Discrete domains of gene expression in germinal layers distinguish the development of gyrencephaly

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1 Manuscript EMBO Discrete domains of gene expression in germinal layers distinguish the development of gyrencephaly Camino De Juan Romero, Carl Bruder, Ugo Tomasello, Jose Miguel Sanz-Anquela and Victor Borrell Corresponding author: Victor Borrell, CSIC - UMH Review timeline: Submission date: 12 February 2015 Editorial Decision: 23 March 2015 Revision received: 26 March 2015 Accepted: 27 March 2015 Editor: Karin Dumstrei Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 23 March 2015 Thank you for submitting your manuscript to the EMBO Journal. I am sorry for the slight delay in getting back to you, but I have now received the referee comments on your paper. Your manuscript has been reviewed by two good experts in the field and as you can see below, both referees find the manuscript exciting, well done and that it makes an important contribution. They raise minor issues with the paper that can be resolved with appropriate text changes - so no new experiments are needed. You can use the link below to upload the revised version. When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process, please visit our website: REFEREE REPORTS Referee #1: The manuscript by De Juan Romero et al. from the lab of Victor Borrell, who is one of the pioneers in the field of neocortex development and evolution, reports the transcriptomes of the various germinal zones of prospective gyri and sulci in the developing neocortex of the ferret. This manuscript is of fundamental importance for two reasons. First, it provides an absolutely unique, rich resource for future studies investigating the molecular mechanisms underlying cortical folding. But it is much more than this. It is, as the authors correctly state, the first time that novel patterns of European Molecular Biology Organization 1

2 gene expression in the germinal layers of the neocortex have been related to gyri versus sulci formation. As such, this is a "must be published without delay" paper for the EMBO J.! This reviewer has only a few minor comments for improvement. 1. While this reviewer enjoys the conceptual style of writing (which is a characteristic of papers from the Borrell lab), there are several instances where the text would benefit from being broken up into multiple paragraphs. The authors are encouraged to go through the pages of their manuscript and introduce paragraphs when the text covers an entire page (or almost a page) without paragraphs. 2. Referencing: the authors should add a few salient references, as follows. 2a. Page 4, ISVZ vs. OSVZ, please add Smart et al also here, which is already in the reference list. 2b. Page 4: when referring to Nonaka-Kinoshita et al. 2013, please clarify that you are talking about the ferret. 2c. It would seem appropriate to add Fietz et al as reference and refer to this work, as it was the first transcriptome analysis of human cortical germinal zones and, for example, may have a bearing on the authors' statements regarding the VZ vs. ISVZ vs. OSVZ (e.g., page 9). It may also be relevant to discuss this work in the context of the GO analysis, as the GO term 'cell adhesion' was also highlighted in Fietz et al Along the same lines: does ECM show up as a GO term in the present analyses? 2d. This reviewer wonders whether two very recent papers (Lui et al from the Kriegstein lab and Florio et al from the Huttner lab) on transcriptome analyses of human cortical progenitors should not be discussed? These papers take absolutely nothing away from the present seminal study, on the contrary: the present study, by focussing on transcriptomes of germinal layers of prospective gyri and sulci, in a certain way goes beyond these recent papers, so the authors should consider citing these works. Referee #2: In this work De Juan Romero and colleagues analyze different germinal zones and different areas of the developing cortex of the gyrencephalic ferret and found new genes that are differentially expressed in those germinal zones and regions. This work is unique and very relevant as it provides, for the first time, evidences of different pattern of expression of genes in the locations of final folds and fissures and in different germinal zones. A functional analysis is missing and it would be great to find out if manipulation of any of these genes in vivo would alter the eventual location of the folds. In my opinion this functional analysis is very important but not necessarily needed for the present manuscript. I have a few minor comments: In general I find the manuscript quite clear but in some case a bit redundant and confusing, for instance Fig.1F and S1A are the same and Fig S1E is not necessary as it is just the same quantification presented in Fig.2. Therefore I would minimize repetitions. In Fig. 2F the colors for the 'cell adhesion' category is not identical and a bit misleading. Fig.4 C-D is not really explained in the text Fig.6 needs some labeling of the stages Fig.8 is not very convincing, it is difficult to appreciate the differences in the expression For clarity the authors should explain in the text why they choose 16 and 21 weeks for the human embryos. 1st Revision - authors' response 26 March 2015 Response to reviews on manuscript EMBOJ European Molecular Biology Organization 2

3 We thank the two reviewers for their careful evaluation and thoughtful comments and suggestions, which overall are very positive and constructive. These have helped us to clarify the data presented and to discuss our results with greater accuracy and perspective, thus strengthening our original manuscript. Below are the point-by-point answers to the comments from each of the reviewers: Reviewer #1 We are very thankful to this reviewer for a very throughout and detailed evaluation of our manuscript, and for unambiguously complimenting the importance, novelty and interest of our findings. We believe that the few minor comments made by this reviewer have significantly contributed to improve the clarity, completeness and scholarship of our manuscript, for which we are particularly thankful. A detailed response to each of his/her comments follows below: The manuscript by De Juan Romero et al. from the lab of Victor Borrell, who is one of the pioneers in the field of neocortex development and evolution, reports the transcriptomes of the various germinal zones of prospective gyri and sulci in the developing neocortex of the ferret. This manuscript is of fundamental importance for two reasons. First, it provides an absolutely unique, rich resource for future studies investigating the molecular mechanisms underlying cortical folding. But it is much more than this. It is, as the authors correctly state, the first time that novel patterns of gene expression in the germinal layers of the neocortex have been related to gyri versus sulci formation. As such, this is a "must be published without delay" paper for the EMBO J.! We are very pleased about this Reviewer s very positive evaluation of our findings and extreme enthusiasm for the value and interest of our manuscript. This reviewer has only a few minor comments for improvement. 1. While this reviewer enjoys the conceptual style of writing (which is a characteristic of papers from the Borrell lab), there are several instances where the text would benefit from being broken up into multiple paragraphs. The authors are encouraged to go through the pages of their manuscript and introduce paragraphs when the text covers an entire page (or almost a page) without paragraphs. We are very thankful to this reviewer for the nice compliment, and agree that some sections of our text may benefit from being broken down into various paragraphs. Following this suggestion we have now changed this and separated various sections of the text into different paragraphs. This has been done in the following points: Page 6 new paragraph starting: Due to the location of the ISVZ Page 9 new paragraph starting: Unsupervised hierarchical clustering Page 10 new paragraph starting: Taken together, our analyses Page 11 new paragraph starting: The location and extent of domains Page 12 new paragraph starting: In further support that modular expression Page 16 new paragraph starting: In agreement with the remarkable size Page 17 new paragraph starting: Importantly, previous reports had already 2. Referencing: the authors should add a few salient references, as follows. 2a. Page 4, ISVZ vs. OSVZ, please add Smart et al also here, which is already in the reference list. We apologize for having missed including this reference, as we completely agree that this seminal paper must be acknowledged for being the first to distinguish ISVZ from OSVZ. This reference has now been added, as suggested, in the following sentence: European Molecular Biology Organization 3

4 In gyrencephalic species (with a folded cerebral cortex) the SVZ is extraordinarily large and split into inner (ISVZ) and outer subventricular zone (OSVZ) (Smart et al, 2002). 2b. Page 4: when referring to Nonaka-Kinoshita et al. 2013, please clarify that you are talking about the ferret. This comment refers to the fact that the Nonaka-Kinoshita paper included work in mouse and ferret, and our reference to that paper is solely regarding the ferret experiments, precisely as indicated by this reviewer. Accordingly, following this reviewer s suggestion we have modified the relevant sentence to clarify this fact: Previous work has demonstrated that, in the gyrencephalic ferret, local overexpression in OSVZ of the cell cycle-promoting genes Cdk4 and CyclinD1 increases cortical surface area and folding, whereas reduction of cell proliferation in OSVZ has the opposite effect (Nonaka-Kinoshita et al, 2013; Reillo et al, 2011). 2c. It would seem appropriate to add Fietz et al as reference and refer to this work, as it was the first transcriptome analysis of human cortical germinal zones and, for example, may have a bearing on the authors' statements regarding the VZ vs. ISVZ vs. OSVZ (e.g., page 9). We thank the reviewer for pointing out the absence of this reference, which indeed is directly relevant to our study and findings. In page 9 of our revised manuscript we have added a sentence to highlight this study and the difference with our present findings in ferret: Intriguingly, previous transcriptomic analyses of human cortical germinal zones concluded that VZ is the most different germinal zone, while ISVZ and OSVZ are relatively similar to each other (Fietz et al, 2012). It may also be relevant to discuss this work in the context of the GO analysis, as the GO term 'cell adhesion' was also highlighted in Fietz et al Along the same lines: does ECM show up as a GO term in the present analyses? Indeed, the GO term cell adhesion, which appears in our various microarray comparisons, was highlighted in Fietz et al., 2012, where Extracellular Matrix was highlighted as well very prominently. Upon re-examination of our GO analysis results we confirm that ECM is also a GO term frequently represented in the ferret cortical germinal zones. This is illustrated in the graph below, which indicates the enrichment score of this term for each of the different comparisons we performed: across germinal layers within SG or within LS, between SG and LS along VZ, ISVZ or OSVZ (very enriched), and simultaneously between germinal layers and cortical areas. Layers vs. Areas OSVZ ISVZ VZ LS SG Extracellular matrix Enrichment score European Molecular Biology Organization 4

5 Accordingly, and following this reviewer s suggestion, we have added the Fietz et al reference in the following modified sentence of page 9: GO analysis of DEGs in our microarray dataset revealed that some of the most highly-enriched categories among DEGs between germinal layers were common to both SG and LS, including regulation of transcription, cell adhesion, extracellular matrix, cytoskeleton and neuron differentiation, as previously also found in the human embryo cortical germinal zones (Fietz et al, 2012). Following the same principle, we have also modified the description of our GO analysis in the previous section, when referring to genes differentially-expressed between cortical areas along a given germinal layer (page 7): The categories most highly represented were common to all three germinal layers and included regulation of transcription, cell adhesion, extracellular matrix, cytoskeleton and cell cycle (i.e. Cdk4, CyclinD1; Figure 2F), all directly related to regulating the proliferation of the various types of cortical progenitor cells and their lineage relationships (Borrell & Reillo, 2012; Fietz et al, 2010; Gotz & Huttner, 2005; Lui et al, 2011). 2d. This reviewer wonders whether two very recent papers (Lui et al from the Kriegstein lab and Florio et al from the Huttner lab) on transcriptome analyses of human cortical progenitors should not be discussed? These papers take absolutely nothing away from the present seminal study, on the contrary: the present study, by focussing on transcriptomes of germinal layers of prospective gyri and sulci, in a certain way goes beyond these recent papers, so the authors should consider citing these works. We very much agree with this reviewer s opinion and advice. Following this suggestion, we have added two new sentences at the beginning of the Discussion section (page 15) to refer to these papers and others directly related, and to highlight the relevance of our study on this respect. Recent landmark studies using whole-tissue and single cell RNA sequencing have identified a reduced number of genes with key importance in human-specific cortical expansion and progenitor cell heterogeneity (Florio et al, 2015; Johnson et al, 2015; Lui et al, 2014). Yet in large-brained mammals like humans, macaques and ferrets, the embryonic cerebral cortex is highly regionalized, including the formation of folds and fissures (Welker, 1990), and many of the critical features defining cortical progenitor cells (cell cycle, amplification, neurogenesis) vary significantly across such regions (Dehay et al, 1993; Dehay & Kennedy, 2007; Lukaszewicz et al, 2005; Reillo et al, 2011). Here we have compared for the first time the transcriptomes of germinal layers between prospective folds and fissures of the developing cerebral cortex, and describe the existence of a novel pattern of gene expression along these germinal layers. Referee #2: In this work De Juan Romero and colleagues analyze different germinal zones and different areas of the developing cortex of the gyrencephalic ferret and found new genes that are differentially expressed in those germinal zones and regions. This work is unique and very relevant as it provides, for the first time, evidences of different pattern of expression of genes in the locations of final folds and fissures and in different germinal zones. A functional analysis is missing and it would be great to find out if manipulation of any of these genes in vivo would alter the eventual location of the folds. In my opinion this functional analysis is very important but not necessarily needed for the present manuscript. We are pleased about this Reviewer s very positive appreciation of our manuscript and high enthusiasm for the unique value and broad interest of our findings. Whereas a functional analysis of some of our identified genes will be an important next step, we agree that this should not delay communicating our rich genetic resource to the scientific community. European Molecular Biology Organization 5

6 In general I find the manuscript quite clear but in some case a bit redundant and confusing, for instance Fig.1F and S1A are the same and Fig S1E is not necessary as it is just the same quantification presented in Fig.2. Therefore I would minimize repetitions. We agree with this reviewer that Fig 1F and Supplementary Fig S1A display the exact same data, so we have removed S1A altogether as suggested by the reviewer. However we disagree regarding the duplicity of Supplementary Fig S1E and Fig 2B. Whereas Supplementary Fig S1E is a histogram showing the exact percentage of genes with higher expression in LS versus SG within each germinal layer, this information is not present in Fig 2B, which is a color-coded hierarchical clustering of differentially-expressed genes only offering a visual notion of these proportions, but not quantitative. Therefore we have retained Fig 2B and Suppl Fig S1E (now Figure E1D) as they were. In Fig. 2F the colors for the 'cell adhesion' category is not identical and a bit misleading. We agree that in the VZ pie chart the brown color corresponding to Cell adhesion is a bit lighter than in the other charts. We have changed this tone to make it now identical in all three pie charts and avoid any confusion. Fig.4 C-D is not really explained in the text We agree and apologize for this omission, which was due to the fact that panels C and D of Fig 4 are basically validations of microarray data by qrt-pcr and in situ hybridization. We have now added the following sentence in page 10: Microarray results were validated for a selection of genes by qrt-pcr using ferret-specific primers, and also by in situ hybridization (ISH) using ferret-specific probes (Figure 4C and D, and Tables E2 and E5). Fig.6 needs some labeling of the stages Labeling of the postnatal stage in each case was already present in the legend of this figure. However, for better clarity we have now added this information also in each panel of Fig 6. Fig.8 is not very convincing, it is difficult to appreciate the differences in the expression Forclarity the authors should explain in the text why they choose 16 and 21 weeks for the human embryos. To highlight the differences in expression levels shown in the images of Fig 8, we have added brackets to indicate the locations to compare in each case where differences are apparent. We believe that with these indications the differences in expression are now quite evident. These differences highlighted with brackets are also explained and indicated in the legend for this figure. Regarding the reasons for choosing the gestational stages of human embryos, we agree with the reviewer that no reason or explanation was offered in our previous manuscript version. In the revised version we have added two sentences to explain these reasons: We performed ISH stains on brain sections from human embryos at 16 and 21 gestational weeks (gw), which correspond to stages of mid- and late neurogenesis and immediately prior to the emergence of the first folds and fissures. Thus, analysis of these stages should help us define the genetic profile of germinal layers preceding the appearance of the cortical folds. European Molecular Biology Organization 6