Pdx1 Is Expressed in the Skin Figure 6. Biochemical analysis of K5;N1ko and K5;N2ko keratinocytes. A: Western blot analysis of primary keratinocytes isolated from different genotypes indicates correct Notch1 and Notch2 ablation and shows distinct modulation of b-catenin signaling and p21 expression. B: Quantitative RT-PCR show Hes1 and p21 transcripts levels in primary keratinocytes of the indicated genotypes. C: Luciferase reporter assay reveals that N1IC is a more potent inhibitor of b-catenin-LEF/TCF-sensitive TOP plasmid than N2IC. FOP plasmid is b-catenin-LEF/TCF-insensitive and serves as a specificity control. Both N1IC and N2IC induce Hes1 in a comparable manner as quantified using a Hes1-luc reporter. doi:10.1371/journal.pone.0013578.g006 are present only in partially overlapping layers of the epidermis. Consistent with previous studies, Notch1 is present throughout all skin layers including the tumor-prone basal layer of the skin, whereas Notch2 is expressed exclusively in suprabasal keratino- cytes [37]. These findings were confirmed using immunohisto- chemical staining as well as Notch1-GFP and Notch2LacZ reporter mice (Fig. 5A). This divergent expression pattern is very likely at least partially responsible for the downregulation of p21 in Notch1- but not Notch2-deficient keratinocytes and in line with previous studies [37,40]. p21 is a cyclin-dependent kinase inhibitor that induces cell cycle arrest [35] and its loss is commonly associated with skin malignancies, particularly in an active Ras context [36]. In KrasG12D-induced tumorigenesis inhibition of p21 via Myc activation, observed in Pdx1-Cre;Kras;N1ko papillomas (Fig. 4), is a critical step for malignant transformation [34]. Thus, the observed differences in p21 induction by Notch1 and 2 receptors (Fig. 6A and B) could partially explain the observed phenotypes. The second notable difference between Notch1 and Notch2 was their ability to inhibit b-catenin-mediated signaling. b-catenin is responsible for hair-follicle morphogenesis and epidermal stem cell maintenance [54], whereas the disruption of the b-catenin signaling has been associated with several malignancies of the skin [18,42,43]. Notch1 deficiency leading to accumulation of b- catenin in the nucleus has been associated with tumorigenesis [14]. Surprisingly, we did not observe a similar effect when the Notch2 Figure 7. Model of epidermal Pdx1 expression and Cre-mediated epidermal recombination. Recombination rarely occurs in basal layer keratinocytes but leads to papilloma formation in Pdx1-Cre;Kras;N1ko mice: (SC) Stratum Corneum, (GL) Granular Layer, (SL) Spinous Layer, (BL) Basal Layer, (BM) Basement Membrane, (D) Dermis. doi:10.1371/journal.pone.0013578.g007 PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13578