A dysmorphic mouse model reveals developmental interactions of chondrocranium and dermatocranium

  1. Susan M Motch Perrine
  2. M Kathleen Pitirri
  3. Emily L Durham
  4. Mizuho Kawasaki
  5. Hao Zheng
  6. Danny Z Chen
  7. Kazuhiko Kawasaki
  8. Joan T Richtsmeier

  • Curated by eLife

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    Evaluation Summary:

    This work combines imaging and quantitative analyses to address the conclusions that the chondrocranium and dermatocranium form an integrated unit of development and alterations in the chondrocranium drive changes in the dermatocranium. However, the manuscript in the current form suffers from data uncertainty because raw data was not provided, and the title and the conclusions go too much beyond the results they have gathered.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

The cranial endo and dermal skeletons, which comprise the vertebrate skull, evolved independently over 470 million years ago and form separately during embryogenesis. In mammals, much of the cartilaginous chondrocranium is transient, undergoing endochondral ossification or disappearing, so its role in skull morphogenesis is not well studied and it remains an enigmatic structure. We provide complete 3D reconstructions of the laboratory mouse chondrocranium from embryonic day (E) 13.5 through E17.5 using a novel methodology of uncertainty-guided segmentation of phosphotungstic enhanced 3D micro-computed tomography images with sparse annotation. We evaluate the embryonic mouse chondrocranium and dermatocranium in 3D, and delineate the effects of a Fgfr2 variant on embryonic chondrocranial cartilages and on their association with forming dermal bones using the Fgfr2c C342Y/+ Crouzon syndrome mouse. We show that the dermatocranium develops outside of and in shapes that conform to the chondrocranium. Results reveal direct effects of the Fgfr2 variant on embryonic cartilage, on chondrocranium morphology, and on the association between chondrocranium and dermatocranium development. Histologically, we observe a trend of relatively more chondrocytes, larger chondrocytes, and/or more matrix in the Fgfr2c C342Y/+ embryos at all timepoints before the chondrocranium begins to disintegrate at E16.5. The chondrocrania and forming dermatocrania of Fgfr2c C342Y/+ embryos are relatively large, but a contrasting trend begins at E16.5 and continues into early postnatal (P0 and P2) timepoints, with the skulls of older Fgfr2c C342Y/+ mice reduced in most dimensions compared to Fgfr2c +/+ littermates. Our findings have implications for the study and treatment of human craniofacial disease, for understanding the impact of chondrocranial morphology on skull growth, and potentially on the evolution of skull morphology.

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  1. Version published to 10.7554/elife.76653 on eLife
  2. eLife

    Author Response

    Reviewer #3 (Public Review):

    The aim of the study is to tangle the over 400 million years' cooperation between chondrocranium and dermatocranium development. Mice with Crouzon syndrome were chosen for the study. The strength of this study is the novel application of machine learning techniques to segment the mouse cranium, which can be applied to a variety of vertebrates. The figures are very appealing. The major drawback of this study is that it only focuses on the Curzon mouse, even though the goal of the study is to investigate the relationship between the chondrocranium and dermatocranium. The authors emphasize that this study was undertaken to study the 400-million-year history of the cranium of Osteichthyes, which includes bony fishes, amphibians, lizards, and birds, in addition to mammals. In order to "untangle the over 400 million years' cooperation between chondrocranium and dermatocranium" as the title states, it is too obvious that they must include bony fish, amphibians, lizards, and birds. It is also unclear throughout the manuscript why the study of Curzon mice would provide insight into the relationship between the chondrocranium and dermatocranium. This study is only a descriptive study of the Curzon mouse and does not provide any insight into the "evolution" of the chondrocranium and dermatocranium. The results appear to be too much exaggerated. Again, it needs to be clearly stated why the cranial suture model is suitable for discussing the association between the chondrocranium and dermatocranium.

    We agree that we have not presented adequate data to treat the topic of 400+ million years of cooperation between the chondrocranium and dermatocranium adequately and have changed our title and specific text. Still, what is known about the evolutionary appearance and association of these two cranial skeletons that combine to form the modern vertebrate skull is relevant to our study. We have changed the title to reflect our change of focus and significantly decreased the number of words used to discuss the evolution of these structures.

    The use of the Fgfr2c Crouzon mouse represents an ideal experimental setting to determine the direct effect of a specific Fgfr2 mutation on cartilage formation and the indirect effect of chondrocranial morphology on the formation of cranial dermal bone. Our group has used various Fgfr2 mouse models to demonstrate that craniosynostosis is more than a cranial suture disease and that analysis of mouse models for craniosynostosis that carry Fgfr2 mutations reveal growth disorders of several tissues. The submitted paper builds on this body of work providing further evidence of this conclusion - but the work submitted to eLife is novel in showing that this specific Fgfr2 mutation affects cartilage cellular processes that produce quantifiable changes in the composite, 3D, cartilaginous structure of the chondrocranium, as well as revealing the impact of a malformed chondrocranium on dermatocranial morphology (as summarized in the Public Evaluation Summary).

    There is also a need to cite and review work in the fields of evolutionary anatomy and palaeontology; it is a shame that the authors ignore important contributions by evolutionary anatomists such as Parker, Wolfgang Maier, Sánchez-Villagra, and Koyabu. In its present form, it has little relevance to evolutionary biology.

    Recent collaborations by Koyabu, Sanchez-Villagra and others focus on the link between cranial development and brain size (https://doi.org/10.1038/ncomms4625), a topic of interest to our lab but not particularly relevant to the current study. Other recent work by these authors demonstrate the derivation of the interparietal bone from both neural crest and mesodermal cells (https://doi.org/10.1073/pnas.1208693109), also interesting and useful in general but not particularly relevant to our study. Since our paper no longer focuses on the co-evolution of dermatocranium and chondrocranium, these fine publications are not relevant to our discussion.

    Their conclusion that chondrocranium and dermatocranium development are associated is also not a novel finding, either. Apert mouse which exhibit the same abnormality previously reported showed that chondrocyte-specific changes in Fgfr2 alone produce an Apert-like cranial morphology suggesting that changes in Fgfr2 expression in chondrocytes may lead to the formation of membranous bone. It has already been reported that changes in Fgfr2 expression in chondrocytes have a significant effect on overall cranial morphology, including membranous bone. This study neglects such previous studies and exaggerates their results.

    Other reviewers state that our work is novel, and we agree. As stated in our abstract, “This is the first study providing fully complete three-dimensional (3D) reconstructions of the mouse embryonic chondrocranium using a novel methodology of uncertainty guided segmentation of microcomputed tomography images with sparse annotation.” Our findings are novel because they go beyond a histological demonstration of a localized effect of Fgfr2 mutations on specific cartilages. The study by Kim et al., to which we believe Reviewer 3 is referring (https://doi.org/10.1038/s41598-021-87260-5 ), is a fine study that shows effects of an alternate Fgfr2 mutation on the postnatal nasal septal cartilage and states that “Morphological and histological examination revealed that the presence of increased septal chondrocyte hypertrophy and abnormal thickening of nasal septum is causally related to midface deformities in nasal septum-associated structures” adding to the evidence that Fgfr variants affect cartilage and bone (reviewed by by Ornitz and Marie doi:10.1101/gad.990702 and updated 10.1101/gad.266551.115Genes & Dev. 2015. 29: 1463-1486). Kim et al. focused on a relationship between specific olfactory cartilages including the nasal septum and facial bones as our group did in a paper published in 2018 (doi:10.1242/dev.166488). In this study we used various Fgfr2 mouse models and we found that different Fgfr2 variants associated with human craniosynostosis syndromes affect the nasal cartilages differently. We have now included a discussion of these papers (see lines 577-586) and include a caution against the assumption that all Fgfr2 mutations have similar effects on multiple tissue types across developmental time (lines 577-581). This relationship does not only hold in mouse models for human disease but is an aspect of normal development as we demonstrated in a paper published in 2020 that specifies the association between specific chondrocranial cartilages and specific dermal bones (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644101/pdf/nihms-1641877.pdf - see especially Table 1B).

    What the submitted manuscript shows that is novel is that specific, quantifiable, 3D morphological alterations in chondrocranial morphology are produced by the effect of this Fgfr2c variant on the function of cartilage cells that in turn alter the shape and positioning of dermal bones of the prenatal dermatocranium – but not the postnatal dermatocranium.

    This study suffers from data uncertainty because raw data was not provided. The authors seem to want to keep their data to themselves and are against open science and data transparency. "3D coordinates of landmark data...will be made available to interested parties upon request." These raw coordinates MUST be fully provided as supplementary material, otherwise no one can re-evaluate their results. I am so surprised that even basic statics (PC scores, loadings, eigen values, explained variance) are not provided. Data availability and transparency are very important. 3D models are also not provided in the review, so at this point I cannot be sure of the accuracy of their segmentation. They have stated that they will make it available at https://www.facebase.org/ and/or https://scholarsphere.psu.edu/, but it should be accessible now for reviewers. Facebase is fine, but it should not be provided on their own institute's server that may go out of service at any time. It should be provided through a permanent public archive.

    We failed to make some data available at the time of review. Reasons include errors on our part in understanding what was useful to the readers (e.g., 3D coordinates of landmark data that we did not make available originally are now available) and the inability of available repositories to accommodate data sets of the size of our CT images (10-15 GB per CT study). Since review, we have found that Scholarsphere (https://scholarsphere.psu.edu/) is the only data repository that can economically and efficiently handle our data requirements and size of our PTA-enhanced microCTs. All data have been made available.

    Percent of variance explained was provided in the original submitted Figure 4. Principal components analysis (PCA) is not a statistical test but only provides the results of a clustering algorithm utilized by PCA that shows how individuals group together. As we already know group membership, we thought it unnecessary to include the other values that the reviewer seeks. We understand that this was an error of omission on our part and are happy to provide PC scores and the rest of this type of descriptive output from PCA. We also provide the individual scores for suture patency for all individuals.
    All data available at: DOI 10.26207/qgke-r185

  3. Version published to 10.1101/2021.11.24.469914v2 on bioRxiv
  4. eLife

    Evaluation Summary:

    This work combines imaging and quantitative analyses to address the conclusions that the chondrocranium and dermatocranium form an integrated unit of development and alterations in the chondrocranium drive changes in the dermatocranium. However, the manuscript in the current form suffers from data uncertainty because raw data was not provided, and the title and the conclusions go too much beyond the results they have gathered.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  5. eLife

    Reviewer #1 (Public Review):

    The interaction or potential correspondence between chondrocranial elements and dermal skull bones has been in debate for decades. Based on 3D reconstructions of samples of laboratory mouse chondrocranial anatomy for embryonic days 13.5- 17.5, the authors reveal an embryonic relationship between the chondrocranium and forming bones of the dermatocranium in vertebrates, and provide critical data to demonstrate the role of the chondrocranium in normal craniofacial development. This will add to our understanding of the correspondence between chondrocranial elements and dermal skull bones, and the homology of dermal skull bones across major vertebrate groups.

  6. eLife

    Reviewer #2 (Public Review):

    The objective of the research performed was to assess the role of the chondrocranium in directing the morphogenesis of the skull. In this work a new method was developed to visualize the cartilages that comprise the chondrocranium, which is used to assess how these cartilage elements change over time in wild type embryos and in embryos harboring a mutation that causes premature suture closure-a model of craniosynostosis. The major strengths of this work are the combination of imaging and quantitative analyses that drive the conclusions that the chondrocranium and dermatocranium form an integrated unit of development and alterations in the chondrocranium drive changes in the dermatocranium. The methods appear robust and the authors significantly advance the field by providing results that strongly support these conclusions. Prior to this work, the changes in the skull associated with craniosynostosis were attributed to alterations in osteoblasts that form the sutures, but this work provides significant evidence that dysmorphology of the skull occurs earlier and is due to altered molecular signaling in the chondrocytes that form the chondrocranium.

  7. eLife

    Reviewer #3 (Public Review):

    The aim of the study is to tangle the over 400 million years' cooperation between chondrocranium and dermatocranium development. Mice with Crouzon syndrome were chosen for the study. The strength of this study is the novel application of machine learning techniques to segment the mouse cranium, which can be applied to a variety of vertebrates. The figures are very appealing. The major drawback of this study is that it only focuses on the Curzon mouse, even though the goal of the study is to investigate the relationship between the chondrocranium and dermatocranium. The authors emphasize that this study was undertaken to study the 400-million-year history of the cranium of Osteichthyes, which includes bony fishes, amphibians, lizards, and birds, in addition to mammals. In order to "untangle the over 400 million years' cooperation between chondrocranium and dermatocranium" as the title states, it is too obvious that they must include bony fish, amphibians, lizards, and birds. It is also unclear throughout the manuscript why the study of Curzon mice would provide insight into the relationship between the chondrocranium and dermatocranium. This study is only a descriptive study of the Curzon mouse and does not provide any insight into the "evolution" of the chondrocranium and dermatocranium. The results appear to be too much exaggerated. Again, it needs to be clearly stated why the cranial suture model is suitable for discussing the association between the chondrocranium and dermatocranium.

    There is also a need to cite and review work in the fields of evolutionary anatomy and palaeontology; it is a shame that the authors ignore important contributions by evolutionary anatomists such as Parker, Wolfgang Maier, Sánchez-Villagra, and Koyabu. In its present form, it has little relevance to evolutionary biology.

    Their conclusion that chondrocranium and dermatocranium development are associated is also not a novel finding, either. Apert mouse which exhibit the same abnormality previously reported showed that chondrocyte-specific changes in Fgfr2 alone produce an Apert-like cranial morphology suggesting that changes in Fgfr2 expression in chondrocytes may lead to the formation of membranous bone. It has already been reported that changes in Fgfr2 expression in chondrocytes have a significant effect on overall cranial morphology, including membranous bone. This study neglects such previous studies and exaggerates their results.

    This study suffers from data uncertainty because raw data was not provided. "3D coordinates of landmark data...will be made available to interested parties upon request." These raw coordinates MUST be fully provided as supplementary material, otherwise no one can re-evaluate their results. I am so surprised that even basic statics (PC scores, loadings, eigen values, explained variance) are not provided. Data availability and transparency are very important. 3D models are also not provided in the review, so at this point I cannot be sure of the accuracy of their segmentation. They have stated that they will make it available at https://www.facebase.org/ and/or https://scholarsphere.psu.edu/, but it should be accessible now for reviewers. Facebase is fine, but it should not be provided on their own institute's server that may go out of service at any time. It should be provided through a permanent public archive.

  8. Version published to 10.1101/2021.11.24.469914v1 on bioRxiv