Review Reports - Spatiotemporal Frameworks of Morphogenesis and Cell Lineage Specification in Pre- and Peri-Implantation Mammalian Embryogenesis: Insights and Knowledge Gaps from Mouse Embryo

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall

I went back and forth about whether to reject this article outright or send it back for major corrections. I’m deciding to do the latter, but I found this to be a frustrating read because the author does not seem to have a clear idea of what this article should be or how it should be organized.

Throw out everything except Section 4. Most of the rest of this article is textbook-level summaries of early embryogenesis and feels incoherent with the portion of this article that examines cell fate determination. The introduction should provide just enough information to understand your discussion of first and second cell lineage specification. The title should also reflect this, as it is too vague as stated.

You also need to expand your discussion of the different models. In each case, you explain the model briefly and move on to another topic. Explain each of these more clearly and take the time to show how they are similar and different. You declare that you are synthesizing findings into a new model, but you spend a single paragraph on that model. If this is truly a contribution to the field that is uniquely made by this paper, it needs to be discussed in greater detail. A single paragraph is insufficient to outline a new model for cell lineage specification in early embryogenesis.

Figures

For figures like figure 3, don’t separate them into 3 panels. It’s a single figure that is directly comparing two things. Panel A should be moved to the bottom, and the labels for B and C should be changed to the name of the model being illustrated. Same for figure 4.

Author Response

Dear the Reviewer,

I would like to express my sincere gratitude to you for your reading and insightful feedback, which have significantly improved the manuscript. I have carefully revised the manuscript and provided detailed point-by-point responses to each of your suggestions. Note: For line numbers, please refer to the attached PDF. Due to formatting differences in the tracked-changes version of the Word document, line numbers may vary by ±1.

Responses to Reviewer 1’s Comments

Comments and Suggestions for Authors

Overall

Re: First, I appreciate your reading and valuable comments. In response to your feedback, I have thoroughly revised the manuscript, incorporating insights from both reviewers. The revisions include refining and restructuring the content to improve logical flow, as well as clarifying key concepts to ensure greater focus on the main topic of the manuscript.

Specifically, I have made the following improvements: 1) ‌I have restructured the content in the Simple Summary (lines 8-25), Abstract (lines 26-46), and sections on Cell Migration (page 13 paragraph 1) and PrE Cell Differentiation (page 14 the last paragraph), which increased and enhanced clarity and readability. 2) I have corrected technical terms and ensured consistency in terminology throughout the manuscript. 3) I have addressed all specific concerns raised by the reviewers and incorporated their suggestions to strengthen the manuscript. 4) Where essential context was needed, I have streamlined explanations to ensure they are concise and directly relevant to the aims and topic of the manuscript.

The revised manuscript now presents a more coherent narrative with improved flow and clarity.

Re: Thank you for your important feedback. While the relevant sections (beyond the first/second cell lineage specification) may seem at 'textbook' level for experienced experts in the field, they are also designed to engage readers from interdisciplinary backgrounds, including emerging researchers. These sections also provide the context of the unresolved gaps of knowledge about morphological events and cell lineage specification during pre- and peri-implantation stages. Therefore, I believe these sections are important to retain, as they align with the manuscript's goal of accessibility.

Incorporating both reviewers’ comments, I have revised introduction to make it clearer and more specified with neutral critical for divergent data interpretations for pre- and peri-implantation morphogenesis and cell lineage specification (current lines 55, 61-69).

The title has been specified as ‘Spatiotemporal frameworks of morphogenesis and cell lineage specification in pre- and peri-implantation mammalian embryogenesis: insights and knowledge gaps from mouse embryo’, as you suggested.

Re: Thank you for your valuable suggestions regarding the discussion of the different models. In response to your feedback, I have expanded and clarified the presentation of these models.

For the first cell lineage models, I have integrated the relevant discussions and extended them with additional details on polarity-induced mechanisms, by incorporating both examiners’ comments. (lines 283, 288-295)

For the models regarding the second cell lineage specification, I have thoroughly evaluated each model, delineating what is currently known and what remains to be elucidated (lines 311-314; lines 341-343; lines 356-359). I also have provided a comparative table to highlight the similarities and differences between these models. (line 375)

Additionally, as you suggested, I have incorporated a new subsection (current Section 4.1.2.2) that synthesises the discussed three models to explain Epi and PrE segregation and explores the potential mechanisms underlying this process (lines 379-407).

Figures

Re: Thank you very much for your valuable insights and suggestions regarding improving the clarity and visual presentation of Figures 3 and 4.

I have modified these two Figures by incorporating both reviewers’ insights and comments. In response to your feedback, for Figure 3, I have moved Panel A to the bottom, and changed labels B and C to "Inside-outside model" and "Polarity model", respectively. For Figure 4, I have updated labels A, B, and C to the corresponding model names. I have also updated the legend for Figures 3 and 4 accordingly.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a useful review that summarizes the events in mouse development immediately following fertilization and leading up to the first lineage differentiation events. The diagrams are pretty, although sometimes the colours a bit too subtle (pls see below). Belor are several issues that should be addressed to improve this manuscript:

The Simple Summary remains fairly obscure and implies a highly controversial area of research, which is not really so. Please phrase the Abstract more clearly along the lines of what the manuscript addresses, and not some perceived challenges to dogmas.
Along the same line, please also change the title to make clearer what this article actually addresses ("early mammalian embryogenesis" is a very broad field).
Line 72, "fragmented intellectual landscape" - unclear what this means, and also in general the article portrays the field as being more controversial than it actually is.
Line 98, "transgenic strains" - perhaps say "genetically manipulated" so to include knockouts etc.
Line 105, what "anatomical constraints"? This does not seem to be a genuine impediment.
Figure 1A: "Through to three layers" - What does this possibly mean. Please clarify.
Figure 1B: "two cell clock"??? Is this meant to read "block"? Also, specify what "work in Wang lab" refers to more precisely.
Lines 140/141: following the breakdown of germinal vesicle (a large and round nucleus) - this is not a correct or valid definition of a germinal vesicle. The sentence says that the GV is a round nucleus, this is fundamentally incorrect.
Line 144: "encomapssing" is the wrong word here, it should say "surrounding" or similar.
Line 162, "mitotic division without cell growth in size", this is incorrect, the entity that does not grow is the embryo as a whole.
Line 170: E-Cadherin transport channels? E-Cadherin is a cytoskeletal protein.
Line 179: Change to "blastomeres begin structural and functional..."
Line 235: "35-cell stage", this is incorrect and should read the 32-cell stage
Line 235, "hydraulic pressure" - incorrect terminology
Line 253 ff, describe that Sox2 is not only ICM-specific, but that it switches to become a marker of the trophoblast (stem cell state) just a day later. Sox2 mutants die of trophoblast defects, not of embryonic defects (Yamanaka 1999)
Line 245: that will "form"
Important: Lines 277 ff, the polarity model is not distinct from the inside-outside model, it is perhaps an extension of it. Hence it is not valid to describe these as competing hypotheses, and accordingly Figure 3 should also be changed.
Line 302, "100-cell stage" - usually, the stages are multiples of 2, so it is either the 64-cell stage or the 128-cell stage, unless the cell number accuracy is not as solid at these stages.
Important: Lines 317 onward and Figure 4, it is unclear how the division numbers relate to the events, i.e. how can the 5th division lead to "cells on the surface of the ICM" when this occurs at the compacted morula stage. It is also unclear what colour descriptions belong to which cells.
Line 370: Correct sentence structure.
Important: Figure 5: The VE/VE label seems to point to the parietal trophoblast giant cell layer, and also the colour-coding cannot distinguish between TE-derived and PrE-derived cells. Please amend.
PE cells are very migratory at any stage, so they should not be included in the EMT description as there are, at best, only very subtle changes related to this process in the PE lineage (line 420).
Line 458 and Figure 6: Highlight the MZE and describe better what this is, as it is not as commonly known.
Line 465: wingless integration site is not correct if referring to WNT as a cytokine. Also specify which WNT(s) are involved.
Lines 495-497: Fgf/Erk attenuation with consecutive Erf nuclear localisation and function and by lysine acetyltransferase 8-mediated histone H4, lysine 16 (H4K16) acytylation, which upregulates Cdx2 expression -- there is a lot of "stuff" in here that is not connected and that also cannot be correct. what does attenuated FGF signaling have to do with ERF translocation to the nucleus? Describe/specify. H4K16 acetylation (spelling!), why would that up-regulate Cdx2? Gene expression is usually coupled to H3K4me3, or H3K27ac. Also, why would Cdx2 be up-regulated in the EPC when it is a stem cell marker, as described earlier? More detail is needed.
Following lines, the use of "featured" is not the correct terminology.
Line 511, "of" (sp)
Important: Figure 7 and text, the "Why" should be take out and rephrased. Why is never a valid question in developmental biology, i.e. we only describe what happens or how some process might work, but not "why" it happens. Please rephrase. Also "What is the result".

Author Response

Dear the Reviewer,

I would like to deeply express my sincere gratitude to you for your thoughtful reading and insightful feedback, which have significantly improved the manuscript. I have carefully revised the manuscript and provided detailed point-by-point responses to each of your suggestions. Note: For line numbers, please refer to the attached PDF. Due to formatting differences in the tracked-changes version of the Word document, line numbers may vary by ±1.

Responses to Reviewer 2’s Comments

Comments and Suggestions for Authors

Re: Thank you very much for your thorough review and insightful comments on the issues raised. I sincerely appreciate your efforts in carefully evaluating the manuscript and providing valuable feedback.

The Simple Summary remains fairly obscure and implies a highly controversial area of research, which is not really so. Please phrase the Abstract more clearly along the lines of what the manuscript addresses, and not some perceived challenges to dogmas.

Re: Thank you for the feedback on the clarity and focus of the Abstract and Simple Summary. I have revised and clarified the Simple Summary, with the original implication of a "controversial" research area now modified to a relative "competitive" one. I have also refined the Abstract to focus more directly on the content of this manuscript, with improved structure and organisation (current lines 9-25).

Along the same line, please also change the title to make clearer what this article actually addresses ("early mammalian embryogenesis" is a very broad field).

Re: Thank you for your suggestion regarding the article title. In response to both reviewers’ comments, I have changed the title as ‘Spatiotemporal frameworks of morphogenesis and cell lineage specification in pre- and peri-implantation mammalian embryogenesis: insights and knowledge gaps from mouse embryo’.

Line 72, "fragmented intellectual landscape" - unclear what this means, and also in general the article portrays the field as being more controversial than it actually is.

Re: Thank you for the feedback. I have removed the ‘fragmented intellectual landscape’ as the preceding context of ‘the integration’ already implies the overcome of divergence. This revision also helps present a more neutral critique of controversial data in the article. Furthermore, in the same paragraph, I have replaced ‘deepened’ with ‘sparked’ (current line 71) and removed ‘counterproductively’ to further align with a neutral tone in discussing divergent data.

Line 98, "transgenic strains" - perhaps say "genetically manipulated" so to include knockouts etc.

Re: Thank you for your suggestion. I have changed ‘transgenic strains’ to ‘genetically manipulated strains’. (line 98)

Line 105, what "anatomical constraints"? This does not seem to be a genuine impediment.

Re: Thank you for your feedback. After careful consideration, I agree with this comment. Therefore, I have removed ‘anatomical constraints’. (line 106)

Figure 1A: "Through to three layers" - What does this possibly mean. Please clarify.

Re: Thank you for your comment. I have rephrased "through to three layers" in Figure 1A as ‘Early embryos & Three germ layers’. For further clarity, the legend now explicitly defines the three germ layers as endoderm, mesoderm, and ectoderm (current line 127).

Figure 1B: "two cell clock"??? Is this meant to read "block"? Also, specify what "work in Wang lab" refers to more precisely.

Re: Thank you very much for pointing out this error. I have corrected ‘clock’ to ‘blcok’. Additionally, I have clarified ‘Work in Wang lab’ in the legend by specifying that it refers to ‘developed IVC system using matrigel-coated petri dishes with adapted blastocyst culture medium’. (lines 136-138)

Lines 140/141: following the breakdown of germinal vesicle (a large and round nucleus) - this is not a correct or valid definition of a germinal vesicle. The sentence says that the GV is a round nucleus, this is fundamentally incorrect.

Re: Thank you for identifying this inaccuracy. I have corrected the GV definition as ‘oocyte nucleus in prophase I of meiosis I’ (current lines 141-142).

Line 144: "encomapssing" is the wrong word here, it should say "surrounding" or similar.

Re: Thank you for your suggestion. I have changed ‘encompassing’ to ‘surrounding’. (line 145/146)

Line 162, "mitotic division without cell growth in size", this is incorrect, the entity that does not grow is the embryo as a whole.

Re: Thank you very much for your careful reading and pointing out this. I agree with this comment. I have corrected it and the rephrased text now reads: ‘mitotic division without growth of embryo size as a whole’. (line 164/165)

Line 170: E-Cadherin transport channels? E-Cadherin is a cytoskeletal protein.

Re: Thanks for your feedback. You are absolutely right that E-cadherin is a cytoskeletal protein, not a transport channel. I have now corrected the phrase as "modulates the transport of adhesion molecule E-cadherin to cell membranes" (current lines 172-173).

Line 179: Change to "blastomeres begin structural and functional..."

Re: Thank you for your comment. I have removed ‘were thought to’ before ‘begin’ as suggested. (line 181)

Line 235: "35-cell stage", this is incorrect and should read the 32-cell stage

Re: Thank you for your comment (I guessed you mean original line 205). I have changed the phrase as ’32-cell stage’ to align with conventional stage designations, as you suggested. (Line 206)

Line 235, "hydraulic pressure" - incorrect terminology

Re: Thank you for your meticulous review. I have revised "hydraulic pressure" to "fluid pressure" to ensure accurate and consistent terminology. (line 237)

Line 253 ff, describe that Sox2 is not only ICM-specific, but that it switches to become a marker of the trophoblast (stem cell state) just a day later. Sox2 mutants die of trophoblast defects, not of embryonic defects (Yamanaka 1999)

Re: Thank you for your valuable feedback. I have carefully revised the text to clarify the Sox2 expression and roles. The updated description now reads: " It is worth noting that Sox2 is not only Epi/ICM-specific but also marks early TE-derived trophoblast, and Sox2 mutant embryos die of TE defects rather than embryonic defects" (current lines 255-257). Additionally, I have adjusted the structural order of the PrE and Epi markers descriptions to improve flow and logical coherence (current lines 252-254). The related citation numbers have been updated accordingly.

Line 245: that will "form"

Re: Thank you for catching this grammar typo. I have corrected ‘forms’ to ‘form’. (lien 246)

Important: Lines 277 ff, the polarity model is not distinct from the inside-outside model, it is perhaps an extension of it. Hence it is not valid to describe these as competing hypotheses, and accordingly Figure 3 should also be changed.

Re: Thank you for your insightful suggestion on the relations of these models. I have now revised the descriptions of the models. The rephrased text reads now: ‘The “inside-outside model” has been extended by the “polarity model”, which highlights the influences of cell polarity on cell fates’ (current line 283). I have also modified Figure 3, incorporating both reviewers’ comments. Additionally, I have updated the legend of Figure 3 to correspond the text of integrating two models (current lines 273-274) and figure changes.

Line 302, "100-cell stage" - usually, the stages are multiples of 2, so it is either the 64-cell stage or the 128-cell stage, unless the cell number accuracy is not as solid at these stages.

Re: Thank you for your comment. You are correct that cell number accuracy is less precise at these stages. After careful consideration, I have revised "100-cell stage" to "128-cell stage" (current line xxx) to align with embryological staging conventions. (line 311)

Important: Lines 317 onward and Figure 4, it is unclear how the division numbers relate to the events, i.e. how can the 5th division lead to "cells on the surface of the ICM" when this occurs at the compacted morula stage. It is also unclear what colour descriptions belong to which cells.

Re: Thank you very much for raising this critical question regarding the relationship between division numbers and cell lineage specification, which is still under ongoing investigation and debate. Your insightful feedback has promoted me to revise this section. I have included a brief discussion of this open question in the revised manuscript (lines 311-312). Furthermore, incorporating both reviewers’ comment, I have included a new section of ‘Synthesised theoretical model for the second cell fate decision’, which ellustrated the potential mechansims of the raltions between cell division rounds and cell linage specification. (page 11, lines 379-407)

Regarding ‌Figure 4‌, we have made the following improvements: 1) I have added ‌lines and text‌ directly in the figure to clarify the color-coded cell types; 2) I have expanded the ‌figure legend‌ to further explain the color-coded proteins and cells (current lines 316-318, 321-324, 328-329).

Line 370: Correct sentence structure.

Re: I have revised and replaced ‘re-appraise’ and ‘concomitantly with the first and second cell fate decisions’ respectively with ‘re-evaluation’, and ‘during preimplantation stage’. Furthermore, I have broken the sentence into short ones to enhance readability (current lines 420-423)

Important: Figure 5: The VE/VE label seems to point to the parietal trophoblast giant cell layer, and also the colour-coding cannot distinguish between TE-derived and PrE-derived cells. Please amend.

Re: Thank you for bringing this to my attention. I have made the following improvements in response to your comments: 1) The "VE/VE" label has been removed, leaving only the color-coded text to improve label clarity; 2) The color of TE-derived cells (including late mural TE) has been changed to wood brown with text labels to make them more distinguishable from PrE-derived cells; 3) The legend has been updated accordingly (current lines 455-456).

PE cells are very migratory at any stage, so they should not be included in the EMT description as there are, at best, only very subtle changes related to this process in the PE lineage (line 420).

Re: Thank you very much for your insightful comment. I have removed the reference to EMT in the context of PE cells (current lines 478-479). The related citation has been removed accordingly.

Line 458 and Figure 6: Highlight the MZE and describe better what this is, as it is not as commonly known.

Re: Thank you for your valuable suggestion regarding the clarification of the MZE. I have made the following improvements to address this: 1) In Figure 6B, I have highlighted MZE with black arrows or asterisks, and the figure legend has been updated to include the MZE definition (541-543). 2) ‌After careful revision, I have first introduced the MZE concept in the cell migration section (line 468-469) where I discuss its proposed relationship with PrE/PE migration. The MZE is then further elaborated in the PrE differentiation section with details on molecular regulation (line 519-524).

Line 465: wingless integration site is not correct if referring to WNT as a cytokine. Also specify which WNT(s) are involved.

Re: Thank you for pointing this out. I have corrected ‘wingless integration site’ as ‘Wnt’, and specified Wnt family members as Wnt 6, 7 (current line 528).

Lines 495-497: Fgf/Erk attenuation with consecutive Erf nuclear localisation and function and by lysine acetyltransferase 8-mediated histone H4, lysine 16 (H4K16) acytylation, which upregulates Cdx2 expression -- there is a lot of "stuff" in here that is not connected and that also cannot be correct. what does attenuated FGF signaling have to do with ERF translocation to the nucleus? Describe/specify. H4K16 acetylation (spelling!), why would that up-regulate Cdx2? Gene expression is usually coupled to H3K4me3, or H3K27ac. Also, why would Cdx2 be up-regulated in the EPC when it is a stem cell marker, as described earlier? More detail is needed.

Re: Thank you very much for your insightful comments. I have carefully revised this section to improve clarity, accuracy, and logical flow. The revised text now reads: ‘The related content now reads: EPC formation also requires the regulation of Kat8 (lysine acetyltransferase 8)-mediated H4K16 acetylation, which upregulates Cdx2 expression to maintain the stemness of TE during implantation (i.e., trophoblast) and potentially promote TE dif-ferentiation into EPC in turn [153]. Furthermore, the closure of EPC cavity (detailed mechanism of this cavity formation remains unclear)-requires the regulation of Ex-Ec-derived Efr, which is produced upon Fgf/Erk attenuation and in turn reproduces Fgf [154].’ (current liens 561-567).

Following lines, the use of "featured" is not the correct terminology.

Re: Thank you for bringing this to my attention. I have corrected ‘featured’ to ‘expressing’. (lines 568-569)

Line 511, "of" (sp)

Re: Thank you for your careful reading and for pointing out this typo. I have corrected this error. (line 581)

Important: Figure 7 and text, the "Why" should be take out and rephrased. Why is never a valid question in developmental biology, i.e. we only describe what happens or how some process might work, but not "why" it happens. Please rephrase. Also "What is the result".

Re: Thank you for your insightful comments. I have revised and rephrased ‘Why’ to ‘How’ and ‘what is the result’ to ‘outcome’. I have also updated Figure 7, legend and the related text accordingly. (current lines 600, 601, 606/607, 620, 624)