Ets1 in Squamous Neoplasia Figure 4. Ets1 expression disrupts the normal differentiation pattern of the skin epidermis. Immunofluorescent staining of epidermal differentiation markers on dorsal skin of induced Ets1 BT mice and littermate wild type mice. Each section was stained with antibodies to b4- integrin to mark the position of the basement membrane and with DAPI to detect nuclei. Epidermal differentiation was assessed by staining for (A) the basal layer marker keratin 14 (K14), (B) the spinous layer marker keratin 10 (K10) and (C) the granular layer marker loricrin (Lor). (D) Western blot analysis of dorsal skin extracts to confirm results obtained in immunofluorescence. GAPDH serves as a loading control for Western blots. doi:10.1371/journal.pone.0004179.g004 Figure 5. Ets1 expression induces hyperproliferation of the skin epidermis. Immunostaining for epidermal hyperplasia with antibodies to specific markers found in hyperplastic tissues. (A) Staining with anti-keratin 6 (K6) (counterstained with DAPI to detect nuclei). (B) Staining with anti-DNp63 (counterstained with nuclear fast red (NFR)). (C) Staining with anti-Ki67 (counterstained with hematoxylin). (D) Staining with anti-PCNA (counterstained with anti-K14). Note that the proliferating cells are located predominantly in the basal layer in both BT and control samples. doi:10.1371/journal.pone.0004179.g005 (Figure 6B), which was confirmed by staining for an endothelial marker, PECAM1 (CD31) (Figure 6C). Because the Ets1 transgene is not expressed in endothelial cells (although the endogenous Ets1 gene is expressed in this cell type), this increased angiogenesis is likely due to the release of pro-angiogenic molecules from keratinocytes in the BT epidermis. Ets1 affects epithelial homeostasis in a reversible and age-dependent fashion When BT animals that were severely affected at an early age (4– 12 weeks) were returned to a Dox diet to suppress the transgene expression, they recovered completely within six weeks and became indistinguishable from their wild type or single transgenic littermates (Figure 7A). This overall reversal of skin phenotype was also clearly apparent upon microscopic examination of biopsies taken from BT mice before and after Dox rescue (Figure 7B). Within 6 weeks of turning the expression of HA-Ets1 transgene off, the epidermis regressed back to its wild type appearance, but for a residual sebaceous hyperplasia and some dermal fibrosis. The studies described above were performed on bi-transgenic mice at an early age (4–12 weeks). To test the effects of Ets1 induction in older animals, we withdrew Dox supplementation from animals that were older than six months of age. The animals that underwent a late induction presented with a milder phenotype including minimal hyperplasia and focal hair loss (Figure 7C). Thus, there is an age-dependent severity of the phenotype observed. Re-induction of the Ets1 transgene leads to localized lesions To determine whether repeat induction of the transgene would result in a more severe phenotype, we induced young adult BT mice, followed by transgene suppression (by Dox supplementation) and then reinduction at a later age (Figure 8A). The mice chosen for these analyses had a severe visible phenotype in the first induction within a short period, but surprisingly exhibited a more localized phenotype in the second induction only after an extended period of induction (4–7 months). The re-induced mice developed discrete papillomatous or deep ulcerative lesions often with a distinct crateriform appearance, whereas the bulk of the PLoS ONE | www.plosone.org 6 January 2009 | Volume 4 | Issue 1 | e4179