Figures and data
Sox10-Cre EedFl/Fl embryos develop craniofacial malformations.
(a) Genotyping frequencies of postnatal (top) and embryonic (bottom) Sox10-Cre EedFl/Fl mice. (b) Sagittal brightfield images of Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl embryos from E10.5 to E17.5 showing craniofacial malformations appearing at E12.5 that were observed with 100% penetrance. Scale bar, 1mm. (c) Coronal brightfield images of E16.5 Sox10-Cre EedFl/WTor Sox10-Cre EedFl/Flembryos showing craniofacial malformations. Scale bar, 1mm. (d) Whole-mount fluorescence microscopy of DAPI-stained Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads at E12.5, E14.5, or E16.5. Scale bar, 1mm. All images are representative of ≥3 biological replicates.
Eed is required for craniofacial skeletal development from the neural crest.
(a) Sagittal brightfield images of whole-mount skeletal stains of E18.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl embryos. Alcian blue and alizarin red identify cartilage or bone, respectively. Scale bar, 1mm. (b) Magnified sagittal brightfield images of whole-mount skeletal stains of E18.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl embryos. Bones structures are annotated (I, interparietal; P, parietal; Mx, maxilla; Mb, mandible; Tr, tympanic ring; F, frontal; N, nasal; T, temporal; S, supraoccipital). Scale bar, 1mm. (c) Micro-CT images of E19.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads. Scale bar, 1mm. (d) Micro-CT images of E19.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl neural crest-derived craniofacial bones shaded in orange (frontal), grey (maxilla), or green (mandible). Scale bar, 1mm. (d) Magnified micro-CT images of E19.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl neural crest-derived craniofacial bones. Scale bars, 1mm.
Eed regulates craniofacial differentiation and proliferation from the neural crest.
(a) Brightfield coronal H&E images of E15.5 Sox10-Cre EedFl/WTor Sox10-Cre EedFl/Flheads. Anatomic structures are annotated (Ct, cortex; CP, cartilage primordium; CS, conjunctival sac; Gg, genioglossus muscle; HC, hyaloid cavity; Hg, hyoglossus muscle; L, lens; LV, lateral ventricle; Mb, midbrain; MM, masseter muscle; Np, nasopharynx; NC, nasal cavity; OB, olfactory bulb; OE, olfactory epithelium; OM, ocular muscle; RNL, retina neural layer; Sm/St, sublingual and submandibular ducts; T, tongue; TV, third ventricle; VF, vibrissa follicles). Scale bar, 1mm. (b) Coronal immunofluorescence images of E15.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads stained for ALPL (green) and Sox9 (red). Scale bars, 1mm and 100µm. (c) Coronal immunofluorescence images of E15.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads stained for Ki67 (green) and Vimentin (red). Scale bars, 1mm and 100µm. (d) Quantification of Ki67 immunofluorescence labeling index from E15.5 heads. (e) Coronal immunofluorescence images of E12.5 Sox10-Cre EedFl/WTor Sox10-Cre EedFl/Flheads stained for Runx2 (red) and Sox9 (green). Intracranial tissues demonstrate ex vacuo dilation and anterior herniation in Sox10-Cre EedFl/Fl heads. Scale bars, 500µm and 100µm. (f) Quantification of immunofluorescence imaging intensity from primary craniofacial cell cultures from E12.5 Sox10-Cre EedFl/WTor Sox10-Cre EedFl/Flembryos. All images are representative of ≥3 biological replicates. DNA is marked by DAPI. Lines represent means and error bars represent standard error of the means. Student’s t-tests, **p≤0.01, ***p≤0.0001.
Eed regulates craniofacial mesenchymal stem cell, osteoblast, and proliferating mesenchymal cell fate from the neural crest.
(a) Integrated UMAP of 63,730 transcriptomes from single-cell RNA sequencing of litter-matched E12.5 Sox10-Cre EedFl/WT (n=3) or Sox10-Cre EedFl/Fl (n=3) heads. (b) Integrated UMAP overlaying cell cluster distribution from single-cell RNA sequencing. (c) Quantification of mesenchymal cell cluster distribution from single-cell RNA sequencing. Lines represent means and error bars represent standard error of the means. Student’s t-tests, **p≤0.01, ***p≤0.0001. (d) Gene expression feature plots of mesenchymal lineages from single-cell RNA sequencing. (e) Volcano plot showing differentially expressed genes between C0 (osteoblasts) and C4 (mesenchymal stem cells). (f) Volcano plot showing differentially expressed genes between C0 and C5 (mesenchymal stem cells). (g) Volcano plot showing differentially expressed genes between 2 clusters of mesenchymal stem cells from single-cell RNA sequencing of litter-matched E12.5 Sox10-Cre EedFl/WT (n=3) or Sox10-Cre EedFl/Fl(n=3) heads. (h) Volcano plot showing differentially expressed genes in differentiating osteoblasts (C0), mesenchymal stem cells (C4, C5), and proliferating mesenchymal cells (C7) from single-cell RNA sequencing of litter-matched E12.5 Sox10-Cre EedFl/WT (n=3) versus Sox10-Cre EedFl/Fl (n=3) heads.
Sox10-Cre EedFl/Fl embryos develop craniofacial malformations.
(a) Craniofacial measurement quantification of pupillary distance (left) or craniofacial width (right) from E12.5 Sox10-Cre EedFl/WTor Sox10-Cre EedFl/Flembryos. (b) Body measurement quantification of crown-rump length from E12.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl embryos. Lines represent means and error bars represent standard error of the means. (c) Craniofacial measurement quantification of pupillary distance (left) or craniofacial width (right) from E16.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl embryos. (d) Body measurement quantification of crown-rump length (left) or body weight (right) from E16.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl embryos. Lines represent means and error bars represent standard error of the means. Student’s t-tests, ***p≤0.0001.
Sox10-Cre EedFl/Fl embryos develop subtle cardiac malformations.
(a) Sagittal view of E16.5 Sox10-Cre EedFl/WT (top) or Sox10-Cre EedFl/Fl (bottom) embryos in B-mode echocardiography (FL, frontal lobe; NB, nasal bridge; N, nose; UL, upper lip; M, mandible). Scale bar, 1mm. (b) Transversal view of B-mode echocardiography with measurements of left ventricle internal and external areas of an E16.5 embryo. (c) M-mode echocardiography of E16.5 Sox10-Cre EedFl/WT (left) or Sox10-Cre EedFl/Fl (right) embryos. Four cardiac cycles are included per trace and all cardiac segments are visible in each embryo, as labeled. (d) Quantitative echocardiography measurements from Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl embryos. Lines represent means and error bars represent standard error of the means. Student’s t-tests, *p≤0.05, **p≤ .01.
Sox10-Cre EedFl/Fl embryos develop significant craniofacial mesenchymal differentiation phenotypes and subtle apoptotic phenotypes.
(a) Coronal brightfield images of whole-mount skeletal stains of E18.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl embryos. Scale bar, 1mm. (b) Brightfield sagittal H&E images of E16.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads in series from lateral (left) to medial (right). Vibrissa (V) were reduced in Sox10-Cre EedFl/Fl heads compared to controls. Scale bar, 1mm. (c) Coronal immunofluorescence images of E15.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads stained for osteocalcin (green). Scale bar, 1mm. (d) Coronal immunofluorescence images of E15.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl mandibles stained for Cleaved Caspase 3 (red). Scale bar, 100µm. (e) Quantification of Cleaved Caspase 3 immunofluorescence labeling index from E15.5 Sox10-Cre EedFl/WTor Sox10-Cre EedFl/Flmandibles. Lines represent means and error bars represent standard error of the means. Student’s t-test, **p≤0.01. (f) Coronal immunofluorescence images of E15.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads stained for Runx2 (red). Scale bar, 1mm. All images are representative of ≥3 biological replicates. DNA is marked by DAPI.
Eed regulates craniofacial differentiation and proliferation in vivo and in primary cell cultures.
(a) Coronal immunofluorescence images of E12.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads stained for BrdU (red). Scale bars, 500µm and 50µm. (b) Quantification of BrdU immunofluorescence labeling index from E12.5 heads. (c) Immunofluorescence images of primary craniofacial cell cultures from E12.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl embryos stained for Eed, Runx2, Ki67, or Sox10. Scale bar, 100µm. Quantification of ALPL immunofluorescence imaging intensity from primary E15.5 craniofacial cell cultures. (d) Coronal immunofluorescence images of E12.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads stained for Sox10 (red). Scale bars, 500µm and 50µm. All images are representative of ≥3 biological replicates. DNA is marked by DAPI. Lines represent means and error bars represent standard error of the means. Student’s t-tests, ***p≤0.0001.
Cell clusters from single-cell RNA sequencing of E12.5 Sox10-Cre EedFl/WT or Sox10-Cre EedFl/Fl heads.
(a) Heat map of differentially expressed genes across 23 cell clusters.
Cell cluster marker genes from single-cell RNA sequencing of E12.5 Sox EedFl/WT or Sox10-Cre EedFl/Fl heads.
(a) Gene expression feature plots for differentially expressed across 23 cell clusters.
Single-cell transcriptome differential expression analyses among mesenchymal cell clusters.
(a) Gene expression feature plots for differentially expressed genes between C0 and C4. (b) Gene expression feature plots for differentially expressed genes between C0 and C5. (c) Gene expression feature plots for differentially expressed genes between mesenchymal stem cell clusters.
