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  1. Author Response

    Reviewer #2 (Public Review):

    In this interesting and beautifully illustrated study, the authors are addressing the question of the emergence of craniofacial tissues by dissecting the interplay between skeletal muscle progenitors and associated connective tissue cells. By combining sophisticated lineage-tracing single cell RNA-seq experiments with potent computational analysis tools followed by in situ validations, the authors have identified a population of Myf5+ bipotent progenitors that give rise to both muscle and connective tissue. However, some conclusions are solely based on the RNA-Seq data that would require further experimental validations.

    We thank the Reviewer for evaluating our work and their encouraging comments. We agreed with the assessment and have now added more in-situ validations and quantifications to support the in-silico analyses.

    Reviewer #3 (Public Review):

    In this manuscript, Grimaldi et al. present evidence for the existence of Myf5+ bipotent progenitors for myogenic and connective lineages in the dorsal regions of the mouse head, which is not populated by neural crest cell-derived connective tissue. The study relies heavily on scRNA-seq dataset obtained from cell populations sorted at defined time points, and refined computational analysis, including trajectory and gene network inference using the established tools RNA velocity and SCENIC, respectively. The proposed model is partially validated by in situ staining experiments, including genetic labeling, which identified Pdgfra+ non-myogenic cells within the Myf5+ lineages, notably in association with extraocular muscles (EOM). The authors propose a myogenic origin for the connective tissue, in regions devoid of neural crest cells, and show that loss of Myf5 function causes an increase in the proportion of Sox9+ cells among Myf5+ lineage cells, which is consistent with a binary fate choice from Myf5+ progenitors. The authors tentatively identify signaling molecules and transcription regulators underlying both fate decisions and cell-cell communications between myogenic and non-myogenic cell populations.

    The general message of the study offers a potentially new paradigm to study neural crest cell-independent mesodermal fate decision in the vertebrate head, and is thus poised to augment our understanding of craniofacial development, and potential diseases.

    Unfortunately, there are shortcomings that strongly reduce enthusiasm for this manuscript. Strictly speaking, there is no clear demonstration for the existence of bipotent progenitors in the absence of clonal analysis. The study relies excessively on computational analysis of descriptive scRNA-seq datasets, with a general paucity of secondary experimental validation. The manuscript would benefit from a refined focus on the key point, and addition of validation for the initial conclusions, at the expense of somewhat convoluted analyses (e.g. Figs. 6 and 7)

    We thank the Reviewer for their constructive comments. We have now provided additional in-situ validations on embryos and quantifications to support the in-silico analyses.

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  2. Evaluation Summary:

    The paper will be of interest to a broad audience of developmental biologists, as it provides evidence for a population of novel bipotent cells, which possess a signature of both muscle and connective tissue. This work implies an adjustment to our current understanding of cell fate decision in myogenesis and fibrogenesis. Combining the sophisticated lineage tracing and single-cell RNAseq analysis, the key claims of the paper are well supported.

    (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 #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    This manuscript describes the identification of a group of Myf5-expressing cells which can give rise to not only myogenic cells but also non-myogenic connective tissue cells. Single-cell sequencing and trajectory analysis of mouse Mesp1-derived lineage at embryonic stage E10.5 identify a potential cell fate transition from myogenic to non-myogenic cells. This transition and the presence of bipotent Myf5+ cells are further confirmed by lineage tracing experiments in mouse demonstrating the presence of a significant number of Pdgfra-expressing cells in the Myf5 lineage in anterior muscles. Interestingly, these Myf5-derived cells are mainly restricted in anterior regions where the muscle connective tissue is not derived from neural crest but from the mesoderm. Analysis of the Myf5 lineage in extraocular muscle at different embryonic stages reveals a signaling network involving PDGFA and its receptor between myogenic and non-myogenic cell populations.

    Overall, this is a very interesting study and the conclusions of the paper are consistent with their presented data.

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  4. Reviewer #2 (Public Review):

    In this interesting and beautifully illustrated study, the authors are addressing the question of the emergence of craniofacial tissues by dissecting the interplay between skeletal muscle progenitors and associated connective tissue cells. By combining sophisticated lineage-tracing single cell RNA-seq experiments with potent computational analysis tools followed by in situ validations, the authors have identified a population of Myf5+ bipotent progenitors that give rise to both muscle and connective tissue. However, some conclusions are solely based on the RNA-Seq data that would require further experimental validations.

    Read the original source
    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    In this manuscript, Grimaldi et al. present evidence for the existence of Myf5+ bipotent progenitors for myogenic and connective lineages in the dorsal regions of the mouse head, which is not populated by neural crest cell-derived connective tissue. The study relies heavily on scRNA-seq dataset obtained from cell populations sorted at defined time points, and refined computational analysis, including trajectory and gene network inference using the established tools RNA velocity and SCENIC, respectively. The proposed model is partially validated by in situ staining experiments, including genetic labeling, which identified Pdgfra+ non-myogenic cells within the Myf5+ lineages, notably in association with extraocular muscles (EOM). The authors propose a myogenic origin for the connective tissue, in regions devoid of neural crest cells, and show that loss of Myf5 function causes an increase in the proportion of Sox9+ cells among Myf5+ lineage cells, which is consistent with a binary fate choice from Myf5+ progenitors. The authors tentatively identify signaling molecules and transcription regulators underlying both fate decisions and cell-cell communications between myogenic and non-myogenic cell populations.

    The general message of the study offers a potentially new paradigm to study neural crest cell-independent mesodermal fate decision in the vertebrate head, and is thus poised to augment our understanding of craniofacial development, and potential diseases.

    Unfortunately, there are shortcomings that strongly reduce enthusiasm for this manuscript. Strictly speaking, there is no clear demonstration for the existence of bipotent progenitors in the absence of clonal analysis. The study relies excessively on computational analysis of descriptive scRNA-seq datasets, with a general paucity of secondary experimental validation. The manuscript would benefit from a refined focus on the key point, and addition of validation for the initial conclusions, at the expense of somewhat convoluted analyses (e.g. Figs. 6 and 7)

    Read the original source
    Was this evaluation helpful?