Figure 4.

Behavior of Differentiating p53^∗/wt Keratinocytes

(A) Rendered confocal z stacks showing p53^∗/wt clone area. Lateral view shows the section of projected image (red line). White line, basement membrane; blue, DAPI; green, GFP; red, EdU. Scale bars, 20 μm.

(B) Side view of p53^∗/wt clone at the 12-week time point. White line, basement membrane; yellow line, epidermal surface; green line, outer limit of GFP-expressing cells. Green, GFP; blue, DAPI; white, F-actin. Scale bar, 20 μm, arrowheads, basal layer cells.

(C) Schematic: typical p53^∗/wt clone with GFP expression (green) confined to basal and first suprabasal cell layers.

(D) Multicolor lineage tracing in Ahcre^ERTRosa26^{flConfetti/wt}p53^∗/wt animals. Left: the multicolor confetti reporter allele encodes four fluorescent proteins. Different cre-mediated inversion and excision recombination events result in the heritable expression of one of the four fluorescent proteins depicted, nuclear GFP, yellow fluorescent protein (YFP) red fluorescent protein (RFP), or membrane cyan fluorescent protein (CFP). The fate of p53 mutant progenitors can be tracked in double-labeled clones even if the p53 locus becomes inactive in the differentiating progeny. Right: rendered z stacks of typical Confetti-p53^∗/wt double-labeled clone 12 weeks post induction of Ahcre^ERTRosa26^Confettip53^∗/wt mice. Images representative of 8 clones. Yellow arrowhead, cornified cell. Scale bars, 20 μm.

(E) Schematic: RFP expression reveals differentiation of p53^∗/wt clone.

(F) Projected area of labeled epidermis (%) in induced p53^∗/wt (open green circles) and p53^wt/wtRYFP (solid black circles) mice. Data are from 5 fields per animal. n = 3–5 mice per time point/group. Red and blue lines, mean area for p53^∗/wt and p53^wt/wtRYFP, respectively.

(G) Mean cell density in immediate suprabasal layer. Data are means of 5 fields per mouse. n = 3 mice at each time point/group except 4 fields from 1 animal are shown for YFP. ^∗p = 0.0236, ^∗∗p = 0.0035 by two-tailed unpaired t test.

(H) Single z-slice image of immediate suprabasal layer from p53^∗/wt mice at indicated time point. Schematic: typical p53^∗/wt colonized area at indicated time point. Scale bar, 20 μm.

(I) Rendered confocal z stacks showing side view of epidermal whole mounts. Yellow bars indicate the thickness of epidermis. Green, GFP; blue, DAPI; white, F-actin. Scale bar, 20 μm. White line, basement membrane; yellow line, epidermal surface.

(J) Detail analysis of change in epidermal thickness. Top, confocal images (side view) showing changes in p53^∗/wt epidermis over time. Yellow bars, length of cells in first suprabasal layer; asterisks, each cell layers. Arrowhead indicates cell in between layers. Scale bar, 20 μm. Bottom, mean thickness of epidermis at time points indicated. n ≥ 3 mice at each time point/group. ^∗p = 0.016, ^∗∗p = 0.0043 by Mann-Whitney test.

See also Figure S6 and Table S2–S4 and S7.