Spatial Distribution and Temporal Dynamics of Neomycin-Induced Neuromast Cell Damage and Regeneration in the Mexican tetra (Astyanax mexicanus)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled "Spatial Distribution and Temporal Dynamics of Neomycin-Induced Neuromast Cell Damage and Regeneration in the Mexican tetra (Astyanax mexicanus)”, by Gandhrav Goel and co-workers, is an interesting study that analyzes the effects of aminoglycoxides, specifically neomycin, on hairy cells of cephalic and trunk neuromasts of the lateral line of Astyanax mexicanus, and their cycle  of degeneration-regeneration.

Classically, neuromast hair cells have been used as models to study mammalian inner ear equivalents and to analyze models of ototoxicity.

In the present work, the authors study a topic that is not new and on which there is abundant previous scientific literature on zebrafish and other fishes, although the model proposed in their study is new (Astyanax mexicanus).

The manuscript is well-written, well-structured, and easy to follow. However, there are some issues that need to be clarified:

1.- Abstract.- It is correct and contains all the information.

2.- Introduction.- It contains essential information about the structure of the neuromasts of the lateral line and introduce the studies of ototoxicity by aminoglycosides. Nevertheless, some references have not been included, which are essential as they highlight that similar experimental studies have already been carried out previously. Just to mention a few Montalbano G, et al. 2014;196(4):236-40. doi: 10.1016/j.aanat.2014.01.005.; Venuto A et al. J Assoc Res Otolaryngol. 2003;4(2):219-34. doi: 10.1007/s10162-002-3022-x.; Fan C, et al. J Comp Neurol. 2016;524(7):1443-56. doi: 10.1002/cne.23918. Authors should take all these works into account and discuss them.

On the other hand, in the introduction they do not clarify what are the advantages offered by the Astyanax mexicanus over other models such as the Danio rerio. The differences between the two morphs (surface and cave) are described in detail but the advantages of this model are not discussed. And they should also clarify why they chose the surface morph vs. the cave morph. Are there differences between the two in the structure of neuromasts and/or in their response to ototoxicants?

Materials and Methods.- Are sound

Results.- In general they are well developed and exposed, but the results shown in figures 2 and 4 are not convincing and it is practically impossible to identify the cells of the neuromasts and separate them from the red background. These two images need to be improved. On the other hand, the results would be much clearer and more evident if information were provided on the structure and/or ultrastructure of the changes in neuromasts.

Discussion and Conlcusions.- Are corrects.

 

In summary, it is a well-planned work, on a new model (?) but adequate and well-documented cell quantification studies are needed. I strongly encourage authors to carry out, if possible, the studies I suggest.

Author Response

Review report 1

Thank you very much for reviewing our manuscript. We appreciate your thoughtful feedback and have carefully addressed each of your comments, please see our detailed responses attached.

 

The manuscript entitled "Spatial Distribution and Temporal Dynamics of Neomycin-Induced Neuromast Cell Damage and Regeneration in the Mexican tetra (Astyanax mexicanus)”, by Gandhrav Goel and co-workers, is an interesting study that analyzes the effects of aminoglycoxides, specifically neomycin, on hairy cells of cephalic and trunk neuromasts of the lateral line of Astyanax mexicanus, and their cycle  of degeneration-regeneration.

Classically, neuromast hair cells have been used as models to study mammalian inner ear equivalents and to analyze models of ototoxicity.

In the present work, the authors study a topic that is not new and on which there is abundant previous scientific literature on zebrafish and other fishes, although the model proposed in their study is new (Astyanax mexicanus).

The manuscript is well-written, well-structured, and easy to follow. However, there are some issues that need to be clarified:

1.- Abstract.- It is correct and contains all the information.

2.- Introduction.- It contains essential information about the structure of the neuromasts of the lateral line and introduce the studies of ototoxicity by aminoglycosides. Nevertheless, some references have not been included, which are essential as they highlight that similar experimental studies have already been carried out previously. Just to mention a few Montalbano G, et al. 2014;196(4):236-40. doi: 10.1016/j.aanat.2014.01.005.; Venuto A et al. J Assoc Res Otolaryngol. 2003;4(2):219-34. doi: 10.1007/s10162-002-3022-x.; Fan C, et al. J Comp Neurol. 2016;524(7):1443-56. doi: 10.1002/cne.23918. Authors should take all these works into account and discuss them.

We have incorporated these references along with the relevant discussions into the revised manuscript, and they are cited at the appropriate locations indicated by line # 141 - 150

On the other hand, in the introduction they do not clarify what are the advantages offered by the Astyanax mexicanus over other models such as the Danio rerio. The differences between the two morphs (surface and cave) are described in detail but the advantages of this model are not discussed. And they should also clarify why they chose the surface  morph vs. the cave morph. Are there differences between the two in the structure of neuromasts and/or in their response to ototoxicants?

Thank you for the suggestion—we’ve now incorporated the advantages of using surface fish over both zebrafish and cavefish in the revised version. Line # 151-164.

Materials and Methods.- Are sound

Results.- In general they are well developed and exposed, but the results shown in figures 2 and 4 are not convincing and it is practically impossible to identify the cells of the neuromasts and separate them from the red background. These two images need to be improved. On the other hand, the results would be much clearer and more evident if information were provided on the structure and/or ultrastructure of the changes in neuromasts.

Thank you for your thoughtful feedback regarding Figures 2 and 4. We appreciate your observation about the difficulty in distinguishing neuromast cells from the red background in the original images. In response, we have improved the  images to improve clarity. Additionally, we have included supplementary information (Line # 595 )  featuring confocal imaging that highlights the structure of neuromast cells in control samples. These enhancements aim to provide a clearer representation of the cellular organization. Please note that confocal imaging was not performed on the treated samples in this study, as our focus was on establishing baseline structural features in the control group.

Discussion and Conlcusions.- Are corrects.

 In summary, it is a well-planned work, on a new model (?) but adequate and well-documented cell quantification studies are needed. I strongly encourage authors to carry out, if possible, the studies I suggest.

We sincerely appreciate your thoughtful and constructive feedback on this manuscript once again.

Reviewer 2 Report

Comments and Suggestions for Authors

The study explores neomycin-induced damage and subsequent regeneration of neuromast cells in Astyanax mexicanus, emphasizing its usefulness as a new model for sensory hair cell recovery. The authors combine histological observation, imaging, and gene expression analysis to describe the time-dependent regenerative changes. The topic is of clear importance to both fish sensory physiology and research on ototoxic injury and repair.

Nonetheless, some areas of the experimental design, data presentation, and interpretation need clarification or additional support before the manuscript is suitable for publication.

Comments

• The qPCR section includes a very small number of biological replicates (n = 2 for control, n = 3 for treated groups), which limits the reliability of the statistical analysis.

• Although the gene expression results were not statistically significant, the discussion interprets them as biologically meaningful. These conclusions should be moderated or validated through additional approaches, such as protein-level assays or analysis at more time points.

• Please indicate the developmental stage of the juveniles used for neomycin exposure (in days post-fertilization or standard length).

• The method for counting neuromasts in Figures 3 and 5 should be described in more detail. Clarify how individual neuromasts were defined and whether counts were normalized to body area or fish length.

• The representative micrographs should include scale bars and information on fluorescence intensity calibration.

• In lines 307–310, the statement “slightly brighter fluorescence” should be supported by quantitative measurement of fluorescence intensity.

• Confirm that untreated and solvent controls were processed in parallel for all assays, including DASPEI staining and RNA extraction.

• Consider adding a positive control for regeneration (such as a mechanical injury model) or a negative control (for example, inhibition of regenerative signaling) to strengthen the conclusions.

• The choice of only fgf1, axin2, and sox2 for gene expression analysis should be better justified. Explain why other well-known regulators of neuromast regeneration, such as atoh1, notch1, or myc, were not included.

• The text would benefit from careful language editing to improve clarity and consistency (for instance, ensure uniform use of “neuromast cells” or “hair cells,” and correct typographical errors such as “DASPIE” → “DASPEI,” “ffgf1” → “fgf1,” etc.).

Author Response

Review report 2

Thank you very much for taking the time to review our manuscript. We’ve carefully considered your comments and provided detailed responses for your review.

The study explores neomycin-induced damage and subsequent regeneration of neuromast cells in Astyanax mexicanus, emphasizing its usefulness as a new model for sensory hair cell recovery. The authors combine histological observation, imaging, and gene expression analysis to describe the time-dependent regenerative changes. The topic is of clear importance to both fish sensory physiology and research on ototoxic injury and repair.

Nonetheless, some areas of the experimental design, data presentation, and interpretation need clarification or additional support before the manuscript is suitable for publication.

Comments

• The qPCR section includes a very small number of biological replicates (n = 2 for control, n = 3 for treated groups), which limits the reliability of the statistical analysis.

We have increased the sample number and redone the statistical analysis. Please see the new Figure 6 . Line # 326.

• Although the gene expression results were not statistically significant, the discussion interprets them as biologically meaningful. These conclusions should be moderated or validated through additional approaches, such as protein-level assays or analysis at more time points.

We agree that further validation through protein-level assays or expanded time-point analyses would strengthen these conclusions; however, such experiments were beyond the scope of the current study. We have revised the discussion to moderate our interpretation and clearly indicate the need for future studies to explore these aspects.

• Please indicate the developmental stage of the juveniles used for neomycin exposure (in days post-fertilization or standard length).

Thank you for this comment the standard length of the fish has been added. Line # 176-177

• The method for counting neuromasts in Figures 3 and 5 should be described in more detail. Clarify how individual neuromasts were defined and whether counts were normalized to body area or fish length.

We have added this in the material and methods section line # 196-204

• The representative micrographs should include scale bars and information on fluorescence intensity calibration.

We have added this information into the new images.

• In lines 307–310, the statement “slightly brighter fluorescence” should be supported by quantitative measurement of fluorescence intensity.

Thank you for the suggestion. To avoid ambiguity and ensure clarity, we have removed the phrase ‘slightly brighter fluorescence,’ as it was not supported by quantitative intensity data. Line # 374-381.

• Confirm that untreated and solvent controls were processed in parallel for all assays, including DASPEI staining and RNA extraction.

Thank you for this important clarification request. All samples used in this study were derived from a single pool of fish treated with neomycin. Following treatment, the fish were post-stained with DASPEI. From this common pool, one subset was used for imaging, while the other was processed for RNA extraction.

 

• Consider adding a positive control for regeneration (such as a mechanical injury model) or a negative control (for example, inhibition of regenerative signaling) to strengthen the conclusions.

Thank you for this valuable suggestion. We acknowledge that the inclusion of a positive control (e.g., mechanical injury model) or a negative control (e.g., inhibition of regenerative signaling) would have strengthened the conclusions. However, these control experiments were not incorporated into the current study design.

• The choice of only fgf1, axin2, and sox2 for gene expression analysis should be better justified. Explain why other well-known regulators of neuromast regeneration, such as atoh1, notch1, or myc, were not included. (Garrry can you write the answer for this?)

Thank you for your valuable insight, we had initially planned to look for the following genes that you had suggested but due to the novelty of the organism current genomic database could not clearly located the locations of the following genes. In the case of myc an abundance of variants were found within the database, which we could not note toward the true gene of interest. In relation to atoh1 this was once again another gene we could not locate, similarly with notch1. Therefore, future work needs to be conducted on the current model at hand to be able to advance current knowledge and tools available for future research.

 

• The text would benefit from careful language editing to improve clarity and consistency (for instance, ensure uniform use of “neuromast cells” or “hair cells,” and correct typographical errors such as “DASPIE” → “DASPEI,” “ffgf1” → “fgf1,” etc.).

Thank you very much for highlighting these typographical errors. We have corrected them in the revised manuscript.

Once again, Thank you for your valuable comments and constructive input on the manuscript.

 

Reviewer 3 Report

Comments and Suggestions for Authors

• Language & Style:

  • Minor grammatical errors throughout (e.g., “neuro-mast” inconsistently hyphenated; “ffgf1” typo). Manuscript requires careful proofreading.

  • Abstract should be condensed for clarity.

• Methods:

  • Details on RNA quality (RIN scores) should be included for reproducibility.

  • More detail on statistical analysis (ANOVA assumptions, corrections) is needed.

• Discussion:

  • While Wnt/Fgf cross-talk is well described, integration of Sox2 findings is underdeveloped. Why was Sox2 stable while fgf1/axin2 showed changes? Possible biological explanations should be explored.

  • Recent literature (2023–2025) on mammalian attempts at pharmacological hair cell regeneration (e.g., Edge et al., JCI 2024)  could be integrated for translational relevance.

 

Author Response

Review report  3

• Minor grammatical errors throughout (e.g., “neuro-mast” inconsistently hyphenated; “ffgf1” typo). Manuscript requires careful proofreading.

• Thank you very much for highlighting these typographical errors. We have corrected them in the revised manuscript.

 

• Abstract should be condensed for clarity.
• Methods:
• Details on RNA quality (RIN scores) should be included for reproducibility.

Thank you for pointing this out. We have not included this information into the main text but can be introduced as a supplementary table.

Sample #	Control	24 hours	72 hours
1	96.2	161.2	270.7
2	52.9	193.4	292.5
3	241.6	244.4	135.2
4	244.04

 

 

• • More detail on statistical analysis (ANOVA assumptions, corrections) is needed.  

Thank you for this comment and we have incorporated the analysis in the relevant results section

 

• Discussion:
• While Wnt/Fgf cross-talk is well described, integration of Sox2 findings is underdeveloped. Why was Sox2 stable while fgf1/axin2 showed changes? Possible biological explanations should be explored.

Thank you for your thoughtful observation regarding the integration of Sox2 findings. We appreciate your insight and have incorporated this consideration into our revised discussion. While Wnt/Fgf cross-talk is well characterized, the relative stability of Sox2 expression compared to the dynamic changes observed in fgf1 and axin2 suggests distinct regulatory mechanisms may be at play. Possible biological explanations include differential sensitivity to upstream signaling gradients, temporal buffering of transcriptional responses, or context-dependent roles of Sox2 in maintaining progenitor identity.

Recent literature (2023–2025) on mammalian attempts at pharmacological hair cell regeneration (e.g., Edge et al., JCI 2024) could be integrated for translational relevance.

We have incorporated the reference in Line #13

 

Once again, Thank you for your valuable comments and constructive input on the manuscript

 

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately responded to the questions raised by this reviewer.

Reviewer 2 Report

Comments and Suggestions for Authors

All the requested changes were done by the authors. I recommended publication of this manuscript in the present form.