Calcium and Ca²⁺-Binding Proteins Regulate Microtubule and Cytoskeletal Dynamics During Mammalian Corticogenesis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Journal: Brain Sciences (ISSN 2076-3425)

Manuscript ID: brainsci-4239849

Type: Review

Title: Calcium Signaling as a Master Regulator of Microtubule Dynamics in Mammalian Corticogenesis

Authors: Diana Sarahi De la Merced-García , Rocío Valle-Bautista , Rebeca Hernández-García , Néstor Fabián Díaz , Anayansi Molina-Hernández *

Section: Molecular and Cellular Neuroscience

1: Absence of an explicit new concept or hypothesis. The abstract summarizes the literature but does not state whether this review presents a novel conceptual model or synthesis. Provide a clear integrative contribution, such as linking Ca²⁺ oscillation codes to microtubule polarity transitions during corticogenesis.

2: No evidence of Ca²⁺ specificity in RGC phases. The three modes of Ca²⁺ waves are described but not linked to specific downstream targets or outcomes in experimental studies.

3: S100 section:  Excessive descriptive detail, with a weak developmental linkage. The biochemical description takes preference over developmental mechanisms, which are tangential to the review's focus. Condense to structural essentials and emphasize how S100B/A4 influence microtubule stability in NSCs.

4: There is an incomplete link between the structural and functional roles of PCDH19 (900-950 (Cadherins). The structure is detailed, but the pathway from Ca²⁺ binding to spindle orientation is not elaborated.

5: Typographical difference between "cytoarchitecture" and "citoarchitecture" (40-60).

6: line no. 122:  "phosphorilates" is misspelled.

7: Figure 4 caption is too dense.

8: line no. 467: S1000B instead of S100B (typo: extra zero).

9: The table of abbreviations contains inconsistencies in capitalization and numbers (for example, "CaMK (I, II, III, IV)" vs "CaMKs"). Editorial consistency. Harmonize all abbreviations and eliminate redundant numbering.

10: Expand the "Conclusions" section to highlight translational implications and open questions.

11: Ensure consistent editorial and typographic styles (e.g., Ca²⁺, italic gene names, figure numbering).

12: Emphasize novelty- what conceptual advancements this review makes over previous calcium/microtubule papers.

Author Response

1. Comment: Absence of an explicit new concept or hypothesis. The abstract summarizes the literature but does not state whether this review presents a novel conceptual model or synthesis. Provide a clear integrative contribution, such as linking Ca²⁺ oscillation codes to microtubule polarity transitions during corticogenesis.

Author's response: We thank the reviewer for this important suggestion. In response, we have revised the Abstract and relevant sections of the Introduction and main text to explicitly articulate the integrative conceptual framework proposed in this review. Specifically, we now emphasize the link between Ca²⁺ oscillatory dynamics and microtubule polarity transitions during corticogenesis, highlighting this relationship as a central conceptual contribution of the manuscript (Abstract pp 1 lines 29-31, Introduction pp 2 in lines 87-95; pp 6 lines 232-242; and pp 8 lines 350 and pp 362-336).

2. Comment: No evidence of Ca²⁺ specificity in RGC phases. The three modes of Ca²⁺ waves are described but not linked to specific downstream targets or outcomes in experimental studies.

Author's response: We appreciate this observation. To address it, we have revised the text to more clearly connect distinct Ca²⁺ signaling patterns with experimentally supported downstream outcomes in radial glial cells, including effects on proliferation, differentiation, and migration. These additions clarify the functional relevance of Ca²⁺ dynamics in specific phases of cortical development(pp 2, lines 60-76 and 87-91).

3. Comment: S100 section: Excessive descriptive detail, with a weak developmental linkage. The biochemical description takes preference over developmental mechanisms, which are tangential to the review's focus. Condense to structural essentials and emphasize how S100B/A4 influence microtubule stability in NSCs.

 Author's response: We thank the reviewer for this helpful suggestion. We have condensed the biochemical description of the S100 proteins and revised this section to focus more directly on their developmental relevance, particularly their roles in microtubule regulation, tau-associated processes, and neural stem cell biology(pp 4 and 5, lines 171–196).

4. Comment: There is an incomplete link between the structural and functional roles of PCDH19 (900-950 (Cadherins). The structure is detailed, but the pathway from Ca²⁺ binding to spindle orientation is not elaborated.

Author's response: We appreciate this comment. We have revised the relevant sections to strengthen the mechanistic connection between the structural properties of PCDH19 and its functional implications in corticogenesis. In particular, we now better explain how Ca²⁺-dependent PCDH19-associated signaling may influence cytoskeletal organization, centrosome-related processes, and spindle orientation (Section 3.1; pp 7 to 9 from line 319-335; pp 8 and 9 lines 350-362; section 3.2 pp 10 and 11, lines 438-454, and 3.3 section pp 14 lines 611-612-592).

5 and 6. Comment: Typographical difference between "cytoarchitecture" and citoarchitecture" (40-60)."phosphorilates" is misspelled.

Author's response: We thank the reviewer for noting these errors. These typographical issues have been corrected throughout the manuscript.

7. Comment: Figure 4 caption too dense.

Author's response: We appreciate this suggestion. We have revised the Figure 4 legend to make it more concise and easier to follow, while preserving the essential explanatory content.

8. Comment: line no. 467: S1000B instead of S100B (typo: extra zero).

Author's response: We thank the reviewer for identifying this error. It has been corrected.

9. Comment: The table of abbreviations contains inconsistencies in capitalization and numbers (for example, "CaMK (I, II, III, IV)" vs "CaMKs"). Editorial consistency. Harmonize all abbreviations and eliminate redundant numbering.

Author's response: We appreciate this comment. We have revised the abbreviation table and the main text to standardize capitalization and nomenclature throughout the manuscript, and we removed abbreviations that were used only once or were unnecessary.

10. Comment: Expand the "Conclusions" section to highlight translational implications and open questions.

Author's response: We thank the reviewer for this valuable recommendation. The Conclusions section has been substantially expanded to include a more critical discussion of open questions in the field as well as the potential translational implications of Ca²⁺-dependent regulation of microtubule dynamics in cortical development and disease.

11. Comment: Ensure consistent editorial and typographic styles (e.g., Ca²⁺, italic gene names, figure numbering).

Author's response: We appreciate the reviewer’s attention to editorial detail. We have carefully revised the manuscript to improve typographic consistency, including ion notation, gene and protein formatting where appropriate, and figure numbering/style.

12. Comment: Emphasize novelty- what conceptual advancements this review makes over previous calcium/microtubule papers.

Author's response: We thank the reviewer for this important point. We have strengthened the discussion of the manuscript’s novelty in the Introduction and Conclusions by more clearly defining the conceptual advance of this review, namely, the integration of Ca²⁺ signaling dynamics with microtubule regulation as a unifying framework for understanding mammalian corticogenesis.

Reviewer 2 Report

Comments and Suggestions for Authors

The present manuscript highlights in a very effective and comprehensive way the role of Ca2+ in corticogenesis, specifying the distinct pathways is with calcium has a significant role in glial and neurons development and fate with focus on cytoskeletal/microtubules dynamics. Overall, the presented information is precise and accurate with scientific literature and is presented in a well-structured manner, evidencing the different molecular targets and pathways in which calcium is essential to attain the projected outcomes. The figures also provide a clear illustration of the processes described, which enriches the manuscript’s content. With that in mind, I have only a few brief comments that may help further improve the manuscript’s content:

- In the introduction section, the role of other ions (e.g. Na+, K+, Cl-) in corticogenesis could also be briefly explained in order to highlight even more the differentiating role of Ca2+

- In section 3, the most common brain regions where proliferation and differentiation occur could be mentioned

- The role of Ca2+ in olfactory bulbs and related migratory areas known for specific role in the rostral migratory stream, could be included in the manuscript

- A discussion section could be added where the authors present their critical and constructive assessment of the role of calcium in the issues addressed, and how new lines of research can contribute to scientific progress in this area and even lead to the development of new therapies for neurodegenerative diseases in which neurogenesis may play a significant role.

Author Response

1. Comment: In the introduction section, the role of other ions (e.g. Na+, K+, Cl-) in corticogenesis could also be briefly explained in order to highlight even more the differentiating role of Ca^2+.

Author's response: We thank the reviewer for this suggestion. We have added a paragraph to the Introduction that briefly contextualizes the roles of Na⁺, K⁺, Cl⁻, and other biologically relevant ions during corticogenesis to better highlight the distinctive regulatory functions of Ca²⁺ in the processes discussed in this review (Section 1, Page 3, lines 108-118).

2. Comment: In section 3, the most common brain regions where proliferation and differentiation occur could be mentioned
   Author's response: We appreciate this comment. Because the review focuses specifically on cortical development, we have clarified the relevant proliferative and differentiative regions within the developing cortical neuroepithelium, rather than expanding into adult neurogenic niches. This clarification has been incorporated at the beginning of Section 3.1 to better orient the reader.
3. Comment: The role of Ca²⁺ in olfactory bulbs and related migratory areas known for specific role in the rostral migratory stream, could be included in the manuscript
   Author's response: We thank the reviewer for this thoughtful suggestion. We agree that Ca²⁺ signaling also has important roles in the rostral migratory stream and olfactory bulb-associated neurogenesis. However, because the present review is centered on mammalian corticogenesis, we chose to maintain a defined scope and not expand the discussion substantially into these regions.
4. Comment: A discussion section could be added where the authors present their critical and constructive assessment of the role of calcium in the issues addressed, and how new lines of research can contribute to scientific progress in this area, and even lead to the development of new therapies for neurodegenerative diseases in which neurogenesis may play a significant role.

Author's response: We appreciate this valuable recommendation. In response, we have strengthened the critical discussion across the manuscript, particularly in the concluding section, to better address unresolved questions, emerging research directions, and the broader translational relevance of Ca²⁺-dependent mechanisms for neurodevelopmental and neurodegenerative conditions.

Reviewer 3 Report

Comments and Suggestions for Authors

This review addresses an interesting and relevant topic. The interplay between calcium signaling, cytoskeletal regulation, and cortical development is clearly important, and the manuscript has the potential to be of interest to readers working in developmental neurobiology. The overall organization is reasonable, and the figures are helpful in guiding the reader through the main biological processes discussed in the text.

That said, I believe the manuscript still requires substantial revision before it can be considered for publication.

My main concern is that the review is presented as being specifically focused on calcium signaling as a regulator of microtubule dynamics in mammalian corticogenesis, whereas in several sections the discussion becomes broader and shifts toward more general calcium-dependent mechanisms, including adhesion, actin remodeling, membrane trafficking, and later aspects of neuronal maturation. These topics are not irrelevant, but in several places the connection to microtubule regulation is only indirect, which makes the central message less focused than the title suggests.

A second point is that the manuscript often reads as a descriptive overview of molecules and pathways, whereas it would benefit from a more critical discussion of the evidence. In particular, the authors should make clearer which mechanisms have been directly demonstrated in mammalian corticogenesis and which are instead inferred from other systems or from broader cell biological studies. At present, those different levels of evidence are not always sufficiently distinguished. This is particularly important in sections that rely on non-neural cell lines or experimental systems outside cortical development.

I would also strongly encourage the authors to carefully re-check the reference list and in-text citations throughout the manuscript. I noticed at least some cases in which the cited reference does not appear to match the statement in the text. For example, the section discussing PCDH19 deficiency in E14.5 cortical neurosphere assays cites reference [75], but reference 75 in the bibliography corresponds to a paper on the relationship between free Ca2+ and Ca2+-calmodulin concentrations in intact cells, which does not appear to support that statement. Similarly, in the section on centrins, reference [37] seems inconsistent with the topic discussed, as it corresponds in the bibliography to a study on Pcdh19 loss-of-function rather than centrin biology. Since this is a review article, citation accuracy is especially important and the references should be checked carefully throughout.

Another issue is the tone of some mechanistic statements. In a few places, the manuscript appears more definitive than the available evidence would justify. This is especially relevant where conclusions are drawn from transformed cell lines, non-mammalian systems, or preprint data. These studies can certainly be discussed, but the wording should better reflect the strength and limitations of the available evidence.

The language also requires further revision. Although the manuscript is generally understandable, there are multiple grammatical problems, awkward formulations, and typographical errors throughout the text. A careful language revision would improve the overall readability considerably.

Overall, I think the topic is interesting and the manuscript could become a useful review after revision, but at this stage it would benefit from a clearer focus, a more critical treatment of the literature, a thorough check of citation accuracy, and substantial language polishing.

Author Response

Reviewer Comment: This review addresses an interesting and relevant topic. The interplay between calcium signaling, cytoskeletal regulation, and cortical development is clearly important, and the manuscript has the potential to be of interest to readers working in developmental neurobiology. The overall organization is reasonable, and the figures are helpful in guiding the reader through the main biological processes discussed in the text.

That said, I believe the manuscript still requires substantial revision before it can be considered for publication.

My main concern is that the review is presented as being specifically focused on calcium signaling as a regulator of microtubule dynamics in mammalian corticogenesis, whereas in several sections the discussion becomes broader and shifts toward more general calcium-dependent mechanisms, including adhesion, actin remodeling, membrane trafficking, and later aspects of neuronal maturation. These topics are not irrelevant, but in several places the connection to microtubule regulation is only indirect, which makes the central message less focused than the title suggests.

A second point is that the manuscript often reads as a descriptive overview of molecules and pathways, whereas it would benefit from a more critical discussion of the evidence. In particular, the authors should make clearer which mechanisms have been directly demonstrated in mammalian corticogenesis and which are instead inferred from other systems or from broader cell biological studies. At present, those different levels of evidence are not always sufficiently distinguished. This is particularly important in sections that rely on non-neural cell lines or experimental systems outside cortical development.

I would also strongly encourage the authors to carefully re-  and in-text citations throughout the manuscript. I noticed at least some cases in which the cited reference does not appear to match the statement in the text. For example, the section discussing PCDH19 deficiency in E14.5 cortical neurosphere assays cites reference [75], but reference 75 in the bibliography corresponds to a paper on the relationship between free Ca2+ and Ca2+-calmodulin concentrations in intact cells, which does not appear to support that statement. Similarly, in the section on centrins, reference [37] seems inconsistent with the topic discussed, as it corresponds in the bibliography to a study on Pcdh19 loss-of-function rather than centrin biology. Since this is a review article, citation accuracy is especially important and the references should be checked carefully throughout.

Another issue is the tone of some mechanistic statements. In a few places, the manuscript appears more definitive than the available evidence would justify. This is especially relevant where conclusions are drawn from transformed cell lines, non-mammalian systems, or preprint data. These studies can certainly be discussed, but the wording should better reflect the strength and limitations of the available evidence.

The language also requires further revision. Although the manuscript is generally understandable, there are multiple grammatical problems, awkward formulations, and typographical errors throughout the text. A careful language revision would improve the overall readability considerably.

Overall, I think the topic is interesting and the manuscript could become a useful review after revision, but at this stage it would benefit from a clearer focus, a more critical treatment of the literature, a thorough check of citation accuracy, and substantial language polishing.

Author's response: We sincerely thank the reviewer for this careful, constructive, and insightful evaluation of our manuscript. We have substantially revised the review in response to these comments.

First, to address the concern regarding conceptual focus, we revised multiple sections to more consistently center the discussion on Ca²⁺-dependent regulation of microtubule dynamics during mammalian corticogenesis. Topics such as adhesion, actin remodeling, membrane trafficking, and neuronal maturation are now discussed more selectively and only insofar as they contribute directly to microtubule organization, centrosome-related functions, or cytoskeletal mechanisms relevant to cortical development. In addition, we revised the title and subsection framing to better reflect the manuscript's scope.

Second, in response to the concern that the review was at times overly descriptive, we incorporated a more critical treatment of the literature throughout the manuscript. We now more clearly distinguish mechanisms that have been directly demonstrated in mammalian corticogenesis from those inferred from non-neural, in vitro, or broader cell biological systems, and we explicitly indicate these differences where relevant.

Third, we carefully reviewed the in-text citations and the reference list to correct any mismatches and ensure that each cited study appropriately supports its corresponding statement. The specific inconsistencies noted by the reviewer were corrected, and the reference list was checked more broadly for accuracy and consistency.

Fourth, we revised the tone of mechanistic statements throughout the manuscript to better reflect the strengths and limitations of the available evidence, especially in cases involving transformed cell lines, non-mammalian models, or indirect experimental systems.

Finally, the manuscript underwent language revision to improve grammar, clarity, and overall readability.

We are grateful for these comments, which significantly improved the focus, rigor, and presentation of the manuscript.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the revised version of the manuscript. The review has improved substantially and the main concerns raised in the previous round have been addressed. In particular, the manuscript is now more clearly framed around the relationship between calcium signaling and microtubule dynamics during mammalian corticogenesis, and the distinction between directly supported mechanisms and more indirect or extrapolated interpretations is better acknowledged across the text.

I also appreciate the effort to revise the mechanistic tone and to improve the overall coherence of the review. The manuscript is now more focused and more balanced than the original submission.

However, I do not think the manuscript is ready for acceptance in its current form. A final round of minor revision is still needed. The main remaining issue is language and editorial polishing. There are still several grammatical inaccuracies, awkward formulations, and typographical errors scattered throughout the text. In addition, some sections remain somewhat broader than the specific focus suggested by the title, particularly where the discussion moves toward more general aspects of actin remodeling, adhesion, or membrane trafficking.

Overall, I consider the scientific revision satisfactory, but I recommend one final careful revision of the language and style before publication.

Author Response

Round 2

Reviewer 3 Comments

Thank you for the revised version of the manuscript. The review has improved substantially, and the main concerns raised in the previous round have been addressed. In particular, the manuscript is now more clearly framed around the relationship between calcium signaling and microtubule dynamics during mammalian corticogenesis, and the distinction between directly supported mechanisms and more indirect or extrapolated interpretations is better acknowledged across the text.

I also appreciate the effort to revise the mechanistic tone and to improve the overall coherence of the review. The manuscript is now more focused and more balanced than the original submission.

However, I do not think the manuscript is ready for acceptance in its current form. A final round of minor revision is still needed. The main remaining issue is language and editorial polishing. There are still several grammatical inaccuracies, awkward formulations, and typographical errors scattered throughout the text. In addition, some sections remain somewhat broader than the specific focus suggested by the title, particularly where the discussion moves toward more general aspects of actin remodeling, adhesion, or membrane trafficking.

Overall, I consider the scientific revision satisfactory, but I recommend one final careful revision of the language and style before publication.

Author's response:

We thank the reviewer for the careful reassessment of our manuscript and for recognizing the substantial improvements achieved in response to the previous round of comments. We particularly appreciate the acknowledgment that the manuscript's conceptual focus, mechanistic clarity, and balance have been significantly strengthened.

In response to the remaining points, we have thoroughly revised the manuscript to address language and stylistic issues. This included correcting grammatical inaccuracies, refining sentence structure for clarity, and eliminating typographical errors throughout the text (Changes are in red). We have also ensured consistency in terminology and notation across all sections.

Regarding the concern that certain sections remain broader than the scope suggested by the title, we would like to emphasize that these elements have been deliberately retained where they are directly relevant to the functional interplay between Ca²⁺ signaling and microtubule dynamics. In the revised version, we have further clarified these connections to avoid potential ambiguity and maintain coherence with the review's central theme.

Importantly, the scope and level of detail of the manuscript are constrained by the current state of the field. In areas where direct experimental evidence remains limited, we have intentionally avoided overinterpretation and have explicitly distinguished between well-supported mechanisms and those inferred from related systems. We believe this reflects a rigorous and responsible synthesis of the literature rather than a limitation of the manuscript.

Given the extent of the revisions already undertaken, including substantial conceptual restructuring, improved critical analysis, careful verification of references, and comprehensive language editing, we respectfully consider that the manuscript meets the scientific and editorial standards required for publication. We therefore believe that no further substantive revisions are necessary.

We thank the reviewer again for their constructive input, which has meaningfully strengthened the manuscript.