Figure 7.
Long-Term UV Exposure Depletes Epidermis of p53∗/wt Cells
(A) Protocol: AhcreERT-p53∗/wt mice were induced (green arrow), treated with a single dose of dimethylbenzanthracine (DMBA, black arrow), followed by repetitive sub-minimal erythema doses of UVB (red bars); blue arrows indicate sampling.
(B) Confocal z stacks showing projected views of UV-irradiated and an adjacent unexposed area of IFE. Blue, DAPI; green, GFP, reporting p53∗ transcription; red, p53 indicating a p53 protein-stabilizing mutation. Scale bar, 20 μm.
(C) Labeled projected area of p53∗/wt IFE in UV-exposed (purple circles; red line indicates mean value) and adjacent unexposed areas (green circles; red lines indicate mean value). n = 3 at the 12-week time point and n = 5 at the 28- to 36-week time point. Comparison between different time points, within same animal (unexposed versus UV irradiate area), ∗∗p = 0.0046, ∗p = 0.036 by unpaired two-tailed t test, ∗∗p = 0.0071 by paired t test.
(D) Proportion of labeled basal cells in UV-irradiated IFE at indicated time points. Values are mean percentage from 5–8 fields per animal. n = 3 mice per time point. Red bars, mean value. ∗p = 0.042 by unpaired two-tailed t test.
(E) Hypothesis: effect of prolonged low-dose UV on p53∗/wt clonal dynamics. Following induction, in the absence of UV, p53∗/wt clones (green) expand progressively in a background of wild-type cells (beige). In UV-exposed IFE, a wide variety of different mutant clones arises, indicated by multiple colors, some of which may expand, outcompete, and displace p53∗/wt cells from the IFE.
(F) Simulation of clone competition under ongoing mutagenesis (see Video S1). A transgenic mutant (green) is induced at 1% frequency in a background of wild-type cells (yellow). Subsequently, new mutations (red cross, shades of green if in transgenic cells, other colors if in wild-type cells) are induced at random and assigned a fitness value as described in STAR Methods.
(G) The number of mutations detected per square millimeter by the Shearwater algorithm in each 16-mm2 biopsy of mouse back skin: 3 induced and 2 non-induced mice at 12 weeks, 4 induced and 3 non-induced mice at 36 weeks. Solid circles, non-induced samples; open circles, induced samples. Green, non-exposed skin; purple, UV irradiated. ∗∗p < 0.01, ∗p = 0.02 by two-tailed Mann-Whitney test. Note that this method does not detect the induced and uninduced p53∗ allele.
(H) Confocal z stacks showing p53∗/wt clone in direct contact with p53-immunopositive clone in UV-irradiated area (36 weeks post-induction). Blue, DAPI; green, GFP; red, p53. Scale bar, 20 μm. Dashed lines indicate outline of clones in top-down image and basement membrane in lateral view. Graph on the right shows proportion of p53 immunopositive area in UV-irradiated skin. Measurement of 8 fields of view per mouse are plotted individually. n = 3 mice per time point. Red lines indicate mean values. ∗∗∗∗p < 0.0001 by two-tailed Mann-Whitney test.