Figure 5.

(A) Stacked barplot showing percentages of cell types at each time point from 12 to 24 hpf. Color code corresponds to Figure 2C.

(B) New larger scRNA-seq dataset generated at 18 hpf. Dimensionality reduction and unbiased clustering revealed 5 distinct clusters: 2 skeletal-like clusters; a pigment cluster; a transitional cluster; and a cluster showing signs of cell stress. Color code distinct from (A).

(C) Heatmap showing top 10 marker genes for each cluster; colored bar across the top indicates color code corresponding to (B).

(D) Boxplots showing early NC, pigment, and skeletal module scores for each cluster. Color coding corresponds to (B) and (C).

(E) Confocal micrographs of isHCR for known markers of early NC (foxd3, white), pigment (mitfa, green), and skeletal (dlx2a, blue) progenitors in tg(sox10:-lynTdTomato) embryos where NC plasma membranes are marked by tdTomato (red). Expression of foxd3 overlaps strongly with mitfa and to a lesser extent with dlx2a. Although mitfa and dlx2a mostly do not overlap, a few cells do have overlapping expression (indicated by white arrows).

(F) RNA velocity showing differentiation trajectories (arrows) for all cells. The transitional population (inset) is split between progressing toward the skeletal (blue) and pigment (green) clusters.

(G) Heatmaps for pigment and skeletal branches with cells ordered by pseudotime. One set of genes marks the transition from early NC to skeletal, and another marks the transition from early NC to pigment. Delineation between early and late genes along these trajectories is marked by horizontal black lines.

(H) Local regression graphs showing expression scores for early and late pigment and skeletal markers with cells ordered by pseudotime. A clear branch point marks emergence of pigment (green) and skeletal (blue) lineages.

For micrographs, scale bars represent 50 μm.