Author response:
Public reviews:
Reviewer #1:
Epigenetic regulation complex (PRC2) is essential for neural crest specification, and its misregulation has been shown to cause severe craniofacial defects. This study shows that Eed, a core PRC2 component, is critical for craniofacial osteoblast differentiation and mesenchymal proliferation after neural crest induction. Using mouse genetics and single-cell RNA sequencing, the researcher found that conditional knockout of Eed leads to significant craniofacial hypoplasia, impaired osteogenesis, and reduced proliferation of mesenchymal cells in post-migratory neural crest populations.
Overall, the study is superficial and descriptive. No in-depth mechanism was analyzed and the phenotype analysis is not comprehensive.
We thank the reviewer for sharing their expertise and for taking the time to provide a helpful suggestion to improve our study. We are gratified that the striking phenotypes we report from Eed loss in post-migratory neural crest craniofacial tissues were appreciated. The breadth and depth of our phenotyping techniques, including skeletal staining, micro-CT, echocardiogram, immunofluorescence, histology, and unbiased single-cell gene expression analysis, provide comprehensive data in support our conclusion that PRC2 is required for craniofacial osteoblast differentiation. We hypothesize that epigenetic regulation of chromatin accessibility downstream of PRC2 activity is the molecular mechanism that underlies these phenotypes. To test this hypothesis in our revision, we are using CUT&Tag to profile H3K27me3 epigenetic modifications genome-wide and at the loci encoding the differentially expressed genes revealed by our single-cell transcriptomics in developing craniofacial structures. We anticipate that these experiments will reveal an epigenetic mechanism underlying the phenotypes we report from Eed loss in post-migratory neural crest craniofacial tissues.
Reviewer #2:
Summary:The role of PRC2 in post-neural crest induction was not well understood. This work developed an elegant mouse genetic system to conditionally deplete EED upon SOX10 activation. Substantial developmental defects were identified for craniofacial and bone development. The authors also performed extensive single-cell RNA sequencing to analyze differentiation gene expression changes upon conditional EED disruption.
Strengths:
(1) Elegant genetic system to ablate EED post neural crest induction.
(2) Single-cell RNA-seq analysis is extremely suitable for studying the cell type-specific gene expression changes in developmental systems.
We thank the reviewer for their generous and helpful comments on our study. We are pleased that our mouse genetic and single-cell RNA sequencing approaches were appropriate in pairing the craniofacial phenotypes we report with distinct gene expression changes in post-migratory neural crest tissues upon Eed deletion.
Weaknesses:
(1) Although this study is well designed and contains state-of-the-art single-cell RNA-seq analysis, it lacks the mechanistic depth in the EED/PRC2-mediated epigenetic repression. This is largely because no epigenomic data was shown.
Thank you for this suggestion. As described in response to Reviewer #1, we will include H2K27me3 CUT&Tag data in craniofacial tissue harvested from E12.5 and E16.5 Sox10-Cretg+ Eedfl/fl and Sox10-Cretg+ Eedfl/wt embryos in our revision. Our analyses will including genome-wide and targeted metaplot visualizations across genotypes and developmental timepoints and assess how H3K27me3 occupancy relates to gene expression changes in our single-cell RNA sequencing data.
(2) The mouse model of conditional loss of EZH2 in neural crest has been previously reported, as the authors pointed out in the discussion. What is novel in this study to disrupt EED? Perhaps a more detailed comparison of the two mouse models would be beneficial.
We acknowledge the study the reviewer has indicated (Schwarz et al. Development 2014). This elegant investigation uses Wnt1-Cre to delete Ezh2 and found a similar phenotype to ours in the form of catastrophic craniofacial hypoplasia. We sought to add depth to the study of PRC2’s vital role in neural crest development by ablating Eed, which has a unique function in the PRC2 complex by binding to H3K27me3 and allosterically activating Ezh2. In this sense, we sought to test if phenotypes arising from deletion of Eed, the PRC2 “reader”, differ from phenotypes arising from deletion of Ezh2, the PRC2 “writer”, in neural crest derived tissues. Due to limitations associated with the Wnt1-Cre transgene (Lewis et al. Developmental Biology 2013), we used the Sox10-Cre allele which targets the migratory neural crest and is completely recombined by E10.5, instead of Wnt1-Cre which targets pre-migratory neural crest cells. A more detailed comparison of these mouse models will be included in the Discussion section of our revised manuscript, and we thank the reviewer for this thoughtful suggestion.
(3) The presentation of the single-cell RNA-seq data may need improvement. The complexity of the many cell types blurs the importance of which cell types are affected the most by EED disruption.
We agree with the reviewer’s critique of the scRNA-seq data presentation. Because Sox10+ cells were not sorted (via FACS, for example) from craniofacial tissues before single-cell RNA sequencing, we identified a breath of cell types in UMAP space unrelated to epigenetic disruption of neural crest derived tissues. We will include subcluster visualization plots in the figures of our revised manuscript to highlight specific changes in clusters, such as osteoblasts and mesenchymal stem cells, that arise from Eed loss in post-migratory neural crest craniofacial tissues.
(4) While it's easy to identify PRC2/EED target genes using published epigenomic data, it would be nice to tease out the direct versus indirect effects in the gene expression changes (e.g Figure 4e).
We agree with the reviewer that our single-cell RNA sequencing data do not provide insight into direct versus indirect changes in gene expression downstream of PRC2. We hope that the aforementioned CUT&Tag experiment will provide the necessary mechanistic insight into H3K27me3 occupancy and direct effects on gene expression resulting from PRC2 inactivation in our mouse model.
