Pdx1 Is Expressed in the Skin Figure 3. Mosaic Cre-mediated recombination in Pdx1-Cre mice. A: Pdx1-Cre;ROSA26R-LacZ reporter mice reveal patchy X-Gal staining as surrogate for the Pdx1 cell lineage in the pancreas (i) and in whole mount skin (ii). Analysis of X-Gal+ areas of the epidermis indicates that recombined keratinocytes are localized primarily in suprabasal layers of the skin (iii). Early cutaneous hyperplasia sections demonstrate that X-Gal+ cells are also located in the basal layer of the epidermis (iv). Asterisks indicate non-recombined areas of pancreatic tissue; arrowheads point to recombined X-Gal+ cells and regions; arrows show positive basal layer keratinocytes. B: Cre-mediated recombination of the Notch1 locus occurs predominantly in suprabasal keratinocytes (K10+) with a small fraction of recombined basal cells (K14+). Schematic depiction of areas of possible Pdx1-Cre driven recombination in the epidermis (right): (SC) Stratum Corneum, (GL) Granular Layer, (SL) Spinous Layer, (BL) Basal Layer, (BM) Basement Membrane, (D) Dermis, (KC) Keratinocytes. C: Immunohistochemical staining of healing wound epidermis indicates increased expression of PDX1 in keratinocytes comparing to normal skin. D: Expression of Pdx1 along with Keratin6 is induced in wounded skin as revealed by qRT-PCR. The scale bars represent 50 mm. doi:10.1371/journal.pone.0013578.g003 B). These results support the hypothesis that p21 is mainly regulated by Notch1 but not by Notch2 potentially due to cell- and context-specific differences. Notch1 but not Notch2 is a suppressor of b-catenin in the skin As an increased level of active b-catenin is commonly associated with skin malignancies [18,42,43], we investigated the regulation of this pathway in Notch1 and Notch2 ablated epidermis. Immunohistochemical analysis revealed increased levels of nuclear localized b-catenin (active b-catenin) in K5;N1ko mice in agreement with previous studies [14]. Remarkably, neither wildtype nor K5;N2ko mice showed strong epidermal active b- catenin staining (Fig. 5D). Furthermore, immunoblot analysis of primary keratinocytes isolated from K5;N1ko and K5;N2ko mice exhibited a similar pattern (Fig. 6A). Differences in expression of Notch1 and Notch2 in the epidermal layers as well as receptor-specific regulatory mechanisms may contribute to distinct and potentially tumorigenic alterations of b- PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13578