Hedgehog signaling patterns the oral-aboral axis of the mandibular arch
- Cincinnati Children’s Hospital Medical Center, United States
- University of Cincinnati College of Medicine, United States
- Shriners Hospitals for Children – Cincinnati, United States
- University of Colorado, United States
Figures
Figure 1 with 9 supplements
Analysis of scRNA-seq data of E10.5 mouse mandibular arch.
(A) tSNE plot of all cells with high quality RNA-seq results shows clustering of the major cell types. (B) Unsupervised clustering of the neural crest-derived mandibular mesenchyme cells shows six …
Figure 1—figure supplement 1
Heatmap showing transcriptome profiles of the major cell types in the E10.5 mouse mandibular arch, with select marker genes exhibiting more than 1.5-fold difference in expression levels across all clusters.
NC1 – NC3, neural crest derived mesenchyme cells; Epi, epithelial cells; Endo, endothelial cells; HM, head mesoderm cells. Each row shows the pattern of expression of one marker gene in all cells of …
Figure 1—figure supplement 2
Dot plot showing the number of unique genes whose expression was detected in the single cells in the six different clusters.
NC1 – NC3, neural crest derived mesenchyme cells; Epi, epithelial cells; Endo, endothelial cells; HM, head mesoderm cells. The box marks the number of genes detected in 25% to 75% of the cells in …
Figure 1—figure supplement 3
Dot plot showing the number of transcripts detected in the single cells in the six different clusters.
NC1 – NC3, neural crest derived mesenchyme cells; Epi, epithelial cells; Endo, endothelial cells; HM, head mesoderm cells. The box marks the number of transcripts detected in 25% to 75% of the cells …
Figure 1—figure supplement 4
Comparison of the levels of transcripts for selected marker genes in tSNE maps of the E10.5 mandibular arch scRNA-seq data.
The tSNE cluster map as shown in Figure 1A in the manuscript is used to show the marker gene expression in the individual cells, with transcript levels shown in red color (darker color indicating …
Figure 1—figure supplement 5
Distribution of the principal components by JackStraw plot.
The red line marks the cutoff for identifying the most significant PCs used for tSNE implementation.
Figure 1—figure supplement 6
Analysis of the top eight principal components identified in the scRNA-seq data.
Most of the cells showed similar distribution by principal component analysis, with no apparent spatial segregation.
Figure 1—figure supplement 7
Developmental trajectories of all of the neural crest cells derived from analysis of the E10.5 mandibular arch scRNA-seq data using Monocle-2.
The cellular states are color coded. Two branch points were identified. Gene ontology analysis showed that States 3 and 4 were enriched with the cell differentiation terms ‘osteoblast …
Figure 1—figure supplement 8
Distribution of the five cellular states from the developmental trajectory analysis of the neural crest cells in the E10.5 mandibular arch on tSNE plot of the six subgroups derived from iterative clustering.
The color code for the cell subgroups is the same as shown in Figure 1B in the manuscript. The developmental trajectories did not correlate with obvious spatial axis.
Figure 1—video 1
A .mov file showing three dimensional tSNE projection of the six subgroups of neural crest derived mandibular arch mesenchyme cells.
The color codes for the six subgroups are the same as in Figure 1B (Cluster 0, red; Cluster 1, green; Cluster 2, blue; Cluster 3, yellow; Cluster 4, purple; Cluster 5, orange).
Figure 2
Distribution of E10.5 mandibular arch cells expressing select marker genes in the neural crest compartments in tSNE maps.
(A–D) Cells expressing Pou3f3 (A), Dlx5 (B), Hand2 (C), and Hand1 (D) in the neural crest cell population, detected by scRNA-seq, are shown in red color in tSNE plot, with the brightness of the red …
Figure 3
Expression of Shh, Bmp4, Fgf8, and their target genes in the developing mandibular arch.
(A–C) Frontal views of E10.5 embryonic mouse heads after whole mount in situ hybridization detecting Fgf8 (A), Msx1 (B) and Foxf1 (C). Green dashed lines mark the rostral and caudal sides of the …
Figure 4 with 2 supplements
Smoc/c;Wnt1-Cre embryos display tongue agenesis and ectopic ossification.
(A, B) Lateral view of skeletal preparations of heads of E18.5 Smoc/c (A) and Smoc/c;Wnt1-Cre (B) embryos (n = 3 for each genotype). (C, D) Comparison of the mandibular skeletons of E18.5 Smoc/c (C) …
Figure 4—figure supplement 1
Smoc/c;Wnt1-Cre display agenesis of the oral tongue structure.
(A–F) Immunofluorescent detection of muscle actin (red) on sagittal sections (A–B), and frontal sections (C–F) of E15.5 Smoc/c (A, C, E) and Smoc/cWnt1-Cre (B, D, F) embryos. Sections were …
Figure 4—figure supplement 2
Mouse embryos with tissue-specific inactivation of Smo signaling in the neural crest lineage exhibit altered pattern of expression of BMP target genes in the mandibular arch.
(A–F) Rostral views of microdissected E10.5 mandibles from Smoc/c control (A, C, E) and Smoc/c;Wnt1-Cre (B, D, F) embryos showing differential expression of Msx1 (A, B), Msx2 (C, D), and Alx4 (E, F) …
Figure 5 with 1 supplement
Smoc/c;Hand2-Cre embryos exhibit oropharyngeally expanded BMP signaling activity in the developing mandibular arch.
(A–H) Whole mount in situ hybridization detection of Bmp4 (A, B), Msx1 (C, D), Msx2 (E, F), and Alx4 (G, H) mRNAs in the mandibular arches in E10.5 Smoc/c (A, C, E, G), and Smoc/c;Hand2-Cre (B, D, …
Figure 5—figure supplement 1
Tissue specificity of Cre-mediated activation of lacZ expression in the developing craniofacial complex of Hand2-Cre;R26R-LacZ embryos from E9.5 to E12.5.
(A–G) X-gal staining of whole mount heads of Hand2-Cre;R26R-LacZ embryos at E9.5 (A, E), E10.5 (B, F), E11.5 (C, G) and E12.5 (D). (H) X-gal staining on frontal sections from the middle region of Han…
Figure 6 with 1 supplement
Smoc/c;Hand2-Cre embryos display tongue agenesis and ectopic ossification.
(A, B) Skeletal preparations of heads of E18.5 Smoc/c (A) and Smoc/c;Hand2-Cre (B) embryos (n = 5 for each genotype). Arrow in B indicates the shortened mandible in the Smoc/c;Hand2-Cre embryo. (C, D…
Figure 6—figure supplement 1
Smoc/c;Hand2-Cre display agenesis of the oral tongue structure.
(A–B) Frontal views of E10.5 embryos from Smoc/c control (A) and Smoc/c;Hand2-Cre (B) embryos showing expression of Myf5. Arrows show the Myf5 positive tongue muscle precursor cells. (C–H) …
Figure 7
Ectopic activation of Smo signaling inhibits BMP signaling activity in the mandibular arch mesenchyme.
(A–F) Rostral views of the mandibular arches following whole mount in situ hybridization detection of Foxf1 (A, B), Msx2 (C, D), and Alx4 (E, F) mRNAs in the E10.5 R26SmoM2 (A, C, E), and R26SmoM2;Ha…
Figure 8
Smoc/c;Hand2-Cre embryos showed Bmp4-dependent increase in apoptosis of distal mandibular mesenchyme.
(A–C) Immunofluorescent detection of active-Caspase3 (red) in E10.5 Smoc/c (A), Smoc/c;Hand2-Cre (B), and Smoc/c;Hand2-Cre;Bmp4+/- (C) embryos. Mandibles are outlined with white dash lines. (D) …
Figure 9 with 1 supplement
Foxf1 and Fox2 mediate Shh-Smo signaling function in mandible and tongue development.
(A–F) Rostral views of the mandibular arches of E10.5 Smoc/c (A, C, E) and Smoc/c;Hand2-Cre (B, D, F) embryos following whole mount in situ hybridization detection of Ptch1 (A, B), Foxf1 (C, D), and …
Figure 9—figure supplement 1
Foxf1c/c; Foxf2c/c;Wnt1-Cre display agenesis of the oral tongue structure.
(A–B) Frontal views of E10.5 embryos from control (littermates that are Cre negative) (A) and Foxf1c/c; Foxf2c/c;Wnt1-Cre (B) embryos showing expression of Myf5. Arrows show the Myf5 positive tongue …
Figure 10
Foxf1c/c;Foxf2c/c;Wnt1-Cre embryos exhibit oropharyngeally expanded BMP signaling activity in the developing mandibular arch.
(A, B) Immunofluorescent detection of phospho-Smad1/5/9 (pSmad1/5/9, red) on sagittal sections through the distal region of the mandibular arches in E10.5 control (littermates that are Cre negative) …
Additional files
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Supplementary file 1
List of marker genes with more than 1.5-fold enrichment in the individual clusters.
Column A lists gene name. Column B lists p value of differential expression. Column C lists average fold change over all other clusters. Column D list the percentage of cells in the corresponding cluster expressing the marker gene. Column E list the percentage of cells in all other clusters combined expressing the marker gene. Column F list the Bonferroni corrected p value. Column G lists the cluster name. NC1 – NC2, neural crest derived mesenchyme cells; Epi, epithelial cells; Endo, endothelial cells; HM, head mesoderm cells.
- https://doi.org/10.7554/eLife.40315.026
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Supplementary file 2
List of marker genes exhibiting differential expression (at least 1.3-fold) between cells in the NC3 cluster and cells in the NC1 and NC2 clusters.
Column A list gene name. Column B list p value of differential expression. Column C lists average fold change of expression of the marker gene in NC1/2 cells over NC3 cells. Positive value in Column C indicates higher levels of expression in NC1/2 than in NC3. Column D lists percentage of cells in NC1/2 clusters expressing the gene. Column E list percentage of cells in NC3 cluster expressing the gene. Column F list Bonferroni corrected p value of differentiation expression. Genes whose expression pattern is shown in Figure 1—figure supplement 4 are highlighted in yellow.
- https://doi.org/10.7554/eLife.40315.027
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Supplementary file 3
List of marker genes exhibiting more than 1.3-fold enrichment in expression levels in a specific neural crest subgroup over all other five subgroups.
Genes that are shown in Figure 1B are highlighted in yellow color. Column A lists gene name. Column B lists p value of differential expression. Column C lists average fold change over all other subgroups. Column D list the percentage of cells in the corresponding subgroup expressing the marker gene. Column E list the percentage of cells in all other subgroups combined expressing the marker gene. Column F list the Bonferroni corrected p value of differential expression. Column G lists the subgroup number corresponding to Figure 1B.
- https://doi.org/10.7554/eLife.40315.028
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Supplementary file 4
Top 50 hits from gene ontology (GO) analyses of marker genes of Subgroup 0 of the neural crest cells shown in Figure 1B.
- https://doi.org/10.7554/eLife.40315.029
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Supplementary file 5
Top 100 hits from gene ontology (GO) analyses of marker genes of Subgroup 1 of neural crest cells shown in Figure 1B.
GO analysis was performed using Toppgene (https://toppgene.cchmc.org/enrichment.jsp).
- https://doi.org/10.7554/eLife.40315.030
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Supplementary file 6
Top 50 hits from gene ontology (GO) analyses of marker genes of State three from developmental trajectory analysis shown in Figure 1—figure supplement 7.
- https://doi.org/10.7554/eLife.40315.031
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Supplementary file 7
Top 20 hits from gene ontology (GO) analyses of marker genes of State four from developmental trajectory analysis shown in Figure 1—figure supplement 7.
- https://doi.org/10.7554/eLife.40315.032
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Transparent reporting form
- https://doi.org/10.7554/eLife.40315.033
