Fig. 5: Sequential transcriptional programs drive motile ciliogenesis.

a Histological overview of human basal cell ALI mucociliary differentiation. Image outlines: shades of black = submerged culture, shades of blue to red = polarized differentiation and maintenance of ALI human epithelial cell culture. Representative brightfield or H&E stained images of indicated timepoints are shown, scale bars in far left panels are both 50 μm. b tSNE plot depicts the distribution of inferred clusters of in vitro cells transcriptionally sampled from across the entire differentiation time course. Cluster identities based on expressed markers are shown at the right. Ciliated cell clusters are boxed. c Proportion of cells in each cell state (corresponding to clusters in b) present at each timepoint over differentiation. Time course black/blue/red gradient coloring at bottom corresponds to colors in a. d Heat map depicts gene signatures of three ciliated cell states (function summarized in schematic above) in human airway epithelial ALI cultures sampled across differentiation. Genes plotted were those with known ciliogenic function that were characteristic of one of the three states, as indicated. e Similarities and differences in transcriptional programs between distinct pseudotime lineages constructed with Slingshot⁸¹ that lead to mature secretory and ciliated cells in vitro. Select markers or genes correlated with pseudotime are indicated. f Dot plots reveal TFs exhibiting expression associated with ciliated states in vitro. g Heat map illustrates differences in proportions of spliced or unspliced transcripts for a given gene (one per row) between later ciliating and mature ciliated cells that exhibit non-zero expression for the gene. mRNA splice status was inferred using the Velocyto⁷⁸ pipeline. Genes listed are cilia-related genes with non-zero expression (ignoring splicing) in at least 10% of cells for at least one of the later ciliating or mature ciliated cell populations. See Supplementary Fig. 7.