Eed controls craniofacial osteoblast differentiation and mesenchymal proliferation from the neural crest
- Department of Radiation Oncology, University of California San Francisco, United States
- Department of Neurosurgery, University of California San Francisco, United States
- Department of Pathology, University of California San Francisco, United States
- Gladstone Institutes, United States
- Roddenberry Center for Stem Cell Biology at Gladstone Institutes, United States
- Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, United States
- Department of Cell and Tissue Biology, University of California San Francisco, United States
- Department of Medicine, Division of Cardiology, University of California San Francisco, United States
- Cardiovascular Resarch Institute, University of California San Francisco, United States
Figures
Figure 1 with 2 supplements
Sox10-Cre; EedFl/Fl embryos develop craniofacial malformations.
(a) Genotyping frequencies of postnatal (top) and embryonic (bottom) Sox10-Cre; EedFl/Fl mice. (b) Sagittal bright-field 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, 1 mm. (c) Coronal brightfield images of E16.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl embryos showing craniofacial malformations. Scale bar, 1 mm. (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, 1 mm. All images are representative of ≥3 biological replicates.
Figure 1—figure supplement 1
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/WT or Sox10-Cre; EedFl/Fl embryos. (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.
Figure 1—figure supplement 2
Sox10-Cre; EedFl/Fl embryos develop subtle cardiac malformations.
(a) Sagittal view of embryonic day (E)16.5 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, 1 mm. (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≤0.01.
Figure 2 with 1 supplement
Eed is required for craniofacial skeletal development from the neural crest.
(a) Sagittal bright-field images of whole-mount skeletal stains of embryonic day (E)18.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl embryos. Alcian blue and Alizarin Red identify cartilage or bone, respectively. Scale bar, 1 mm. (b) Magnified sagittal bright-field images of whole-mount skeletal stains of E18.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl embryos. Bone structures are annotated (I, interparietal; P, parietal; Mx, maxilla; Mb, mandible; Tr, tympanic ring; F, frontal; N, nasal; T, temporal; S, supraoccipital). Scale bar, 1 mm. (c) Micro-computed tomography (microCT) images of E19.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl heads. Scale bar, 1 mm. (d) MicroCT images of E19.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl neural crest-derived craniofacial bones shaded in orange (frontal), gray (maxilla), or green (mandible). Scale bar, 1 mm. (e) Magnified microCT images of E19.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl neural crest-derived craniofacial bones. Scale bars, 1 mm.
Figure 2—figure supplement 1
Sox10-Cre; EedFl/Fl embryos develop significant craniofacial mesenchymal differentiation phenotypes and subtle apoptotic phenotypes.
(a) Coronal bright-field images of whole-mount skeletal stains of embryonic day (E)18.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl embryos. Scale bar, 1 mm. (b) Bright-field 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, 1 mm. (c) Coronal immunofluorescence images of E15.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl heads stained for osteocalcin (green). Scale bar, 1 mm. (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, 100 µm. (e) Quantification of Cleaved Caspase 3 immunofluorescence labeling index from E15.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl mandibles. 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, 1 mm. All images are representative of ≥3 biological replicates. DNA is marked by DAPI.
Figure 3 with 1 supplement
Eed regulates craniofacial differentiation and proliferation from the neural crest.
(a) Bright-field coronal H&E images of E15.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl heads. 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, 1 mm. (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, 1 mm and 100 µm. (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, 1 mm and 100 µm. (d) Quantification of Ki67 immunofluorescence labeling index from E15.5 heads. (e) Coronal immunofluorescence images of E12.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl heads stained for Runx2 (red) and Sox9 (green). Intracranial tissues demonstrate ex vacuo dilation and anterior herniation in Sox10-Cre; EedFl/Fl heads. Scale bars, 500 µm and 100 µm. (f) Quantification of immunofluorescence imaging intensity from primary craniofacial cell cultures from E12.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl embryos. 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.
Figure 3—figure supplement 1
Eed regulates craniofacial differentiation and proliferation in vivo and in primary cell cultures.
(a) Coronal immunofluorescence images of embryonic day (E)12.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl heads stained for BrdU (red). Scale bars, 500 µm and 50 µm. (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, 100 µm. (d) Quantification of ALPL immunofluorescence imaging intensity from primary E15.5 craniofacial cell cultures. (e) Coronal immunofluorescence images of E12.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl heads stained for Sox10 (red). Scale bars, 500 µm and 50 µm. 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.
Figure 4 with 4 supplements
Eed regulates craniofacial mesenchymal stem cell, osteoblast, and proliferating mesenchymal cell fate from the neural crest.
(a) Integrated uniform manifold approximation and projection (UMAP) of 63,730 transcriptomes from single-cell RNA sequencing of litter-matched embryonic day (E)12.5 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 two 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.
Figure 4—figure supplement 1
Cell clusters from single-cell RNA sequencing of embryonic day (E)12.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl heads.
(a) Heat map of differentially expressed genes across 23 cell clusters.
Figure 4—figure supplement 2
Cell cluster marker genes from single-cell RNA sequencing of embryonic day (E)12.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl heads.
(a) Gene expression feature plots for differentially expressed genes across 23 cell clusters.
Figure 4—figure supplement 3
Mesenchymal cell cluster distribution from single-cell RNA sequencing.
(a) Integrated uniform manifold approximation and projection (UMAP) of transcriptomes from single-cell RNA sequencing of litter-matched embryonic day (E)12.5 Sox10-Cre; EedFl/WT (n=3) or Sox10-Cre; EedFl/Fl (n=3) heads with shading of mesenchymal cell clusters C0 (differentiating osteoblasts), C4 (mesenchymal stem cells), C5 (mesenchymal stem cells), and C7 (proliferating mesenchymal stem cells) across genotypes. Differentiating and proliferating mesenchymal cells are enriched in Sox10-Cre; EedFl/WT heads, and mesenchymal stem cells are enriched in Sox10-Cre; EedFl/Fl heads.
Figure 4—figure supplement 4
Single-cell transcriptome differential expression analyses among mesenchymal cell clusters.
(a) Gene expression feature plots for differentially expressed genes between C0 (differentiating osteoblasts) and C4 (mesenchymal stem cells). (b) Gene expression feature plots for differentially expressed genes between C0 and C5 (mesenchymal stem cells). (c) Gene expression feature plots for differentially expressed genes between mesenchymal stem cell clusters C4 and C5.
Figure 5 with 1 supplement
Eed regulates H3K27me3 deposition at transcription factor loci that are required for craniofacial development.
(a) Metaplots of H3K27me3 occupancy within 10 kb of all genes or within 10 kb of differentially expressed mesenchymal cell cluster (C0, C4, C5, C7) from CUT&Tag sequencing of embryonic day (E)12.5 Sox10-Cre; EedFl/WT (n=3) or Sox10-Cre; EedFl/Fl (n=3) craniofacial tissues. (b) Metaplots of H3K27me3 occupancy within 10 kb of all genes or within 10 kb of differentially expressed mesenchymal cell cluster genes (C0, C4, C5, C7) from CUT&Tag sequencing of E16.5 Sox10-Cre; EedFl/WT (n=3) or Sox10-Cre; EedFl/Fl (n=3) craniofacial tissues. (c) Distribution of H3K27me3 signal identifying 115 methylation-rich regions (MRRs, shaded) in E12.5 Sox10-Cre; EedFl/WT (n=3) and 111 MRRs in Sox10-Cre; EedFl/Fl (n=3) craniofacial tissues. (d) Distribution of H3K27me3 signal identifying 147 MRRs in E16.5 Sox10-Cre; EedFl/WT (n=3) and 123 MRRs in Sox10-Cre; EedFl/Fl (n=3) craniofacial tissues. (e) H3K27me3 tracks at selected loci overlayed from E12.5 or E16.5 Sox10-Cre; EedFl/WT (n=3 per timepoint) or Sox10-Cre; EedFl/Fl (n=3 per timepoint) craniofacial tissues.
Figure 5—figure supplement 1
Gene ontology analyses of H3K27me3 rich regions from embryonic day (E)12.5 or E16.5 Sox10-Cre; EedFl/WT or Sox10-Cre; EedFl/Fl craniofacial tissues.
(a) Gene ontology biological process (2025) terms from E12.5 Sox10-Cre; EedFl/WT (n=3) or Sox10-Cre; EedFl/Fl (n=3) methylation-rich regions (MRRs). (b) Gene ontology biological process (2025) terms from E16.5 Sox10-Cre; EedFl/WT (n=3) or Sox10-Cre; EedFl/Fl (n=3) MRRs.
Videos
Video 1
B-mode echocardiography movie of an embryonic day (E)16.5 Sox10-Cre; EedFl/WT heart, transversal view at medium level.
Both ventricles, external walls, and interventricular septum are visible. Right ventricle is up, and left ventricle is down. The difference in size between ventricles is visible. The ventricular contraction is rhythmic, symmetric, and complete.
Video 2
B-mode echocardiography movie of an embryonic day (E)16.5 Sox10-Cre; EedFl/Fl heart, transversal view at medium level.
Both ventricles, external walls, and interventricular septum are visible. Right ventricle is up, and left ventricle is down. The ventricular contraction is rhythmic and symmetric.
Video 3
Three-dimensional reconstruction of micro-computed tomography (microCT) imaging of an embryonic day (E)19.5 Sox10-Cre; EedFl/WT head.
Video 4
Three-dimensional reconstruction of micro-computed tomography (microCT) imaging of an embryonic day (E)19.5 Sox10-Cre; EedFl/Fl head.
Additional files
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Supplementary file 1
Cell count per embryo and per cell cluster from single-cell RNA sequencing.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp1-v1.xlsx
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Supplementary file 2
Single-cell RNA sequencing cluster marker genes.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp2-v1.xlsx
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Supplementary file 3
Differential gene expression between C4 and C0 from single-cell RNA sequencing.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp3-v1.xlsx
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Supplementary file 4
Differential gene expression between C5 and C0 from single-cell RNA sequencing.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp4-v1.xlsx
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Supplementary file 5
Differential gene expression between C4 and C5 from single-cell RNA sequencing.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp5-v1.xlsx
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Supplementary file 6
Differential gene expression between genotypes in C0, C4, C5, and C7 from single-cell RNA sequencing.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp6-v1.xlsx
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Supplementary file 7
Methylation rank and nearest gene coordinates from CUT&Tag H3K27me3 sequencing of E12.5 Sox10-Cre; EedFl/WT craniofacial tissues.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp7-v1.xlsx
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Supplementary file 8
Methylation rank and nearest gene coordinates from CUT&Tag H3K27me3 sequencing of E12.5 Sox10-Cre; EedFl/Fl craniofacial tissues.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp8-v1.xlsx
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Supplementary file 9
Methylation rank and nearest gene coordinates from CUT&Tag H3K27me3 sequencing of E16.5 Sox10-Cre; EedFl/WT craniofacial tissues.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp9-v1.xlsx
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Supplementary file 10
Methylation rank and nearest gene coordinates from CUT&Tag H3K27me3 sequencing of E16.5 Sox10-Cre; EedFl/Fl craniofacial tissues.
- https://cdn.elifesciences.org/articles/100159/elife-100159-supp10-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/100159/elife-100159-mdarchecklist1-v1.pdf
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Eed controls craniofacial osteoblast differentiation and mesenchymal proliferation from the neural crest
eLife 13:RP100159.
https://doi.org/10.7554/eLife.100159.3
