Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum
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Abstract
The corpus callosum (CC) is the large axon bundle connecting the telencephalic hemispheres. The CC is formed exclusively in placental mammals, and the lack of comparable structures in other amniotes obscures the evolutionary origin of the CC. We here demonstrate that interhemispheric remodeling, a prior developmental step for CC formation, is highly conserved in non-mammalian amniotes, such as reptiles and birds. In these animal groups, the spatiotemporal dynamics of interhemispheric remodeling are tightly connected with distinct commissural formations. We observed a high degree of similarity between the mammalian CC and reptilian rostral pallial commissure, (RPC) and significant modifications in the avian pallial projection. Furthermore, we determined that Satb2 plays crucial roles in interhemispheric remodeling, which is associated with proper formation of both the CC and RPC in mice and geckoes, via the use of CRISPR-mediated gene-targeting. Our findings suggest that developmental mechanisms for midline remodeling were already present in the common ancestor of amniotes, which contributed to the evolution of eutherian-specific CC formation.
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Major comments
Unfortunately the major conclusions of the article are not well supported by the provided data. Including:
- * That interhemispheric remodelling occurs in non-mammalian amniotes. It would not surprise me that this may be the case, however, the major evidence for this is a series of horizontal insets that do not evidence* this point well. There are broad morphological changes during development that can change the proportions and regionalisation of tissue, and therefore the IHF becoming apparently smaller as development progresses (qualitatively, in single sectioning planes, and without clear n numbers) may easily be explained by sutble …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Major comments
Unfortunately the major conclusions of the article are not well supported by the provided data. Including:
- * That interhemispheric remodelling occurs in non-mammalian amniotes. It would not surprise me that this may be the case, however, the major evidence for this is a series of horizontal insets that do not evidence* this point well. There are broad morphological changes during development that can change the proportions and regionalisation of tissue, and therefore the IHF becoming apparently smaller as development progresses (qualitatively, in single sectioning planes, and without clear n numbers) may easily be explained by sutble differences in sectioning planes, or, for example, more caudal territories of the brain expanding at faster rates than the rostral territories. Quantification of the ratio between the IHF and total midline length across ages and between species may go some way to helping to clarify the degree of potential midline remodelling. Very high quality live imaging of the process would be the definitive way to evidence the claim, although I appreciate this is highly technically difficult and may not be possible. A key opportunity seems to be missed in the Satb2 knockout geckoes, where midline remodelling is purported to not occur. This is shown only qualitatively in a single plane of sectioning and again is not convincing. If the IHF length in these animals was quantified to be longer than wildtype at a comparable age, this would help to evidence the claim that remodelling occurs in these species.
Our responses
We take seriously the critique that the series of horizontal section images in the figures do not sufficiently substantiate our claim that interhemispheric remodeling occurs in non-mammalian amniotes. To address this, we plan to create a simplified atlas composed of adjacent sections of various wild-type amniotes as well as Satb2-knockout geckos.
Additionally, in response to the suggestion that the IHF (interhemispheric fissure) should be quantified relative to the total midline length across developmental stages and species, we note that Figure 1 already presents such an analysis. Specifically, we have quantified changes in the midline collagen content using Principal Component Analysis (PCA) in Satb2 Crispants in geckos(FigureS4). However, if necessary, we also plan to perform a similar analysis on wild-type soft-shelled turtles at developmental stages before and after interhemispheric remodeling.
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* That similar cell types contribute to remodelling in non-mammalian* amniotes as mice/eutherian mammals. The microphotographs presented are not of very high quality, and it is often difficult to be convinced that the data is showing the strong claims made in the paper. For instance the "MZG-like cells" may in fact be astrocytes or another cell type as it is hard to visualise morphology, and the "intercalation of GFP-positive radial glial fibres" is very unclear from the photos. The colocalization of MMPsense with laminin positive cells is very hard to appreciate from the figure, and again not quantified. Similarly, there is a claim that there was degeneration of laminin-positive leptomeninges during astroglial intercalation, which is an active process that is difficult to infer from a single microphotograph. From the data, I can appreciate that some of the similar broad categories of cell types that exist at the mouse midline (glia, radial glia) are also present in non-mammalian amniote midlines, but it is difficult to be convinced of much more than this from the data presented.
Our responses
We take seriously the critique that the degeneration of Laminin-positive leptomeninges close to astroglial components is not accepted and that the evidence for glial fiber intercalation is insufficient.
Verifying the degeneration of Laminin-positive leptomeninges is highly challenging. However, we have recently developed a method to visualize collagen in the pia mater using μCT and a CHP probe (3Helix Inc.). Preliminary experiments have already revealed pan-collagen deposition in the midline of the telencephalon (with lower amounts in the fusion region) and degeneration of the collagen composing the pia mater. We plan to incorporate these findings into the revised manuscript.
* That the gecko RPC and CPC connect distinct parts of the brain* (rostral and caudal). These tracer injections lacked visualisation of the deposition site to confirm specificity, as well as appropriate quantification. Importantly, the absence of axons in the CPC following the rostral dye deposition (and vice versa) was not shown, which is essential to make the claim that these commissures carry axons from specific parts of the brain. The alternative hypothesis is that all axons are intermixed and traverse both commissures, independent of brain area of origin, which is not at all tested or disproved by the data presented.
Our responses
Thank you for the valuable critique suggestion. To support our claim that the pallial commissure in geckos consists of axons derived from specific brain regions, we should carefully eliminate the possibility that all axons are intermixed and cross both RPC and CPC regardless of brain region.
To address this, we are planning additional experiments and will include a schematic diagram clearly indicating the labeling sites.
Overall, the major conclusions of the study are not well supported by the data. A major effort to quantify phenomena and/or dramatically soften conclusions would be needed in order to make the conclusions well supported.
Our responses
We will thoroughly reconsider our conclusions and make significant efforts to revise the manuscript.
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Minor comments
- * The n numbers are not always clearly reported* * *
Our responses
We plan to address the clarification of quantitative data and the exact number of replicates.
* At times important points reference reviews or articles that do not support the statements as well as the most important primary articles* might.
Our responses
We plan to carefully review the manuscript and, in addition to citing the most important primary papers, revise any descriptions that are not sufficiently supported by the cited reviews or articles, as per the suggestions.
* Figures showing the entire section that insets were taken from would help to convince that sectioning planes were equivalent, and also show the deposition site of neurovue experiments.*
Our response
We will add a schematic showing the locations labeled in NeuroVue and additional experiments as a similar point made in Major comment 3.
* The fibre direction of GFAP+ fibres in figure 6 is confusing - It* seems from the labelling on the figures as if red is used for the WT condition in mouse, but for the Satb2del condition in Gecko? If this is the case, then it would appear that the fibres are more specifically oriented in the del condition in mice, but in the WT condition of geckoes? There are several instances of this where clearer description and labelling would help the reader to interpret the results.
Our response
We plan to add clarification and indication of the direction of GFAP+ fibers in Figure 6 to make it easier to understand.
Reviewer #1 (Significance (Required)):
This study attempts to address a highly significant, novel and important question, that, if well achieved, would be publishable at a high degree of interest and impact to the basic research fields of brain development and evolution. Unfortunately the major conclusions made by the study are stronger than the data provided is able to evidence, and I remain unconvinced by many of them.
Our responses
We take seriously the suggestion that the major claims made by this study are excessive and so strong that they cannot be proven with the data provided. We will revise the manuscript as necessary.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary
The authors provide a comparative analysis of interhemispheric (IHF) remodeling and its potential role in the generation of commissural axons. Based on histological material from mice, chickens, turtles, and geckos, the IHF remodeling of the midline is divided in two events: caudal and rostral. It is suggested that the rostral event is a preliminary step to the crossing of commissural axons, as it is characteristic of eutherian mammals with a corpus callosum (CC). However, the authors describe similar histologic features in other amniotes during development, particularly reptiles. This is in contrast with the case of the chick, which does not show signs of IHF remodeling nor a rostral pallial commissure. Additionally, deficient transgenic mice and geckos illustrate a potential role of Satb2 in rostral IHF remodeling and subsequent commissural formation. Whereas the topic and the conclusions of the analysis are interesting and provide new knowledge to the evo-devo field, several issues should be addressed prior to publication, such as data precision and presentation to support the main statements in the manuscript.
Major comments:
* ____-A central point of this article is the splitting of the IHF into rostral* and caudal events. The authors suggest that each one can be regulated differentially, and they attribute the rostral remodeling as a step prior to corpus callosum (CC) formation, in contrast to the caudal remodeling. In my opinion, these two events are not sufficiently characterized either in the figures or the manuscript. It is necessary to better describe these two processes that the authors mention. For instance, the authors could add or re-organize information in Figures 1-3 to include wide-field images showing the whole septum from rostral to caudal, and representative dorsoventral sections at important stages (with insets pointing at specific features). Otherwise, a table summarizing the rostral and caudal events would also be helpful to the reader.
Our responses
We take the suggestion seriously that the distinction between rostral and caudal remodeling may not be clear, especially regarding rostral remodeling, which is prior to the stage of corpus callosum (CC) formation, in contrast to caudal remodeling. Specifically, we plan to add or restructure the information from Figures 1 to 3 by including wide-field images that show the entire septum from rostral to caudal, as well as representative sagittal sections along the dorsal-ventral axis at key stages, with insets highlighting specific features. These will be added to the Supplementary data. Additionally, a table summarizing the events in both the rostral and caudal regions will also be created and included in the revised manuscript.
When the authors refer to the reptilian rostral pallial commissure (RPC) and caudal pallial commissure (CPC), are these the same structures as the pallial commissure and anterior commissure described by Lanuza and Halpern (1997), Butler and Hodos (2005) and Puelles et al. (2019)? It is necessary to clarify the nomenclature, given that they are providing data from several species. Also, structures with the same names among species may not be truly homologous. A simple atlas with some horizontal and transverse planes highlighting anatomical landmarks and important structures (commissural tracts in this case) of the non-mammalian species would be extremely useful for the reader.
Our responses
As suggested by the reviewer, we are considering to provide a more detailed definition of the nomenclature of the pallial commissure in the revised manuscript, specifically in the introduction. Additionally, as mentioned earlier, we plan to create a simplified atlas with several horizontal and transverse sections, emphasizing anatomical landmarks and important structures (in this case, the commissural pathways) in species other than mammals.
* ____I wonder if the authors tested Fgf8 as marker on any of their sauropsidian tissue samples, as this gene has a known role in murine MZG* development, which is required for IHF remodeling (Gobius et al. 2016, already cited in the manuscript). It would be beneficial to test this marker for the study, and if positive, it would open the possibility of designing loss-of-function experiments in avian or reptilian development models to identify mechanisms common to eutherians and support the statements of this work.
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Our responses
We plan to verify the gene expression necessary for mouse MZG development and IHF remodeling, including Fgf8, DCC, and MMP2, through immunohistochemical staining as suggested.
It would be really interesting to provide a more elaborate discussion on whether authors consider the sauropsidian IHF as a homologous process to eutherian IHF, and the reptilian RPC as an homologous of the CC.
Our responses
Since 3 out of the 4 reviewers consider IHF remodeling in sauropods to be homologous to that in placental mammals, we plan to further emphasize this claim in the revised manuscript. Additionally, we will expand on the discussion regarding whether the process of RPC formation in reptiles is considered homologous to that of the corpus callosum, and I will approach this from the context of character identity mechanisms claimed by Dr. Günter Wagner.
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Data and methods are presented in such a way that, in principle, they could be reproduced. Authors should indicate the number of animals/replicates of each species used in each experiment.
Our responses
As suggested, we plan to provide more detailed descriptions of the methods to ensure reproducibility. This will include adding the number of samples and trial repetitions for each animal species used in the experiments, including those for the additional experiments, in the revised manuscript.
Minor comments:
In the results section, paragraph 2, line 3: "We detected the accumulation of GFAP-positive cells and phosphorylated vimentin (Ser55) -positive mitotic radial glia in the IHF and telencephalic hinge in developing turtles, geckoes and chicks (Figure 2A)". Figure 2A shows sections from the four analyzed species labeled with radial glia markers at the end of the IHF remodeling. It would be beneficial to have analogous sections at several time points (perhaps before or after the process) to compare and show more clearly the accumulation of glial cells at that location.
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Our responses
We have prepared serial sections before and after the developmental stages when interhemispheric remodeling occurs, in order to compare and more clearly show the accumulation of glial cells at their respective locations in mice, geckos, and soft-shelled turtles. I plan to add these results to Figure 2A in the revised manuscript.
The article will improve its quality by adding more comparative information in the introduction about the analyzed sauropsidian structures (rostral pallial commissure and caudal pallial commissure), their relations with the pallial and anterior commissures, the structures/cells connected by them, and homologies previously proposed.
Our responses
We will add comparative information regarding the brain structures in sauropod, including the rostral and caudal pallial commissures and their relationship to the pallial commissure and anterior commissure, and the structures they connect, such as pyramidal cells, along with previously proposed homologies. This information will be included in the introduction and summarized in a table.
In Figure 1 panels A-D, there is a lot of disparity in brain sizes and scales both between sections of the same species and between species. Placing the insets next to their source images is very necessary for clarity.
Our responses
As mentioned earlier, I will create a simplified atlas using adjacent sections and continuous μCT tomography images. Additionally, I will adjust the placement of the inset images in the revised manuscript to more visually accessible positions, improving their visibility.
In the results section, paragraph 2, line 11: "In addition, it was suggested that astroglial intercalation occurs in conjunction with the aforementioned regression of the IHF from st.21 to st.26 in the developing turtle (Figure 2C)." In Figure 2C, all images are at different scales,
which makes it very hard to properly compare between stages.
Our responses
By creating inset images based on the low-magnification images in the upper panel, we will enhance the visibility of GFAP intercalation. Additionally, we will improve the visibility in the revised manuscript by adding scale bars, referencing the simplified atlas in the figure legends, and standardizing the tissue specimen scale. we also plan to correct any typographical errors in the figures.
In Figure 2D, the authors show the presence of MMP around the leptomeninges, suggesting MMP-mediated degradation. In the images, MMP labeling is revealed in dark blue, which is largely invisible against the black background. Colors should be used properly to allow visualization of this MMP labeling.
Our responses
In Figure 2D, we will reconsider the selection of pseudo-colors and use cyan to represent MMPsense.
In Figure 4, it would really help if the authors provided wide-field images and DAPI counterstaining of the anterograde and retrograde tracings, to provide anatomical landmarks that help readers to identify the midline and understand the orientation of images.
Our responses
In addition to the previously mentioned schematic diagram of the gecko's pallial commissure and the additional experiments, we plan to include wide-field images along with forward and retrograde tracing using Hoechst counterstaining.
In Figure 5B, I understand that the images in the red and blue squares correspond to brain areas in the squares in A. However, some confusion remains, especially with the image in B, which does not seem to be at the same angle as in the diagram representation. This makes it difficult to understand the results.
Our responses
According to the comment, we will revise the design of the Figure 5B to be more easily understand, and modify the scheme to match the angle of sections with actual figures.
In Figure 6D, to better visualize defects in the RPC formation, the asterisk in the middle of the deficient structure needs to be replaced with a more lateral arrow pointing to the malformation.
Our responses
To better visualize the absence of RPC formation in Figure 6D, we will replace the asterisk in the center of the missing structure with a horizontal arrow indicating the malformation.
In Figure S5, violin plots in panel C do not correspond with data in A and B. This needs correction or clarification.
Our responses
In Figure S5, the inconsistency between the violin plot in panel C and the data in panels A and B is a clear error, and we will correct this in the revised manuscript.
In the article, a section appears solely to explain spatial transcriptomics results in a chick coronal section. The conclusion of this experiment is that three markers associated with midline remodeling are present in chick, suggesting that interhemispheric remodeling is conserved between mouse and chick. As these are complementary results and are not deeply analyzed in this manuscript, I think it would be better to summarize these findings in a dedicated paragraph and transfer some of the key images from Figure S2 to one of the main figures. Other problems with Figure S2: color contrast between clusters in the tSNE projection in B is very poor, should be enhanced; color intensity in FeaturePlots of panels D-F is too weak, and it seems that there is not really much expression at all in any cluster for any of these genes.
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Our responses
In the revised manuscript, we will move some of the key images from Figure S2 to Main Figure 3 to demonstrate that the three markers related to midline remodeling are also present in chickens, showing that interhemispheric remodeling is conserved between mice and chickens. Additionally, we will enhance the contrast between clusters in the tSNE projection of the FeaturePlots in S2B and D-F by increasing the pseudo-color intensity or adjusting the intensity levels to emphasize the color contrast, and incorporate this updated figure into the revised manuscript.
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Reviewer #2 (Significance (Required)):
The authors identify in the developing brain of sauropsids an event similar to IHF remodeling in eutherians, and suggest a causal relation between the rostral IHF remodeling and the formation of the pallial commissure in reptilian brains. This implies a potential homology between the pallial commissure and the corpus callosum of placental mammals. If this is the intention of the authors, this conclusion should be addressed explicitly and at length in the Discussion section. Whereas the results and conclusions described in the manuscript will be valuable in the field, the data presented in the manuscript needs quite some improvement, particularly for some of the images in the previously mentioned figures. Otherwise, the original data cannot be properly judged and may set reasonable doubt to readers.
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Advance: The findings described in this report are new to my knowledge. The description of the IHF remodeling event prior to corpus callosum development in mice has been published (Gobius et al. 2016, Cell Reports), but not in other mammalian branches or non-mammalian vertebrates. For this reason, the data in this report should be very convincing and better presented.
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Audience: This research will be interesting for a specialized and basic research audience, particularly for researchers in the evo-devo fields.
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My expertise: neuroanatomy, development, evolution, brain, cerebral cortex
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Our responses
Thank you for your positive feedback on the novelty and high evaluation of identifying phenomena in reptilian development that resemble interhemispheric fissure (IHF) remodeling in placental mammals and demonstrating a causal relationship between rostral IHF remodeling and the formation of the reptilian pallial commissure. we will incorporate the concept of the potential homology between the corpus callosum in placental mammals and the brain commissures in reptiles into the revised manuscript, reflecting this in the context of character Identity mechanisms claimed by Dr. Günter Wagner. This will be clearly and thoroughly discussed in the discussion section. Additionally, we sincerely appreciate the constructive comment about the room for significant improvement, particularly in some of the figures, and we will address these points in the revised manuscript.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Conserved interhemispheric morphogenesis in amniotes preceded the evolution
of the corpus callosum. Noji Kaneko et al., 2025
The CC is formed exclusively in placental mammals. In other amniotes species, the communication of the two hemispheres is mediated by other structures such as the anterior commissure or the hippocampal commissure. The authors perform anatomical comparisons between species to conclude that interhemispheric fissure remodeling, a prior developmental step for CC formation, is highly conserved in non-mammalian amniotes, such as reptiles and birds. They suggest that might have contributed to the evolution of eutherian-specific CC formation. In an attempt to test their hypothesis, the authors investigate the role of Satb2 in interhemispheric fissure remodeling. They show IH fissure defects in both mice and geckoes. This is a nice manuscript that bridges a gap in the current understanding of CC formation. The study is mostly anatomical and directed at a specialized community.
Our response
We appreciate for positive comments on the manuscript.
I suggest some changes that might contribute to improving the manuscript.
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Main
- * Much of the most important conclusions are extracted from the* anatomical observation of the dynamics of IHF closure and the emergence of the Hinge. It is very clear that the researchers are specialists in the field but for a broader audience, the images they provide are not always easy to interpret. It takes a lot of effort to visualize the anatomical data they use for their conclusions. As an example, perhaps the authors can find ways to explain how to identify the hinge specifically. It is very clear what the hinge is in the schemes (drawings)but forms one picture to the other at different developmental stages neither in the same animal species nor from different species. In Figure 1, it is difficult to see how the hinge in the mouse is similar (i.e. the same structure) to the hinge in the Gecko and chick. Moreover, in panels C , chick brain sections are shown at much greater magnification than the gecko, and thus is very difficult. In addition, in the manuscript text, the authors refer to sequential sectioning, but only one image for each stage is shown. They can show more images in supplementary Figures, otr they can just explain that they show the relevant images of the sectioning. As another example, in Fig2A, in the text, the authors explain that they detect the same specific glial components, but the images show very different co-localizations and distributions. In Figures 1 and 3, there are lines indicating Dorsal to ventral. This refers to the sectioning but in reality, what the sections are illustrating is the anterior-to-posterior differences in the IHF. maybe they can clarify it, because at quick sight it can be confusing.
Our responses
We sincerely appreciate the feedback regarding the interpretation of images that show the dynamics of interhemispheric remodeling and the emergence of the hinge, which is central to the most important conclusions of this study, as it may not always be easy to interpret. In the revised manuscript, we plan to address this by making the following revisions. For example, to clarify how the hinge corresponds across different species, we will create a simplified atlas to explain that the sections from the main figure are at the same level within the continuous slices.
* The authors have to revise the manuscript text to be more precise. For* example, In the result section quote "To address whether the interhemispheric remodeling in non-mammalian amniotes is dependent on midline glial activities, we next examined the expression of several glial markers in the reptilian and avian midline regions". the anatomical comparison does not address the role of glial.
Our responses
Thank you for your feedback. I will correct the expression "midline glial activities" to "midline glial components" and incorporate this more accurate terminology into the revised manuscript.
* As an option to increase the relevance of their work, the authors might want to consider to describe in more detail and moving the results of the RNAseq and the analysis of the Stab2 mutants to the main figures.*
Our responses
Thank you for your feedback. we will move the RNAseq results and the analysis of Satb2 mutants to the main figures and will describe them in more detail to enhance the relevance of the study. Specifically, we plan to separate Figure 6A-C as independent figures and add Supplementary Figure 5, corresponding to mice and geckos, to the main figures in the revised manuscript.
Minor:
Please indicate the length of the scale bars in the figure legends, and not only in the figure panels Fig5. Indicate the animal model in the panel Perhaps they can draw a model of the different mechanisms of caudal and anterior remodeling.
Our responses
Thank you for your feedback. I plan to revise the figure legend for Figure 5 by clearly indicating the scale bar length and increasing the font size, as well as including the information in each panel. Additionally, I will add a graphical abstract that illustrates the different mechanisms of caudal and rostral remodeling to enhance visual comprehension.
Reviewer #3 (Significance (Required)):
The study addresses a gap in knowledge from an evolutionary perspective. It provides novel hypotheses and an innovative framework for the understanding of cortical development and evolution. however, most of the conclusions are inferred from anatomical observations and the experimental testing of the hypothesis (Mutants and RNAseq analysis) are minor part of the study that could be further developed. The study is interesting for investigators with expertise in brain development and evolution but requires familiarity with comparative anatomy and even then it is difficult to go through the work.
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Reviewer #4 (Evidence, reproducibility and clarity (Required)):
* *
Overall, this is a well-written manuscript focusing on the evolution of mid-line interhemispheric fusion related to corpus callosum development and evolution from amniotes to eutherian species. The authors also demonstrated that Satb2 plays a critical role in interhemispheric remodeling, which is essential for corpus callosum development. This is a nicely organized and interesting study and the data are compelling. The following are suggestions for improvement, mostly for clarity:
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Minor comments:
- * Figure 1A: While the E14 and E17 horizontal sections are informative, the addition of the E12 horizontal section does not provide further information. It would be better to place the inset and the whole image side by side, rather than having them far apart across the whole figure. For* Figures 1C-D, is it possible to include horizontal sections for chick at
E14 and Gecko at 45 dpo, as shown in the subsequent images?
Our responses
In Figure 1A, we will replace the current figure with a new one that visually enhances the comparison by placing the inset and the full image side by side. we will also add new horizontal sections of the whole image for chicken E14 and gecko 45 dpo, obtained from μCT tomography images and HE staining, to improve visibility between the images.
* When comparing across species it is sometimes helpful to use a standard staging system so that different developmentally staged tissue can be compared. A timeline of how embryonic day or dpo equates to stage might be helpful.*
* *
Our response
To clarify the developmental stages, I plan to incorporate a time scale into the graphical abstract in the revised manuscript.
* Figure 2B: It is difficult to discern the perspective without a full,* lower power section of Gecko at 45 dpo. Adding a full image with an inset would be helpful. In Figure 1C, it would be helpful to define the magnified area by placing a box on the low magnification image.
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Our responses
We plan to add a low-magnification μCT tomography image or HE-stained whole image of the gecko at 45 dpo in the revised manuscript. As for Figure 1C, it has already been included in the preprint.
* Figures 3B-E: Include the staining methods used for these sections.*
Our response
We plan to add a note specifying that the image is stained with HE.
* Figure 4B: Add a low magnification image with an inset. The current image is a bit confusing as it is unclear what is being shown.*
Our responses
We plan to add a low-magnification image showing the entire section and use an inset to indicate the positional relationship of the section's plane in a schematic diagram.
* Figures 6A-E: It would be helpful to denote the genotype as Satb2+/- or heterozygous, rather than Satb2 WT/del, which can be confusing. Ensure consistency in genotyping notation throughout all figures. It is noted that some are CRISPR knockdown and could be denoted as such.*
Our responses
For CRISPR knockdown, I will adopt the term "CRISPANT" in the revised manuscript. This terminology will be used consistently throughout all figures to avoid confusion in genotype notation.
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Reviewer #4 (Significance (Required)):
The corpus callosum evolved only in eutherian mammals and its development relies critically on an earlier developmental process known as interhemispheric remodeling. Nomura and colleagues investigate the evolution of these processes and identify that interhemispheric remodeling occurs in reptiles and birds and was therefore already present in the common ancestor of amniotes. This highly conserved developmental process likley evolved early and provided a substrates for major commissures to form throughout evolution.
* 3.*____Description of the revisions that have already been incorporated in the transferred manuscript.
Currently we do not incorporate the revision in the transferred manuscript.
__ Description of analyses that authors prefer not to carry out__
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
*Major *
* That similar cell types contribute to remodelling in non-mammalian* amniotes as mice/eutherian mammals. The microphotographs presented are not of very high quality, and it is often difficult to be convinced that the data is showing the strong claims made in the paper. For instance the "MZG-like cells" may in fact be astrocytes or another cell type as it is hard to visualise morphology, and the "intercalation of GFP-positive radial glial fibres" is very unclear from the photos. The colocalization of MMPsense with laminin positive cells is very hard to appreciate from the figure, and again not quantified. Similarly, there is a claim that there was degeneration of laminin-positive leptomeninges during astroglial intercalation, which is an active process that is difficult to infer from a single microphotograph. From the data, I can appreciate that some of the similar broad categories of cell types that exist at the mouse midline____ ____(glia, radial glia) are also present in non-mammalian amniote midlines, but it is difficult to be convinced of much more than this from the data presented.
Our responses
We are confident that this paper provides sufficient evidence that cell types similar to those in non-mammalian amniotes, mice, and placental mammals contribute to interhemispheric remodeling and that glial fiber intercalation occurs. This point is also supported by other reviewers.
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In the present study, we have not conducted the MMPsense experiments with the aim of showing the co-localization of MMPsense and laminin-positive cells or pia mater. Contrary to the reviewer's claim, it is important that the non-continuous regions of MMPsense and laminin-positive areas (pia mater), which are extracellular components, are adjacent to each other. Unfortunately, establishing a quantification system using MMPsense is practically impossible.
Major
* The implication that Satb2 expression at the midline is necessary for appropriate interhemispheric remodeling. Alternative hypotheses for an* inappropriately remodeled midline upon whole-brain Satb2 knockout is that it is not dependent on expression at the midline region. Rather, it could be that, for example, the appropriately timed interaction between ingrowing callosal axons and the midline territory is needed for the timely differentiation and/or behavior of midline cells. Other alternatives include that the lack of axonal midline crossing changes the morphology of the midline territory, including potentially "unfusing" the midline. Given the high prevalence of midline remodelling defects concomitant with callosal agenesis referred to be the authors in the literature, it seems like these alternatives would be worth considering. Indeed, the only article the authors reference in their statement that "several studies implicated that agenesis of CC in Satb2-deficient mice is also associated with defects in midline fusion" is an article where Satb2 was knocked out specifically in the cortex and hippocampus. This result is difficult to interpret, as some Emx1 promotors do label some of the midline territory, however the point stands that it is difficult to interpret solely that Satb2 action at the midline is responsible for the effects. I understand that this is a hard question to investigate, so I would suggest allusion to the alternative hypotheses/interpretations as the main priority when interpreting the data.
Our responses
This study does not aim to demonstrate the detailed molecular function of Satb2 in the developmental processes of the corpus callosum or pallial commissure. We plan to clearly state this point in the revised manuscript and focus on the findings obtained as a result. Based on the histological relationships, we will classify interhemispheric remodeling and consider adding a section in the Discussion to identify the common character identity mechanisms underlying the development of the pallial commissure and corpus callosum. This addition will help provide a more detailed understanding of the remodeling mechanisms. As is well known, discussions of homology are complex, and we understand that providing concrete evidence is even more challenging. When discussing homology, we will emphasize that it must be handled cautiously, and that discussions on molecular features and homology will rely heavily on future research. As an alternative, we plan to position the results of Satb2 Crispants in mice and geckos as evidence of the disruption of character identity mechanisms. By incorporating this perspective into the revised manuscript, we believe it will deepen our understanding of the role of Satb2 and its molecular mechanisms.
Reviewer4
Minor comment 7. There is very valuable data in the supplementary figures. As suggestion is to incorporate Supp. figures S1, S2 and S5 in the main figures.
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Our responses
Due to space constraints, we plan to move only Supplementary Figure S5 to the supplementary section, and Figures S1 and S2 will not be included in the main figures of the revised manuscript.
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Referee #4
Evidence, reproducibility and clarity
Overall, this is a well-written manuscript focusing on the evolution of mid-line interhemispheric fusion related to corpus callosum development and evolution from amniotes to eutherian species. The authors also demonstrated that Satb2 plays a critical role in interhemispheric remodeling, which is essential for corpus callosum development. This is a nicely organized and interesting study and the data are compelling. The following are suggestions for improvement, mostly for clarity:
Minor comments:
- Figure 1A: While the E14 and E17 horizontal sections are informative, the addition of the E12 horizontal section does not provide further …
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Referee #4
Evidence, reproducibility and clarity
Overall, this is a well-written manuscript focusing on the evolution of mid-line interhemispheric fusion related to corpus callosum development and evolution from amniotes to eutherian species. The authors also demonstrated that Satb2 plays a critical role in interhemispheric remodeling, which is essential for corpus callosum development. This is a nicely organized and interesting study and the data are compelling. The following are suggestions for improvement, mostly for clarity:
Minor comments:
- Figure 1A: While the E14 and E17 horizontal sections are informative, the addition of the E12 horizontal section does not provide further information. It would be better to place the inset and the whole image side by side, rather than having them far apart across the whole figure. For Figures 1C-D, is it possible to include horizontal sections for chick at E14 and Gecko at 45 dpo, as shown in the subsequent images?
- When comparing across species it is sometimes helpful to use a standard staging system so that different developmentally staged tissue can be compared. A timeline of how embryonic day or dpo equates to stage might be helpful.
- Figure 2B: It is difficult to discern the perspective without a full, lower power section of Gecko at 45 dpo. Adding a full image with an inset would be helpful. In Figure 1C, it would be helpful to define the magnified area by placing a box on the low magnification image.
- Figures 3B-E: Include the staining methods used for these sections.
- Figure 4B: Add a low magnification image with an inset. The current image is a bit confusing as it is unclear what is being shown.
- Figures 6A-E: It would be helpful to denote the genotype as Satb2 +/- or heterozygous, rather than Satb2 WT/del, which can be confusing. Ensure consistency in genotyping notation throughout all figures. It is noted that some are CRISPR knockdown and could be denoted as such.
- There is very valuable data in the supplementary figures. As suggestion is to incorporate Supp. figures S1, S2 and S5 in the main figures.
Significance
The corpus callosum evolved only in eutherian mammals and its development relies critically on an earlier developmental process known as interhemispheric remodeling. Nomura and colleagues investigate the evolution of these processes and identify that interhemispheric remodeling occurs in reptiles and birds and was therefore already present in the common ancestor of amniotes. This highly conserved developmental process likley evolved early and provided a substrates for major commissures to form throughout evolution.
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Referee #3
Evidence, reproducibility and clarity
Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum. Kaneko et al., 2025
The CC is formed exclusively in placental mammals. In other amniotes species, the communication of the two hemispheres is mediated by other structures such as the anterior commissure or the hippocampal commissure. The authors perform anatomical comparisons between species to conclude that interhemispheric fissure remodeling, a prior developmental step for CC formation, is highly conserved in non-mammalian amniotes, such as reptiles and birds. They suggest that might have contributed to the evolution of …
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Referee #3
Evidence, reproducibility and clarity
Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum. Kaneko et al., 2025
The CC is formed exclusively in placental mammals. In other amniotes species, the communication of the two hemispheres is mediated by other structures such as the anterior commissure or the hippocampal commissure. The authors perform anatomical comparisons between species to conclude that interhemispheric fissure remodeling, a prior developmental step for CC formation, is highly conserved in non-mammalian amniotes, such as reptiles and birds. They suggest that might have contributed to the evolution of eutherian-specific CC formation. In an attempt to test their hypothesis, the authors investigate the role of Satb2 in interhemispheric fissure remodeling. They show IH fissure defects in both mice and geckoes. This is a nice manuscript that bridges a gap in the current understanding of CC formation. The study is mostly anatomical and directed at a specialized community.
I suggest some changes that might contribute to improving the manuscript.
Main
- Much of the most important conclusions are extracted from the anatomical observation of the dynamics of IHF closure and the emergence of the Hinge. It is very clear that the researchers are specialists in the field but for a broader audience, the images they provide are not always easy to interpret. It takes a lot of effort to visualize the anatomical data they use for their conclusions. As an example, perhaps the authors can find ways to explain how to identify the hinge specifically. It is very clear what the hinge is in the schemes (drawings)but forms one picture to the other at different developmental stages neither in the same animal species nor from different species. In Figure 1, it is difficult to see how the hinge in the mouse is similar (i.e. the same structure) to the hinge in the Gecko and chick. Moreover, in panels C , chick brain sections are shown at much greater magnification than the gecko, and thus is very difficult In addition, in the manuscript text, the authors refer to sequential sectioning, but only one image for each stage is shown. They can show more images in supplementary Figures, otr they can just explain that they show the relevant images of the sectioning. As another example, in Fig2A, in the text, the authors explain that they detect the same specific glial components, but the images show very different co-localizations and distributions. In Figures 1 and 3, there are lines indicating Dorsal to ventral. This refers to the sectioning but in reality, what the sections are illustrating is the anterior-to-posterior differences in the IHF. maybe they can clarify it, because at quick sight it can be confusing.
- The authors have to revise the manuscript text to be more precise. For example, In the result section quote "To address whether the interhemispheric remodeling in non-mammalian amniotes is dependent on midline glial activities, we next examined the expression of several glial markers in the reptilian and avian midline regions". the anatomical comparison does not address the role of glial.
- As an option to increase the relevance of their work, the authors might want to consider to describe in more detail and moving the results of the RNAseq and the analysis of the Stab2 mutants to the main figures.
Minor:
Please indicate the length of the scale bars in the figure legends, and not only in the figure panels
Fig5 .Indicate the animal model in the panel
Perhaps they can draw a model of the different mechanisms of caudal and anterior remodeling.
Significance
The study addresses a gap in knowledge from an evolutionary perspective. It provides novel hypotheses and an innovative framework for the understanding of cortical development and evolution. however, most of the conclusions are inferred from anatomical observations and the experimental testing of the hypothesis (Mutants and RNAseq analysis) are minor part of the study that could be further developed. The study is interesting for investigators with expertise in brain development and evolution but requires familiarity with comparative anatomy and even then it is difficult to go through the work.
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Referee #2
Evidence, reproducibility and clarity
Summary
The authors provide a comparative analysis of interhemispheric (IHF) remodeling and its potential role in the generation of commissural axons. Based on histological material from mice, chickens, turtles, and geckos, the IHF remodeling of the midline is divided in two events: caudal and rostral. It is suggested that the rostral event is a preliminary step to the crossing of commissural axons, as it is characteristic of eutherian mammals with a corpus callosum (CC). However, the authors describe similar histologic features in other amniotes during development, particularly reptiles. This is in contrast with the case of the …
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Referee #2
Evidence, reproducibility and clarity
Summary
The authors provide a comparative analysis of interhemispheric (IHF) remodeling and its potential role in the generation of commissural axons. Based on histological material from mice, chickens, turtles, and geckos, the IHF remodeling of the midline is divided in two events: caudal and rostral. It is suggested that the rostral event is a preliminary step to the crossing of commissural axons, as it is characteristic of eutherian mammals with a corpus callosum (CC). However, the authors describe similar histologic features in other amniotes during development, particularly reptiles. This is in contrast with the case of the chick, which does not show signs of IHF remodeling nor a rostral pallial commissure. Additionally, deficient transgenic mice and geckos illustrate a potential role of Satb2 in rostral IHF remodeling and subsequent commissural formation. Whereas the topic and the conclusions of the analysis are interesting and provide new knowledge to the evo-devo field, several issues should be addressed prior to publication, such as data precision and presentation to support the main statements in the manuscript.
Major comments:
- A central point of this article is the splitting of the IHF into rostral and caudal events. The authors suggest that each one can be regulated differentially, and they attribute the rostral remodeling as a step prior to corpus callosum (CC) formation, in contrast to the caudal remodeling. In my opinion, these two events are not sufficiently characterized either in the figures or the manuscript. It is necessary to better describe these two processes that the authors mention. For instance, the authors could add or re-organize information in Figures 1-3 to include wide-field images showing the whole septum from rostral to caudal, and representative dorsoventral sections at important stages (with insets pointing at specific features). Otherwise, a table summarizing the rostral and caudal events would also be helpful to the reader.
- When the authors refer to the reptilian rostral pallial commissure (RPC) and caudal pallial commissure (CPC), are these the same structures as the pallial commissure and anterior commissure described by Lanuza and Halpern (1997), Butler and Hodos (2005) and Puelles et al. (2019)? It is necessary to clarify the nomenclature, given that they are providing data from several species. Also, structures with the same names among species may not be truly homologous. A simple atlas with some horizontal and transverse planes highlighting anatomical landmarks and important structures (commissural tracts in this case) of the non-mammalian species would be extremely useful for the reader.
- I wonder if the authors tested Fgf8 as marker on any of their sauropsidian tissue samples, as this gene has a known role in murine MZG development, which is required for IHF remodeling (Gobius et al. 2016, already cited in the manuscript). It would be beneficial to test this marker for the study, and if positive, it would open the possibility of designing loss-of-function experiments in avian or reptilian development models to identify mechanisms common to eutherians and support the statements of this work
- It would be really interesting to provide a more elaborate discussion on whether authors consider the sauropsidian IHF as a homologous process to eutherian IHF, and the reptilian RPC as an homologous of the CC.
- Data and methods are presented in such a way that, in principle, they could be reproduced. Authors should indicate the number of animals/replicates of each species used in each experiment.
Minor comments:
- In the results section, paragraph 2, line 3: "We detected the accumulation of GFAP-positive cells and phosphorylated vimentin (Ser55) -positive mitotic radial glia in the IHF and telencephalic hinge in developing turtles, geckoes and chicks (Figure 2A)". Figure 2A shows sections from the four analyzed species labeled with radial glia markers at the end of the IHF remodeling. It would be beneficial to have analogous sections at several time points (perhaps before or after the process) to compare and show more clearly the accumulation of glial cells at that location.
- The article will improve its quality by adding more comparative information in the introduction about the analyzed sauropsidian structures (rostral pallial commissure and caudal pallial commissure), their relations with the pallial and anterior commissures, the structures/cells connected by them, and homologies previously proposed.
- In Figure 1 panels A-D, there is a lot of disparity in brain sizes and scales both between sections of the same species and between species. Placing the insets next to their source images is very necessary for clarity.
- In the results section, paragraph 2, line 11: "In addition, it was suggested that astroglial intercalation occurs in conjunction with the aforementioned regression of the IHF from st.21 to st.26 in the developing turtle (Figure 2C)." In Figure 2C, all images are at different scales, which makes it very hard to properly compare between stages.
- In Figure 2D, the authors show the presence of MMP around the leptomeninges, suggesting MMP-mediated degradation. In the images, MMP labeling is revealed in dark blue, which is largely invisible against the black background. Colors should be used properly to allow visualization of this MMP labeling.
- In Figure 4, it would really help if the authors provided wide-field images and DAPI counterstaining of the anterograde and retrograde tracings, to provide anatomical landmarks that help readers to identify the midline and understand the orientation of images.
- In Figure 5B, I understand that the images in the red and blue squares correspond to brain areas in the squares in A. However, some confusion remains, especially with the image in B, which does not seem to be at the same angle as in the diagram representation. This makes it difficult to understand the results.
- In Figure 6D, to better visualize defects in the RPC formation, the asterisk in the middle of the deficient structure needs to be replaced with a more lateral arrow pointing to the malformation.
- In Figure S5, violin plots in panel C do not correspond with data in A and B. This needs correction or clarification.
- In the article, a section appears solely to explain spatial transcriptomics results in a chick coronal section. The conclusion of this experiment is that three markers associated with midline remodeling are present in chick, suggesting that interhemispheric remodeling is conserved between mouse and chick. As these are complementary results and are not deeply analyzed in this manuscript, I think it would be better to summarize these findings in a dedicated paragraph and transfer some of the key images from Figure S2 to one of the main figures. Other problems with Figure S2: color contrast between clusters in the tSNE projection in B is very poor, should be enhanced; color intensity in FeaturePlots of panels D-F is too weak, and it seems that there is not really much expression at all in any cluster for any of these genes.
Significance
The authors identify in the developing brain of sauropsids an event similar to IHF remodeling in eutherians, and suggest a causal relation between the rostral IHF remodeling and the formation of the pallial commissure in reptilian brains. This implies a potential homology between the pallial commissure and the corpus callosum of placental mammals. If this is the intention of the authors, this conclusion should be addressed explicitly and at length in the Discussion section. Whereas the results and conclusions described in the manuscript will be valuable in the field, the data presented in the manuscript needs quite some improvement, particularly for some of the images in the previously mentioned figures. Otherwise, the original data cannot be properly judged and may set reasonable doubt to readers.
Advance: The findings described in this report are new to my knowledge. The description of the IHF remodeling event prior to corpus callosum development in mice has been published (Gobius et al. 2016, Cell Reports), but not in other mammalian branches or non-mammalian vertebrates. For this reason, the data in this report should be very convincing and better presented.
Audience: This research will be interesting for a specialized and basic research audience, particularly for researchers in the evo-devo fields.
My expertise: neuroanatomy, development, evolution, brain, cerebral cortex
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Referee #1
Evidence, reproducibility and clarity
Summary
The authors study several valuable developmental series of non-mammalian amniotes, reaching the conclusion that interhemispheric remodeling occurs in these species and that it is dependent on transcription factor Satb2
Major comments
Unfortunately the major conclusions of the article are not well supported by the provided data. Including:
- That interhemispheric remodelling occurs in non-mammalian amniotes. It would not surprise me that this may be the case, however, the major evidence for this is a series of horizontal insets that do not evidence this point well. There are broad morphological changes during development that …
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Referee #1
Evidence, reproducibility and clarity
Summary
The authors study several valuable developmental series of non-mammalian amniotes, reaching the conclusion that interhemispheric remodeling occurs in these species and that it is dependent on transcription factor Satb2
Major comments
Unfortunately the major conclusions of the article are not well supported by the provided data. Including:
- That interhemispheric remodelling occurs in non-mammalian amniotes. It would not surprise me that this may be the case, however, the major evidence for this is a series of horizontal insets that do not evidence this point well. There are broad morphological changes during development that can change the proportions and regionalisation of tissue, and therefore the IHF becoming apparently smaller as development progresses (qualitatively, in single sectioning planes, and without clear n numbers) may easily be explained by sutble differences in sectioning planes, or, for example, more caudal territories of the brain expanding at faster rates than the rostral territories. Quantification of the ratio between the IHF and total midline length across ages and between species may go some way to helping to clarify the degree of potential midline remodelling. Very high quality live imaging of the process would be the definitive way to evidence the claim, although I appreciate this is highly technically difficult and may not be possible. A key opportunity seems to be missed in the Satb2 knockout geckoes, where midline remodelling is purported to not occur. This is shown only qualitatively in a single plane of sectioning and again is not convincing. If the IHF length in these animals was quantified to be longer than wildtype at a comparable age, this would help to evidence the claim that remodelling occurs in these species.
- That similar cell types contribute to remodelling in non-mammalian amniotes as mice/eutherian mammals. The microphotographs presented are not of very high quality, and it is often difficult to be convinced that the data is showing the strong claims made in the paper. For instance the "MZG-like cells" may in fact be astrocytes or another cell type as it is hard to visualise morphology, and the "intercalation of GFP-positive radial glial fibres" is very unclear from the photos. The colocalization of MMPsense with laminin positive cells is very hard to appreciate from the figure, and again not quantified. Similarly, there is a claim that there was degeneration of laminin-positive leptomeninges during astroglial intercalation, which is an active process that is difficult to infer from a single microphotograph. From the data, I can appreciate that some of the similar broad categories of cell types that exist at the mouse midline (glia, radial glia) are also present in non-mammalian amniote midlines, but it is difficult to be convinced of much more than this from the data presented.
- That the gecko RPC and CPC connect distinct parts of the brain (rostral and caudal). These tracer injections lacked visualisation of the deposition site to confirm specificity, as well as appropriate quantification. Importantly, the absence of axons in the CPC following the rostral dye deposition (and vice versa) was not shown, which is essential to make the claim that these commissures carry axons from specific parts of the brain. The alternative hypothesis is that all axons are intermixed and traverse both commissures, independent of brain area of origin, which is not at all tested or disproved by the data presented.
- The implication that Satb2 expression at the midline is necessary for appropriate interhemispheric remodeling. Alternative hypotheses for an inappropriately remodeled midline upon whole-brain Satb2 knockout is that it is not dependent on expression at the midline region. Rather, it could be that, for example, the appropriately timed interaction between ingrowing callosal axons and the midline territory is needed for the timely differentiation and/or behavior of midline cells. Other alternatives include that the lack of axonal midline crossing changes the morphology of the midline territory, including potentially "unfusing" the midline. Given the high prevalence of midline remodelling defects concomitant with callosal agenesis referred to be the authors in the literature, it seems like these alternatives would be worth considering. Indeed, the only article the authors reference in their statement that "several studies implicated that agenesis of CC in Satb2-deficient mice is also associated with defects in midline fusion" is an article where Satb2 was knocked out specifically in the cortex and hippocampus. This result is difficult to interpret, as some Emx1 promotors do label some of the midline territory, however the point stands that it is difficult to interpret solely that Satb2 action at the midline is responsible for the effects. I understand that this is a hard question to investigate, so I would suggest allusion to the alternative hypotheses/interpretations as the main priority when interpreting the data.
Overall, the major conclusions of the study are not well supported by the data. A major effort to quantify phenomena and/or dramatically soften conclusions would be needed in order to make the conclusions well supported.
Minor comments
- The n numbers are not always clearly reported
- At times important points reference reviews or articles that do not support the statements as well as the most important primary articles might.
- Figures showing the entire section that insets were taken from would help to convince that sectioning planes were equivalent, and also show the deposition site of neurovue experiments.
- The fibre direction of GFAP+ fibres in figure 6 is confusing - It seems from the labelling on the figures as if red is used for the WT condition in mouse, but for the Satb2del condition in Gecko? If this is the case, then it would appear that the fibres are more specifically oriented in the del condition in mice, but in the WT condition of geckoes? There are several instances of this where clearer description and labelling would help the reader to interpret the results.
Significance
This study attempts to address a highly significant, novel and important question, that, if well achieved, would be publishable at a high degree of interest and impact to the basic research fields of brain development and evolution. Unfortunately the major conclusions made by the study are stronger than the data provided is able to evidence, and I remain unconvinced by many of them.
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