Reduction in N-Acetylglucosaminyltransferase-I Activity Decreases Survivability and Delays Development of Zebrafish

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Reduction of N-acetylglucosaminyltransferase-I activity de- 2 creases survivability and delays development of zebrafish" by Hall et al. deals with important topic glycobiology. Glycobiology is clearly a understudied field especially in developmental biology although glycosylation post-translational modifications play a key role in many biological processes and diseases. The manuscript was written in clear and logical manner, and I have only few minor comments to improve the manuscript.

Specific comments:

1) Figure 1A: One could add the gene name also in the figure and figure legend, so that the link to the gene and biochemistry would be clearer.

2) Figure 2A. This figure should have graphical legend to give names to the symbols (eg. blue box, red triangle).

3) Figure 3C. This mRNA decay could be a result of non-sense mediated decay mechanism regulating dysfunctional mRNAs. This should be discussed.

4) Figure 4. The viability of zebrafish mutants in the early developmental stages could be due to maternal contribution of functional mRNA. Please discuss this.

5) Page2, line 76. The line created is a mutant and not knock-down.

 

Author Response

Responses to Reviewers

Reviewer 1

Comments and Suggestions for Authors

The manuscript "Reduction of N-acetylglucosaminyltransferase-I activity de- 2 creases survivability and delays development of zebrafish" by Hall et al. deals with important topic glycobiology. Glycobiology is clearly a understudied field especially in developmental biology although glycosylation post-translational modifications play a key role in many biological processes and diseases. The manuscript was written in clear and logical manner, and I have only few minor comments to improve the manuscript.

Specific comments:

1) Figure 1A: One could add the gene name also in the figure and figure legend, so that the link to the gene and biochemistry would be clearer.

Mgat1b‑ is used to identify the mutant strain in the figure. Also we have added the following edit for clarification, “Depiction of N-glycan types and the sequential processing initiated via GnT-I (Mgat1) from the simplest N-glycan type (oligomannose) to more processed hybrid and complex.”

2) Figure 2A. This figure should have graphical legend to give names to the symbols (eg. blue box, red triangle).

We have added a legend to figure 2A.

3) Figure 3C. This mRNA decay could be a result of non-sense mediated decay mechanism regulating dysfunctional mRNAs. This should be discussed.

A sentence has been added in the results with the following reference (Lejeune F. (2022) Biomedicines 10(1):141.

The decay in Mgat1b mRNA throughout zebrafish development may be due to the non-sense mediated decay mechanism (Lejeune 2022), as Mgat1b gene editing resulted in a premature termination codon.

4) Figure 4. The viability of zebrafish mutants in the early developmental stages could be due to maternal contribution of functional mRNA. Please discuss this.

A sentence has been added in the discussion section, including the following reference (Abrams EW and Mullins MC. Curr Opin Genet Dev (2009) 4:396-403)

5) Page2, line 76. The line created is a mutant and not knock-down.

Changed knock-down to mutant strain here and some other inconsistencies. 

 

Thank you for your suggestions as they were helpful!

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a detailed study on the role of N-glycans, specifically complex types, in the survival and development of zebrafish (Danio rerio). Using CRISPR/Cas9 technology, the authors knocked down the Mgat1b gene, responsible for the enzymatic step converting oligomannose to hybrid and complex N-glycans. The results showed decreased levels of complex N-glycans and consequent adverse effects on the zebrafish's survivability and development. Overall, the manuscript is scientifically rigorous and adds valuable knowledge to the understanding of glycosylation disorders and developmental biology.

 

Below are some suggestions to enhance the quality of this manuscript:

 

Abstract

 

1.  Your mention of CRISPR/Cas9 is good, but you might consider briefly stating what kind of assays were used for developmental and locomotor evaluations to give a fuller picture.

 

2. You've summarized the findings well, but it would add weight if you could explicitly state the significance of these findings, particularly in relation to existing literature or potential medical applications.

 

3. Be cautious with terms that might need more context or explanation. For example, you could consider defining or explaining what "incomplete penetrance and variable expressivity" mean for readers less familiar with genetic terminology.

 

Introduction

 

4. While you explain the types of N-glycans well, you might want to consider defining or elaborating on other specific terms like "epiboly" or "anlagen" for readers less familiar with developmental biology.

 

5. You've done well in referencing prior studies, but make sure you synthesize what these prior studies mean for your research. For instance, why is the prior information on Mgat1 in mice relevant to your zebrafish model?

 

6. Your objective ("In the present study, we created a Mgat1b knockdown fish strain...") could be made a bit more prominent. You could preface it with "The objective of this study is to..." for additional clarity.

 

Results

 

7. The authors should further clarify why zebrafish were chosen as a model, especially considering the differences in N-acetylglucosaminyltrases between zebrafish and mammals.

 

8. The authors should consider showing representative sequencing chromatograms to better visualize the 5 nucleotide deletion and the premature stop codon (Figure 1C).

 

9. The manuscript might benefit from a more detailed explanation or a supplementary table showing the specific N-glycan structures identified.

 

10. including effect sizes and confidence intervals, in addition to p-values, to provide a more comprehensive statistical analysis.

 

11. What are the functional consequences of having higher amounts of oligomannose N-glycans and lower levels of complex N-glycans in the mutant line? This could be discussed in more detail.

 

12. The finding that most deaths in Mgat1b-/- embryos occur between 75% to 90% epiboly is intriguing. Are there any theories or hypotheses that the authors can provide to explain this?

 

13. Since the Mgat1 gene in mice had effects on neural structure, is there evidence of similar neural changes in the zebrafish mutant line?

 

14. The text ends abruptly while discussing motor-sensory function. This seems to be a critical part of the study, and more information or data should be included.

 

15. For ease of reading, all abbreviations like GnT-I, Wt AB, and Mgat1b-/- should be defined upon first use in both the abstract and main body of the manuscript.

 

16. There are some issues with sentence structure and clarity. For instance, "e.g.,GnT-Ib, is a type II" should be cleaned up for readability and proper spacing.

 

17. Please clarify the methodology used to monitor heartbeat in the zebrafish embryos. Is it done via direct observation or some sort of sensor?

 

18. How was the birefringence normalized? And could you explain why lower light intensity in mutant embryos at 58 hpf is indicative of delayed muscle development?

 

19. While you specify "n≥22 fish" and "n≥5 plates," consider providing exact sample sizes for each group and experiment, as this will lend more weight to the statistical analysis.

 

20. You've reported a p-value but have not mentioned the effect size and confidence intervals. Including these could provide a more robust statistical picture.

 

21. The manuscript states that the birefringence in mutant fish "approached that of the Wt AB larval fish over the time course." Could you provide a statistical comparison between the rates at which this happens in Wt AB and Mgat1b-/- fish?

 

22. The delay in cardiac muscle development has obvious clinical relevance. Can you discuss the implications of this finding for understanding human congenital heart defects or other muscle-related diseases?

 

23. The manuscript notes that all embryos had heartbeats at 28 hpf. Is there a particular developmental milestone that is crossed at 28 hpf, making it a crucial point for cardiac development?

 

Discussion

 

24. Given the extensive findings, it would be beneficial to place the current study in a broader context. How do the results compare with previous research in the field? What novel insights are offered?

 

25. You compare the embryonic development timelines between zebrafish and mice. Could you elaborate on the implications of this comparison? What can be inferred regarding the conservation of certain genetic functions or developmental stages?

 

26. The discussion indicates a drastic decline in embryonic survivability from 8-12 hpf. Is this a typical timeframe for observing the effects of such mutations in zebrafish? Why or why not?

 

27. While the study focuses on the outcomes, the mechanisms behind these outcomes are less discussed. For example, why would inhibition in brain anlagen formation specifically occur due to GnT-Ib knockout? Hypotheses or speculations could add depth to the discussion.

 

28. Your study investigates complex N-glycans in relation to locomotor activity and muscle development. It would be beneficial to explain why these complex N-glycans are particularly important in these contexts, backed by citations if possible.

 

29. Since the Discussion mentions individuals suffering from congenital disorders of glycosylation, could you elaborate more on how your findings might relate to these conditions? Are there potential therapeutic avenues that your research could inform?

 

30. While the study appears comprehensive, it might be beneficial to acknowledge any limitations. For example, are there limitations in the methodology, or are there uncertainties that should be addressed in future studies?

 

31. The last paragraph could benefit from a more explicit section on future directions. Are there particular experiments or models that should be used to further validate your findings?

 

32. Be cautious with word choices to maintain a scientific tone. For instance, "ecotopic" seems to be a typographical error and should be corrected to "ectopic."

 

Conclusion

 

33. You mentioned that the study could offer additional potential diagnostic targets and treatment options. However, the conclusion could benefit from further elaboration on this point. For example, could your findings inform early diagnostic tests for Congenital Disorders of Glycosylation, or suggest new areas of therapeutic research?

 

34. A sentence or two indicating what the next steps in this research could be would round out the conclusion nicely. For instance, are there additional experiments that should be performed? Are there other models or systems where these findings should be tested for validation?

 

35. Emphasize why your findings are unique and why they matter, especially considering the limited treatment options available for the diseases discussed.

 

36. Make sure that the conclusion is free of typographical errors. For example, it's "Congenital Disorders of Glycosylation," not "Congenital Orders of Glycosylation."

Comments on the Quality of English Language

While the manuscript seems well-written, ensuring a smooth transition between sentences and paragraphs can improve readability and flow. There may be a need to maintain consistency in terminology. For example, if "Mgat1b-/-" is used to describe the knockdown strain, this nomenclature should be consistently applied throughout the paper.

Author Response

Reviewer 2

 

Comments and Suggestions for Authors

The manuscript presents a detailed study on the role of N-glycans, specifically complex types, in the survival and development of zebrafish (Danio rerio). Using CRISPR/Cas9 technology, the authors knocked down the Mgat1b gene, responsible for the enzymatic step converting oligomannose to hybrid and complex N-glycans. The results showed decreased levels of complex N-glycans and consequent adverse effects on the zebrafish's survivability and development. Overall, the manuscript is scientifically rigorous and adds valuable knowledge to the understanding of glycosylation disorders and developmental biology.

 

Below are some suggestions to enhance the quality of this manuscript:

 

Abstract

 

1.  Your mention of CRISPR/Cas9 is good, but you might consider briefly stating what kind of assays were used for developmental and locomotor evaluations to give a fuller picture.

 

Assays utilized for developmental and locomotor evaluations has been added, see abstract.

 

2. You've summarized the findings well, but it would add weight if you could explicitly state the significance of these findings, particularly in relation to existing literature or potential medical applications.

 

Significance as it relates to human disorders has been added to abstract.

 

3. Be cautious with terms that might need more context or explanation. For example, you could consider defining or explaining what "incomplete penetrance and variable expressivity" mean for readers less familiar with genetic terminology.

 

Explanation of incomplete penetrance and variable expressivity has been added, see abstract.

 

Introduction

 

4. While you explain the types of N-glycans well, you might want to consider defining or elaborating on other specific terms like "epiboly" or "anlagen" for readers less familiar with developmental biology.

 

Anlagen has been further defined, along with an explanation of epiboly, see introduction.

 

5. You've done well in referencing prior studies, but make sure you synthesize what these prior studies mean for your research. For instance, why is the prior information on Mgat1 in mice relevant to your zebrafish model?

 

The relevance of Mgat 1 knockdown in mice as it relates to our zebrafish studies has been added to introduction.

 

6. Your objective ("In the present study, we created a Mgat1b knockdown fish strain...") could be made a bit more prominent. You could preface it with "The objective of this study is to..." for additional clarity.

 

Text has been modified to clarify the objective of the study, see introduction.

 

Results

 

7. The authors should further clarify why zebrafish were chosen as a model, especially considering the differences in N-acetylglucosaminyltrases between zebrafish and mammals.

 

Clarification for use of zebrafish has been added to results.

 

8. The authors should consider showing representative sequencing chromatograms to better visualize the 5 nucleotide deletion and the premature stop codon (Figure 1C).

 

See updated figure 1.

 

9. The manuscript might benefit from a more detailed explanation or a supplementary table showing the specific N-glycan structures identified.

 

Table S2, Table S3, Fig S1, and Fig S2 depict the relative abundancies and intensities of the permethylated N-glycans of each of the N-glycans detected. Additionally, MSMS CID (collision-induced dissociation) fragmentation offers robust identification of core fucosylation, antennal fucosylation, alpha-gal structures etc.

 

10. including effect sizes and confidence intervals, in addition to p-values, to provide a more comprehensive statistical analysis.

 

Since samples were treated with PNGase F enzyme, the relative abundance of a particular glycan maybe a result of more efficient digestion, not a result of glycan amount in the sample. Hence relative abundancies are interpreted with that in mind. As such, comprehensive statistical analysis was not performed on the mass spec data.

 

11. What are the functional consequences of having higher amounts of oligomannose N-glycans and lower levels of complex N-glycans in the mutant line? This could be discussed in more detail.

 

Text has been added to specify the consequences of higher amounts of oligomannose N-glycans and lower levels of complex N-glycans has been added to results.

 

12. The finding that most deaths in Mgat1b-/- embryos occur between 75% to 90% epiboly is intriguing. Are there any theories or hypotheses that the authors can provide to explain this?

 

Text has been added to expand upon the results.

 

13. Since the Mgat1 gene in mice had effects on neural structure, is there evidence of similar neural changes in the zebrafish mutant line?

 

Our future plans will be to conduct more detailed studies of neurons in Mgat1b-/-, as well as Mgat1a-/- and (Mgat1a-/-)/( Mgat1b-/-) double knockdown,  since this study showed the overall effect of lowered complex N-glycans at the neural, skeletal, and muscular systems.

 

14. The text ends abruptly while discussing motor-sensory function. This seems to be a critical part of the study, and more information or data should be included.

 

Additional text has been included to expand upon the results.

 

15. For ease of reading, all abbreviations like GnT-I, Wt AB, and Mgat1b-/- should be defined upon first use in both the abstract and main body of the manuscript.

 

All abbreviations have been defined in both the abstract and upon first use in the main body, however; Wt AB is the actual strain of fish and not an abbreviation

 

16. There are some issues with sentence structure and clarity. For instance, "e.g.,GnT-Ib, is a type II" should be cleaned up for readability and proper spacing.

 

Sentence has been changed for clarity in the results.

 

17. Please clarify the methodology used to monitor heartbeat in the zebrafish embryos. Is it done via direct observation or some sort of sensor?

 

Clarification of the methodology to monitor heartbeat of zebrafish embryos, see materials and methods.

 

18. How was the birefringence normalized?

 

Text added explaining normalization has been added to results.

 

And could you explain why lower light intensity in mutant embryos at 58 hpf is indicative of delayed muscle development?

 

Explanation explaining light emission has been added to text (lines 419-421)

 

19. While you specify "n≥22 fish" and "n≥5 plates," consider providing exact sample sizes for each group and experiment, as this will lend more weight to the statistical analysis.

 

See figure legend 6 and methods under 2.13.

 

20. You've reported a p-value but have not mentioned the effect size and confidence intervals. Including these could provide a more robust statistical picture.

 

We have included p-values.

 

21. The manuscript states that the birefringence in mutant fish "approached that of the Wt AB larval fish over the time course." Could you provide a statistical comparison between the rates at which this happens in Wt AB and Mgat1b-/- fish?

 

This statement is based on the result that the means are not significantly different at 4 dpf.

 

22. The delay in cardiac muscle development has obvious clinical relevance. Can you discuss the implications of this finding for understanding human congenital heart defects or other muscle-related diseases?

 

The conclusion has been rewritten to emphasize highlight how the developmental studies specifically may contribute to a better understanding of congenital defects and thus the potential diagnostic and therapeutic implications (lines 506-521).

 

23. The manuscript notes that all embryos had heartbeats at 28 hpf. Is there a particular developmental milestone that is crossed at 28 hpf, making it a crucial point for cardiac development?

 

The conclusion has been rewritten to emphasize highlight how the developmental studies specifically may contribute to a better understanding of congenital defects and thus the potential diagnostic and therapeutic implications.

 

 

Discussion

 

24. Given the extensive findings, it would be beneficial to place the current study in a broader context. How do the results compare with previous research in the field? What novel insights are offered?

Conclusion paragraph has been rewritten to indicate deficiencies of studies in the field, the novelty of our current study, and our goals to enhance future studies.

 

25. You compare the embryonic development timelines between zebrafish and mice. Could you elaborate on the implications of this comparison? What can be inferred regarding the conservation of certain genetic functions or developmental stages?

 We have added additional text to emphasize the significance of the comparison between mice and zebrafish, see discussion.

 

26. The discussion indicates a drastic decline in embryonic survivability from 8-12 hpf. Is this a typical timeframe for observing the effects of such mutations in zebrafish? Why or why not?

 

Although we do not have any citations for similar mutations in zebrafish resulting in death between 8-12 hpf, we postulate that the impact of lessened complex type N-glycans impedes cell migration to such an extent that epiboly does not proceed due to the necessity of coordinated cell movement during this period (75% epiboly occurring around 8 hpf, and 100% epiboly around 10.5 hpf).

 

27. While the study focuses on the outcomes, the mechanisms behind these outcomes are less discussed. For example, why would inhibition in brain anlagen formation specifically occur due to GnT-Ib knockout? Hypotheses or speculations could add depth to the discussion.

 

Text has been added to the discussion to address inhibition of brain anlagen formation in the GnT-Ib knockout.

 

28. Your study investigates complex N-glycans in relation to locomotor activity and muscle development. It would be beneficial to explain why these complex N-glycans are particularly important in these contexts, backed by citations if possible.

 

 Text, with citation has been added to discussion.

 

29. Since the Discussion mentions individuals suffering from congenital disorders of glycosylation, could you elaborate more on how your findings might relate to these conditions? Are there potential therapeutic avenues that your research could inform?

 

The conclusion has been rewritten to emphasize the potential diagnostic and therapeutic implications.

 

30. While the study appears comprehensive, it might be beneficial to acknowledge any limitations. For example, are there limitations in the methodology, or are there uncertainties that should be addressed in future studies?

 

Text has been added to the conclusion to acknowledge limitations of the study and suggestions for enhancement.

 

31. The last paragraph could benefit from a more explicit section on future directions. Are there particular experiments or models that should be used to further validate your findings?

 

The conclusion paragraph has been rewritten to address future directions.

 

32. Be cautious with word choices to maintain a scientific tone. For instance, "ecotopic" seems to be a typographical error and should be corrected to "ectopic."

 

Above mentioned typographical error has been corrected. Further, manuscript has again been edited for typos.

 

Conclusion

33. You mentioned that the study could offer additional potential diagnostic targets and treatment options. However, the conclusion could benefit from further elaboration on this point. For example, could your findings inform early diagnostic tests for Congenital Disorders of Glycosylation, or suggest new areas of therapeutic research?

 

The conclusion has been rewritten to emphasize the potential diagnostic and therapeutic implications.

 

34. A sentence or two indicating what the next steps in this research could be would round out the conclusion nicely. For instance, are there additional experiments that should be performed? Are there other models or systems where these findings should be tested for validation?

 

Text has been added concerning future directions, to include additional models and experiments, see conclusion.

 

35. Emphasize why your findings are unique and why they matter, especially considering the limited treatment options available for the diseases discussed.

 

Text has been included to emphasize novelty of the study, see conclusion.

 

36. Make sure that the conclusion is free of typographical errors. For example, it's "Congenital Disorders of Glycosylation," not "Congenital Orders of Glycosylation."

 

The conclusion has been reviewed to ensure no typographical errors.

 

Comments on the Quality of English Language

While the manuscript seems well-written, ensuring a smooth transition between sentences and paragraphs can improve readability and flow. There may be a need to maintain consistency in terminology. For example, if "Mgat1b-/-" is used to describe the knockdown strain, this nomenclature should be consistently applied throughout the paper.

Thank you for your constructive comments throughout. We have utilized your suggestions to strengthen and emphasize the novelty and potential of the study. Transition sentences have been added for a smooth transition between sentences/paragraphs, and terminology has been checked for consistency.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors present a study dedicated to the evaluation of decreased N acetylglucosaminyltransferase-I activity on zebrafish development through the development of a mutant line. Different molecular techniques were applied to evaluate the decreased activity and the impact on development and survivability of zebrafish embryos. The authors conclude that inadequate levels of complex type N-glycans can impair skeletal muscle development which can have impact on survivability of embryos.

Overall, the study is new, novel and present interesting findings which can have significant impact of the filed. However, there are some minor changes that need to be addressed:

-          L74, is this comparison with mice straightforward?

-          L113, how many embryos, larvae and adults were used? How many replicates? Review along the manuscript.

-          L115, which speed was used?

-          L157, how many larvae were pooled?

-          L160, what was the concentration of DTT?

-          L167, what was the dilution of these probes?

-          L171, how many larvae per replicate?

-          L225, how was dechorionation achieved?

-          L231, SD should be used instead of SE.

-          L244, what is egg water?

-          L273, SD should be used instead of SE.

-          L393, y-axis is missing the units in B and C figure.

 

-          Supplementary files should be mentioned in the main text.

Author Response

Reviewer 3

Comments and Suggestions for Authors

The authors present a study dedicated to the evaluation of decreased N acetylglucosaminyltransferase-I activity on zebrafish development through the development of a mutant line. Different molecular techniques were applied to evaluate the decreased activity and the impact on development and survivability of zebrafish embryos. The authors conclude that inadequate levels of complex type N-glycans can impair skeletal muscle development which can have impact on survivability of embryos.

Overall, the study is new, novel and present interesting findings which can have significant impact of the filed. However, there are some minor changes that need to be addressed:

-          L74, is this comparison with mice straightforward?

In terms of formation of the neural tube a direct comparison could be made. However, mice do not undergo epiboly so there is not a direct comparison at this point.

-          L113, how many embryos, larvae and adults were used? How many replicates? Review along the manuscript.

This was addressed in the methods under 2.13, and figure legend 6.

-          L115, which speed was used?

This has been clarified in methods 2.2.

-          L157, how many larvae were pooled?

About 90 larvae were used in the study, section 2.5.

-          L160, what was the concentration of DTT?

200 mM concentration of DTT, section 2.5.

-          L167, what was the dilution of these probes?

10 ug/ml, see section 2.6

-          L171, how many larvae per replicate?

About ninety-five larval zebrafish were collected, see section 2.7

-          L225, how was dechorionation achieved?

Dechorionation was achieved using needles, see section 2.10

-          L231, SD should be used instead of SE.

Changed to SD, see section 2.10

The touch-evoked escape response assay was conducted to simultaneously determine the ability of Wt (2 dpf, 1.71 ± 1.92, n=100; 3 dpf, 1.3 ± 0.99, n=100) and mutant (2 dpf, 4.03 ± 3.63, n=100; 3 dpf, 2.76 ± 2.90, n=90) larval zebrafish to sense tactile stimuli on their tail region and respond via locomotor activity (Figure 5A).

-          L244, what is egg water?

5.03 mM NaCl, 0.17mM KCl, 0.33mM CaCl₂•2H₂O, 0.33mM MgSO₄ •7H₂O, 0.05% Methylene in 1 Liter system water) explanation added in line 94

-          L273, SD should be used instead of SE.

Changed to SD

The touch-evoked escape response assay was conducted to simultaneously determine the ability of Wt (2 dpf, 1.71 ± 1.92, n=100; 3 dpf, 1.3 ± 0.99, n=100) and mutant (2 dpf, 4.03 ± 3.63, n=100; 3 dpf, 2.76 ± 2.90, n=90) larval zebrafish to sense tactile stimuli on their tail region and respond via locomotor activity (Figure 5A).

-          L393, y-axis is missing the units in B and C figure.

 Units have been added, see figure

-          Supplementary files should be mentioned in the main text.

Mentioned in text of results and/or figure legend

Thanks for your comments as they were helpful.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you to the authors for their comprehensive revisions. The manuscript has significantly improved from its first submission, and it is evident that the authors have diligently worked to address all the issues I highlighted.