Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this manuscript, Azlan et al. identified a novel maternal factor called Sakura that is required for proper oogenesis in Drosophila. They showed that Sakura is specifically expressed in the female germline cells. Consistent with its expression pattern, Sakura functioned autonomously in germline cells to ensure proper oogenesis. In Sakura KO flies, germline cells were lost during early oogenesis and often became tumorous before degenerating by apoptosis. In these tumorous germ cells, piRNA production was defective and many transposons were derepressed. Interestingly, Smad signaling, a critical signaling pathway for GSC maintenance, was abolished in sakura KO germline stem cells, resulting in ectopic expression of Bam in whole germline cells in the tumorous germline. A recent study reported that Bam acts together with the deubiquitinase Otu to stabilize Cyc A. In the absence of sakura, Cyc A was upregulated in tumorous germline cells in the germarium. Furthermore, the authors showed that Sakura co-immunoprecipitated Otu in ovarian extracts. A series of in vitro assays suggested that the Otu (1-339 aa) and Sakura (1-49 aa) are sufficient for their direct interaction. Finally, the authors demonstrated that the loss of otu phenocopies the loss of sakura, supporting their idea that Sakura plays a role in germ cell maintenance and differentiation through interaction with Otu during oogenesis.
Strengths:
To my knowledge, this is the first characterization of the role of CG14545 genes. Each experiment seems to be well-designed and adequately controlled.
Weaknesses:
However, the conclusions from each experiment are somewhat separate, and the functional relationships between Sakura's functions are not well established. In other words, although the loss of Sakura in the germline causes pleiotropic effects, the cause-and-effect relationships between the individual defects remain unclear.
Reviewer #2 (Public review):
In this study, the authors identified CG14545 (and named it Sakura), as a key gene essential for Drosophila oogenesis. Genetic analyses revealed that Sakura is vital for both oogenesis progression and ultimate female fertility, playing a central role in the renewal and differentiation of germ stem cells (GSC).
The absence of Sakura disrupts the Dpp/BMP signaling pathway, resulting in abnormal bam gene expression, which impairs GSC differentiation and leads to GSC loss. Additionally, Sakura is critical for maintaining normal levels of piRNAs. Also, the authors convincingly demonstrate that Sakura physically interacts with Otu, identifying the specific domains necessary for this interaction, suggesting a cooperative role in germline regulation. Importantly, the loss of otu produces similar defects to those observed in Sakura mutants, highlighting their functional collaboration.
The authors provide compelling evidence that Sakura is a critical regulator of germ cell fate, maintenance, and differentiation in Drosophila. This regulatory role is mediated through the modulation of pMad and Bam expression. However, the phenotypes observed in the germarium appear to stem from reduced pMad levels, which subsequently trigger premature and ectopic expression of Bam. This aberrant Bam expression could lead to increased CycA levels and altered transcriptional regulation, impacting piRNA expression. Given Sakura's role in pMad expression, it would be insightful to investigate whether overexpression of Mad or pMad could mitigate these phenotypic defects (UAS-Mad line is available at Bloomington Drosophila Stock Center).
As suggested reviewer 1, we tested whether overexpression of Mad could rescue or mitigate the loss of sakura phenotypic defects, by using nos-Gal4-VP16 > UASp-Mad-GFP in the background of sakuranull. As shown in Fig S11, we did not observe any mitigation of defects.
Then, we also tested whether expressing a constitutive active form of Tkv, by using UAS-Dcr2, NGT-Gal4 > UASp-tkv.Q235D in the background of sakuraRNAi. As shown in Fig S12, we did not observe any mitigation of defects by this approach either.
A major concern is the overstated role of Sakura in regulating Orb. The data does not reveal mislocalized Orb; rather, a mislocalized oocyte and cytoskeletal breakdown, which may be secondary consequences of defects in oocyte polarity and structure rather than direct misregulation of Orb. The conclusion that Sakura is necessary for Orb localization is not supported by the data. Orb still localizes to the oocyte until about stage 6. In the later stage, it looks like the cytoskeleton is broken down and the oocyte is not positioned properly, however, there is still Orb localization in the ~8-stage egg chamber in the oocyte. This phenotype points towards a defect in the transport of Orb and possibly all other factors that need to localize to the oocyte due to cytoskeletal breakdown, not Orb regulation directly. While this result is very interesting it needs further evaluation on the underlying mechanism. For example, the decrease in E-cadherin levels leads to a similar phenotype and Bam is known to regulate E-cadherin expression. Is Bam expressed in these later knockdowns?
We examined Bam and DE-Cadherin expression in later RNAi knockdowns driven by ToskGal4. As shown in Fig S9, Bam was not expressed in these later knockdowns compared with controls. DE-Cadherin staining suggested a disorganized structure in late-stage egg chambers.
We agree that we overstated a role of Sakura in regulating Orb in the initial manuscript. We changed the text to avoid overstating.
The manuscript would benefit from a more balanced interpretation of the data concerning Sakura's role in Orb regulation. Furthermore, a more expanded discussion on Sakura's potential role in pMad regulation is needed. For example, since Otu and Bam are involved in translational regulation, do the authors think that Mad is not translated and therefore it is the reason for less pMad? Currently the discussion presents just a summary of the results and not an extension of possible interpretation discussed in context of present literature.
We changed the text to avoid overstating a role of Sakura in regulating Orb localization.
Based on our newly added results showing that transgenic overexpression of Mad could not rescue or mitigate the phenotypic defects of sakuranull mutant (Fig S11), we do not think the reason for less pMad is less translation of Mad.
Reviewer #3 (Public review):
In this very thorough study, the authors characterize the function of a novel Drosophila gene, which they name Sakura. They start with the observation that sakura expression is predicted to be highly enriched in the ovary and they generate an anti-sakura antibody, a line with a GFP-tagged sakura transgene, and a sakura null allele to investigate sakura localization and function directly. They confirm the prediction that it is primarily expressed in the ovary and, specifically, that it is expressed in germ cells, and find that about 2/3 of the mutants lack germ cells completely and the remaining have tumorous ovaries. Further investigation reveals that Sakura is required for piRNA-mediated repression of transposons in germ cells. They also find evidence that sakura is important for germ cell specification during development and germline stem cell maintenance during adulthood. However, despite the role of sakura in maintaining germline stem cells, they find that sakura mutant germ cells also fail to differentiate properly such that mutant germline stem cell clones have an increased number of "GSC-like" cells. They attribute this phenotype to a failure in the repression of Bam by dpp signaling. Lastly, they demonstrate that sakura physically interacts with otu and that sakura and otu mutants have similar germ cell phenotypes. Overall, this study helps to advance the field by providing a characterization of a novel gene that is required for oogenesis. The data are generally high-quality and the new lines and reagents they generated will be useful for the field. However, there are some weaknesses and I would recommend that they address the comments in the Recommendations for the authors section below.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
General Comments:
(1) The gene nomenclature: As mentioned in the text, Sakura means cherry blossom and is one of the national flowers of Japan. I am not sure whether the phenotype of the CG14545 mutant is related to Sakura or not. I would like to suggest the authors reconsider the naming.
The striking phenotype of sakura mutant is tumorous and germless ovarioles. The tumorous phenotype, exhibiting lots of round fusome in germarium visualized by anti-Hts staining, looks like cherry blossom blooming to us. Also, the germless phenotype reminds us falling of the cherry blossom, especially considering that the ratio of tumorous phenotype decreases and that of germless decreases over fly age. Furthermore, “Sakura” symbolizes birth and renewal in Japanese culture (the last author of this manuscript is Japanese). Our findings indicated that the gene sakura is involved in regulation of renewal and differentiation of GSCs (which leads to birth). These are the reasons for the naming, which we would like to keep.
(2) In many of the microscopic photographs in the figures, especially for the merged confocal images, the resolution looks low, and the images appear blurred, making it difficult to judge the authors' claims. Also, the Alpha Fold structure in Figure 10A requires higher contrast images. The magnification of the images is often inadequate (e.g. Figures 3A, 3B, 5E, 7A, etc). The authors should take high-magnification images separately for the germarium and several different stages of the egg chambers and lay out the figures.
We are very sorry for the low-resolution images. This was caused when the original PDF file with high-resolution images was compressed in order to meet the small file size limit in the eLife submission portal. In the revised submission, we used high-resolution images.
Specific Comments
(1) How Sakura can cooperate with Otu remains unanswered. Sakura does not regulate deubiquitinase activity in vitro. Both sakura and otu appear to be involved in the Dpp-Smad signaling pathway and in the spatial control of Bam expression in the germarium, whereas Otu has been reported to act in concert with Bam to deubiquitinate and stabilize Cyc A for proper cystoblast differentiation. Therefore, it is plausible that the stabilization of Cyc A in the Sakura mutant is an indirect consequence of Bam misexpression and independent of the Sakura-Otu interaction. The authors may need to provide much deeper insight into the mechanism by which Sakura plays roles in these seemingly separable steps to orchestrate germ cell maintenance and differentiation during early oogenesis.
Yes, it is possible that the stabilization of CycA in the sakura mutant is an indirect consequence of Bam misexpression and independent of the Sakura-Otu interaction. To test the significance and role of the Sakura-Otu interaction, we have attempted to identify Sakura point mutants that lose interaction with Otu. If such point mutants were successfully obtained, we were planning to test if their transgene expression could rescue the phenotypes of sakura mutant as the wild-type transgene did. However, after designing and testing the interaction of over 30 point mutants with Otu, we could not obtain such mutant version of Sakura yet. We will continue making efforts, but it is beyond the scope of the current study. We hope to address this important point in future studies.
(2) Figure 3A and Figure 4: The authors show that piRNA production is abolished in Sakura KO ovaries. It is known that piRNA amplification (the ping-pong cycle) occurs in the Vasa-positive perinuclear nuage in nurse cells. Is the nuage normally formed in the absence of Sakura? The authors provide high-magnification images in the germarium expressing Vas-GFP. How does Sakura, and possibly Out, contribute to piRNA production? Are the defects a direct or indirect consequence of the loss of Sakura?
We provided higher magnification images of germarium expressing Vasa-EGFP in sakura mutant background (Fig 3A and 3B). The nuage formation does not seem to be dysregulated in sakura mutant. Currently, we do not know if the piRNA defects are direct or indirect consequence of the loss of Sakura. This question cannot be answered easily. We hope to address this in future studies.
(3) Figure 7 and Figure 12: The authors showed that Dpp-Smad signaling was abolished in Sakura KO germline cells. The same defects were also observed in otu mutant ovaries (Figure 12B). How does the Sakura-Otu axis contribute to the Dpp-Smad pathway in the germline?
As we mentioned in the response to comment (1), we attempted to test the significance and role of the Sakura-Otu interaction, including in the Dpp-Smad pathway in the germline, but we have not yet been able to obtain loss-of-interaction mutant(s) of Sakura. We hope to address this in future studies.
(4) Figure 9 and Fig 10: The authors raised antibodies against both Sakura and Otu, but their specificities were not provided. For Western blot data, the authors should provide whole gel images as source data files. Also, the authors argue that the Otu band they observed corresponds to the 98-kDa isoform (lines 302-304). The molecular weight on the Western blot alone would be insufficient to support this argument.
When we submitted the initial manuscript, we also submitted original, uncropped, and unmodified whole Western blot images for all gel images to the eLife journal, as requested. We did the same for this revised submission. I believe eLife makes all those files available for downloading to readers.
In the newly added Fig S13B, we used very young 2-5 hours ovaries and 3-7 days ovaries. 2-5 days ovaries contain only mostly pre-differentiated germ cells. Older ovaries (3-7 days in our case here) contain all 14 stages of oogenesis and later stages predominate in whole ovary lysates.
As reported in previous literature (Sass et al. 1995), we detected a higher abundance of the 104 kDa Otu isoform than the 98 kDa isoform in from 2-5 hours ovaries and predominantly the 98 kDa isoform in 3-7 days ovaries (Fig S13B). These results confirmed that the major Otu isoform we detected in Western blot, all of which uses old ovaries except for the 2-5 hours ovaries in Fig S13B, is the 98 kDa isoform.
(5) Otu has been reported to regulate ovo and Sxl in the female germline. Is Sakura involved in their regulation?
We examined sxl alternative splicing pattern in sakura mutant ovaries. As shown in Fig S6, we detected the male-specific isoform of sxl RNA and a reduced level of the female-specific sxl isoform in sakura mutant ovaries. Thus Sakura seems to be involved in sxl splicing in the female germline, while further studies will be needed to understand whether Sakura has a direct or indirect role here.
(6) Lines 443-447: The GSC loss phenotype in piwi mutant ovaries is thought to occur in a somatic cell-autonomous manner: both piwi-mutant germline clones and germline-specific piwi knockdown do not show the GSC-loss phenotype. In contrast, the authors provide compelling evidence that Sakura functions in the germline. Therefore, the Piwi-mediated GSC maintenance pathway is likely to be independent of the Sakura-Otu axis.
We changed the text accordingly.
Reviewer #2 (Recommendations for the authors):
Overall, this is a cleanly written manuscript, with some sentences/sections that are confusing the way they are constructed (i.e. Line 37-38, 334, section on Flp/FRT experiments).
We rewrote those sections to avoid confusion.
Comment for all merged image data: the quality of the merged images is very poor - the individual channels are better but should also be reprocessed for more resolved image data sets. Also, it would be helpful to have boundaries drawn in an individual panel to identify the regions of the germarium, as cartooned in Figure S1A (which should be brought into Figure 1) F-actin or Vsg staining would have helped throughout the manuscript to enhance the visualization of described phenotypes.
We are very sorry for the low-resolution images. This was caused when the original PDF file with high-resolution images was compressed in order to meet the small file size limit in the eLife submission portal. In the revised submission, we used high-resolution images.
We outlined the germarium in Fig 1E.
We brought the former FigS1 into Fig 1A.
We provided Phalloidin (F-Actin) staining images in Fig S7.
All p-values seem off. I recommend running the data through the student t-test again.
We used the student t-test to calculate p-values and confirmed that they are correct. We don’t understand why the reviewer thinks all p-values seem off.
In the original manuscript, as we mentioned in each figure legends, we used asterisk (*) to indicate p-value <0.05, without distinguishing whether it’s <0.001, <0.01< or <0.05.
Probably reviewer 2 is suggesting us to use ***, **, and *, to indicate p-value of <0.001, <0.01, and <0.05, respectively? If so, we now followed reviewer2’s suggestions.
Figure 1
(1) Within the text, C is mentioned before A.
We updated the text and now we mentioned Fig 1A before Fig 1C.
(2) B should be the supplemental figure.
We moved the former Fig 1B to Supplemental Figure 1.
(3) C - How were the different egg chamber stages selected in the WB? Naming them 'oocytes' is deceiving. Recommend labeling them as 'egg chambers', since an oocyte is claimed to be just the one-cell of that cyst.
We changed the labeling to egg chambers.
(4) Is the antibody not detecting Sakura in IF? There is no mention of this anywhere in the manuscript.
While our Sakura antibody detects Sakura in IF, it seems to detect some other proteins as well. Since we have Sakura-EGFP fly strain (which fully rescues sakuranull phenotypes) to examine Sakura expression and localization without such non-specific signal issues, we relied on Sakura-EGFP rather than anti-Sakura antibodies for IF.
(5) Expand on the reliance of the sakura-EGFP fly line. Does this overexpression cause any phenotypes?
sakura-EGFP does not cause any phenotypes in the background of sakura[+/+] and sakura[+/-].
(6) Line 95 "as shown below" is not clear that it's referencing panel D.
We now referenced Fig 1D.
(7) Re: Figures 1 E and F. There is no mention of Hts or Vasa proteins in the text.
"Sakura-EGFP was not expressed in somatic cells such as terminal filament, cap cells, escort cells, or follicle cells (Figure 1E). In the egg chamber, Sakura-EGFP was detected in the cytoplasm of nurse cells and was enriched in developing oocytes (Figure 1F)". Outline these areas or label these structures/sites in the images. The color of Merge labels is confusing as the blue is not easily seen.
We mentioned Hts and Vasa in the text. We labeled the structures/sites in the images and updated the color labeling.
Figure 2
(1) Entire figure is not essential to be a main figure, but rather supplemental.
We don’t agree with the reviewer. We think that the female fertility assay data, where sakura null mutant exhibits strikingly strong phenotype, which was completely rescued by our Sakura-EGFP transgene, is very important data and we would like to present them in a main figure.
(2) 2A- one star (*) significance does not seem correct for the presented values between 0 and 100+.
In the original manuscript, as we mentioned in each figure legends, we used asterisk (*) to indicate p-value <0.05, without distinguishing whether it’s <0.001, <0.01< or <0.05.
Probably reviewer 2 is suggesting us to use ***, **, and *, to indicate p-value of <0.001, <0.01, and <0.05, respectively? If so, we now followed reviewer2’s suggestions.
(3) 2C images are extremely low quality. Should be presented as bigger panels.
We are very sorry for the low-resolution images. This was caused when the original PDF file with high-resolution images was compressed in order to meet the small file size limit in the eLife submission portal. In the revised submission, we used high-resolution images. We also presented as bigger panels.
Figure 3
(1) "We observed that some sakuranull /null ovarioles were devoid of germ cells ("germless"), while others retained germ cells (Fig 3A)" What is described is, that it is hard to see. Must have a zoomed-in panel.
We provided zoomed-in panels in Fig 3B
(2) C - The control doesn't seem to match. Must zoom in.
We provided matched control and also zoomed in.
(3) For clarity, separate the tumorous and germless images.
In the new image, only one tumorous and one germless ovarioles are shown with clear labeling and outline, for clarity.
(4) Use arrows to help clearly indicate the changes that occur. As they are presented, they are difficult to see.
We updated all the panels to enhance clarity.
(5) Line 158 seems like a strong statement since it could be indirect.
We softened the statement.
Figure 4
(1) Line 188-189 - Conclusion is an overstatement.
We softened the statement.
(2) Is the piRNA reduction due to a change in transcription? Or a direct effect by Sakura?
We do not know the answers to these questions. We hope to address these in future studies.
Figure 5
(1) D - It might make more sense if this graph showed % instead of the numbers.
We did not understand the reviewer’s point. We think using numbers, not %, makes more sense.
(2) Line 213 - explain why RNAi 2 was chosen when RNAi 1 looks stronger.
Fly stock of RNAi line 2 is much healthier than RNAi line 1 (without being driven Gal4) for some reasons. We had a concern that the RNAi line 1 might contain an unwanted genetic background. We chose to use the RNAi 2 line to avoid such an issue.
(3) In Line 218 there's an extra parenthesis after the PGC acronym.
We corrected the error.
(4) TOsk-Gal4 fly is not in the Methods section.
We mentioned TOsk-Gal4 in the Methods.
Figure 6:
(1) The FLP-FRT section must be rewritten.
We rewrote the FLP-FRT section.
(2) A - include statistics.
We included statistics using the chi-square test.
(3) B - is not recalled in the Results text.
We referred Fig 6B in the text.
(4) Line 232 references Figure 3, but not a specific panel.
We referred Fig 3A, 3C, 3D, and 3E, in the text.
Figure 7/8 - can go to Supplemental.
We moved Fig 8 to supplemental. However, we think Fig 7 data is important and therefore we would like to present them as a main figure.
(1) There should be CycA expression in the control during the first 4 divisions.
Yes, there is CycA expression observed in the control during the first 4 divisions, while it’s much weaker than in sakuranull clone.
(2) Helpful to add the dotted lines to delineate (A) as well.
We added a dotted outline for germarium in Fig 7A.
(3) Line 263 CycA is miswritten as CyA.
We corrected the typo.
Figure 9
(1) Otu antibody control?
We validated Otu antibody in newly added Fig 10C and Fig S13A.
(2) Which Sakura-EGFP line was used? sakura het. or null background? This isn't mentioned in the text, nor legend.
We used Sakura-EGFP in the background of sakura[+/+]. We added this information in the methods and figure legend.
(3) C - Why the switch to S2 cells? Not able to use the Otu antibody in the IP of ovaries?
We can use the Otu antibody in the IP of ovaries. However, in anti-Sakura Western after anti-Otu IP, antibody light chain bands of the Otu antibodies overlap with the Sakura band. Therefore, we switched to S2 cells to avoid this issue by using an epitope tag.
Figure 10
(1) A- The resolution of images of the ribbon protein structure is poor.
We are very sorry for the low-resolution images. This was caused when the original PDF file with high-resolution images was compressed in order to meet the small file size limit in the eLife submission portal. In the revised submission, we used high-resolution images.
(2) A table summarizing the interactions between domains would help bring clarity to the data presented.
We added a table summarizing the fragment interaction results.
(3) Some images would be nice here to show that the truncations no longer colocalize.
We did not understand the reviewer’s points. In our study, even for the full-length proteins.
We have not shown any colocalization of Sakura and Otu in S2 cells or in ovaries, except that they both are enriched in developing oocytes in egg chambers.
Figure 12
(1) A - control and RNAi lines do not match.
We provided matched images.
(2) In general, since for Sakura, only its binding to Otu was identified and since they phenocopy each other, doesn't most of the characterization of Sakura just look at Otu phenotypes? Does Sakura knockdown affect Otu localization or expression level (and vice versa)?
We tested this by Western (Fig S15) and IF (Fig 12). Sakura knockdown did not decrease Otu protein level, and Otu knockdown did not decrease Sakura protein level (Fig S15). In sakuranull clone, Otu level was not notably affected (Fig 12). In sakuranull clone, Otu lost its localization to the posterior position within egg chambers.
Figure S6
(1) It is Luciferase, not Lucifarase.
We corrected the typo.
Reviewer #3 (Recommendations for the authors):
(1) It is interesting that germless and tumorous phenotypes coexist in the same population of flies. Additional consideration of these essentially opposite phenotypes would significantly strengthen the study. For example, do they co-exist within the same fly and are the tumorous ovarioles present in newly eclosed flies or do they develop with age? The data in Figure 8 show that bam knockdown partially suppresses the germless phenotype. What effect does it have on the tumorous phenotype? Is transposon expression involved in either phenotype? Do Sakura mutant germline stem cell clones overgrow relative to wild-type cells in the same ovariole? Does sakura RNAi driven by NGT-Gal4 only cause germless ovaries or does it also cause tumorous phenotypes? What happens if the knockdown of Sakura is restricted to adulthood with a Gal80ts? It may not be necessary to answer all of these questions, but more insight into how these two phenotypes can be caused by loss of sakura would be helpful.
We performed new experiments to answer these questions.
do they co-exist within the same fly and are the tumorous ovarioles present in newly eclosed flies or do they develop with age?
Tumorous and germless ovarioles coexist in the same fly (in the same ovary). Tumorous ovarioles are present in very young (0-1 day old) flies, including newly eclosed (Fig S5). The ratio of germless ovarioles increases and that of tumorous ovarioles decreases with age (Fig S5).
The data in Figure 8 show that bam knockdown partially suppresses the germless phenotype. What effect does it have on the tumorous phenotype?
bam knockdown effect on tumorous phenotype is shown in Fig S10. bam knockdown increased the ratio of tumorous ovarioles and the number of GSC-like cells.
Is transposon expression involved in either phenotype?
Since our transposon-piRNA reporter uses germline-specific nos promoter, it is expressed only in germ line cells, so we cannot examine in germless ovarioles.
Do Sakura mutant germline stem cell clones overgrow relative to wild-type cells in the same ovariole?
Yes, Sakura mutant GSC clones overgrow. Please compare Fig 6C and Fig S8.
Does sakura RNAi driven by NGT-Gal4 only cause germless ovaries or does it also cause tumorous phenotypes?
Fig S10 and Fig S12 show the ovariole phenotypes of sakura RNAi driven by NGT-Gal4. It causes both germless and tumorous phenotypes.
What happens if the knockdown of Sakura is restricted to adulthood with a Gal80ts?
Our mosaic clone was induced at the adult stage, so we already have data of adulthood-specific loss of function. Gal80ts does not work well with nos-Gal4.
(2) The idea that the excessive bam expression in tumorous ovaries is due to a failure of bam repression by dpp signaling is not well-supported by the data. Dpp signaling is activated in a very narrow region immediately adjacent to the niche but the images in Figure 7A show bam expression in cells that are very far away from the niche. Thus, it seems more likely to be due to a failure to turn bam expression off at the 16-cell stage than to a failure to keep it off in the niche region. To determine whether bam repression in the niche region is impaired, it would be important to examine cells adjacent to the niche directly at a higher magnification than is shown in Figure 7A.
We provided higher magnification images of cells adjacent to the niche in new Fig 7A.
We found that cells adjacent to the niche also express Bam-GFP.
That said, we agree with the reviewer. A failure to turn bam expression off at the 16-cell stage may be an additional or even a main cause of bam misexpression in sakura mutant. We added this in the Discussion.
(3) In addition, several minor comments should be addressed:
a. Does anti-Sakura work for immunofluorescence?
While our Sakura antibody detects Sakura in IF, it seems to detect some other proteins as well. Since we have Sakura-EGFP fly strain to examine Sakura expression and localization without such non-specific signal issues, we relied on Sakura-EGFP rather than anti-Sakura antibodies.
b. Please provide insets to show the phenotypes indicated by the different color stars in Figure 3C more clearly.
We provided new, higher-magnification images to show the phenotypes more clearly.
c. Please indicate the frequency of the expression patterns shown in Figure 4D (do all ovarioles in each genotype show those patterns or is there variable penetrance?).
We indicated the frequency.
d. An image showing TOskGal4 driving a fluorophore should be provided so that readers can see which cells express Gal4 with this driver combination.
It has been already done in the paper ElMaghraby et al, GENETICS, 2022, 220(1), iyab179, so we did not repeat the same experiment.
