alms1 mutant zebrafish do not show hair cell phenotypes seen in other cilia mutants

Lauren Parkinson; Tamara M. Stawicki

doi:10.1371/journal.pone.0246844

Peer Review History

Original SubmissionDecember 8, 2020
13 Jan 2021 Decision Letter - Hemant Khanna, Editor PONE-D-20-38618 alms1 mutant zebrafish do not show hair cell phenotypes seen in other cilia mutants PLOS ONE Dear Dr. Stawicki, 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 points raised during the review process. Please submit your revised manuscript by Feb 06 2021 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. 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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Parkinson and Stawicki present data that suggest loss of alms1 in zebrafish does not affect vestibular hair cell function. Mutations in Alms1 are associated with Alstrom Syndrome in humans, which is a ciliopathy associated with loss of hearing and vision, as well as heart disease and diabetes. The zebrafish mutant was previously shown to have several phenotypes consistent with Alstrom Syndrome but the effects on hair cells had not been characterized. Here, the authors use imaging and functional assays to show that hair cell development and function in the lateral line were grossly normal. Although brief, the manuscript is well-written and the figures are clearly presented and organized. The results are somewhat unexpected, given that prior studies has demonstrated functional conservation of alms1 in other zebrafish tissues. This study adds to the body of knowledge for alms1 function, but a few weaknesses remain. The zebrafish lateral line is considered an easily accessible tissue for vestibular hair cell study, but it may not fully recapitulate phenotypes limited to auditory hair cells. As such, the lack of a phenotype in the lateral line must be interpreted conservatively. The authors would be well served to consider additional experiments on auditory hair cells to fully rule out any possible phenotypes. Major concerns • The authors demonstrate that zebrafish alms1 mutants lack phenotypes typically associated with hair cell dysfunction and speculate that genetic compensation, lack of conservation with the mammalian homolog, or for a different role in different hair cell types. What the authors have not done, however, is show that alms1 is expressed in lateral line hair cells during development. If alms1 isn’t expressed in lateral line hair cells, then the absence of a phenotype would not be surprising. • More information should also be included that discusses potential paralogs, including a rigorous homology search for any paralog/homologs that may not be listed in Ensembl. • Lines 170-172: While the authors state that cilia look grossly normal, additional quantitative metrics would strengthen their argument. Are cilia lengths normal? Is there some way to assess cilia polarity or the degree of splaying that occurs in other mutants? While the authors are likely correct, having additional evidence that is less subjective would add rigor. • To more fully support the argument that alms1 may have a role in auditory hair cells, the authors should examine cilia during auditory hair cell development or maintenance in alms1 larvae by imaging cilia within the otic vesicle (see early work from Jarema Malicki or Bruce Riley for examples). Minor concerns • Line 127: please correct the dilution value for the otoferlin antibody. Reviewer #2: Full Title: alms1 mutant zebrafish do not show hair cell phenotypes seen in other cilia mutants Since Alms1 is a basal body protein which mutations have been involved in Alström Syndrome patient’s hearing loss, the aim of this study was to elucidate the effect of Alsm1 mutation on hair cell phenotypes and function in the previously described Alsm1 mutant zebrafish. The paper focused on hair cells morphology via staining in Alms1 zebrafish larvae and didn’t observed any morphology change. To study the effect of such mutation on hair cell properties, authors conducted escaping reflex experiments and vital dye staining (FM1-43) and showed no impact on mechanotransduction function in Alms1 mutant hair cells. Finally, authors conducted hair cell death analysis after neomycin or gentamicin treatment in adult and zebrafish larvae and couldn’t observe any increase in death sensitivity due to the loss of Alms1. This study concluded that Alms1 mutant zebrafish does not show hair cell defects. This data suggests that Alms1, one of the major cilia-associated protein involve in human auditory hear cells does not show any phenotype in zebrafish hair cells, convincing that the Alsm1 mutant zebrafish might not be a proper model to study Alms1 auditory function. I consider the manuscript valuable and acceptable for publication with minor revisions: - Even if the final message of this publication is the lack of phenotype, one concern is the absence of additional analysis. As an example, in figure 1, I would appreciate to know the number of ciliated cells even if change will not be observed. Also, authors should draw arrows to point the lateral line and cells of the olfactory pit in both control and Alms1 mutant zebrafish. - Microscopy material and method details associated with the antibody staining are lacking in the method paragraph. Number of cells/cilia counted should also be precise. - Escaping approach used to study mechanostransduction is not described in the method and no quantification of the escaping movement is provided with the supplemental video. - Discussion paragraph on the lack of conservation with the N terminal mammalian isoform is pretty unclear and might need to be clarified or reformulated. - Final paragraph questioning the potential specific auditory vs vestibular cell type expression and function bring an interesting point on the role of Alms1 and the importance to use the proper animal model to study auditory functions. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
25 Jan 2021 Author Response Below please find our response to the concerns brought up by the reviewers. We have put our responses in blue. Reviewer #1 Major concerns • The authors demonstrate that zebrafish alms1 mutants lack phenotypes typically associated with hair cell dysfunction and speculate that genetic compensation, lack of conservation with the mammalian homolog, or for a different role in different hair cell types. What the authors have not done, however, is show that alms1 is expressed in lateral line hair cells during development. If alms1 isn’t expressed in lateral line hair cells, then the absence of a phenotype would not be surprising. We have investigated existing transcriptome data from zebrafish hair cells to confirm alms1 expression. We found it was identified as being expressed in studies that looked at the total hair cell transcriptome in both larvae and adults and also clustered with mature hair cells in a lateral line specific single cell RNA dataset. We have added this information to the discussion (lines 313-316). • More information should also be included that discusses potential paralogs, including a rigorous homology search for any paralog/homologs that may not be listed in Ensembl. Searching for known ohnologues of alms1 that may have resulted from the genome duplication in teleost fish did not identify any hits. We also performed searches for homologous proteins and found only one, a C10orf90 homologue which is also a known mammalian ALMS motif containing gene. We have added this information to the discussion (lines 336-345). • Lines 170-172: While the authors state that cilia look grossly normal, additional quantitative metrics would strengthen their argument. Are cilia lengths normal? Is there some way to assess cilia polarity or the degree of splaying that occurs in other mutants? While the authors are likely correct, having additional evidence that is less subjective would add rigor. We have added quantification of cilia length for all cell types we examined. This is in the new version of Figure 1. • To more fully support the argument that alms1 may have a role in auditory hair cells, the authors should examine cilia during auditory hair cell development or maintenance in alms1 larvae by imaging cilia within the otic vesicle (see early work from Jarema Malicki or Bruce Riley for examples). We imaged the otic vesicle during development as the viewer suggested and included this in the new version of Figure 1. Minor concerns • Line 127: please correct the dilution value for the otoferlin antibody. We have corrected this. The antibody was diluted at 1:100. Reviewer #2 - Even if the final message of this publication is the lack of phenotype, one concern is the absence of additional analysis. As an example, in figure 1, I would appreciate to know the number of ciliated cells even if change will not be observed. Also, authors should draw arrows to point the lateral line and cells of the olfactory pit in both control and Alms1 mutant zebrafish. We have quantified the length of the cilia and noted the number of animals we did this in. The close contact of the cells we are examining at 5dpf make it difficult to distinguish which cilia come from which cell to get an exact cilia/cell count. However, in the 24 hpf fish where we are only looking at 2 ciliated hair cells in the otic vesicle we do not note any cells without cilia which we have noted in the results section (lines 191-193). We have also added arrows and brackets to figure 1 to label the cilia of the hair cells in 1A & 1B. We were not sure how to do this for the olfactory pit as the entire structure imaged is the olfactory pit. - Microscopy material and method details associated with the antibody staining are lacking in the method paragraph. Number of cells/cilia counted should also be precise. We have changed the “antibody labeling” methods section to “antibody labeling, imaging, and cilia length measurement” and included details about how the images were generated (lines 130-133). As we have quantified cilia length as mentioned above we also have the information as to the number of animals we did that for in the figure legend for that data. - Escaping approach used to study mechanotransduction is not described in the method and no quantification of the escaping movement is provided with the supplemental video. We have added a section in the methods on the techniques used for “vibrational stimulus response” (lines 237-241). Additionally, we have quantified the number of fish that show a response and included this as a new Figure 2. - Discussion paragraph on the lack of conservation with the N terminal mammalian isoform is pretty unclear and might need to be clarified or reformulated. We have rewritten that section as follows “It is also possible that the lack of phenotype we observe is due to a lack of sequence conservation between the mammalian and zebrafish alms1 gene. The majority of the N-terminus of the human ALMS1 gene, encoding for the tandem repeat domain of the protein, is specific to mammals [67,68] with the first 1560 amino acids of the human gene having no homologous region in the zebrafish gene.” (lines 324-331) Attachments Attachment Submitted filename: Reviewer Response.docx https://doi.org/10.1371/journal.pone.0246844.r002
27 Jan 2021 Decision Letter - Hemant Khanna, Editor alms1 mutant zebrafish do not show hair cell phenotypes seen in other cilia mutants PONE-D-20-38618R1 Dear Dr. Stawicki, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Hemant Khanna Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. 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 requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (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 https://doi.org/10.1371/journal.pone.0246844.r003
Formally Accepted
22 Mar 2021 Acceptance Letter - Hemant Khanna, Editor PONE-D-20-38618R1 alms1 mutant zebrafish do not show hair cell phenotypes seen in other cilia mutants Dear Dr. Stawicki: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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. If we can help with anything else, please email us at plosone@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. Hemant Khanna Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0246844.r004

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