Pdx1 Is Expressed in the Skin Figure 4. Characterization of papillomas developing in Pdx1-Cre;Kras;N1ko mice. Keratin14, Keratin10 and Loricrin expression show well- differentiated stratified squamous neoplasia. Keratin6 expression indicates pathological growth (upper panel). Immunohistochemical analysis of commonly activated pathways and markers expressed in Pdx1-Cre;Kras;N1ko papillomas (lower panel). The scale bars represent 50 mm. doi:10.1371/journal.pone.0013578.g004 catenin activity. Therefore, we examined the capabilities of active Notch1 (N1IC) and Notch2 (N2IC) to inhibit b-catenin signaling activity in primary keratinocytes using a luciferase reporter assay. Both Notch receptors were able to inhibit b-catenin activity but N1IC was a significantly stronger inhibitor. Forced expression of N1IC represses b-catenin signaling by over 90% whereas N2IC overexpression leads only to a modest reduction of 30% (Fig. 6C). At the same time both Notch receptors showed a similar induction of Hes1 promoter activity, serving as a read-out for similar activation of canonical Notch signaling (Fig.6 C). Taken together, these results support a context- and cell-specific function in addition to a distinct expression pattern of Notch and Notch2 in keratinocytes. Discussion Neoplasms originating from cutaneous epithelial cells are the most common cancer-type in the United States with an annual incidence of over 1 million cases [44]. Developmental signaling pathways play a key role in the induction and progression of cancer. Our study reports a previously unrecognized epidermal expression of PDX1 and adds further evidence for a pivotal role of Notch1 but not Notch2 as a tumor suppressor in the skin, which may be particularly interesting in the light of new therapeutic approaches targeting single Notch receptors [45,46]. Epidermal PDX1 expression As PDX1 is mainly expressed in the pancreas and duodenum, the Pdx1 promoter is commonly utilized for pancreas-specific transgenic mouse lines. Surprisingly, we found conditional gene deletion in the skin using a Pdx1-Cre strain [2]. Further research provided strong evidence that PDX1 is physiologically expressed in the suprabasal layers of the skin (Fig. 2A and B; arrowheads) and rarely in basal keratinocytes (Fig. 2A and B; arrows). A similar pattern of Pdx1 expression was observed in differentiation induced cultured keratinocytes (Fig. 2C). This hypothesis is supported by reports indicating a skin phenotype of Pdx1 knockout mice, which survive 6.5 days postpartum and have, among other characteristic features, thin and cracking skin with little or no fur [7]. While these skin abnormalities may be due to indirect effects, they suggest a role of PDX1 during skin development, which should be addressed in further studies, e.g. by analyzing keratinocyte-specific Pdx1 knockout mice, which however is beyond the scope of this report. In contrast to the ubiquitous expression of Pdx1 in the suprabasal layers of the skin, Pdx1-Cre;Kras,N1ko mice developed skin papillomas and other cutaneous lesions only in preferred sites suggesting that Cre-mediated recombination may be mosaic and/or occurs in the cells resistant to neoplastic transformation. Notably, Cre expression in Pdx1-Cre mice is mosaic such that Cre-mediated recombination occurs far less frequently as would be suggested by the observed PDX1 expression. In addition, papillomas and most other skin tumors typically originate from the basal layer; in fact development from the suprabasal layer is a rather unlikely scenario (Fig. 7). Although PDX1 is mainly expressed in the suprabasal keratinocytes, we occasionally found PDX1 expression and Cre- mediated recombination in K14+ cells (Fig. 3A, B and 7). These observations may be the reason for the relatively few tumors developing per animal. Interestingly, tumors of Pdx1-Cre;Kras,N1ko mice usually develop around exposed areas of the skin (Fig. 1D), possibly due to Pdx1 activation in wound and scar associated basal layer keratinocytes (Fig. 3C). We speculate that cutaneous aggravation or micro-wounds due to grooming and scratching may trigger an inflammatory reaction and wound healing processes with upregulated Pdx1 and Notch expression [47], thus forming a tumor-prone environment in Pdx1-Cre;Kras;N1ko mice. Intriguingly, other studies have reported skin phenotypes using Pdx1-Cre mice despite the fact that different transgenic strains were utilized [3,24]. These reports support our finding that Pdx1 is expressed in the skin. However, only defined genetic alterations lead to a cutaneous phenotype. In the most often analyzed Pdx1-Cre;Kras mouse model, skin lesions were only rarely observed (below 5%, Fig.1B and [3,24]). In our study, Pdx1-Cre;Kras;N1ko but not Pdx1-Cre;Kras;N2ko or Pdx1-Cre;Kras developed skin lesions (Fig. 1A and B) which points to the importance of Notch1 but not Notch2 for skin tumor development. PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13578