Spatiotemporal cellular movement and fate decisions during first pharyngeal arch morphogenesis

Abstract

Cranial neural crest (CNC) cells contribute to different cell types during embryonic development. It is unknown whether postmigratory CNC cells undergo dynamic cellular movement and how the process of cell fate decision occurs within the first pharyngeal arch (FPA). Our investigations demonstrate notable heterogeneity within the CNC cells, refine the patterning domains, and identify progenitor cells within the FPA. These progenitor cells undergo fate bifurcation that separates them into common progenitors and mesenchymal cells, which are characterized by Cdk1 and Spry2/Notch2 expression, respectively. The common progenitors undergo further bifurcations to restrict them into osteogenic/odontogenic and chondrogenic/fibroblast lineages. Disruption of a patterning domain leads to specific mandible and tooth defects, validating the binary cell fate restriction process. Different from the compartment model of mandibular morphogenesis, our data redefine heterogeneous cellular domains within the FPA, reveal dynamic cellular movement in time, and describe a sequential series of binary cell fate decision-making process.

Fig. 1. Patterning domains in the first pharyngeal arch.

(A) Scanning electron microscopy scan of mouse embryo at E10.5 shows the general scheme of the intrapharyngeal arch patterning domains (top); histology of the first pharyngeal arch at E10.5 shows the homogeneous cellular morphology (bottom). PA1, first pharyngeal w of E10.5 mandibular primordium identified 20 major clusters representing eight different cell types. (C to F) FeaturePlots of patterning genes representing different domains. Lhx6 is expressed in the oral domain, Gsc is expressed in the aboral domain, Nbl1 is expressed in the proximal domain, and Hand1 is expressed in the distal domain. (G to J) RNAscope analysis of patterning genes representing different domains. (K) DotPlot of signature genes of mesenchymal clusters. (L) Schematic of cluster mapping within the first pharyngeal arch. The dotted circle indicates the mesodermal core. For example, clusters 2, 12, 6, and 7 as shown with Hand1, Alx3, and Lix1 expression are in the distal domain of the first pharyngeal arch.

Fig. 2. Lineage specification of CNC cells within the mandibular primordium at E12.5.

(A) Cell annotation on UMAP plot of E12.5 mandibular primordium indicates different cell types based on known differentiation marker genes expression. (B) Pseudotemporal analysis of mesenchymal population showing the multilineage differentiation process of postmigratory CNC cells within the first pharyngeal arch. (C) Selected marker gene expression of each cell lineage in green. (D) Pseudotime trajectory of chondrogenic lineage. Top left: Pseudotime trajectory colored by cell clusters; bottom left: pseudotime trajectory colored by timeline, and heatmap showing transcriptional dynamics during differentiation. Specific genes are listed on the right to show their expression dynamics. (E and H) FeaturePlot of genes in the intermediate stage of chondrogenic differentiation at E10.5. (F, G, I, and J) Immunofluorescence (F and G) and RNAscope (I and J) detection of indicated protein or mRNA transcripts for genes in the intermediate stage (cluster II) at E10.5 and E12.5, respectively. M, Meckel’s cartilage.

Fig. 3. Integrative analysis of mouse mandibular development reveals sequential fate decisions of postmigratory CNC cells.

(A) Seurat 3 UMAP plot integration of mandibular primordium at E10.5, E12.5, and E14.5. (B) Marker gene expression in the integration analysis showing terminally differentiated lineages. (C) Seurat 3 UMAP plot integration of mandibular primordium at E10.5, E12.5, and E14.5 based on the developmental stages. The red-boxed area is the epithelial compartment, and the blue-boxed area shows the initial fate bifurcation of the postmigratory CNCs. (D) Monocle 3 pseudotime trajectory of CNC-derived mesenchymal cells showing the sequential fate decisions of postmigratory CNC cells. Top: Pseudotime trajectory colored by stage; bottom left: pseudotime trajectory colored by timeline; bottom right: schematic drawing representing the hierarchy of differentiation trajectory.

Fig. 4. Fate decision of postmigratory CNC cells at E10.5.

(A) FeaturePlot of markers representing the first bifurcated lineages toward common progenitors (Cdk1⁺) and stromal cells (Spry2⁺). (B) Cdk1 and Spry2 show largely branch-specific expression patterns, but are coexpressed in some cells at E10.5. X and Y axes indicate the expression level of each gene in the cells. (C) Heatmap showing expression of branch-specific genes in cells assigned to either lineage. (D to F) RNAscope detection of indicated mRNA transcripts showing lineage bifurcation of the postmigratory CNC cells starting from the proximal region of the first pharyngeal arch toward the oral and aboral directions. Arrow points to Spry2 expression in the oral domain, and arrowhead points to the Cdk1 expression in the proximal region. Boxed area highlights the coexpression of Spry2 and Cdk1. (G) E10.5 clusters in Seurat 3 integration embedding showing different differentiation potential of cells within the first pharyngeal arch mesenchyme. (H) RNA velocity analysis projected onto the t-SNE plot. The arrows indicate the predicted future cell states. (I) Heatmap of whole-mount immunofluorescence of Ki67 expression in E10.5 embryo. Arrow points to the strong expression of Ki67 in the proximal region of the first pharyngeal arch. (J to L) Immunofluorescence of Ki67 expression in E10.5 embryo section. Red-boxed area shows the proximal region of the first pharyngeal arch, and yellow-boxed area shows the distal region of the first pharyngeal arch. Arrows point to Ki67-positive cells.

Fig. 5. Dynamic intra-arch cellular movement contributes to postmigratory CNC cell fate decision.

(A) FeaturePlot of Gbx2 expression at E10.5. (B) RNAscope detection of Gbx2 expression at E10.5 in the proximal region of the first pharyngeal arch. (C to G) Whole-mount views of lineage tracing of Gbx2CreER;tdTomato mice at different time points, as indicated. (C, E, and G) Sagittal views. (D and F) Frontal views. Arrows point to tdTomato-positive cells. (H) Section of E15.5 mandible of Gbx2CreER;tdTomato mouse. (I to M) Immunostaining of Sox9 expression on section of Gbx2CreER;tdTomato at E12. Boxed area in (L) is enlarged in (M). Arrows in (M) point to chondroblasts that are derived from Gbx2⁺ cells. FITC, fluorescein isothiocyanate. (N to S) Immunostaining of Runx2 expression on section of Gbx2CreER;tdTomato at E13.5. Yellow and green boxes in (Q) are enlarged in (R) and (S), respectively. Arrows in (S) point to osteoblasts that are derived from Gbx2⁺ cells. (T to X) Mandibular arch ex vivo culture with DiI labeling on day 0 and day 2. Boxed areas in (T) to (W) are enlarged in (T′) to (W′), respectively. Arrows in (U′) point to migrated CNC cells. (X) Quantification of the DiI-positive area of the ex vivo culture after 2 days. Scale bars, 500 μm.

Fig. 6. Disruption of CNC cell patterning leads to impaired lineage commitment.

(A) UMAP visualization of canonical correlation analysis of control and Wnt1Cre;Alk5^fl/fl mice mandible primordia. Arrows point to the clusters representing fibroblasts (2) and osteoblasts (7). (B) Quantification of cell number percentage of Runx2⁺ osteoblasts and Dlk1⁺ fibroblasts in control and Wnt1Cre;Alk5^fl/fl mice mandible primordia at E12.5. (C) Violin plots of Runx2 and Dlk1 expression in each cluster. (D) Pseudotime trajectories of mesenchymal cells of control and Wnt1Cre;Alk5^fl/fl mice mandible primordia. The colors represent the cell clusters in t-SNE embedding. (E to L) Immunofluorescence of Runx2 and Dlk1 expression in control and Wnt1Cre;Alk5^fl/fl mice at E12.5. Boxed areas in (E), (G), (I), and (K) are enlarged in (F), (H), (J), and (L), respectively. * in (H) indicates the reduced expression of Runx2. Dotted line in (J) outlines the mandible bone primordium. Arrows in (L) point to the ectopic expression of Dlk1 in mandible bone primordium in Wnt1Cre;Alk5^fl/fl mice. M, Meckel’s cartilage; T, tongue.