Mouse scalp development requires Rac1 and SRF for the maintenance of mechanosensing mesenchyme

  1. Bharath H. Rathnakar
  2. Alex Rackley
  3. Hae Ryong Kwon
  4. William L. Berry
  5. Lorin E. Olson

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Regulation of essential cellular responses like proliferation, migration, and differentiation is crucial for normal development. Rac1, a ubiquitously expressed small GTPase, executes these responses under the regulation of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GTPases). Mutations in specific GEFs (DOCK6) and GTPases (AHGAP31) that regulate Rac1 are associated with Adams-Oliver syndrome (AOS), a developmental syndrome characterized by congenital scalp defects and limb truncations. Genetic ablation of Rac1 in the mouse embryonic limb ectoderm results in limb truncation. However, the etiology of Rac1-associated cranial defects is unknown. To investigate the origin and nature of cranial defects, we used a mesenchymal Cre line ( Pdgfra-Cre ) to delete Rac1 in cranial mesenchyme. Rac1 -KO mice died perinatally and lacked the apical portion of the calvarium and overlying dermis, resembling cranial defects seen in severe cases of AOS. In control embryos, α-smooth muscle actin (αSMA) expression was spatially restricted to the apical mesenchyme, suggesting a mechanical interaction between the growing brain and the overlying mesenchyme. In Rac1 -KO embryos there was reduced proliferation of apical mesenchyme, and reduced expression of αSMA and its regulator, serum response factor (SRF). Remarkably, Srf -KO mice generated with Pdgfra-Cre recapitulated the cranial phenotype observed in Rac1- KO mice. Together, these data suggest a model where Rac1 and SRF are critical to maintaining apical fibroblasts in a mechano-sensitive and proliferative state needed to complete cranial development.

Article activity feed

  1. Review Commons

    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

    We thank the reviewers for providing valuable comments and suggestions for improving the manuscript.

    Response to reviewer comments:

    Reviewer-1

    Comment 1: Major concern is the study lacks rigor in several areas where n=2, results are not quantified with statistics. They need to run power analysis and increase their samples sizes. Please include statistics on all measurements. Filamentous actin staining and alpha-sma is used to visualize mechanosensing but also in other cell activities such as cell contractility for movement, cell to substrate adhesion, cell division, etc. They need to query more mechanosensing related pathways (Piezo1/2, Yap/taz-Hippo, integrin-Focal Adhesion Kinase, etc) to show that mechanosensing changed.

    Response: We have increased the sample size to a minimum of n=3 in most cases. However, a few experiments will require more time to increase sample size, as mentioned below.

    Our data emphasized the role of Rac1 and SRF. We understand that other molecular players may also be involved in sensing or responding to mechanical forces, but surveying multiple families of candidates without a specific hypothesis or functional experiment is beyond the scope of this study.

    __Comment 2: __Fig. 1: In panel E, the cranial bone area measurement is not normalized to mitigate the possible effect of individual differences.

    Response: We have re-quantified the data with normalization to the length of the skull.

    __Comment 3: __In Fig. 2 the authors mentioned many phenotypical changes (bone length changes, gap thickness change, apex thickness change, etc.) based on histology stain, none of them are quantified to show a significant difference between Rac1-WT and Rac1-KO.

    Response: In Fig. 2A, we present the gross morphology of the Rac1-KO embryos and only discuss the tissue defects like edema, hematoma, and hypoplasia, confirmed through H&E as shown in Fig. 2C. We also show the apical limits of the intact calvaria in Fig. 2D, consistent with the calvaria defects observed at birth. In fact, we do not discuss any “bone length changes, gap thickness, or apex thickness change” in this section as suggested by the reviewer. To address the request for more quantification we have added measurement of the edematous area of the apical mesenchyme at E14.5 (Fig. 2C), and this is now shown in Suppl. Fig. 1E. We also added quantification of embryo genotypes and Chi-square tests, now shown in Suppl. Fig. 1D.

    Comment 4: Fig. 2 In panel D, with only 2 embryos per group is not enough for quantitation

    Response: We plan to increase the number of embryos during the revision period.

    Comment 5: Fig. 2 In panel D, the two arrows in the Rac1-KO mutants are not easy to catch.

    Response: We made the arrows bigger and bolder.

    Comment 6: Fig. 3 The thickness quantification is not performed.

    Response: We added quantification in Fig. 3D.

    Comment 7: Fig. 3 The images show an obvious curve change of the apex between the control and mutant. Such change is not discussed in the results. Is it due to histology issue?

    Response: We do not think it is due to technical issues but reflects a real change in the shape of the apex of the head. We modified the graphical representation in Figure 3E to reflect this change in curvature. We also added the following sentence to the results on page 7: “We also noted a loss of curvature in the apex of the Rac1-KO head at E13.5, which correlated with loss of aSMA+ mesenchymal cells and thinning of the EMM (Fig. 3E).”

    __Comment 8: __The merged layer did not show S100a6. While the authors are showing apical expansion of the mesenchyme toward the dermis and meninges, it is hard to track where they are without a merged image.

    Response: We added merged images.

    Comment 9: Fig. 4 In panel B, 2 biological replicates per genotype are very low.

    __Response: __The effect of Rac1-KO on cell cycle is already known (Moore et al. 1997; Nikolova et al. 2007; Gahankari et al. 2021), and our result is supported by *in vivo *quantification of Tom+Edu+ cells in different regions of the embryonic head shown in Fig. 4A. We prefer not to repeat this assay.

    Comment 10: Fig. 4 There is no cell death data.

    Response: We will generate data on cell death during the revision period.

    __Comment 11: __Fig. 5 In panel B, the GAPDH western plot bands in the mutants seem to be thinner than those of controls.

    Response: We verified equal loading with a Ponceau stain, so this minor change in the GAPDH level could be due to biological differences in the protein level. Nevertheless, by our estimation this minor difference does not explain away the major difference in Rac1 and Srf levels.

    __Comment 12: __Though the immunostain showed a decrease in signal intensity, it is hard to know whether the decrease is significant enough across all Rac1-KO mutants. They need to measure the fluorescence intensity and perform statistics.

    Response: We will generate better images of SRF staining and quantify the difference between Rac1-WT and Rac1-KO during the revision period.

    Comment 13: Fig. 6: Similar as Fig. 2, there is no quantification and n=1 per genotype is not enough

    Response: During the revision period we will increase the number of E12.5 Srf-KO and Srf-WT embryos to n=3 for Figure 6G. All other panels currently have n=7 or greater.

    Comment 14: Fig. 7: Need quantification between Srf-KO and Rac1-KO with statistics to show they are not different, but both significantly different from WTs

    Response: In Figure 7D we have added quantification of aSMA area in Srf-KO and Rac1-KO. These results show that both mutants have a similar phenotype with reduced aSMA expression compared to their respective WT littermates, which supports the conclusion that they work in the same pathway. We do not agree with the reviewer that the two mutants should show no statistical difference, because Rac1 and Srf are different genes with overlapping but also non-overlapping functions. During the revision period we will add more Srf-KO embryos and repeat the statistical analysis.

    Comment 15: Supplement Fig.2: No image showing the time point before E11.5.

    Response: We will add an E10.5 time point during the revision period.

    Comment 16: Supplement Fig.3: The ventral view of Rac1-WT does not have the same angle as it shows in Rac1-KO. Makes harder to see the difference between control and mutant.

    Response: We adjusted the brightness/contrast to make the difference clearer.

    Comment 17: Supplement Fig.4 &7: The alkaline phosphatase stained area needs to be normalized to some other metric because the embryos could be different size.

    Response: We normalized to the width of the eye and is now represented in Suppl. Fig. 4 and 7.

    Comment 18: Supplement Fig 6 A: The legend and figure don't match. Is it E13.5 or 14.5. Panel 6B needs better images without curling of the tissue.

    Response: This has been fixed. The immunostaining images in Suppl. Fig. 6A is E14.5. Panel B is now replaced with better images in the revised manuscript.

    Reviewer-2

    __Comment 1.1: __In Fig. 5, links between Rac1, SRF, αSMA, and contractility in mesenchymal cells are shown. Molecular analyses (Western blot and qPCR) were performed using primary cultured mesenchymal cells (prepared after freed from the epidermal population). Although use of cells prepared from E18.5 embryos may have been chosen by the authors for the safe isolation of the mesenchymal population without contamination of epidermal cells, this reviewer finds that anti-SRF immunoreactivity is weaker at E13.5 than at E12.5 (throughout the section including the mesencephalic wall) and therefore wonder whether SRF expression changes in a stage-dependent manner. So, simply borrowing results obtained from E18.5-derived cells for describing the scenario around E12.5 and E13.5 is a little disappointing point found only here in this study.

    Response: In fact, the reason we chose E18.5 was to get enough cells to do the experiments in Figure 5A-D without extensive passaging and/or immortalization, which would undoubtedly cause the cells to deviate from their in vivo character as they become adapted to growing on plastic with 10% serum. Therefore, we prefer not to change the cells as suggested by the reviewer.

    __Comment 1.2: __In Fig. 5F, it is difficult to clearly see "reduction" of SRF immunoreactivity in Rac1-KO. Therefore, quantification of %SRF+/totalTomato+ would be desired.

    __Response: __We will generate better images of SRF staining and quantify the difference between Rac1-WT and Rac1-KO during the revision period.

    __Comment 1.3: __Separately, direct comparison of spontaneous centripetal shrinkage of the apical/dorsal scalp tissues, which will occur in 30 min when prepared at E12.5 or E13.5 (Tsujikawa et al., 2022), between WT and Rac1-KO would strengthen the results in Fig. 5D. As KO is specific to the mesenchyme, the authors do not have to worry about removal of the epidermal layer (which would be much more difficult at E12.5-13.5 than E18.5). If the degree of centripetal shrinkage of the "epidermis plus mesenchyme" layers were smaller in Rac1-KO, it would be interpreted to be mainly due to poorer recoiling activity and contractility of the Rac1-KO mesenchymal tissue.

    __Response: __We will try to perform the centripetal shrinkage assays as shown by Tsujikawa et al., during the revision period.

    Comment 2: The authors favor "apical" vs. "basolateral" to tell the relative positions in the embryonic head, not only in the adult head. But "apical" vs. "basolateral" should be accompanied with dorsal vs. ventral at least at the first appearance. Apical-to-basal axis or apex vs. basolateral by itself can provide, in many contexts, impressions that epithelial layers/cells are being discussed. Please note that the authors also use "caudal" (in the embryonic head). Usually, a universally defined anatomical axis perpendicular to the rostral-to-caudal axis is the dorsal-to-ventral axis.

    Response: Apologies for confusing terminology. The terminology is now defined uniformly according to the anatomical axis.

    Comment 3: One of the authors' statements in ABSTRACT "In control embryos, α-smooth muscle actin (αSMA) expression was spatially restricted to the apical mesenchyme, suggesting a mechanical interaction between the growing brain and the overlying mesenchyme" and a similar one in RESULTS "αSMA was not detected in the basolateral mesenchyme of either genotype from E12.5-E14.5 (Suppl. Fig. 4A), suggesting restriction of the mechanosensitive cell state to the apical mesenchyme" need to be at least partly revised, taking previous publication about the normal αSMA pattern in the embryonic head into account more carefully. Tsujikawa et al. (2022) described "Low-magnification observations showed superficial immunoreactivity for alpha smooth muscle actin (αSMA), which has been suggested to function in cells playing force-generating and/or constricting roles; this immunoreactivity was continuously strong throughout the dorsal (calvarial) side of the head but not ventrally toward the face, producing a staining pattern similar to a cap (Figure 2A)" . Therefore, in this new paper, descriptions like "we observed ...., consistent with ....(2022)" or "we confirmed .... (2022)" would be more accurate and appropriate regarding this specific point. Such a minor change does not reduce this study's overall novelty at all.

    Response: Thank you for the correction. We have replaced the terminology and cited the article (Tsujikawa et al., 2022) appropriately, crediting their finding.

    Comment 4: It would be very helpful if the authors provide a schematic illustration in which physiological and pathological scenarios (at the molecular, cellular, and tissue levels found or suggested by this study) are shown.

    Response: We have added a schematic representation of the molecular changes happening in the apical head development because of Rac1- and Srf-KO, and it is represented in Suppl. Fig. 7C.

    Comment 5: Despite being put in the title, "mechanosensing" by mesenchymal cells is not directly assessed in this study. If appropriate, something like "mechano-functioning" would be closer to what the authors demonstrated.

    __Response: __We changed the title to refer to “mechano-responsive mesenchyme”. We think this is appropriate because the cells of interest have reduced aSMA and reduced proliferation, both of which are known to occur, at least in part, as responses to mechanical inputs.

    Reviewer-3

    Comment 1: Prrx1-Cre targets calvarial mesenchyme and Suzuki et al., 2009 showed that Prrx1-Cre mediated loss of Rac1 lead to calvarial bone phenotype due to incomplete fusion of the skull. While this phenotype was not studied in detail, the statement in the intro and discussion that the calvarial phenotype has not been recapitulated in mice is incorrect.

    Response: Suzuki et al showed incomplete fusion of the skull. Although the skull is a tissue that is affected in AOS, it is not akin to the scalp and calvaria aplasia that typifies AOS. Our result stands apart from this. We clarified our position as such:

    Introduction (page 4): “Nevertheless, the calvaria phenotype seen in AOS individuals has not been explored in detail or fully recapitulated in mice.”

    Discussion (page 11): “Previous studies have demonstrated the role of Rac1 in mesenchyme-derived tissues, but they did not recapitulate AOS phenotypes.”

    Comment 2: The authors show that Pdgfra-Cre induced knockout of Rac1 leads to lower-than-expected numbers of Rac1-cKO embryos at E18.5 and P1. Phenotypic analysis shows that the earliest phenotype is blebbing and hematoma in the nasal region at E11.5/12.5. It is stated that this was resolved at E18.5. It is unclear if this is truly a resolution of the phenotype or that these embryos fail to survive until E18.5. Do 100% of the Rac1-cKO embryos exhibit the blebbing/hematoma at E11.5/12.5? What is the observed number/percentage of Rac1-cKO embryos at E11.5/12.5? If the observed percentage of Rac1-cKO is similar to that at E18.5 (lower than the expected 25%), this would support resolution. If the observed ratio is as expected at E11.5/12.5, then this would support embryonic loss before E18.5 rather than phenotypic resolution.

    Response: Please note that 100% (n=12) of E12.5 Rac1-KO embryos displayed nasal and mild caudal edema as exhibited in Fig. 2A, but none (n=16) had blebbing/hematoma by E18.5. We added tables for the number of embryos recovered at E12.5 and E18.5 to Supplemental Figure 1. These results show that the percentage of mutants at E12.5 was 21.42%, not significantly different from the expected frequency (p = 0.5371). At E18.5, the percentage dropped slightly to 18.3%, but still not significantly different from expected (p = 0.1545). The significant change in frequency of blebbing/hematoma from E12.5 to E18.5, without any significant change in the frequency of mutants, supports phenotypic resolution of the early blebbing/hematoma.

    Comment 3: It is stated that brain shape is altered in Rac1-cKO embryos at E14.5 and E18.5 and concluded that these shape differences are secondary to the cranial defects. Pdgfra+ cells gives rise to the meninges and if the Pdgfra-Cre line recapitulates this expression, then loss of the ubiquitously expressed Rac1 in the meninges could lead to a primary defect in the brain, which may lead to secondary defects in the calvarium and scalp. Their conclusion should recognize other possibilities.

    Response: We agree it is possible that there are meninges defects that secondarily change the shape of the brain, and we added a mention of this possibility. It is highly unlikely that scalp defects are only secondary to brain changes because the first observable phenotypes are in the EMM that gives rise to the scalp.

    Comment 4: The TdTom staining in wholemount at E13.5 (Supplemental Figure 2B) is difficult to appreciate in the image shown.

    Response: At E11.5 there is good contrast between labeled cranial structures and non-labeled body. At E13.5, Tomato appears in most of the mesenchymal cells in the embryo, so there is not as much contrast. The lack of contrast at E13.5 may cause the reviewer think there is something wrong with the image.

    Comment 5: The idea that the EMM laminates into the meninges and scalp layers is not new and should be properly cited (Vu et al., 2021, Scientific Reports). The following paper should also be cited on the use of alpha-SMA (Acta2) as a marker of the anterior calvaria mesenchyme: Holms et al., 2020 Cell Reports.

    Response: Thank you. We are happy to add those citations.

    Comment 6: It is concluded that meningeal development is maintained in the cKO; however, this conclusion was based on a single marker (S100a6) that is both expressed in the presumptive meninges and dermis and greatly reduced overall in the cKO. This conclusion should be softened or other markers used to show that the meninges is indeed normal.

    Response: We softened the conclusion on the meninges in the revised manuscript, as this part of the phenotype is was not our focus but it would be a good thing to look at in the future.

    Comment 7: The overlap of S100a6 and alpha-SMA is difficult to appreciate in the images shown in Figure 3. Since this is important to the conclusion, co-staining should be done. If co-staining cannot be done due to the primary antibodies' origins, then ISH should be done.

    Response: We added merged images.

    Comment 8: It is concluded that reduced alpha-SMA suggests an early failure of Rac-cKO cells to respond to the mechanical environment. While this is one possibility, the reduction of alpha-SMA may simply be due to a reduction of these cells resulting from failed differentiation, decreased proliferation, or increased apoptosis.

    Response: We think the fact that aSMA is downregulated in cultured cells strongly argues against it being a trivial consequence of reduce proliferation etc. Nevertheless, we softened our conclusion to allow for some of these things to also contribute to the reduced aSMA expression. We will check apoptosis during the revision period.

    Comment 9: The conclusion that alpha-SMA is a transient population only present in apical cranial mesenchyme between E12.5-14.5 is not consistent with prior studies: Holms et al., 2020 Cell Reports; Holms et al., 2021 Nature Communications; Farmer et al., 2021 Nature Communications; Takeshita et al., 2016 JBMR.

    Response: There is no contradiction. Our statements are based on antibody staining where it is very evident that a-SMA-expressing cells are detectable throughout the apical mesenchyme between E12.5 and E14.5. But at E18.5 we do not see this kind of broad aSMA expression the apical head, suggesting a transient and spatially restricted population of cells in the apical mesenchyme. This is consistent with the studies from Tsujikawa et al., 2022 and Angelozzi et al., 2022. The papers mentioned by the reviewer are only focused on the suture mesenchyme. They do not claim there is broad aSMA/Acta2 expression in the apical head, but only in a spatially restricted subpopulation of suture mesenchymal cells.

    Comment 10: In the SRF immunostaining results in control and Rac1-cKO embryos, it is difficult to appreciate the nuclear localization at E12.5 in Figure 5E, as the DAPI is over saturated, and the image quality is poor. The image quality is also poor in Figure 5F.

    Response: We will generate better images of SRF staining and quantify the difference between Rac1-WT and Rac1-KO during the revision period.

    Comment 11: To what extent is the expression/localization of MRTF, the transcriptional co-activator of SRF, altered in the calvarial mesenchyme of Rac1-cKO embryos? Changes in MRTF would strengthen the link between Rac1 and SRF.

    Response: We do not know how MRTF expression/localization changes in the embryo tissue, but western blot data on Rac1-KO fibroblasts revealed a reduction in expression/nuclear localization of MRTF-A/B that mirrored the changes in SRF. We added these blots to Figure 5A. However, as noted at the end of the discussion, MRTF is not always required for SRF function in vivo ( Dinsmore, Elife 2022). The MRTFA/B-KO is a possibility for future work.

    Comment 12: Hypoplasia of the apical mesenchyme (Figure 6G, inset 1) in Srf-cKO is difficult to see.

    Response: During the revision period we will increase the number of E12.5 Srf-KO and Srf-WT embryos to n=3 for Figure 6G and replace the picture with a better one.

    Comment 13: Generally, the organization of the data into many main and supplemental Figures makes the flow difficult to follow.

    __Response____: __We understand the concern, but we have tried our best to organize the most important data into main figures and the relevant but less essential data into supplemental figures.

    Comment 14: SFR interacts with Pdgfra interacts genetically with Srf in neural crest cells in craniofacial development, with Srf being a target of PDGFRa signaling (Vasudevan and Soriano, 2015, Dev Cell). Since the Pdgfra-Cre line used here is hemizygous, is important that the control used to look at SRF expression in the Rac1-cKO is Pdgfra-Cre+.

    Response: It is standard practice to include some Cre+ mice in the control set to reveal whether Cre has toxic effects in the cells of interest. To the reviewer’s concern about genetic interactions between the *Pdgfra *gene and Srf, this should not be relevant here because the Pdgfra-Cre used in our study is a transgene and does not affect the endogenous Pdgfra gene.

    Comment 15: The text size in all figures is too small and varies throughout, making it difficult to read.

    Response: To fit the panel in the Word document, the figure is resized. This should not be an issue in the final manuscript.

    Comment 16: Details about the pulse-chase timing of the EdU experiments should be included in the results. Also, does n = 3 for each stage and each genotype? I would be helpful to include a representative section for a control and cKO littermate pair.

    Response: The details are now included in the methods section. Yes, n=3 in each stage and genotype (Fig. 4A). The representative images are also included.

    Comment 17: The relative sizing of the panels within and between figures is haphazard. Some are very large and others very small (Figure 2, 6, Supplemental Figure 1, 2, 6, 7).

    Response: The image panels are fixed in the revised manuscript.

    Comment 18: In Figure 5A and F, the titles "E12.5" and "E13.5" are in italics.

    Response: The fonts for the figures are fixed in the revised manuscript.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary: This manuscript by Rathnakar et al. examines the role of the small GTPase Rac1 in apical closure of the scalp and skull. Rac1 activity is regulated the guanine nucleotide exchange factor DOCK6 and the GTPase AHGAP31. Loss of function variants in DOCK6 and gain of function variants in AHGAP31 lead to sustained inactivation of Rac1 in Adams-Oliver syndrome (AOS), which is characterized by aplasia cutis congenita, underlying calvarial defects, and limb abnormalities. While Rac1 is thought to be a key in the pathogenesis of AOS, how decreased in Rac1 activity impact development of the head is not well-understood. The authors find that conditional loss of Rac1 in cranial mesenchyme (using Pdgfra-Cre), leads to AOS-like abnormalities in the scalp and skull. They go on to show that these abnormalities are linked to reduced alpha-SMA expression in the early migrating mesenchyme (EMM), decreased osteoprogenitor cells in the supraorbital mesenchyme (SOM), decreased proliferation, and the contractile function of fibroblasts. They also find that Rac1 cKO leads to reduced expression of the mechanosensitive transcription factor SRF. Finally, they show that loss of SRF in cranial mesenchyme (using Pdgfra-Cre) leads to an AOS-like scalp and skull phenotype that has mechanistic overlap with their findings in the Rac1 cKO.

    Major:

    1. Prrx1-Cre targets calvarial mesenchyme and Suzuki et al., 2009 showed that Prrx1-Cre mediated loss of Rac1 lead to calvarial bone phenotype due to incomplete fusion of the skull. While this phenotype was not studied in detail, the statement in the intro and discussion that the calvarial phenotype has not been recapitulated in mice is incorrect.
    2. The authors show that Pdgfra-Cre induced knockout of Rac1 leads to lower-than-expected numbers of Rac1-cKO embryos at E18.5 and P1. Phenotypic analysis shows that the earliest phenotype is blebbing and hematoma in the nasal region at E11.5/12.5. It is stated that this was resolved at E18.5. It is unclear if this is truly a resolution of the phenotype or that these embryos fail to survive until E18.5. Do 100% of the Rac1-cKO embryos exhibit the blebbing/hematoma at E11.5/12.5? What is the observed number/percentage of Rac1-cKO embryos at E11.5/12.5? If the observed percentage of Rac1-cKO is similar to that at E18.5 (lower than the expected 25%), this would support resolution. If the observed ratio is as expected at E11.5/12.5, then this would support embryonic loss before E18.5 rather than phenotypic resolution.
    3. It is stated that brain shape is altered in Rac1-cKO embryos at E14.5 and E18.5 and concluded that these shape differences are secondary to the cranial defects. Pdgfra+ cells gives rise to the meninges and if the Pdgfra-Cre line recapitulates this expression, then loss of the ubiquitously expressed Rac1 in the meninges could lead to a primary defect in the brain, which may lead to secondary defects in the calvarium and scalp. Their conclusion should recognize other possibilities.
    4. The TdTom staining in wholemount at E13.5 (Supplemental Figure 2B) is difficult to appreciate in the image shown.
    5. The idea that the EMM laminates into the meninges and scalp layers is not new and should be properly cited (Vu et al., 2021, Scientific Reports). The following paper should also be cited on the use of alpha-SMA (Acta2) as a marker of the anterior calvaria mesenchyme: Holms et al., 2020 Cell Reports.
    6. It is concluded that meningeal development is maintained in the cKO; however, this conclusion was based on a single marker (S100a6) that is both expressed in the presumptive meninges and dermis and greatly reduced overall in the cKO. This conclusion should be softened or other markers used to show that the meninges is indeed normal.
    7. The overlap of S100a6 and alpha-SMA is difficult to appreciate in the images shown in Figure 3. Since this is important to the conclusion, co-staining should be done. If co-staining cannot be done due to the primary antibodies' origins, then ISH should be done.
    8. It is concluded that reduced alpha-SMA suggests an early failure of Rac-cKO cells to respond to the mechanical environment. While this is one possibility, the reduction of alpha-SMA may simply be due to a reduction of these cells resulting from failed differentiation, decreased proliferation, or increased apoptosis.
    9. The conclusion that alpha-SMA is a transient population only present in apical cranial mesenchyme between E12.5-14.5 is not consistent with prior studies: Holms et al., 2020 Cell Reports; Holms et al., 2021 Nature Communications; Farmer et al., 2021 Nature Communications; Takeshita et al., 2016 JBMR.
    10. In the SRF immunostaining results in control and Rac1-cKO embryos, it is difficult to appreciate the nuclear localization at E12.5 in Figure 5E, as the DAPI is over saturated, and the image quality is poor. The image quality is also poor in Figure 5F.
    11. To what extent is the expression/localization of MRTF, the transcriptional co-activator of SRF, altered in the calvarial mesenchyme of Rac1-cKO embryos? Changes in MRTF would strengthen the link between Rac1 and SRF.
    12. Hypoplasia of the apical mesenchyme (Figure 6G, inset 1) in Srf-cKO is difficult to see.
    13. Generally, the organization of the data into many main and supplemental Figures makes the flow difficult to follow.
    14. SFR interacts with Pdgfra interacts genetically with Srf in neural crest cells in craniofacial development, with Srf being a target of PDGFRa signaling (Vasudevan and Soriano, 2015, Dev Cell). Since the Pdgfra-Cre line used here is hemizygous, is important that the control used to look at SRF expression in the Rac1-cKO is Pdgfra-Cre+.

    Minor:

    1. The text size in all figures is too small and varies throughout, making it difficult to read.
    2. Details about the pulse-chase timing of the EdU experiments should be included in the results. Also, does n = 3 for each stage and each genotype? I would be helpful to include a representative section for a control and cKO littermate pair.
    3. The relative sizing of the panels within and between figures is haphazard. Some are very large and others very small (Figure 2, 6, Supplemental Figure 1, 2, 6, 7).
    4. In Figure 5A and F, the titles "E12.5" and "E13.5" are in italics.

    Significance

    Overall, this is an interesting study that shares mechanistic insight into the scalp and skull deformities in AOS. The overall presentation of the work, particularly the figures, should be improved and streamlined to enhance clarity and better emphasize the novelty of the study. In addition, the conclusions are not always well-supported by the results and the interpretation of the results do not fully consider and cite previous studies.

    Audience: Developmental Biologists

    Expertise: Craniofacial development and disease

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary

    In mice lacking Rac1 in the PDGFRa+ mesenchymal cell lineage, the authors found Adams-Oliver syndrome (AOS)-like defects of the apical/dorsal scalp and calvaria, which was accompanied by the secondary brain protrusion by E18.5. The primary phenotype emerged at E11.5 and worsened from E12.5 to E14.5 in the apical/dorsal region of the embryonic head, with limited lateral expansion as well as reduced thickening/stratification of the mesenchymal layer expressing α-smooth muscle actin (αSMA). Very similar in vivo abnormalities were obtained when serum response factor (SRF), known as a mechanotransducing factor, was removed in PDGFRα+ mesenchymal cells. Rac1-lacking mesenchymal cells proliferated poorly in vivo and contracted weakly in culture, with reduced expression of SRF and αSMA. Based on these results and previously obtained understanding that the developing apical/dorsal mesenchyme is mechanically stretched by the underlying brain, the authors conclude that the mechanosensing-triggered morphogenetic behaviors of the apical/dorsal mesenchymal cells (i.e., proliferation, stratification, and contraction, which all lead to physical stability or mechanical resilience of that layer) is mediated by Rac1 and SRF. The authors also suggest that this molecular mechanism for the physiological maturation of the apical/dorsal mesenchyme may underlie the ventral-to-dorsal progression of osteogenesis, absence of which explains AOS pathogenesis.

    Major comments:

    In Fig. 5, links between Rac1, SRF, αSMA, and contractility in mesenchymal cells are shown. Molecular analyses (Western blot and qPCR) were performed using primary cultured mesenchymal cells (prepared after freed from the epidermal population). Although use of cells prepared from E18.5 embryos may have been chosen by the authors for the safe isolation of the mesenchymal population without contamination of epidermal cells, this reviewer finds that anti-SRF immunoreactivity is weaker at E13.5 than at E12.5 (throughout the section including the mesencephalic wall) and therefore wonder whether SRF expression changes in a stage-dependent manner. So, simply borrowing results obtained from E18.5-derived cells for describing the scenario around E12.5 and E13.5 is a little disappointing point found only here in this study. In Fig. 5F, it is difficult to clearly see "reduction" of SRF immunoreactivity in Rac1-KO. Therefore, quantification of %SRF+/totalTomato+ would be desired. Separately, direct comparison of spontaneous centripetal shrinkage of the apical/dorsal scalp tissues, which will occur in 30 min when prepared at E12.5 or E13.5 (Tsujikawa et al., 2022), between WT and Rac1-KO would strengthen the results in Fig. 5D. As KO is specific to the mesenchyme, the authors do not have to worry about removal of the epidermal layer (which would be much more difficult at E12.5-13.5 than E18.5). If the degree of centripetal shrinkage of the "epidermis plus mesenchyme" layers were smaller in Rac1-KO, it would be interpreted to be mainly due to poorer recoiling activity and contractility of the Rac1-KO mesenchymal tissue.

    Minor comments:

    1. The authors favor "apical" vs. "basolateral" to tell the relative positions in the embryonic head, not only in the adult head. But "apical" vs. "basolateral" should be accompanied with dorsal vs. ventral at least at the first appearance. Apical-to-basal axis or apex vs. basolateral by itself can provide, in many contexts, impressions that epithelial layers/cells are being discussed. Please note that the authors also use "caudal" (in the embryonic head). Usually, a universally defined anatomical axis perpendicular to the rostral-to-caudal axis is the dorsal-to-ventral axis.
    2. One of the authors' statements in ABSTRACT "In control embryos, α-smooth muscle actin (αSMA) expression was spatially restricted to the apical mesenchyme, suggesting a mechanical interaction between the growing brain and the overlying mesenchyme" and a similar one in RESULTS "αSMA was not detected in the basolateral mesenchyme of either genotype from E12.5-E14.5 (Suppl. Fig. 4A), suggesting restriction of the mechanosensitive cell state to the apical mesenchyme" need to be at least partly revised, taking previous publication about the normal αSMA pattern in the embryonic head into account more carefully. Tsujikawa et al. (2022) described "Low-magnification observations showed superficial immunoreactivity for alpha smooth muscle actin (αSMA), which has been suggested to function in cells playing force-generating and/or constricting roles; this immunoreactivity was continuously strong throughout the dorsal (calvarial) side of the head but not ventrally toward the face, producing a staining pattern similar to a cap (Figure 2A)" . Therefore, in this new paper, descriptions like "we observed ...., consistent with ....(2022)" or "we confirmed .... (2022)" would be more accurate and appropriate regarding this specific point. Such a minor change does not reduce this study's overall novelty at all.
    3. It would be very helpful if the authors provide a schematic illustration in which physiological and pathological scenarios (at the molecular, cellular, and tissue levels found or suggested by this study) are shown.
    4. Despite being put in the title, "mechanosensing" by mesenchymal cells is not directly assessed in this study. If appropriate, something like "mechano-functioning" would be closer to what the authors demonstrated.

    Significance

    This study advances understanding of a key aspect of the molecular mechanisms underlying the normal mammalian craniofacial development, unveiling the role of Rac1 and SRF in the apical/dorsal mesenchymal layer which has inter-tissue mechanical relationships with the embryonic brain underneath. This study also advances understanding of Adams-Oliver Syndrome pathogenesis, demonstrating the biological significance of the normal inter-tissue mechanical relationships in the developing mammalian head. This study may have opened a door for the genetic/molecular dissection toward the tissue-level mechano-engineering, which would stimulate development of next-generation organoids or assembloids. Broad audience including developmental biologists/neuroscientists, molecular/cellular biologists, pathologists, clinical geneticists, and pediatricians would be interested in this work.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this paper "Mouse scalp development requires Rac1 and SRF for the maintenance of mechanosensing mesenchyme", the authors demonstrated that deletion of Rac1 (Rac1-KO) with a PDGFRαCreTG mouse model led to absence of skull apex and a blebbing formation while the limbs were not impacted. Rac1-KO mice showed the Rac1 regulated expansion of the apical mesenchyme toward the very apex meningeal and dermis layer and the osteogenic differentiation of supra orbital arch mesenchyme. Rac1 also regulates the proliferation of apical mesenchyme, dermis differentiation, and mechanosensing of the cranial mesenchyme cells. The authors also indicated Rac1 was a regulator of Srf by showing the deletion of Rac1 lead to lower Srf mRNA level and SRF protein expression. Deletion of Srf showed similar phenotypes as Rac1-KO mice.

    Major concern is the study lacks rigor in several areas where n=2, results are not quantified with statistics. They need to run power analysis and increase their samples sizes. Please include statistics on all measurements. Filamentous actin staining and alpha-sma is used to visualize mechanosensing but also in other cell activities such as cell contractility for movement, cell to substrate adhesion, cell division, etc. They need to query more mechanosensing related pathways (Piezo1/2, Yap/taz-Hippo, integrin-Focal Adhesion Kinase, etc) to show that mechanosensing changed.

    Comments by figure.

    Fig. 1: In panel E, the cranial bone area measurement is not normalized to mitigate possible effect of individual differences.

    Fig. 2:

    1. While the authors mentioned many phenotypical changes(bone length changes, gap thickness change, apex thickness change, etc) based on histology stain, none of them are quantified to show a siginificant difference between Rac1-WT and Rac1-KO.
    2. In panel D, with only 2 embryos per group is not enough for quantitation.
    3. In panel D, the two arrows in the Rac1-KO mutants are not easy to catch.

    Fig. 3:

    1. The thickness quantification is not performed.
    2. The images show an obvious curve change of the apex between the control and mutant. Such change is not discussed in the results. Is it due to histology issue?
    3. The merged layer did not show S100a6. While the authors are showing apical expansion of the mesenchyme toward the dermis and meninges, it is hard to track where they are without a merged image.

    Fig.4:

    1. In panel B, 2 biological replicates per genotype are very low
    2. There is no cell death data.

    Fig. 5:

    1. In panel B, the GPDH western plot bands in the mutants seem to be thinner than those of controls.
    2. Though the immunostain showed a decrease in signal intensity, it is hard to know whether the decrease is significant enough across all Rac1-KO mutants. They need to measure the fluorescence intensity and perform statistics.

    Fig. 6: Similar as Fig. 2, there is no quantification and n=1 per genotype is not enough.

    Fig. 7: Need quantification between Srf-KO and Rac1-KO with statistics to show they are not different but both significantly different with WTs.

    Supplement Fig.2: No image showing the time point before E11.5.

    Supplement Fig.3: The ventral view of Rac1-WT does not have the same angle as it shows in Rac1-KO. Makes harder to see the difference between control and mutant.

    Supplement Fig.4 &7: The alkaline phosphatase stained area needs to be normalized to some other metric because the embryos could be different size.

    Supplement Fig 6 A: The legend and figure don't match. Is it E13.5 or 14.5. Panel 6B needs better images without curling of the tissue.

    Significance

    Please see my comments above. This work is broadly of interest to developmental biologist, fracture healing, and human genetics fields.

    The paper is easy to understand and follow. The massive amount of histology and immunostaining images make it easy to identify the point the authors want to show. All the figures are well-labeled and visually informative. The experiment sequence is logic. The gene deletion models provide solid and direct evidence on the necessity of their function during early head development. The discussion is thoughtfully written and clear. The authors discuss the connection of Rac1 and SRF with other signaling pathways, which makes them promising target toward Adams-Oliver syndrome.

  5. Version published to 10.1101/2025.02.11.637680 on bioRxiv