Extended Data Fig. 12: Subclustering analysis and trajectory inference of NC cluster in µNTLS.

a. UMAP of NC cluster from integrated dataset of day 4, day 9, and day 11 µNTLS, color-coded according to time points (left) or cell subcluster identity annotations (right). Seven cell subclusters are identified, including Premigratory NC (Cluster 0), Delaminating NC (Cluster 1), Schwann cell (Cluster 2), Mesenchymal cell (Cluster 3), Sensory neuron (Cluster 4), Sympathetic neuron (Cluster 5), and Melanoblast (Cluster 6). n indicates cell number. b. Dot plot showing expression of key marker genes across all NC subclusters as indicated. Dot sizes and colors indicate proportions of cells expressing corresponding genes and their averaged scaled values of log-transformed expression, respectively. c. Feature plots showing expression of selected markers associated with indicated cell subclusters in UMAP plots of NC cluster. d. Heatmap of average relative expression (Z-score) of top-20 gene signatures distinguishing each cell subcluster in NC cluster. For gene signature information, see Supplementary Table 1. e. Dot plot showing expression of HOX genes in HOX⁻ cranial NC, HOX⁺ cranial NC, vagal NC, and trunk NC. HOX⁻ cranial NC doesn’t express any HOX genes. HOX⁺ cranial NC expresses HOX paralogous group (PG) 1-2 but not HOX PG 3-13. Vagal NC expresses HOX PG 3-5 but not HOX PG 6-13. Trunk NC expresses HOX PG 6-9 but not HOX PG 10-13. Dot sizes and colors indicate proportions of cells expressing corresponding genes and their averaged scaled values of log-transformed expression, respectively. f. Stacked bar plot showing cellular compositions in HOX^- cranial, HOX⁺ cranial, vagal, and trunk NC cells, as indicated. g. Dot plot comparing expression of key marker genes in HOX^- cranial NC, HOX⁺ cranial NC, vagal NC, and trunk NC across different NC clusters as indicated. Dot sizes and colors indicate proportions of cells expressing corresponding genes and their averaged scaled values of log-transformed expression, respectively. h. UMAP of HOX^- cranial NC cells separated from NC cluster shown in a, color-coded according to time points (top) or cell subcluster identity annotations (bottom). Six cell subclusters are identified, including Premigratory NC (Cluster 0), Delaminating NC (Cluster 1), Schwann cell (Cluster 2), Mesenchymal cell (Cluster 3), Sensory neuron (Cluster 4), and Melanoblast (Cluster 6). n indicates cell number. i. UMAP of cell subclusters of HOX^- cranial NC cells associated with lineage developments of Schwann cell, Mesenchymal cell, Sensory neuron, and Melanoblast, color-coded according to pseudotime. Solid lines represent principal curves of each lineage. Pseudotime values are computed by projecting each single cell onto principal curves. j. Heatmap of smoothened expression of all differentially expressed genes (DEGs) along pseudotime of Schwann cell, Mesenchymal cell, Sensory neuron and Melanoblast lineage development trajectories in HOX^- cranial NC cells. Selected genes are highlighted. A gene is considered significant when adjusted p-value based on FDR is <0.05 (see Methods). For DEG information, see Supplementary Table 1.

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