E4F1 KO results in expansion of the basal cell compartment of the epidermis and in abnormal keratinocyte differentiation in vivo. (A) Immunostainings of dorsal skin sections from E4F1^+/flox and E4F1^−/flox;K5-Cre neonates with anti-K14 and anti–α6 integrin antibodies. (B) IHC analysis of dorsal skin sections from E4F1^+/flox and E4F1^−/flox;K5-Cre neonates with anti-K6, anti-K10, or anti-involucrin (invol) antibodies. (Scale bar: 40 μm.)

Deletion of the Ink4a/Arf locus partially rescues E4F1 KO skin defects in vivo and ex vivo. (A) Quantitative RT-PCR analyses of Ink4a and Arf mRNA levels (Upper) and p19^ARF immunoblot analyses (Lower) performed on E4F1^+/flox or E4F1^−/flox;K5-Cre freshly isolated primary keratinocytes. (B) Clonogenic assays performed with E4F1^flox;RERT;Ink4a/Arf^−/− or E4F1^flox;RERT;Ink4a/Arf^+/+ primary keratinocytes in the presence of 4OHT. Shown is one representative experiment of three independent experiments performed in duplicate. Quantitative analyses of the number and size (diameter, in mm) of clones was performed after 15 d in culture. (C) High-magnification photograph of representative clones. (Scale bar: 0.5 mm.) (D) H&E staining of dorsal skin sections prepared from 4OHT-treated E4F1^flox;RERT;Ink4a/Arf^+/+ 12-wk-old mice and E4F1^flox;RERT;Ink4a/Arf^−/− mice.

E4F1 KO triggers transient hyperplasia of the epidermis, followed by permanent loss of epidermal cells and severe skin ulcerations. (A) IHC analysis of E4F1 expression in human skin or murine back skin and HFs. (Scale bar: 40 μm.) The dotted line indicates the dermis (d)–epidermis junction. (B) Macroscopic alterations of back skin of adult E4F1^−/flox;RERT mice 7 (early) or 15 d (late) after Cre-mediated E4F1 KO. (C) E4F1^flox; RERT tails at late time points (6 wk) after 4OHT application. (D) H&E staining (Upper) and Ki67 IHC analyses (Lower) of dorsal skin sections prepared from 4OHT-treated E4F1^−/flox;RERT or E4F1^+/flox;RERT control adult mice at early and late time points. (Scale bar: 20 μm.) (E) IHC analysis of dorsal skin sections from E4F1^+/flox;K5-Cre and E4F1^−/flox;K5-Cre neonates using anti-E4F1 antibody. (Scale bar: 40 μm.) (F) E4F1^+/flox and E4F1^−/flox;K5-Cre neonates (P4) showing acute symptoms of dehydration. (G) H&E staining (Upper) and Ki67 IHC analysis (Lower) of dorsal skin sections prepared from E4F1^flox;K5-Cre P4 neonates. (Scale bar: 20 μm.) (H) H&E-stained sections of back skin from E4F1^+/flox;K5 or E4F1^−/flox;K5-Cre P1 neonates at 2 wk after engraftment onto recipient nude mice. (Scale bar: 40 μm.)

E4F1 KO results in ESC exhaustion in vivo and ex vivo. (A) Clonogenic assays performed with primary murine keratinocytes prepared from back skin of E4F1^flox;RERT adult mice 6 d after 4OHT applications (in vivo), from E4F1^flox;RERT neonatal skins and treated with 4OHT in culture medium (ex vivo), or from E4F1^flox;K5-Cre neonates (P1). Rhodamine B staining was performed after 15 d of culture. Data shown are representative of experiments performed in duplicate and repeated at least three times. (Scale bar: 1 cm.) (B) (Left) Clonogenic assays performed with human primary keratinocytes transduced with retroviral vectors expressing shRNAs targeting either human E4F1 (shE4F1) or a nonrelevant sequence (shCtrl). (Right) Quantification of the total number and size (diameter, in mm) of clones from three independent experiments. (C) FACScan analyses of primary keratinocytes isolated from E4F1^flox;K5-Cre P3 neonates after α6-integrin and CD34 immunostainings. The percentage of α6^high/CD34⁺ epidermal stem cells is indicated (average of three independent experiments ± SD). (D) IHC analyses of back skin sections from E4F1^flox;RERT adult mice at 3 wk after 4OHT application with antibodies directed against CD34 and K15. Arrows indicate K15- or CD34-positive stem cells located in the bulge region of HFs. (Scale bar: 80 μm.) (E) Whole mounts of tail epidermis prepared from E4F1^flox;RERT;K15-GFP mice at 4 wk after 4OHT application. White arrows indicate K15-GFP⁺ ESCs located in the bulge region of HFs in control animals (E4F1^+/flox) treated with 4OHT (Left). (Scale bar: 80 μm.) (F) Analyses of BrdU LRCs in whole mounts of tail epidermis prepared from 16-wk-old mice. Anti-BrdU (green) and anti-Ki67 (red) immunostainings of E4F1^+/flox;RERT and E4F1^−/flox;RERT HFs at 3 wk (early) or 6 wk (late) after E4F1 KO showing transient hyperproliferation followed by a loss of LRCs (white arrows). LRCs are located in the bulge region of HFs from control skin (E4F1^+/flox) treated with 4OHT for 6 wk. (Scale bar: 80 μm.)

Down-regulation of the Bmi1-Arf-p53 axis rescues E4F1 KO ESC defects. (A) Primary keratinocytes isolated from E4F1^flox;RERT neonates were transduced with control (pMSCV) or Bmi1-encoding retroviruses (Bmi1) and used for clonogenic assays in the presence of 4OHT. (B) Clonogenic assays performed with E4F1^flox;RERT primary keratinocytes transduced with control (shCtrl) or p53 (shp53) shRNAs encoding lentiviruses in presence of 4OHT. A and B present photographs (Upper) and quantifications (Lower) of a representative experiment. Three independent experiments were performed in duplicate. Quantitative analyses of the number and size (diameter, in mm) of clones were performed after 10 d. (C) Western blot analyses showing ectopic overexpression of Bmi1 in rescued E4F1 KO clones or shRNA-mediated depletion of p53 in rescued E4F1 KO clones.