(A) Generation of the TAp63 conditional knockout mice. The TAp63 targeting vector was generated by inserting loxP sites (triangles) flanking exon 2 and a neomycin (neo) cassette flanked by frt sites (squares) in intron 2. Primers used for genotyping by PCR of wild-type (blue) and TAp63 conditional knockout (fl) (red) alleles are shown. The targeted region of the floxed allele is depicted by a dashed yellow line. (B) Southern blot analysis of genomic DNA from TAp63fl/+, WT, and TAp63−/− mice. (C) PCR of DNA from wild-type (WT), TAp63+/− (+/−) and TAp63−/− (−/−) mice. (D, E) Quantitative RT-PCR of (D) TAp63 mRNA from wild-type (WT) and TAp63−/− keratinocytes or of (E) ΔNp63 mRNA from wild-type (blue) and TAp63−/− (red) keratinocytes and day 9.5 (E9.5) embryos. GAPDH was used as an internal control. (F, G) Western blot for TAp63 (F) in wild-type (WT) and TAp63−/− passage 2 keratinocytes or for ΔNp63 (G) in wild-type (WT) and TAp63−/− E9.5 and E13.5 embryos and keratinocytes. Actin was used as an internal control. (H) Dorsum of an 8-month TAp63−/− mouse with an ulcerated wound. (I) H&E cross section of skin from a 1 month TAp63−/− mouse with a blister. Arrows indicate the split at the epidermal-dermal junction with entrapped serum. (J, K) Immunocytochemistry for keratin 14 (green) and collagen IV (red) of skin from 1 month wild-type (J) and TAp63−/− mice (K). DAPI was used as a counterstain. Magnification 200X. White arrow indicates the beginning of the dermal/epidermal separation. N=6 mice per group. (L) X-ray of 8 month wild-type (WT) and TAp63−/− mice. (M) Kaplan-Meier survival curve for wild-type (WT) and TAp63−/− mice. Median survival for TAp63−/− and WT mice is 333 and 712 days, respectively, n=29 mice per group, p<0.001.

(A) Confocal images of immunocytochemistry of a hair follicle from a young, wild-type mouse for fibronectin (green), NCAM (dark blue), p63 (red), and Hoechst 33258 (blue). Hatched line denotes the dermal papilla (DP), yellow arrowhead indicates a p63-positive dermal sheath (DS) cell, arrows show a p63-positive dermal papilla cells, and white arrowheads epidermal cells with nuclear p63. Magnification 630X. (B) RT-PCR for TAp63 mRNA in Sox2:EGFP-positive (+ve) and negative (−ve) cells from adult backskin. #1, 2 and 3 are samples from 3 different mice, and the ungated sample is total skin cells from one mouse. (C) SA-β-gal staining (blue) of hair follicles in young wild-type or TAp63−/− skin (left two panels). The arrows indicate SA-β-gal-positive dermal papillae. The right two panels, (a) and (b), are micrographs of sequential sections through the same hair follicle, 40µm apart. The arrowhead indicates an SA-β-gal-positive dermal sheath cell (DS), and the arrows a positive dermal papilla (DP). (D) Quantification of SA-β-gal positive cells in the dermal papillae (DP). n = 3 animals per group. *p<0.05. (E) H&E stained skin sections of 1, 6, and 12 month wild-type (WT) and TAp63−/− mice. Arrows denote hair follicles, and double-arrows indicate regions of TAp63−/− skin depleted of hair follicles. Magnification 100X. (F) Quantification of hair follicles per millimeter (E). N = 8 and 4 mice each for wild-type (WT) and TAp63−/− at 1 month and 12 months, respectively. *p<0.05. (G) Quantification of hair follicles in wild-type (WT) and TAp63−/− mice at 1, 6–9, and 12 months. Only patches of skin with no follicles for greater than 400µm were included. N = 4 mice of each genotype at each time point. *p<0.05.

(A,B) Photomicrographs of wild-type (A) and TAp63−/− (B) H&E stained skin sections 6 days after wounding. Dotted lines indicate the dermal-epidermal boundary and boxes denote area of immunohistochemical analysis on serial sections shown at higher magnification in panels D, E, G, H, I, & J. (C) Bar graph indicating relative wound size 6 days after wounding in wild-type (WT), TAp63−/− (−/−), TAp63fl/fl;K14cre− (K14cre−), and TAp63fl/fl;K14cre+ (K14cre+) mice. N=6 mice per group, *p<0.05. (D,E) Immunohistochemistry for BrdU incorporation 6 days after wounding in wild-type (D) and TAp63−/− (E) mice. (F) Quantification of BrdU incorporation in epidermal cells in sections shown in panels D,E in WT, TAp63−/−, TAp63fl/fl;K14cre−, and TAp63fl/fl;K14Cre+ mice. N=6 mice per group, *p<0.001. (G–J) Immunohistochemistry for keratin 5 (G,H; brown) or TAp63 (I,J; brown) in wild-type (G,I) or TAp63−/− (H,J) skin. (K) Quantification of BrdU incorporation in dermal cells. In panels (D, E, G, H, I, J), tissue was counterstained with hematoxylin. Arrowheads denote BrdU positive dermal and epidermal cells (D, E) and TAp63-positive epidermal cells (I). N=6 mice per genotype. (L) qRT-PCR of mRNA from WT, TAp63−/−, and TAp63fl/fl;K14cre+ epidermal cells extracted from E18.5 skin. N=6 mice per genotype in triplicate. (M) Western blots for TAp63 and p53 in stressed keratinocytes (passage 2) and freshly-isolated keratinocytes (passage 0) from wild-type (WT) and TAp63−/− mice. (N) Western blot for TAp63 in WT, TAp63−/−, and TAp63fl/fl;K14cre+ stressed keratinocytes (passage 2). Actin was used as an internal control. Keratinocytes derived from 3 independent embryos for each genotype and performed in triplicate.

(A) Epidermal colonies from wild-type (WT), TAp63fl/fl, TAp63−/−, TAp63−/−;p53−/− and TAp63fl/fl;K14Cre+ mice cultured for 8 or 12 days on J2 3T3 feeder layers and stained with rhodamine B. (B) Immunostaining for BrdU (green) and K5 (red) in wild-type and TAp63−/− epidermal clones. (C,D) Quantification of BrdU incorporation in colonies after 8 (C) or 20 (D) days in culture. *p<0.05. N = 3 cell lines per genotype in triplicate. (E) Immunostaining for the spinous marker, K10 (green), in wild-type (WT) and TAp63−/− colonies cultured in high calcium (1.2 mM) media for 2 days. DAPI was used as a counterstain.

(A, B) RT-PCR in WT and p63−/− SKPs for (A) TAp63 and ΔNp63 mRNAs and (B) p53, p73, and GAPDH mRNAs. Embryonic day 18 cortical tissue (C) was used as a positive control. (C) Immunocytochemistry for p63 (green) in SKP spheres isolated from WT skin, from reconstituted hair follicles (WT follicle), or from E18 p63−/− skin. Hoechst (blue) used as a counterstain. Magnification 400X. (D) SKP spheres isolated from young WT or TAp63−/− skin, or from E18 p63−/− skin and immunostained for fibronectin, nestin, vimentin, smooth muscle actin (SMA) and versican. Magnification 400X. (E) SKP spheres isolated from neonatal WT skin or from E18 p63−/− skin, immunostained for Ki67 (red), and counterstained with Hoechst (blue). Magnification 400X. (F) Quantification of Ki67 positive cells. n = 3, *p < 0.05. (G) Quantification of cells that form a new sphere at clonal density (2500 cells/ml) in methylcellulose cultures. n = 3, *p < 0.05. (H) RT-PCR analysis for p57^Kip2 and GAPDH mRNAs in E18 cortex (C), young WT and TAp63−/− SKPs and E18 p63−/− SKPs. (I) SKP spheres isolated from young WT and TAp63−/− skin, immunostained for p57^Kip2 (red) and counterstained for Hoechst (blue). Magnification 400X. (J) Quantification of p57^Kip2 positive cells. n = 3, *p < 0.05. (K) ChIP for p63 in young WT or E18 p63−/− SKPs and PCR for the p57^Kip2 core promoter. (L) Immunostaining for p57^Kip2 (red) and Ki67 (green) in TAp63−/− SKPs transfected with or without a p57^Kip2 expression vector. Hoechst (blue) was used as a counterstain. Transfected p57^Kip2-positive cells were negative for Ki67. Magnification 200X, n=3.

(A) SKP spheres from young WT and TAp63−/− SKPs stained for SA-β-gal (blue). Magnification 400X. (B) SKP spheres from young and aged WT and young TAp63−/− skin immunostained for p16^Ink4a (red) and Ki67 (green) and counterstained with Hoechst (blue). Green arrowheads indicate Ki67, white arrowheads indicate p16^Ink4a, and white arrows indicate Ki67 and p16^Ink4a double positive cells. Magnification 400X. (C) Quantification of p16^Ink4a-positive and p16^Ink4a, Ki67 double-positive cells. n = 3, *p < 0.05. (D) High magnification of a TAp63−/− SKP cell nucleus that was labeled for p16^Ink4a (red) and Ki67 (green) and counterstained with Hoechst (blue). (E,F) Quantification of SA-β-gal positive (E) or apoptotic (F) keratinocytes in wild-type (WT), TAp63−/−, and TAp63−/−;p53−/− cultures. N=3 cell lines for each genotype, performed in triplicate. *p<0.0001.

(A) SKP spheres from young and aged WT and TAp63−/− skin, immunostained for γ-H2AX (green) and counterstained for Hoechst (blue). Magnification 400X. (B) Quantification of γ-H2AX positive cells. n = 3, *p < 0.05. (C) Immunostaining for γ-H2AX (green) in aged wild-type and young TAp63−/− skin and counterstained Hoechst (blue). Arrows denote hair follicles, and arrowheads indicate positive cells within the dermal sheath. Magnification 200X. (D) Immunostaining for γ-H2AX foci (red) in wild-type and TAp63−/− keratinocytes. DAPI was used as a counterstain. (E) Metaphase spreads of wild-type and TAp63−/− primary keratinocytes (passage 0). Cytogenetic aberrations in TAp63−/− cells are indicated by colored arrows. Chromosomal fusions (black arrows), fragments (red arrows), breaks (green arrowheads), biarmed chromosomes (blue arrow), ring chromosome (blue arrowhead).