High-Salt Exposure Disrupts Cardiovascular Development in Zebrafish Embryos, Brachyodanio rerio, via Calcium and MAPK Signaling Pathways

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript by Thompson et al describes the effects of high salt exposure to zebrafish embryos on heart development.  Unfortunately, outside of measuring heart rate and a marginal quality image of a zebrafish embryo exposed to high salt, the manuscript does not address how heart development may be impacted by high salt exposure.  Rather, it appears to be just a report of an RNA seq analysis with it being left to other researchers to possibly link the current result to heart development.

The following comments must be addressed to improve the quality of the manuscript and possibly allow it to serve as a resource for investigators interested in hypertension during fetal development:

1. Foremost this is a sloppy manuscript with numerous errors that indicate a lack of quality control.  There are too many typographical errors to count, with calsium, musclue, casclar a few examples.  There are 2 references listed under reference 18, #29 is cited but should be #28 being cited (this may result from the error in numbering), there are many grammatical errors, and numerous errors with spacing between words (2 words appear to be 1 word)
2.  In multiple places the authors indicate glucose rather than NaCl, which makes one wonder if their previous manuscript on glucose was used as the template for this manuscript.  This includes Fig. 1 legend states glucose and not NaCl; Ln 191 states glucose and not NaCl; Ln 316-317 reference glucose and not NaCl.
3. How does 2% NaCl in fish water mimic physiological sodium levels in humans with hypertension?  [Na⁺] should be measured in the embryos.
4. Regarding the limited analysis of zebrafish embryos for the effects of NaCl treatment, how was heart rate quantified?  Was this a double-blind analysis?  The morphological effect s should use a dechorionated embryo, as shown for the Control embryo.
5. A more detailed analysis of heart development and morphology is required for this study.  When was the mRNA extracted for RNA seq, at 120h?  What is critically missing is nothing is known regarding possible effects of NaCl on early heart development.  Were any of the relevant genes from the RNA seq altered in expression during key stages of cardiac development, such as specification etc?  ISH can be conducted for some of the candidate genes during early heart development.  Analyzing early heart morphology could allow the authors to determine how heart development may be delayed due to NaCl exposure.
6. In its current form this manuscript provides little new information on the effects of salt exposure on heart development.  

Comments on the Quality of English Language

See above comments regarding the grammatical and spelling erros throughout the manuscript.

Author Response

Dear Reviewr,

We sincerely thank you for the thoughtful and constructive feedback on our manuscript. We have carefully revised the manuscript to address all comments and suggestions. Below, we provide a point-by-point response, detailing the changes made and clarifying aspects where additional explanation was necessary. We believe these revisions have significantly improved the clarity and quality of the manuscript. Thank you!

We sincerely thank you for the thoughtful and constructive feedback on our manuscript. We have carefully revised the manuscript to address all comments and suggestions. Below, we provide a point-by-point response, detailing the changes made and clarifying aspects where additional explanation was necessary. We believe these revisions have significantly improved the clarity and quality of the manuscript. Thank you!

This manuscript by Thompson et al describes the effects of high salt exposure to zebrafish embryos on heart development.  Unfortunately, outside of measuring heart rate and a marginal quality image of a zebrafish embryo exposed to high salt, the manuscript does not address how heart development may be impacted by high salt exposure.  Rather, it appears to be just a report of an RNA seq analysis with it being left to other researchers to possibly link the current result to heart development.

The following comments must be addressed to improve the quality of the manuscript and possibly allow it to serve as a resource for investigators interested in hypertension during fetal development:

1. Foremost this is a sloppy manuscript with numerous errors that indicate a lack of quality control.  There are too many typographical errors to count, with calsium, musclue, casclar a few examples.  There are 2 references listed under reference 18, #29 is cited but should be #28 being cited (this may result from the error in numbering), there are many grammatical errors, and numerous errors with spacing between words (2 words appear to be 1 word)

             We sincerely thank the reviewer for pointing out these critical issues. We have thoroughly revised the manuscript to correct all typographical, spelling, and grammatical errors, including terms such as calcium, muscle, and vascular. We also addressed formatting issues such as inconsistent spacing between words and citation numbering errors. Reference numbers and formatting have now been double-checked and corrected throughout the manuscript to ensure accuracy. We appreciate your attention to detail and believe these corrections have substantially improved the clarity and professionalism of the manuscript.

1.  In multiple places the authors indicate glucose rather than NaCl, which makes one wonder if their previous manuscript on glucose was used as the template for this manuscript.  This includes Fig. 1 legend states glucose and not NaCl; Ln 191 states glucose and not NaCl; Ln 316-317 reference glucose and not NaCl.

 

We thank the reviewer for noticing these important inconsistencies. You are correct—these references to glucose were inadvertently carried over from our previous manuscript and should have referred to NaCl. We have carefully reviewed the entire manuscript and corrected all instances where “glucose” was mistakenly mentioned, including in the Figure 1 legend, Line 191, and Lines 316–317. We sincerely apologize for this oversight and appreciate your attention to detail.

1. How does 2% NaCl in fish water mimic physiological sodium levels in humans with hypertension?  [Na⁺] should be measured in the embryos.

Previous zebrafish studies modeling high‐salt conditions have used 1–3% NaCl in E3 medium to induce an osmotic load that plausibly mimics systemic hypernatremia (e.g., Jeong et al., 2013; Zhang et al., 2020; Hassan et al., 2021). Although the absolute [Na⁺] in 2% NaCl (≈341 mM) exceeds normal physiological plasma levels in mammals (≈140–150 mM), zebrafish embryos readily equilibrate small ions across their permeable epithelium, and prior work has shown that 2% NaCl reliably elevates internal Na⁺ enough to activate salt‐responsive signaling pathways without causing acute lethality (Jeong et al., 2013; Zhang et al., 2020; Hassan et al., 2021). In other words, the 2% external concentration serves as an experimentally validated “high‐salt challenge” (rather than a direct isomolar match) that triggers cellular responses analogous to those seen in mammalian hypernatremia and salt‐sensitive hypertension.

We agree that direct measurement of embryonic [Na⁺] would strengthen the physiological interpretation. Unfortunately, ion‐selective electrode or flame photometry assays were not available to us at the time of this study. We have now added a statement in the Discussion to acknowledge this limitation and to recommend that future work include quantitative ion assays (e.g., atomic absorption spectroscopy or ion‐selective electrode measurements) in embryos exposed to 1–3% NaCl.

Even without direct [Na⁺] measurements, our goal was to characterize downstream transcriptomic and phenotypic changes induced by a well‐established high‐salt challenge in zebrafish embryos. Prior publications (e.g., Jeong et al., 2013; Zhang et al., 2020; Hassan et al., 2021) have demonstrated that exposing zebrafish embryos to 2% NaCl reliably activates osmoregulatory and cardiovascular stress pathways that parallel those observed in mammalian models of salt‐induced hypertension. By focusing on gene expression, morphology, and heart function, we capture the biologically relevant sequelae of elevated sodium exposure.

1. Regarding the limited analysis of zebrafish embryos for the effects of NaCl treatment, how was heart rate quantified?  Was this a double-blind analysis?  The morphological effect s should use a dechorionated embryo, as shown for the Control embryo.

             We thank the reviewer for these insightful comments. As now described in the revised Methods section, heart rate in zebrafish embryos was assessed by recording short video clips and manually counting heartbeats over a 10-second interval, which were then converted to beats per minute (bpm). This was done for five individual embryos per group at each time point, and average values were reported. While a formal double-blind procedure was not employed, all video recordings were anonymized before analysis to minimize observer bias.

We also appreciate the reviewer’s suggestion regarding morphological imaging. Images were taken using an inverted microscope at 4× and 10× magnification, focusing on the cardiac region to assess structural differences. While some embryos were imaged after dechorionation, others remained within the chorion to maintain structural integrity.

Thank you again for helping us improve the clarity and rigor of our experimental methods.

1. A more detailed analysis of heart development and morphology is required for this study.  When was the mRNA extracted for RNA seq, at 120h?  What is critically missing is nothing is known regarding possible effects of NaCl on early heart development.  Were any of the relevant genes from the RNA seq altered in expression during key stages of cardiac development, such as specification etc?  ISH can be conducted for some of the candidate genes during early heart development.  Analyzing early heart morphology could allow the authors to determine how heart development may be delayed due to NaCl exposure.

We appreciate the reviewer’s thoughtful suggestion regarding the importance of early time point analyses for understanding the onset of developmental defects.  In the current study, mRNA was extracted at 120 hours post-fertilization (hpf), which corresponds to a late developmental stage when the cardiovascular system is functional. Our primary objective was to investigate transcriptomic changes associated with prolonged salt exposure, with an emphasis on pathway-level disruptions relevant to cardiovascular and muscular development.In this study, we focused our transcriptomic analysis at 120 hours post fertilization (hpf), a developmental stage at which phenotypic outcomes such as altered heart rate, morphology, and hatching delay were clearly evident. We agree that evaluating earlier stages using approaches like ISH could offer valuable insight into the temporal dynamics of gene expression changes, particularly those involved in early cardiac specification and morphogenesis. While that was beyond the scope of the current work, we have now included a paragraph in the Discussion section to clarify the rationale behind our time point selection and to acknowledge this limitation (Discussion, paragraph 5). We thank the reviewer for highlighting this opportunity, which will guide future follow-up studies aimed at defining the early molecular events driving salt-induced cardiac developmental abnormalities.Due to resource and time constraints, we were unable to conduct earlier stage sampling, in situ hybridization (ISH), or high-resolution cardiac morphological assessments. However, our RNA-Seq analysis did reveal altered expression of genes involved in cardiac muscle development, specification, and calcium signaling pathways—supporting the hypothesis that salt exposure may disrupt key aspects of heart development.While our study focuses on transcriptional responses at 120 hpf, we acknowledge that earlier developmental windows merit detailed investigation. We have added relevant discussion and citations to highlight published morphological studies in this context and suggest future work to address the temporal dynamics of gene expression and morphological changes in early cardiac development under high-salt conditions

For 5 and this specific question: Were any of the relevant genes from the RNA seq altered in expression during key stages of cardiac development, such as specification etc?

 Thank you for your helpful comment. In our dataset, several differentially expressed genes fall within key pathways related to zebrafish heart development, such as MAPK, calcium signaling, and adrenergic signaling. Notably, we identified alpk2 as one of the top 10 downregulated genes in salt-treated embryos. ALPK2 has been shown to promote cardiogenesis in both zebrafish and human pluripotent stem cells, and more recent work has demonstrated its importance in enhancing the therapeutic potential of human cardiomyocytes. We have now included this information in the Discussion section to emphasize the developmental significance of alpk2 and its potential as a key molecular mediator of salt-induced cardiac disruption

1. In its current form this manuscript provides little new information on the effects of salt exposure on heart development.  

We appreciate the reviewer’s feedback and respectfully offer clarification on the novelty of our findings. While salt exposure has been studied in the context of general embryonic stress, our study provides new insights by specifically focusing on the transcriptomic response of zebrafish embryos to high salinity, with a particular emphasis on cardiac development pathways.Through RNA sequencing, we identified several genes that are differentially expressed in salt-treated embryos, including alpk2, a gene previously shown to play a critical role in heart morphogenesis in zebrafish and human cardiomyocyte differentiation. The significant downregulation of alpk2 in our dataset, along with disruptions in the calcium signaling, MAPK signaling, and cardiac muscle contraction pathways, offers mechanistic insight into how elevated salt may impair heart development at the molecular level.We have revised the Discussion to better highlight these novel contributions and the relevance of our findings to environmental toxicology and human health, particularly in the context of prenatal salt exposure.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Please see my detailed comments below. But initially, the manuscript needs a major revision for the methods and results sections.

The authors should place detailed methodological information in the abstract. Specifics such as the number of treatment groups, salt concentrations, exposure duration, and number of embryos used for RNA analysis should be limited to the Methods section. The abstract should highlight the purpose, key findings, and overall conclusions concisely.

The authors should clarify the number of embryos used in each experimental group. There is inconsistency between different sections—some parts mention 25 embryos across 5 groups, while others mention a total of 100 embryos. A clear, consistent description of sample size per group and total number of replicates should be provided in the Methods section.

All tables, figures, and schematic diagrams should be placed in the manuscript at the point where they are first mentioned in the text, rather than grouped together within the Results section. This would improve the logical flow and readability of the manuscript.

The authors should ensure that figure legends and table captions are sufficiently descriptive and clearly explain what is being shown, including statistical significance indicators and sample sizes where applicable.

The organization of the Results section can be improved by aligning the text more directly with the presented figures and data. Each paragraph should lead naturally into the corresponding visual element.

The authors should specify the characteristics of the salt used in the treatments—whether it contained iodine, its purity level, and the manufacturer/source. This is important for reproducibility and to rule out confounding factors.

An ethical approval statement must be included, confirming that all animal procedures were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals.

Figures showing embryo and heart morphology should be more detailed. Higher-resolution or more anatomically informative images would help visualize the phenotypic alterations described in the results. Inclusion of lateral and dorsal views, scale bars, and anatomical labels would enhance clarity and scientific value.

Figures, tables, and diagrams should be placed where first cited in the text, not grouped separately within the Results section.

While the molecular data is convincing, histological validation (e.g., heart morphology) could strengthen the anatomical interpretation.

Statistical methods should be described more thoroughly (e.g., test types, significance thresholds).

Figure 1 legend is "effect of glucose". Thus, the authors check if glucose or salt was tested and explain why there is confusion about glucose or salt in the figure legend.

Please write how you determined the heart rate or other morphological and physiological characteristics

For introduction and discussion,

Consider referencing more recent literature on salinity stress and cardiovascular development in fish or mammals.

Expand on the potential implications for environmental toxicology and human health relevance, especially regarding prenatal salt exposure.

Author Response

Dear Reviewer,

We sincerely thank you for the thoughtful and constructive feedback on our manuscript. We have carefully revised the manuscript to address all comments and suggestions. Below, we provide a point-by-point response, detailing the changes made and clarifying aspects where additional explanation was necessary. We believe these revisions have significantly improved the clarity and quality of the manuscript. Thank you!

Dear Authors,

Please see my detailed comments below. But initially, the manuscript needs a major revision for the methods and results sections.

The authors should place detailed methodological information in the abstract. Specifics such as the number of treatment groups, salt concentrations, exposure duration, and number of embryos used for RNA analysis should be limited to the Methods section. The abstract should highlight the purpose, key findings, and overall conclusions concisely.

We appreciate the reviewer’s guidance. We have revised the abstract to focus on the purpose, key findings, and overall conclusions. Detailed methodological information has been removed and appropriately placed in the Methods section. Thank you for the helpful suggestion.

The authors should clarify the number of embryos used in each experimental group. There is inconsistency between different sections—some parts mention 25 embryos across 5 groups, while others mention a total of 100 embryos. A clear, consistent description of sample size per group and total number of replicates should be provided in the Methods section.

We appreciate the reviewer’s careful attention to detail. We have revised the Materials and Methods section to clearly state that a total of 125 embryos were used, with 25 embryos per Petri dish. Two dishes (50 embryos) were assigned to salt-treated conditions, and three dishes (75 embryos) served as controls. We have ensured that this information is now consistent throughout the manuscript.

Additionally, we clarified that RNA was extracted at 120 hours post-fertilization (hpf), which was the endpoint of the exposure period and the timepoint for transcriptomic analysis. These revisions aim to improve the clarity and reproducibility of our experimental design.

All tables, figures, and schematic diagrams should be placed in the manuscript at the point where they are first mentioned in the text, rather than grouped together within the Results section. This would improve the logical flow and readability of the manuscript.

Thank you for the helpful suggestion. We are currently following the journal’s submission template, which places all figures and tables at 3.2. We have reached out to the editor to confirm whether in-text placement is acceptable and will revise the formatting accordingly upon receiving guidance.

The authors should ensure that figure legends and table captions are sufficiently descriptive and clearly explain what is being shown, including statistical significance indicators and sample sizes where applicable.

We thank the reviewer for this helpful suggestion. In response, we have revised the Figures legend and table captions to improve clarity and ensure that the data presented in the figure are fully interpretable to readers.

The organization of the Results section can be improved by aligning the text more directly with the presented figures and data. Each paragraph should lead naturally into the corresponding visual element.

Thank you for the helpful suggestion. We are currently following the journal’s submission template, which places all figures and tables at 3.2. We have reached out to the editor to confirm whether in-text placement is acceptable and will revise the formatting accordingly upon receiving guidance.

The authors should specify the characteristics of the salt used in the treatments—whether it contained iodine, its purity level, and the manufacturer/source. This is important for reproducibility and to rule out confounding factors.

Thank you for pointing this out. We have clarified the characteristics of the salt used in our study in the Methods section, specifying that pure sodium chloride (NaCl) without iodine was used, including its concentration, purity level, and manufacturer/source, to ensure reproducibility.

An ethical approval statement must be included, confirming that all animal procedures were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals.

We appreciate the reviewer’s comments. Please see Institutional Review Board Statement in the manuscript. Thank you!

Figures showing embryo and heart morphology should be more detailed. Higher-resolution or more anatomically informative images would help visualize the phenotypic alterations described in the results. Inclusion of lateral and dorsal views, scale bars, and anatomical labels would enhance clarity and scientific value.

Thank you for the suggestion. We have added higher magnification (10×) images  in figure 1 to improve the clarity of the morphological differences, focusing particularly on the cardiac region. Due to the limitations of our imaging equipment, these were the highest-resolution images available.

Figures, tables, and diagrams should be placed where first cited in the text, not grouped separately within the Results section.

Thank you for the helpful suggestion. We are currently following the journal’s submission template, which places all figures and tables at 3.2. We have reached out to the editor to confirm whether in-text placement is acceptable and will revise the formatting accordingly upon receiving guidance.

While the molecular data is convincing, histological validation (e.g., heart morphology) could strengthen the anatomical interpretation.

Thank you for this insightful suggestion. While we agree that histological validation would further strengthen the anatomical interpretations, we did not collect additional morphological or histological data beyond what is included in this manuscript. We have provided our best images available and focused on integrating molecular and phenotypic observations to support our conclusions. We appreciate this recommendation and will consider incorporating histological validation in future studies.

Statistical methods should be described more thoroughly (e.g., test types, significance thresholds).

Thank you very much for the insight. We revised the method section including the statistical methods to make it clear to the readers. Thank you for helping us to improve the methods section. Thank you.

Figure 1 legend is "effect of glucose". Thus, the authors check if glucose or salt was tested and explain why there is confusion about glucose or salt in the figure legend.

We thank the reviewer for noticing this important inconsistencies. We changed glucose to salt in Figure 1.

Please write how you determined the heart rate or other morphological and physiological characteristics

We thank the reviewer for these insightful comments. As now described in the revised Methods section, heart rate in zebrafish embryos was assessed by recording short video clips and manually counting heartbeats over a 10-second interval, which were then converted to beats per minute (bpm). This was done for five individual embryos per group at each time point, and average values were reported. all video recordings were anonymized before analysis to minimize observer bias.

We also appreciate the reviewer’s suggestion regarding morphological imaging. Images were taken using an inverted microscope at 4× and 10× magnification, focusing on the cardiac region to assess structural differences. While some embryos were imaged after dechorionation, others remained within the chorion to maintain structural integrity.

Thank you again for helping us improve the clarity and rigor of our experimental methods.

 

For introduction and discussion,

Consider referencing more recent literature on salinity stress and cardiovascular development in fish or mammals.

Thank you very much for your critical comments. We added 4 new references and one is from 2025 and one is from 2021 and one is from 2020. The references are adjusted accordingly in the text as well. Thank you.

Expand on the potential implications for environmental toxicology and human health relevance, especially regarding prenatal salt exposure.

We thank the reviewer for highlighting this important perspective. In response, we have added a paragraph to the end of the Discussion section that expands on the broader implications of our findings in the context of environmental toxicology and human health. Specifically, we discuss how our zebrafish model offers insight into the potential impact of prenatal salt exposure on developmental programming and cardiovascular health, and how this work supports the relevance of using zebrafish for evaluating the effects of environmental and dietary stressors. We appreciate the opportunity to contextualize our results in this broader framework.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed the comments regarding correction of the numerous errors in the manuscript, but overall they still do not directly address the mechanisms by which the NaCl treatment of embryos may be impacting cardiac development.  For example, there are no studies to directly show that MAPK signaling perturbations lead to the observed phenotypes.  So while the manuscript has been improved, it does not provide substantial new information to the field.

Author Response

Please see the attachment. Thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your thoughtful and thorough revisions to the manuscript. I appreciate the clear and detailed responses provided to each reviewer comment, as well as the improvements made throughout the text. The clarification of methodological details, correction of figure legends, and incorporation of broader implications into the discussion have strengthened the scientific quality and clarity of the work.

While certain limitations remain (such as the absence of histological data), you have acknowledged these transparently and justified the scope of your study appropriately. The enhancements to figure descriptions, statistical reporting, and language usage have further improved readability and rigor.

I am pleased to recommend the manuscript for publication, contingent on final editorial checks.

Best regards,

Author Response

Please see the attachment. Thank you.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed the comments to the best of their ability.