-->PONE-D-25-69197-->-->Reproductive-state dependent changes in inner ear gene expression in female plainfin midshipman fish-->-->PLOS One

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PLOS One

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: No

Reviewer #2: Yes

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-->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. 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: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. 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: The manuscript “Reproductive-state dependent changes in inner ear gene expression in female plainfin midshipman fish” by Jasper et al. uses NanoString nCounter technology to investigate transcriptional dynamics associated with auditory plasticity in the female midshipman inner ear. The authors identify distinct gene expression profiles between the saccule and utricle, with seasonal differences in some genes linked to Wnt and Notch signaling in the saccule. They further report greater seasonal variability in dopamine receptor expression in the utricle compared to the saccule, suggesting dopaminergic modulation of utricular sound sensitivity. Overall, the study is interesting and generally well written with some exceptions:

1. The nCounter data is difficult to evaluate in its current presentation. For transparency, It would be helpful if the authors provided nCounter nSolver output as a supplementary Excel file (eg statistical results and the normalized counts).

The statistical analysis used to identify differential expression is not clear. The Methods section states that the authors sought to compare “rates” of gene expression change between the saccule and utricle by standardizing expression values, calculating log2 fold changes (Line 216), and then applying a two-way ANOVA. It is not clear whether this analysis was performed entirely within nSolver or whether additional custom analyses were conducted outside of nSolver software.

It is unclear how variance or dispersion inherent to count data was examined. NanoString’s nSolver does account for count variability analogous to DESeq2 for RNA-seq. But, the manuscript could benefit with a clear explanation of how might dispersion was dealt with before fold-change and ANOVA comparisons. In the manuscripts current form, its hard to assess whether the reported differences in gene expression is robust or reflects variability across a small number of samples.

2. From a visualization standpoint, the nCounter dataset is underutilized. Theres no global expression visualization or heatmap showing normalized gene expression across tissues and the reproductive states. Since only 37 genes were assayed, a heatmap might be straightforward to include in Figure 1 and aid interpretation of the data. nSolver can generate such heatmaps and other R based tools are available, such as Class et al., Bioinformatics, 2023, NanoTube.

3. The scientific community will benefit from Data Availability. Considering PLOS ONEs data sharing expectation, the authors should deposit the NanoStrong expression dataset in a public repository like NCBI GEO https://www.ncbi.nlm.nih.gov/geo/.

4. Wnt and Notch signaling results. A central hypothesis is that saccule of reproductive females will have increased Wnt and Hsp signaling, and decreased Notch signaling. The results indicate that 3 frizzled receptors are downregulated and the other 10 Wnt genes are unchanged. Similarly, deltaA is the only Notch signaling gene with dynamic expression, but with changes in the opposite direction than expected. The Discussion does acknowledge these discrepancies but the manuscript frames the results as “changes in Wnt/Notch signaling associated with seasonal cell proliferation.” The conclusions can be softened or reframed.

5. Dopamine signaling results/conclusions. No individual dopamine receptor genes have a statistically significant seasonal change in expression. Line 320 states that dopamine receptors had greater variability in reproductive females with counts 5.7 to 301.2. D3 receptor had variability according to the text. But examining the Figure 3 shows D2A and D5 having variability as well. Its unclear how variance was calculated and if nSolver modeled the dispersion for discussed genes with variability. The manuscript can benefit from more clarification on how variability was quantified and how it informs conclusions.

6. Overall assessment. This study addresses a fascinating and very important question and builds on prior work on midshipman fish inner ear physiology. More conservative interpretation of the signaling conclusions is needed and clearer statistical analysis explanations are needed.

Reviewer #2: The study by Jasper et al. examines differential gene expression in the female midshipman inner ear during reproductive and non-reproductive months. Based on previous observations of saccular proliferation and hair cell addition, as well as increased sensitivity of utricular hair cells in the female midshipman inner ear during the reproductive season, the authors focused on measuring changes in several sets of effector genes using Nanostring. More specifically, the Wnt, Notch, and estrogen receptor pathways, as well as heat shock protein genes, dopamine receptors and BK ion channels. Their hypotheses were straightforward and methods and data analysis were appropriate for the study. Despite a lack of sensitivity due to potential variability in their samples of wild-caught midshipman, the authors present a thorough and interesting discussion of their results. I have several comments.

Major comments:

The methods section should be revisited to provide more detail. For example, at Line 179 the authors report “Fish were euthanized as described above, and inner ears were flushed with 4%

paraformaldehyde fixative for a few minutes.” It would greatly benefit the paper to provide specifics in the methods sections (how were ears flushed? How many ~ minutes?) so that readers may utilize the methods for their own future experiments. Furthermore, at Line 186: “First, we generated 30-50 µm vibratome sections of the end organs using sterile conditions and processed them for HCR on the same day.” Please provide specifics: what were tissues embedded in? What parameters were used? What vibratome was used for sectioning?

The raw data for the Nanostring analysis has not been made available. These data should be shared in a publicly accessible repository such as Mendeley Data, and a corresponding DOI should be added to the methods.

Minor comments:

Line 135: please define the “h” abbreviation.

Line 163: “Probes and controls were optically counted to quantify total copy number of each gene and estimate type I and type II error rates.” – I believe this should be “total transcript number of each gene.”

Line 326: “Only three genes exhibited seasonal differences: the Wnt receptors fzl1 and fzl6, and the BK channel gene slo1a”- should this be fzd1 and fzd6? These are the genes shown to be significantly changed in Figure 3 and fzd6 is described in the next sentence.

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Reviewer #1: No

Reviewer #2: No

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Reproductive-state dependent changes in inner ear gene expression in female plainfin midshipman fish

PONE-D-25-69197R1

Dear Dr. Coffin,

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.

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Kind regards,

Alan G. Cheng, M.D.

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

Reviewer #2: All comments have been addressed

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-->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

Reviewer #2: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->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

Reviewer #2: Yes

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-->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

Reviewer #2: Yes

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-->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: The manuscript “Reproductive-state dependent changes in inner ear gene expression in female plainfin midshipman fish” by Jasper et al. uses NanoString nCounter technology to investigate transcriptional dynamics associated with auditory plasticity in the female midshipman inner ear. The authors identify distinct gene expression profiles between the saccule and utricle, with seasonal differences in some genes linked to Wnt and Notch signaling in the saccule. They further report greater seasonal variability in dopamine receptor expression in the utricle compared to the saccule, suggesting dopaminergic modulation of utricular sound sensitivity.

The authors addressed all of my previous concerns in a thorough and satisfactory manner. The authors improved statistical transparency, enhanced data visualizations, made the data publicly available and accessible, and clarified the dopamine receptor findings. The manuscript is significantly improved and I have no further comments. I recommend this manuscript for publication.

Reviewer #2: The authors have sufficiently addressed my previous comments and I recommend acceptance of this manuscript.

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-->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

Reviewer #2: No

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