Zebrafish PRL-3 Regulates Yolk Syncytial Layer Integrity and Actomyosin Contractility During Epiboly

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the study "Zebrafish PRL-3 Regulates Yolk Syncytial Layer Integrity and Actomyosin Contractility during Epiboly," the authors investigated the potential role of zfPRL-3 in epiboly in zebrafish. Epiboly is a conserved process of gastrulation, but it has its own specific features in teleost fishes. Specifically, in zebrafish, coordinated vegetal expansion of the blastoderm (early embryonic cells) and the yolk syncytial layer is observed. Because epiboly is widely used in animal development, zebrafish serve as a good model for epithelial morphogenesis in vertebrates. Mechanical forces generated by contraction of actomyosin in the yolk syncytial layer and yolk cell microtubules are known to play an important role in epithelial expansion during epiboly. It is also important to note that deep cell movements mediated by filopodia formation are also essential for proper morphogenesis. The authors' choice of target (PRL-3) for the functional assay and model is undoubtedly justified. Indeed, PRL-3 phosphatase regulates cytoskeletal remodeling in cancer and other events; however, the role of this factor in cell behavior and its possible involvement in morphogenesis at the early stages of vertebrate embryogenesis remains unexplored. The authors demonstrated that morpholino knockout of zfPRL-3 leads to developmental disruptions and arrest, blastoderm destabilization, and mechanical rupture of the yolk cell. The results of the study indicate a disruption of the yolk syncytial layer (YSL) organization and premature assembly of the contractile actomyosin ring in zfPRL-3 morphants. Based on their own results and literature data, the authors offer their interpretation of the role of zfPRL-3 in epiboly in zebrafish.

The paper is interesting and well-written. However, I have several criticisms.

Major points:

1. The authors presented a standard control MO and showed titration results. However, they used a morpholino designed to target the translational start site of zfPRL-3 mRNA. This means that Western blotting is ideally required to demonstrate reduced protein levels with corresponding phenotypic changes.
2. Figure 1. In the graph (A) and diagram (B), the percentage of embryonic death at 4 and 8 ng MO does not correlate at the 8-9 hour time point.
3. The authors repeatedly mention time-lapse imaging experiments. However, the Methods section does not describe the time-lapse imaging procedure. Furthermore, it is important to distinguish between time-lapse imaging and simple life-imaging. Therefore, the method needs to be clarified and described.
4. The F-actin staining results should be supplemented with images at higher magnification. Although not essential, the work would be significantly improved if tubulin staining results were included.
5. Line 215-216, 292. I am not sure that the results allow us to conclude about the role of zfPRL-3 in coordinating the morphogenetic cell and tissue movements required for normal epiboly progression. The results certainly indicate the involvement of zfPRL-3 in cytoskeletal (re)organization, but do not allow us to conclude about a coordinating role.

Minor points:

1. Line 37-38. During gastrulation, only endoderm and mesoderm cells (i.e., the two germ layers) are internalized. Neuroectoderm is internalized later, during neurulation.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,
This study demonstrates that the phosphatase zfPRL-3 is a vital regulator of epiboly and early morphogenesis in zebrafish embryos. Through targeted gene knockdown, the authors found that the absence of this protein leads to developmental arrest, destabilization of the blastoderm, and lethal yolk rupture. This topic is potentially valuable for understanding the regulation of early embryogenesis in bony fishes. However, several points require significant revision.

The text and figures require careful revision for clarity and consistency. 
General comments on Figures.
1. Please remove all redundancy between the figure legends and the main text. Lines 268–270 and lines 273–276 are identical, for example.
2. Also, some experimental time points and abbreviations are explained only in some figure legends but not on others figures.
3. Please add scale bars to every image.
Specific comments on Figures:
Figure 1:
1."hpf" is already defined in the figure legend; repeating the definition directly on the figure is unnecessary.
2. The figure panel labels refer to "stages," while the legend describes "developmental phenotypes." Are these terms synonymous in this context?
3. What does the percentage in the captions for panels C, D, E, F, G means? And also what is the phenotype of the remaining 2 percents of embryos?
4. The embryo described as "having gross morphology" appears visually comparable to the control. What specific abnormality defines its morphology?
Figure 3:
1. I can suggest to revise the title of article. "Distinct morphogenetic failure modes lead to embryo death in zebrafish PRL-3 morphants." Also this figure not distinguish panels labeled (A, A’, A’’, etc). Therefore I can recommend to consolidate them into simple labels: A, B, C, etc.
Figure 4:
1. Please, rename the figure title to something like this. "Whole-mount in situ hybridization reveals a marked difference in signal distribution."
2. Line 237: "8 hpf" is listed twice.
Figure 5:
1. What is the rationale for presenting the stages in the order 65% epiboly, then 60%, then 75% epiboly? A logical chronological order would be preferable.
2.line 271: I suggest removing “were shown.”

The results section would benefit from reorganization to improve it consistency.
1. Please move the part of text at the beginning of the results (lines 137-141).
2. It would be more logical for readers to first see the illustrations and descriptions of the embryonic stages (dome, sphere, shield) in Figure 2, and only then use these terms in Figure 1B. Additionally, please provide uniform terminology throughout the text is essential for clarity (for example, are "dome" and "dome – 50% epiboly" used as synonyms?).
3. Please specify which experimental groups (2, 4, 6, or 8 ng MO) were analyzed in each subsections of the Results.

The Discussion would be significantly strengthened by including a schematic diagram that integrates the authors' proposed molecular model for the consequences of zfPRL-3 loss with the observed embryonic phenotypes.
A description of the software and methods used for image processing and analysis should be added in Materials and Methods.
Other comments and suggestions.
Lines 86-87: Why are all words in this sentence capitalized? Please use standard sentence case.
Lines 120-121: The consecutive percentage makes the sentence harder to read.
Lines 113-124: Can one conclude that the developmental stage distribution was similar between the 4 ng and 8 ng MO groups? Why was the "Dome-50% epiboly" stage never observed in the 8 ng MO group? What was the sample size for the 8 ng MO group? What biological conclusion can be drawn from the observed stage distribution across different treatment groups?
Line 139: The comma in the percentage value provides no additional information. Ensure number consistency throughout the manuscript.
Lines 148-149: What exactly did the authors observe as "no obvious difference"? In this paragraph and similar ones, I recommend not duplicating the percentages from the figure in the text. Instead, it is better to state the trend and indicate that exact percentages are provided in the figure legend.
Lines 175-177: Was the same embryo selection procedure applied for the experiment described in section 2.2?
Lines 226-229: Please clarify the reasoning that led the authors to this specific conclusion.
Line 313: "Tubulin" should not be capitalized.
Line 405: Replace the dash with commas for better readability.
Line 406: Based on the presented results, the claim of demonstrating a "conserved function of PRL-3 in governing cell motility and tension" appears overinterpreted. This conclusion should be toned down or supported by additional discussion/citations.
Line 448: Since this is the Abbreviations section, "PRL" should also be defined in full.

In summary, although the scientific value of the discovery is obvious, the manuscript can be published after addressing the above issues concerning presentation, clarity, data interpretation, and methodological detail. I therefore recommend Major Revision.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank the authors for their satisfactory response. All concerns have been resolved.

Author Response

Comments for Authors: Thank the authors for their satisfactory response. All concerns have been resolved.

Response: We sincerely thank the reviewer for their positive feedback. We are pleased that all concerns have been satisfactorily addressed and resolved.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, 

You've corrected the Materials and Methods section and improved all the figures. However, I still have some comments that need to be addressed before the article can be published. I'm attaching the comments to the original manuscript that you didn't respond to below. Please double-check them.

The results section would benefit from reorganization to improve it consistency.
1. Please move the part of text at the beginning of the results (lines 137-141).
2. It would be more logical for readers to first see the illustrations and descriptions of the embryonic stages (dome, sphere, shield) in Figure 2, and only then use these terms in Figure 1B. Additionally, please provide uniform terminology throughout the text is essential for clarity (for example, are "dome" and "dome – 50% epiboly" used as synonyms?).
3. Please specify which experimental groups (2, 4, 6, or 8 ng MO) were analyzed in each subsections of the Results.

The Discussion would be significantly strengthened by including a schematic diagram that integrates the authors' proposed molecular model for the consequences of zfPRL-3 loss with the observed embryonic phenotypes.
A description of the software and methods used for image processing and analysis should be added in Materials and Methods.
Other comments and suggestions.
Lines 86-87: Why are all words in this sentence capitalized? Please use standard sentence case.
Lines 120-121: The consecutive percentage makes the sentence harder to read.
Lines 113-124: Can one conclude that the developmental stage distribution was similar between the 4 ng and 8 ng MO groups? Why was the "Dome-50% epiboly" stage never observed in the 8 ng MO group? What was the sample size for the 8 ng MO group? What biological conclusion can be drawn from the observed stage distribution across different treatment groups?
Line 139: The comma in the percentage value provides no additional information. Ensure number consistency throughout the manuscript.
Lines 148-149: What exactly did the authors observe as "no obvious difference"? In this paragraph and similar ones, I recommend not duplicating the percentages from the figure in the text. Instead, it is better to state the trend and indicate that exact percentages are provided in the figure legend.
Lines 175-177: Was the same embryo selection procedure applied for the experiment described in section 2.2?
Lines 226-229: Please clarify the reasoning that led the authors to this specific conclusion.
Line 313: "Tubulin" should not be capitalized.
Line 405: Replace the dash with commas for better readability.
Line 406: Based on the presented results, the claim of demonstrating a "conserved function of PRL-3 in governing cell motility and tension" appears overinterpreted. This conclusion should be toned down or supported by additional discussion/citations.
Line 448: Since this is the Abbreviations section, "PRL" should also be defined in full.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf