(A) Expression of genes over pseudotime reflect predicted kinetics for melanophores and iridophores. Solid lines indicate smoothed expression curves for all cells in the branch. mitfa expression declined only marginally with melanophore differentiation yet decreased markedly with a transition from progenitor to iridophore as expected (Curran et al., 2010). pax3a was expressed in pigment progenitors (magenta) and decreased across pseudotime in melanophores, whereas expression of tfec, a transcription factor expressed in iridophores (Lister et al., 2011), increased over pseudotime. Melanin synthesis enzyme genes, dct and tyrp1b, as well as pmel, encoding a melanosome-associated transmembrane protein, all increased over pseudotime in melanophores. In iridophores, gpnmb and pnp4a showed elevated expression late in pseudotime, as expected (Curran et al., 2010; Higdon et al., 2013). (B) Trends of total transcript UMI counts, scores of expressed cell-cycle (e.g. ccnd1, pcna), pigment cell transcription factors (e.g. mitfa, tfec, pax7b, tfap2a), and pigment synthesis-related genes (e.g. impdh1b, gart, and atic for purine processing in iridophores; tyrp1b, pmela, and tyr for melanin synthesis in melanophores) in bins across pseudotime for melanophore and iridophore trajectory branches (for all score-associated genes, see Supplementary file 2—Table 4). Histograms indicate cell-type specific densities across pseudotime for each branch. For melanophores, total transcript number per cell decreased over pseudotime and expression levels of melanin synthesis genes increased. In iridophores, mRNA levels stayed relatively constant whereas expression of purine synthesis genes increased. The expression score for cell-cycle genes was greater for iridophores than melanophores at the terminal step of pseudotime (p<0.0002; Wilcoxon; pseudotime bin 8; n = 91 iridophores, 319 melanophores), consistent with iridophores continuing to proliferate even after differentiation, and melanophores normally failing to do so (Budi et al., 2011; Darzynkiewicz et al., 1980; McMenamin et al., 2014; Spiewak et al., 2018).