Fig. 1: Epidermal stem cell differentiation occurs over multiple days.
a, Whole-mount staining of K10 protein (red) and K10 reporter (K10rtTA; pTRE-H2BGFP) expression (green) in suprabasal and basal cells from ear epidermis. Cell boundaries are visualized with phalloidin (white). Insets show examples of K10-positive basal cells with very little (1) or average (2) amounts of ECM contact. Scale bars, 25 µm or 5 µm (insets). b, Percentage of basal cells with indicated levels of ECM contact that also express K10 protein. Graph represents average from one independent immunostaining experiment using n = 3 mice. c, Overlap between K10 protein expression and K10 reporter expression in basal and suprabasal cells. Graph represents average from one independent immunostaining experiment using n = 3 mice. d, Revisited basal cell as it induces K10 reporter expression and later exits the basal layer. Scale bar, 10 µm. e, Basal versus suprabasal position in basal cells scored as K10 reporter positive on day 0 and revisited for 5 subsequent days. Graph represents average of n = 3 imaged mice. f, K10 reporter expression levels in the days preceding basal layer exit. Graph represents 90 pooled cells from n = 3 imaged mice (see also tracks from individual cells in Supplementary Fig. 3). One-way ANOVA, P < 0.0001; Tukey’s HSD, P < 0.0001 (−120 h versus −96 h, −96 h versus 72 h, −72h versus −48 h, −24 h versus 0 h). g, Membrane GFP (K14CreER; mTmG) labelled basal cell revisited every 12 h in the days preceding delamination. Top row: xy section from the widest point in the upper half of the cell. Middle row: xy section directly above ECM (blue). Bottom row: lateral re-slice. Scale bar, 10 µm. h, Basal cell–ECM contact in the days preceding basal layer exit. Graph represents 72 pooled cells from n = 3 imaged mice. One-way ANOVA, P < 0.0001; 36 h versus 24 h, 24 h versus 12 h, and 12 h versus 0 h Tukey’s HSD, P < 0.0001. i, Suprabasal spreading, measured as the widest section in the upper half of each cell, in the days preceding delamination. Graph represents 68 pooled cells from n = 3 imaged mice. One-way ANOVA, P < 0.0001; 36 h versus 24 h, 24 h versus 12 h, and 12 h versus 0 h Tukey’s HSD, P < 0.0001. For f, h and i, box centres indicate median, boundaries represent 25th and 75th percentiles, and error bars represent maximum and minimum values. For bar graphs in b, c and e, error bars are mean ± standard deviation (s.d.).
