Sequoia affects Drosophila central nervous system development by regulating axonal extension and guidance

Noor Al-Hajri; Raquel Mendez-Castro; Mar Alujas; Sofia J. Araújo; Guy Tear

doi:10.1371/journal.pone.0333573

Peer Review History

Original SubmissionSeptember 15, 2025
6 Nov 2025 Decision Letter - Renping Zhou, Editor Dear Dr. Tear, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the major and minor concerns raised during the review process. Please submit your revised manuscript by Dec 21 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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Please remove them and upload them with the file type 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. 6. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: This article by Noor Al-Hajri et al. describes the phenotypes of several new mutant alleles of sequoia (seq), a gene encoding a zinc-finger transcription factor. The study extends previous reports on the role of Sequoia in nervous system development by characterizing five mutant alleles in total—two previously reported and three newly generated. Overall, the phenotypic descriptions are clear and the data are potentially valuable; however, the clarity of presentation and the completeness of the analysis could be improved in several areas. Major Comments 1. In most figures, only a subset of the alleles is analyzed. Including all five alleles throughout the figures would strengthen the conclusions and provide a more comprehensive view of the phenotypic variability associated with seq mutations. 2. The manuscript does not specify whether all seq alleles are recessive. No data from heterozygous mutants are shown. Information about dominance or partial dominance could help interpret the phenotypic differences reported, particularly in the final section of the paper. 3. Several statements, especially in the Introduction, lack supporting references. These should be added to properly contextualize the study within previous work on seq and related transcription factors. 4. The discussion does not adequately integrate prior knowledge of seq function. For example, is Seq expressed in all central and peripheral nervous system neurons? Are the reported phenotypes demonstrably cell-autonomous? Addressing these points would clarify how the new alleles relate to the known roles of Sequoia. 5. It would be helpful to include a summary table listing all analyzed phenotypes and the corresponding effects of each allele in the different neuronal types examined. Minor Comments 1. Figure formatting In all figures, the scale bar is too thin and difficult to see. Please increase its thickness. The scale information should remain in the figure legend; currently, it is almost illegible even when zoomed in. 2. Figure 1G Ensure that the order of genotypes or conditions in the plot matches the order shown in Figure 1A and in the graphs in Figures 1H–I for easier comparison. 3. Figures 1H and 1I Replace “um” with the correct unit symbol “μm”. 4. Figure 2D The figure legend lacks information about the antibody used. Moreover, only the mutant is shown. A proper control illustrating the normal phenotype should be included for comparison. 5. Figure 3C The legend should clarify what the light-blue arrow indicates. 6. Figures 4E–G These panels are not mentioned in the main text; please include a reference to them where appropriate. 7. Supplementary Figure 1 Add the genotypes directly to the figure to facilitate interpretation. Indicate which antibodies were used in the legend. 8. line 271 The citation “Figure 5D” should be corrected to “Figure 4D”. 9. The description of how seq alleles were generated is confusing. The text states that chemical mutagenesis was performed on a second chromosome carrying a fasIII mutation, but it is unclear how the fasIII gene was later reintroduced. Was a genomic FasIII rescue construct inserted into the third chromosome of the mutant flies? Please clarify. 10. Discussion. Consider whether some of the observed phenotypes could reflect altered cell fate rather than purely axon-guidance defects. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0333573.r001
Revision 1
13 Jan 2026 Author Response RESPONSE TO REVIEWERS Sequoia affects Drosophila central nervous system development by regulating axonal extension and guidance Dear editor and reviewer, We thank the reviewer for his/her comments and suggestions, which will make our manuscript a much better one. Accordingly, we have already made changes to the manuscript (marked in yellow) and some of the figures, and we have performed all the required experiments. We hope you will find this manuscript suitable for publication at PLOSOne. Below, we answer the reviewers point by point (in blue). Reviewer #1: This article by Noor Al-Hajri et al. describes the phenotypes of several new mutant alleles of sequoia (seq), a gene encoding a zinc-finger transcription factor. The study extends previous reports on the role of Sequoia in nervous system development by characterizing five mutant alleles in total—two previously reported and three newly generated. Overall, the phenotypic descriptions are clear and the data are potentially valuable; however, the clarity of presentation and the completeness of the analysis could be improved in several areas. Major Comments 1. In most figures, only a subset of the alleles is analyzed. Including all five alleles throughout the figures would strengthen the conclusions and provide a more comprehensive view of the phenotypic variability associated with seq mutations. We have analysed the phenotypes of all five alleles with BP102, 1D4 and 22C10 antibodies. We have included these in the new figures 2 and 3. 2. The manuscript does not specify whether all seq alleles are recessive. No data from heterozygous mutants are shown. Information about dominance or partial dominance could help interpret the phenotypic differences reported, particularly in the final section of the paper. All alleles analysed are recessive and embryonic lethal, we have included this information in the text. 3. Several statements, especially in the Introduction, lack supporting references. These should be added to properly contextualize the study within previous work on seq and related transcription factors. We have added the necessary references to the introduction. 4. The discussion does not adequately integrate prior knowledge of seq function. For example, is Seq expressed in all central and peripheral nervous system neurons? Are the reported phenotypes demonstrably cell-autonomous? Addressing these points would clarify how the new alleles relate to the known roles of Sequoia. We have included this information in the results and discussion section. 5. It would be helpful to include a summary table listing all analyzed phenotypes and the corresponding effects of each allele in the different neuronal types examined. We have built a summary table (Table 1) with all the alleles, mutations and phenotypes. Minor Comments 1. Figure formatting In all figures, the scale bar is too thin and difficult to see. Please increase its thickness. The scale information should remain in the figure legend; currently, it is almost illegible even when zoomed in. This has been changed. 2. Figure 1G Ensure that the order of genotypes or conditions in the plot matches the order shown in Figure 1A and in the graphs in Figures 1H–I for easier comparison. We have reordered the alleles in the graphs. 3. Figures 1H and 1I Replace “um” with the correct unit symbol “μm”. This has been changed. 4. Figure 2D The figure legend lacks information about the antibody used. Moreover, only the mutant is shown. A proper control illustrating the normal phenotype should be included for comparison. We apologise for this confusion, Fig. 2D shown allele Z1241 stained with 1D4 like all the others. So the control is the same. 5. Figure 3C The legend should clarify what the light-blue arrow indicates. We have removed the light blue arrowheads as they were confusing and not showing anything different from the black ones. 6. Figures 4E–G These panels are not mentioned in the main text; please include a reference to them where appropriate. This has been changed. 7. Supplementary Figure 1 Add the genotypes directly to the figure to facilitate interpretation. Indicate which antibodies were used in the legend. This has been changed. 8. line 271 The citation “Figure 5D” should be corrected to “Figure 4D”. This has been changed. 9. The description of how seq alleles were generated is confusing. The text states that chemical mutagenesis was performed on a second chromosome carrying a fasIII mutation, but it is unclear how the fasIII gene was later reintroduced. Was a genomic FasIII rescue construct inserted into the third chromosome of the mutant flies? Please clarify. This has been changed. 10. Discussion. Consider whether some of the observed phenotypes could reflect altered cell fate rather than purely axon-guidance defects. We have included this possibility in the results and discussion section. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0333573.r002
3 Feb 2026 Decision Letter - Renping Zhou, Editor Dear Dr. Tear, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but two minor issues have been identified that need to be addressed. Therefore, we invite you to submit a revised version of the manuscript that addresses the minor points raised during the review process. Please submit your revised manuscript by Mar 20 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Renping Zhou Academic Editor PLOS One Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: In this new version of the article, almost all my comments have been addressed. Therefore, I believe the paper is nearly ready for publication. Congratulations to the authors. I have only two minor comments, which could be checked by the editor. 1. The way in which the fasIII gene rescue was performed is still unclear. The description remains the same as in the previous version of the paper: “To test whether the axon guidance phenotype is solely due to a mutation in the seq gene and not caused by the absence of the fasIII gene, the fasIII chromosome was introduced into the background on the third chromosome.” I assume this refers to a fasIII genomic construct inserted on the third chromosome. If so, could the authors indicate whether this fly line was generated for this study or if it had been published previously? 2. There is a misreferenced figure in the section describing the mutant alleles in Figure 2. The authors cite Figure 2K′ when referring to motor axonal defects, but I believe they intended to cite Figure 2M′. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0333573.r003
Revision 2
20 Feb 2026 Author Response Two minor changes were requested by the reviewer. These have been addressed as oulined below. 1. “The way in which the fasIII gene rescue was performed is still unclear.” - we have added further information and provided a reference to the stock that was used to perform the fasIII rescue on page 4. 2. There is a misreferenced figure in the section describing the mutant alleles in Figure 2. The authors cite Figure 2K′ when referring to motor axonal defects, but I believe they intended to cite Figure 2M′. - we have corrected this citation on page 8 to cite Figure 2M’ Attachments Attachment Submitted filename: Response to Reviewer Resub2Al-Hajri.docx https://doi.org/10.1371/journal.pone.0333573.r004
25 Feb 2026 Decision Letter - Renping Zhou, Editor Sequoia affects Drosophila central nervous system development by regulating axonal extension and guidance PONE-D-25-50300R2 Dear Dr. Tear, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Renping Zhou Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0333573.r005
Formally Accepted
Acceptance Letter - Renping Zhou, Editor PONE-D-25-50300R2 PLOS One Dear Dr. Tear, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Renping Zhou Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0333573.r006

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