Fig. 6
Single cell RNA-seq analyses reveal heterogeneity within a McSC-derived melanoma. a, b t-SNE plots showing the segregation of single cells into 5 clusters based on unbiased clustering. a shows the original identity of cells (McSC: red; tumor: cyan), b shows the 5 clusters. Because Tyr and c-Kit promoters target the exact same population of follicular McSCs, we chose to do RNA-seq with McSCs isolated from only one of them. c GSEA plot showing neuronal signatures enriched in cluster 2 and 3 compared to cluster 4 and 5 (Top). Violin plots show the distribution of specific neuronal genes in 4 tumor clusters (Bottom). d GSEA plot showing proliferation signatures enriched in cluster 3 compared to clusters 2, 4, and 5 (Top). Violin plots show the distribution of specific proliferation markers in 4 tumor clusters (Bottom). e GSEA plot showing mesenchymal signatures enriched in cluster 5 compared to clusters 2, 3, and 4 (Top). Violin plots show the distribution of specific mesenchymal genes in 4 tumor clusters (Bottom). f Diagram showing that among the top 400 enriched genes in each cluster, cluster 4 shares 73 common genes with cluster2 and 124 genes with cluster5, while cluster 2 and 5 only share 21 genes. g–i Plots showing monocle-generated pseudo-temporal trajectory. g is colored by pseudotime in a gradient from dark to light blue and start of pseudotime is indicated, h is colored by the original identify of cells, i is colored by the 5 clusters shown in b. Pseudotime ordering on McSCs and melanoma cells arranged them into 3 branches (A, B and C) and 2 paths (A to B, A to C). j Branched heat map showing changes of gene expression along pseudotime in both paths. The beginning of pseudotime (branch A) is in the middle of the heatmap. The path from branch A to B along pseudotime is plotted from the middle of the heatmap to the left, while the path from branch A to C is from the middle to the right. The color scale illustrates the normalized gene expression across cells. k Schematic model illustrating the transformation of McSCs with oncogenic mutations into heterogeneous melanoma. TFs transcription factors, EMT epithelial-mesenchymal transition, McSC melanocyte stem cell, C Cluster
