDs. In particular, the number of LDs is significantly higher in spermatocytes from bmm[1] mutants than from bmm[rev] controls. Testes from males with global loss of bmm (bmm[1]) are shorter than controls and have fewer differentiated spermatids. The zone of bam expression, typically close to the niche/hub in WT, is now many cell diameters away from the hub in bmm[1] mutants. There is an increase in the number of GSCs in bmm[1] homozygotes, but this phenotype is probably due to the enlarged hub. However, clonal analyses of GSCs lacking bmm indicate that a greater percentage of the GSC pool is composed of bmm[1]-mutant clones than of bmm[rev]-clones. This suggests that loss of bmm could impart a competitive advantage to GSCs, but this is not explored in greater detail. Despite the increase in number of GSCs that are bmm[1]-mutant clones, there is a significant reduction in the number of bmm[1]-mutant spermatocyte and post-meiotic clones. This suggests that fewer bmm[1]-mutant germ cells differentiate than controls. To gain insights into triglyceride homeostasis in the absence of bmm, they perform mass spec-based lipidomic profiling. Analyses of these data support their model that triglycerides are the class of lipid most affected by loss of bmm, supporting their model that excess triglycerides are the cause of spermatogenetic defects in bmm[1]. Consistent with their model, a double mutant of bmm[1] and a diacylglycerol Oacyltransferase 1 called midway (mdy) reverts the bmm-mutant germline phenotypes.
There are numerous strengths of this paper. First, the authors report rigorous measurements and statistical analyses throughout the study. Second, the authors ulize robust genetic analyses with loss-of-function mutants and lineage-specific knockdown. Third, they demonstrate the appropriate use of controls and markers. Fourth, they show rigorous lipidomic profiling. Lastly, their conclusions are appropriate for the results. In other words, they don't overstate the results.
We thank the Reviewer for their positive assessment of our paper.
There are a few weaknesses. Although the results support the germline autonomous role of bmm in spermatogenesis, one potential caveat that the mdy rescue was global, i.e., in both somatic and germline lineages. The authors did not recover somatic bmm clones, suggesting that bmm may be required for somatic stem self-renewal and/or niche residency. While this is beyond the scope of this paper, it is possible that somatic bmm does impact germline differentiation in a global bmm mutant.
In the revised manuscript, we made several changes to address these points.
- We now clearly state when we used global versus germline-only loss of mdy to rescue bmm mutant phenotypes in the testis.
“Notably, at least some of the effects of global loss of mdy on bmm1 males can be attributed to the germline:
RNAi-mediated knockdown of mdy in the germline of bmm1 males partially rescued the defects in testis size (Figure 4I; Kruskal-Wallis rank sum test with Dunn’s multiple comparison test) and GSC variance (Figure S5J; p=4.5 x 10-5 and 8.2 x 10-3 by F-test from the GAL4- and UAS-only crosses, respectively).”
“Importantly, testes isolated from males with global loss of both bmm and mdy (mdyQX25/k03902;bmm1) had fewer LD than testes dissected from bmm1 males (Figures 5D, S5I; one-way ANOVA with Tukey multiple comparison test).”
- We also discuss the possibility that somatic bmm may play a role in germline differentiation in a global bmm mutant, and present phenotypic data on somatic bmm1 clones.
“We also reveal a potential non-cell-autonomous role for somatic bmm. While there was no difference in the ratio of Zd-1-positive cells between homozygous clones and heterozygous clones in animals carrying the bmm1 or bmmrev alleles at 14 days post clone induction (Figure S4O; Kruskal-Wallis rank sum test), the distance from the hub to the Zd-1 positive clones reside was significantly decreased in bmm1 homozygous clones (Figure S4P; Kruskal-Wallis rank sum test). Together, these data indicate bmm may play a cell-autonomous role in germline cells, and potentially a non-cell-autonomous role in somatic cells, to regulate spermatogenesis.”
- Finally, we clarify that we were unable to assess somatic LD. Specifically, this was a technical issue as the dye we use to visualize testis LD is incompatible with staining protocols to identify somatic cells. As a result, we were unable to count LD in somatic clones with confidence.
“While we were unable to assess LD in bmm1 somatic clones, our data when taken together reveals a previously unrecognized cell-autonomous role for bmm as a regulator of testis LD in germline cells.”
Regarding data presentation, I have a minor point about Fig. 3L: why aren't all data shown as box plots (only Day 14 bmm[rev] does).
In our revised manuscript Figure 4L does present a boxplot across all genotypes and times; the appearance of ‘no boxes’ is simply due to the large number of datapoints with a value of zero, which compress the box near the X-axis.
Finally, the authors provide a detailed pseudotime analysis of snRNA-seq of the testis in Fig. S2A-D, but this analysis is not sufficiently discussed in the text.
In the revised manuscript we added text to describe our pseudotime analysis of single-cell RNA seq data in more detail.
“Using pseudotime analysis, we arranged the germline (Figure S2A) and the somatic cells (Figure S2B) based on their annotated developmental trajectory. The expression pattern of bmm in the germline matched our observation with bmm-GFP reporter (Figure S2C). While levels of the bmm-GFP reporter were lower in somatic cells, single-cell RNA sequencing data identified bmm expression in the somatic lineage that was higher in cells at later stages of development (Figure S2D). Additional neutral lipid- and lipid droplet-associated genes such as lipid storage droplet-2, Seipin, Lipin, and midway also showed differential regulation during differentiation (Figure S2C, S2D). Combined with our data on the location of testis LD, these data suggest that bmm upregulation in both somatic and germline cells during differentiation corresponds to the downregulation of testis LD. Supporting this, germline GFP levels were negatively correlated with testis LD in bmm-GFP flies (Figure 2A, 2C), suggesting regions with higher bmm expression had fewer LD.”
Overall, the many strengths of this paper outweigh the relatively minor weaknesses. The rigorously quantified results support the major aim that appropriate regulation of triglycerides are needed in a germline cell-autonomous manner for spermatogenesis.
This paper should have a positive impact on the field. First and foremost, there is limited knowledge about the role of lipid metabolism in spermatogenesis. The lipidomic data will be useful to researchers in the field who study various lipid species. Going forward, it will be very interesting to determine what triglycerides regulate in germline biology. In other words, what functions/pathways/processes in germ cells are negatively impacted by elevated triglycerides. And as the authors point out in the discussion, it will be important to determine what regulates bmm expression such that bmm is higher in later stages of germline differentiation.
We agree with the reviewer about the many interesting future directions for this project. We added a model figure in the revised manuscript to visualize our findings and highlight remaining questions about how bmm and triglycerides support normal spermatogenesis in Drosophila (Fig. 6).
REVIEWER 2
Summary:
Here, the authors show that neutral lipids play a role in spermatogenesis. Neutral lipids are components of lipid droplets, which are known to maintain lipid homeostasis, and to be involved in non-gonadal differentiation, survival, and energy. Lipid droplets are present in the testis in mice and Drosophila, but not much is known about the role of lipid droplets during spermatogenesis. The authors show that lipid droplets are present in early differentiating germ cells, and absent in spermatocytes. They further show a cell autonomous role for the lipase brummer in regulating lipid droplets and, in turn, spermatogenesis in the Drosophila testis. The data presented show that a relationship between lipid metabolism and spermatogenesis is congruous in mammals and flies, supporting Drosophila spermatogenesis as an effective model to uncover the role lipid droplets play in the testis.
We thank the Reviewer for their positive assessment of our paper.
Strengths and weaknesses:
The authors do a commendably thorough characterization of where lipid droplets are detected in normal testes: located in young somatic cells, and early differentiating germ cells. They use multiple control backgrounds in their analysis, including w[1118], Canton S, and Oregon R, which adds rigor to their interpretations. The authors employ markers that identify which lipid droplets are in somatic cells, and which are in germ cells. The authors use these markers to present measured distances of somatic and germ cell-derived lipid droplets from the hub. Because they can also measure the distance of somatic and germ cells with age-specific markers from the hub, these results allow the authors to correlate position of lipid droplets with the age of cells in which they are present. This analysis is clearly shown and well quantified.
The quantification of lipid droplet distance from the hub is applied well in comparing brummer mutant testes to wild type controls. The authors measure the number of lipid droplets of specific diafteters, and the spatial distribution of lipid droplets as a function of distance from the hub. These measurements quantitatively support their findings that lipid droplets are present in an expanded population of cells further from the hub in brummer mutants. The authors further quantify lipid droplets in germline clones of specified ages; the quantitative analysis here is displayed clearly, and supports a cell autonomous role for brummer in regulating lipid droplets in spermatocytes.
Data examining testis size and number of spermatids in brummer mutants clearly indicates the importance of regulating lipid droplets to spermatogenesis. The authors show beautiful images supported by rigorous quantification supporting their findings that brummer mutants have both smaller testes with fewer spermatids at both 29 and 25C. There is also significant data supporting defects in testis size for 14-day-old brummer mutant animals compared to controls. The comparison of number of spermatids at this age is not significant, which does not detract from the story but does not support sperm development defects specifically caused by brummer loss at 14 days. Their analysis clearly shows an expanded region beyond the testis apex that includes younger germ cells, supporting a role for lipid droplets influencing germ cell differentiation during spermatogenesis.
We thank the reviewer for pointing out this inaccuracy in our manuscript. In the revised manuscript we chose more precise language to describe defects in 14-day-old bmm mutants:
“Defects in testis size were also observed at 14-day post eclosion; suggesting testis size defects persist later into the life course (Figure S4C; Welch two-sample t-test). In contrast, the number of spermatid bundles per testis was not significantly different between bmm1 and bmmrev males at this age (Figure S4D; Welch two-sample ttest), potentially due to a large decrease in the number of spermatid bundles in 14-day-old bmmrev males (Figure 4C, S4D).”
The authors present a series of data exploring a cell autonomous role for brummer in the germline, including clonal analysis and tissue specific manipulations. The clonal data indicating increased lipid droplets in spermatocyte clones, and a higher proportion of brummer mutant GSCs at the hub are convincing and supported by quantitation. The authors also show a tissue specific rescue of the brummer testis size phenotype by knocking down mdy specifically in germ cells, which is also supported by statistically significant quantitation. The authors present data examining the number of spermatocyte and post-meiotic clones 14 days aeer clonal induction. While data they present is significant with a 95% confidence interval and a p value of 0.0496, its significance is not as robust as other values reported in the study, and it is unclear how much information can be gained from that specific result.
We thank the reviewer for raising this point. In the revised manuscript we displayed the p-value clearly in the text and on the figure to ensure our statistical output is clear for readers to evaluate our conclusions regarding bmm mutant clones 14 days after clone induction. We also state that the finding should be reproduced by others given that the statistical significance of this result was not as strong as our other data.
“Because we observed significantly fewer bmm1 spermatocyte and spermatid clones at 14 days after clone induction (Figure 4K,4L; p = 0.0496, Kruskal-Wallis rank sum test), these effects on germline development may represent a cell-autonomous role in regulating spermatogenesis for bmm in this cell type. Given that the statistical significance of this finding was not as strong as for our other data, future studies should repeat this experiment with more samples.”
The authors do a beautiful job of validating where they detect brummer-GFP by presenting their own pseudotime analysis of publicly available single cell RNA sequencing data. Their data is presented very clearly, and supports expression of brummer in older somatic and germline cells of the age when lipid droplets are normally not detected. The authors also present a thorough lipidomic analysis of animals lacking brummer to identify triglycerides as an important lipid droplet component regulating spermatogenesis.
Impact:
The authors present data supporting the broad significance of their findings across phyla. This data represents a key strength of this manuscript. The authors show that loss of a conserved triglyceride lipase impacts testis development and spermatogenesis, and that these impacts can be rescued by supplementing diet with medium chain triglycerides. The authors point out that these findings represent a biological similarity between Drosophila and mice, supporting the relevance of the Drosophila testis as a model for understanding the role of lipid droplets in spermatogenesis. The connection buttresses the relevance of these findings and this model to a broad scientific community.
We thank the Reviewer very much for their positive assessment of our paper!
REVIEWER 3
In this manuscript, Chao et al seek to understand the role of brummer, a triglyceride lipase, in the Drosophila testis. They show that Brummer regulates lipid droplet degradation during differentiation of germ and somatic cells, and that this process is essential for normal development to progress. These findings are interesting and novel, and contribute to a growing realisation that lipid biology is important for differentiation.
We thank the Reviewer for their positive comments about our manuscript.
Major comments:
- The data in Figs 1 and 2, while helpful in setting the scene, do not add much to what was previously shown by the same group, namely that lipid droplets are present in both early germ cells and early somatic cells in the testis, and that Bmm regulates their degradation (PMID: 31961851). Measuring the distance of lipid droplets from the hub, while helpful in quantifying what is apparent, that only stem and early differentiated stages have lipid droplets, is not as informative as the way data are presented later (Fig. 2I), where droplets in specific stages are measured. Much of this could be condensed without much overall loss to the manuscript.
We thank the reviewer for this comment. In our revised manuscript we edited the first part of the paper while still preserving the detailed characterization that builds upon our previous paper.
- It would be important to show images of the clones from which the data in Fig. 2I are generated. The main argument is that Bmm regulates lipid droplets in a cell autonomous manner; these data are the strongest argument in support of this and should be emphasised at the expense of full animal mutants (which could be moved to supplementary data).
We thank the reviewer for this comment. In the revised manuscript we added a figure showing lipid droplets in control and bmm mutant spermatocyte clones in Fig. 3A, 3B with a quantification of this data in Figure 3C.
Similarly, the title of Fig. S2 ("brummer regulates lipid droplets in a cell autonomous manner") should be changed as the figure has no experiments with cell (or cell-type)-specific knockdowns/mutants. This figure does show changes in lipid droplets in both lineages in bmm mutants, so an appropriate title could be "brummer regulates lipid droplets in both germ and soma".
We thank the reviewer for this comment, we adjusted the Figure 2 legend title in the revised manuscript to “brummer regulates lipid droplets in both germline and somatic cells of the testis”.
- Interestingly, the clonal data show that bmm is dispensable in germ cells until spermatocyte stages, as no increase in lipid droplet number is seen until then. This should be more clearly stated, as it indicates that the important function of Bmm is to degrade lipid droplets at the transition from spermatogonial to spermatocyte stages. This is consistent with the phenotypes observed in which late stage germ cells are reduced or missing. However, the effect on niche retention of the mutant GSCs at the expense of neighbouring wildtype GSCs is hard to explain. Are lipid droplets in mutant GSCs larger than in control? Is there any discernible effect of bmm mutation on lipids in GSCs? Additionally, bam expression is delayed, suggesting that bmm may have roles on cell fate in earlier stages than its roles that can be detected on lipid droplets.
We thank the reviewer for this comment. We included more text in the revised manuscript to clarify the key role bmm plays in regulating lipid droplets at the spermatogonia-spermatocyte transition.
“Because we observed no significant effect of cell-autonomous bmm loss on LD at any other stage of germline development (Figure 3C), this suggests bmm function is not required to regulate LD at early stages of germ cell development. Instead, our data suggests bmm plays a role in regulating LD at the spermatogonia-spermatocyte transition.”
We also added more detail to our description of how bmm affects lipid droplets in cells at the earliest stages of germline development.
“Given that we detected no effect of cell-autonomous bmm loss on the number of GSC LD (Fig. 3C), more work will be needed to understand how bmm regulates GSC at a stage prior to its effects on LD number.”
- The bmm loss-of-function phenotype could be better described. Some of the data is glossed over with little description in the text (see for example the reference to Fig. 3A-C). For instance, in the discussion, the text states "loss of bmm delays germline differentiation leading to an accumulation of early-stage germ cells" (p13, l.25960). However, this accumulation has not been clearly shown, or at least described in the manuscript. Most of the data show a reduction (or almost complete absence) of differentiated cell types. This could indeed be due to delayed differentiation, or alternatively to a block in differentiation or to death of the differentiated cells. The clonal data presented show a decrease in the number of cells recovered, but do not allow inferences as to the timing of differentiation, making it hard to distinguish between the various possibilities for the lack of differentiated spermatids. Apart from data showing that GSCs are more likely to remain at the niche, no further data are shown to support the fact that mutant germ cells accumulate in early stages. While additional experiments could help resolve some of these issues, much of this could also be resolved by tempering the conclusions drawn in the text.
We thank the reviewer for these comments. In the revised manuscript we temper our conclusions regarding bmm’s precise role in spermatogenesis by discussing different mechanisms (e.g. differentiation or death) that could lead to the phenotypes we observe.
“This regulation is important for sperm development, as our data indicates that loss of bmm causes a decrease in the number of differentiated cell types. This reduction in differentiated cell types may be attributed to a delay in differentiation, a block in differentiation, or to a loss of differentiated cells through cell death. Future studies will therefore be essential to resolve why bmm loss causes a reduction in differentiated cell types.”
- In the discussion (p.14, l-273 onwards), the authors suggest that products of triglyceride breakdown are important for spermatogenesis. However, an alternative interpretation of the results presented here (especially those using the midway mutant) could be that triglycerides impede normal differentiation directly. Indeed, preventing the cells' ability to produce triglycerides in the first place can rescue many of the defects observed. A better discussion of these results with a model for the function of triglycerides and their by-products would be a great improvement to this manuscript.
We thank the reviewer for this comment. To ensure our data is clearly communicated with readers, we added a model to the paper suggesting how triglyceride and its by-products influence spermatogenesis (Fig. 6) and text to clarify that triglyceride could potentially impeded differentiation.
“It will also be important to determine whether it is the loss of metabolites produced by bmm’s enzymatic action, or an increase in triglycerides, that leads to the reduction in differentiated cell types during spermatogenesis. Together, these experiments will provide critical insight into how triglyceride stored within testis LD contributes to overall cellular lipid metabolism during spermatogenesis.”
Together, these changes will strengthen our overall finding that bmm-mediated regulation of testis triglyceride is important for normal sperm development. Because our findings in flies align with and extend data from rodent models, the developmental mechanisms we uncovered about how triglyceride lipase bmm regulates testis lipid droplets and sperm development will likely operate in other species.
Reviewer #1 (Recommendations For The Authors):
I have a minor concern about methodology: how were spermatocytes identified? I ask because data in Figure 3 indicate that there is a significant delay in germline differentiation in the bmm[1] mutant, with relatively smaller germ cells throughout the apical half of the testis. Typical large spermatocyte-like cells are not clearly obvious to me in Fig. 3.
We thank the Reviewer for suggesting we add more clarity to how we identified spermatocytes. We state in the revised manuscript how we identify spermatocytes:
“Cells in the testis region occupied by primary spermatocytes were identified by their large cell size and decondensed chromosome staining occupying three nuclear domains [120].”
Also, we note that while it is difficult to see where the bmm1 testis have spermatocytes in Fig. 4E, this is due to the large number of early-stage cells in this close-up image. The spermatocytes can be more easily seen in Fig. 4I and 4I’ when the whole testis is included in the image.
Reviewer #2 (Recommendations For The Authors):
• Lines 197-198 mention "Boule-positive area," "individualization complexes," and "waste bags." It would be helpful to the reader to explain what these measurements are to help contextualize the data shown related to these statements.
We thank the Reviewer for this comment. We added the following text to the revised manuscript:
“Because Boule-positive area, individualization complexes, and waste bags are all markers for later stages in sperm development, these data indicate the loss of bmm causes a reduction in differentiated cell types.”
• Line 162 states a defect in sperm development observed in 14-day-old bmm[1] males, but the data presented in Figure S3D does not show a significant difference. The words "sperm development" should be removed from this sentence.
We thank the Reviewer for pointing out this inaccurate statement. We fixed the statement as follows in the revised manuscript:
“Defects in testis size were also observed at 14-day post eclosion; suggesting testis size defects persist later into the life course (Figure S4C; Welch two-sample t-test). In contrast, the number of spermatid bundles per testis was not significantly different between bmm1 and bmmrev males at this age (Figure S4D; Welch two-sample ttest), potentially due to a large decrease in the number of spermatid bundles in 14-day-old bmmrev males (Figure 4C, S4D).”
• Line 294 has a typo: "regulating" should likely be "regulated"
We thank the Reviewer for pointing out this mistake, which we corrected.
• Line 456 should include the length of time for heat shock
We thank the Reviewer for pointing out this omission. We now include these details:
“Adult males were collected at 3-5 days post-eclosion and heat-shocked three times at 37°C for 30 min followed by a 10 min rest period at room temperature between heat shocks.”
• Methods section beginning on Line 442 might include an explanation of how hub area was quantified.
We thank the Reviewer for this suggestion. We now include the following information:
“Hub size was measured by quantifying FasIII-positive area of the testis.”
• Figure 1 legend could benefit from adding a statement on how spermatocytes (arrowheads) were identified
We thank the Reviewer for this suggestion, we now refer the reader to the more detailed description in the methods section.
• Figure 2A should present the merged panel in A' first. The legend states that Panel A shows Lipid Droplets, but LipidTox is not shown until A'.
We thank the Reviewer for this suggestion, we now clarify that the text refers to panels A-A''''.
• Figure 2I would benefit from a key, to emphasize that these are individual cell clones, highlighting the idea of cell autonomous effects of bmm in the spermatocytes. Showing example images of spermatocyte clones with increased lipid droplets could also emphasize this result. The legend for this panel should note the statistical test done to confirm significance in the SC result.
We agree with the Reviewer and have added images of the LD in bmm1 spermatocyte clones in Figure 3B, and the quantification in Figure 3C. We explicitly state the significance of this result and the statistical test in Figure 3 legend.
• In Figure 3, the cell autonomous data clearly indicates that there are higher proportions of bmm mutant GSCs occupying the hub compared to control GSCs. It could be worth stating whether this observation indicates an increased ability of bmm mutant GSCs to compete for occupying space at the hub.
We thank the Reviewer for pointing out this potential implication of our data, which we acknowledge in the revised version of our manuscript:
“Future studies will also need to confirm whether bmm1 mutant GSCs show an increased ability to occupy space at the hub.”
• In Figure 4, I suggest changing the title of Panel B to "Proportion of significant species in each lipid class" for clarity.
We made this change in the Figure 5 legend (Figure 5 is the corresponding figure in the revised manuscript).
• It could be valuable to quantify the number of spermatids in the germline specific mdy knockdown, which would lend additional support to a cell autonomous requirement for bmm in spermatogenesis
We added a sentence to the revised manuscript recognizing that this is an interesting experiment for studies on the role of germline triglyceride in promoting spermatogenesis.
“While future studies will need to test whether germline-specific loss of mdy also rescues spermatid number defects in bmm1 males, our data suggest bmm-mediated regulation of testis triglyceride plays a previously unrecognized role in regulating sperm development.”
Reviewer #3 (Recommendations For The Authors):
- bmm-GFP does not show expression in somatic cells yet previous work by the same group has shown a requirement for bmm in the testis soma using C587-Gal4.
We thank the Reviewer for raising this issue. While the reporter shows low GFP expression in the somatic cells, the single-cell RNA sequencing data we analyze suggests bmm is expressed in these cells. We address this issue in the revised manuscript as follows:
“While levels of the bmm-GFP reporter were lower in somatic cells, single-cell RNA sequencing data identified bmm expression in the somatic lineage that was higher in cells at later stages of development (Figure S2D).”
- p.11 l.200-202 "Because we recovered fewer bmm1 spermatocyte and spermatid clones 14 days after clone induction (Figure 3K,3L; Kruskal-Wallis rank sum test), this effect on germline development represents a cell-autonomous role for bmm." This sentence should be rephrased as the phenotype could be a combination of autonomous roles within the germline and non-autonomous roles in supporting cyst cells.
“We also reveal a potential non cell-autonomous role for somatic bmm. While there was no difference in the ratio of Zd-1-positive cells between homozygous clones and heterozygous clones in animals carrying the bmm1 or bmmrev alleles at 14 days post clone induction (Figure S4O; Kruskal-Wallis rank sum test), the distance from the hub to the Zd-1 positive clones reside was significantly decreased in bmm1 homozygous clones (Figure S4P; Kruskal-Wallis rank sum test). Together, these data indicate bmm may play a cell-autonomous role in germline cells, and potentially a non-cell-autonomous role in somatic cells, to regulate spermatogenesis.”
- The labelling in Fig. 3 is confusing - presumably the graph in 3C refers to spermatid bundles [this comment applies to other figures showing spermatid bundle numbers], not individual spermatids, while the graph in 3G refers to the proportion of the total GSC pool that is contained within the clone. The data in Fig. 3C are not described in the main text.
We adjusted the confusing labelling to ‘spermatid bundles’ from ‘number of spermatids’, as suggested. We also changed the title of panel Fig. 3G (now 4G) as suggested and men5oned Fig. 3C (now Fig. 4C) in the text.
- On p.9, comments are speculative or seek to draw comparisons with the broader literature and would seem to belong more to the discussion (eg "our data suggests flies are a good model to study how bmm/ATGL influences sperm development" - also there is a typo, it should be "suggest").
We thank the Reviewer for raising concern about our speculative statement; we changed the text as follows in the revised manuscript:
“This identifies similarities between flies and mice in fertility-related phenotypes associated with whole-body loss of bmm/ATGL.”
- The length of the heat shocks used for clone induction should be specified in the methods (rather than just the period in between heat shocks).
We now include more information on clone induction:
“Adult males were collected at 3-5 days post-eclosion and heat-shocked three times at 37°C for 30 min followed by a 10 min rest period at room temperature between heat shocks. Amer heat-shock, the flies were incubated at room temperature until dissection.”
- p.8 l.132 "bmm-GFP accurately reproduces changes to bmm mRNA levels". This sentence should be rephrased.
We thank the Reviewer for this comment and rephrased the sentence:
“We first examined bmm expression in the testis by isolating this organ from flies carrying a bmm promoter driven GFP transgene (bmm-GFP) that recapitulates many aspects of bmm mRNA regulation [77].”
- p.9 l.172 "we used germline-specific marker" should read "we used an antibody against the germline-specific marker".
We corrected this inaccurate statement in our revised manuscript.
- p.10 several lines, "GSC" should be "GSCs".
We corrected this inaccurate use of GSC in our revised manuscript.
- p.13 l.247 should read "variance in GSC numbers".
Thank you, this error was fixed.
