Fig. 1: Description of the mouse TSC differentiation time course experiment.

a In vitro differentiation of mouse TSCs was induced by withdrawal of the stemness-maintaining components FGF and embryonic fibroblast conditioned medium (CM; Remove dataset) or by inhibition of MEK with PD0325901 (Inhibit dataset). Remove conditions are expected to favour initial lineage entry towards junctional zone precursors, whereas the continued presence of CM in Inhibit conditions is expected to enrich for labyrinth precursors. Samples were collected at t0 (stem cell state, n = 2 independent experiments), 1 h (n = 4 independent experiments Inhibit and Remove), 4 h (n = 4 independent experiments Inhibit and n = 3 independent experiments Remove), 24 h (n = 4 independent experiments Inhibit and n = 2 Remove), 36 h (n = 4 independent experiments Inhibit and Remove) and 48 h (n = 4 independent experiments Inhibit and Remove). Cells were dissociated and single cell barcoded transcriptome libraries were generated using Drop-seq and sequenced. UMI = Unique molecular identifier. b Phase contrast images of cells cultured over the outlined time course in Remove and Inhibit conditions. Images are representative of three independent experiments. c RT-qPCR for junctional zone (JZ) and labyrinth markers after 24 h of differentiation in Remove or Inhibit conditions. Data are normalised to Remove conditions at 24 h and plotted as mean +/- SEM of n = 3 independent replicates. Statistical significance was calculated using two-sided unpaired t-test. * p < 0.05, ** P < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file which contains exact P values. Rel. expression = relative expression. d Immunofluorescence staining of cells grown for 48 h in Remove and Inhibit conditions for JZ marker NCAM1 (red) and labyrinth (Lab) marker STRA6 (green). Image is representative of three independent experiments.