and differentiation processes [9]. Notch receptors are expressed in the skin, although their precise functions remain uncertain (reviewed in [10,11]). Gain- and loss-of-function studies have suggested various functions for Notch including proliferation control, differentiation switch of developing epidermis and formation of hair follicles [12–17]. Mice with epidermal loss of Notch1 as well as Presenilin-deficient mice develop epidermal hyperplasia and skin cancers [14,18]. Of note, most studies have focused on Notch1 and downstream signaling members such as Rbpj or Hes1 [19,20]. Very little is known about the function of Notch2 and other receptors in skin physiology and carcinogenesis. Here, we investigate the role of Notch1 and Notch2 using two different Cre expression systems. Our results provide evidence for different roles of Notch1 and Notch2 in skin development and carcinogenesis. Results Notch1 but not Notch2 deletion increases susceptibility to KrasG12D induced skin carcinogenesis in Pdx1-Cre mice To analyze the effect of Notch1 and Notch2 deficiency during pancreas carcinogenesis, we crossed previously described Pdx1-Cre [2], Notch1fl/fl [21], Notch2fl/fl [22] and Kras+/LSL-G12D [3] mice for generation of Pdx1-Cre;Kras+/LSL-G12D, Pdx1-Cre;Kras+/LSL-G12D;Notch1fl/fl and Pdx1- Cre;Kras+/LSL-G12D;Notch2fl/fl mice (referred to as Pdx1-Cre;Kras, Pdx1- Cre;Kras;N1ko and Pdx1-Cre;Kras;N2ko, respectively). These mice were born at the expected Mendelian ratio and successful recombination of the floxed loci in the pancreas was confirmed by PCR (Fig. 1C). Surprisingly, Pdx1-Cre;Kras;N1ko mice developed focal skin hyperplasia at 10–15 days of age and as early as 4 weeks of age developed massive skin papillomas (Fig. 1D). These lesions and tumors showed recombination of the floxed loci (Fig. 1C) pointing to epidermal Cre expression, which was further corroborated using Pdx1-Cre;Kras;N1- ko;ROSA26R-LacZ reporter mice (Fig. 1F) [23]. The penetrance of the skin papilloma development was 78%. In contrast, Pdx1-Cre;Kras;N2ko mice rarely developed any skin phenotype. However, double Notch1 and Notch2 knockout mice (Pdx1-Cre;Kras;N1ko;N2ko) featured an accelerated skin tumor formation (Fig. 1A and B) suggesting an essential role of Notch1 ablation in epidermal lesion development and a promoting role of Notch2 deletion. Pdx1-Cre;Kras mice manifested a skin phenotype with low penetrance, which has been observed previously [3,24]. Most tumors encountered in Pdx1-Cre;Kras;N1ko mice were benign papillomas but often grew large and ulcerating, requiring euthanasia of animals for ethical reasons. Hence, the intended pancreatic carcinogenesis study was inconclusive (data not shown). Pdx1-Cre;Kras;N1ko mice developed the following skin patholo- gies: squamous papillomas involving the ear, neck, lips, anal and vulvo-vaginal skin, epidermal cysts, and sebaceous gland hyper- plasia and cutaneous horns to lesser extend (Fig. 1D and E). Moreover, 32% of the animals developed squamous cell carcinomas (SCC), (Fig. 1E), supporting the previous observations that papillomas progressing to SCC are a common manifestation of activated Ras signaling [25–27]. Mice without oncogenic KrasG12D but ablation of Notch1 and Notch2 (Pdx1-Cre;N1ko, Pdx1- Cre;N2ko) only very rarely developed skin abnormalities (not shown). Evidence of Pdx1 expression in vivo and in vitro The observation that Pdx1-Cre;Kras;N1ko mice develop skin neoplastic lesions with high penetrance and undergo Cre-mediated recombination are evidence of Cre expression in the epidermis possibly due to Pdx1-Cre transgene misexpression or physiological PDX1 expression in the skin. To test both hypotheses, immuno- Pdx1 Is Expressed in the Skin histochemical expression analysis was performed in the skin of wildtype and Pdx1-Cre mice, which showed a small subset of PDX1+ cells (Fig. 2A). Thus, the observed phenotype is due to physiological PDX1 expression in the skin rather than transgenic misexpression of Cre recombinase. Immunofluorescent staining of PDX1 shows that the intensity of staining was comparable to that in the duodenum and much lower than in pancreatic islet cells (Fig. 2Bi and ii). Double immuno- fluorescent staining revealed that PDX1 co-localizes with Kera- tin10 (K10) in the spinous layer of the epidermis (Fig. 2Biii; arrowheads). Noteworthy, a very small fraction of PDX1+ cells was located in the basal layer of the epidermis suggesting that PDX1 expression may be initiated also in this layer (Fig. 2Bi and iii; arrows). Above-mentioned experiments demonstrate that PDX1 is predominantly present in differentiated keratinocytes of the skin. To test whether PDX1 expression is regulated during keratinocyte differentiation we induced terminal differentiation in cultured wildtype keratinocytes by calcium as described [28]. As early as 12 hours after calcium addition growth arrest and a switch in keratin expression occurred. As expected, treated keratinocytes showed a three-fold induction of the differentiation markers Keratin10 and Loricrin and a five fold reduction of p63 associated with amplifying keratinocytes in the basal layer of the epidermis. In addition, we found a robust 10-fold induction of Pdx1 transcript expression in treated keratinocytes (Fig. 2C). These findings strongly support the hypothesis that Pdx1 is predominantly expressed in suprabasal layers of the epidermis (Fig. 2D). Mosaic epidermal Cre expression in Pdx1-Cre mice Physiological PDX1 expression in the epidermis does not explain the stochastic character of papilloma formation in the Pdx1-Cre;Kras,N1ko mice. Hence, we speculated that Cre expression has a mosaic character or alternatively may be induced by mechanical skin irritation. To address the first hypothesis we examined X-Gal expression in Pdx1-Cre;ROSA26R-LacZ reporter mice [23]. Consistent with previous studies, we found that Pdx1- Cre mice showed a mosaic recombination pattern in the pancreas [1] (Fig. 3Ai). Interestingly, similar mosaic staining was observed in the skin (Fig. 3Aii). Microscopic evaluation of X-Gal positive areas indicated that suprabasal keratinocytes underwent recombination (Fig. 3Aiii; arrowheads), supporting the hypothesis that PDX1 is mainly expressed in differentiated keratinocytes. However, we found sporadically X-Gal+ keratinocytes residing in the basal layer (Fig. 3Aiii; arrow). All examined skin hyperplasia had X-Gal+ basal layer cells suggesting that neoplastic structures originate from the basal keratinocytes of the skin (Fig. 3Aiv; arrow). To further asses the scale of recombination in the basal layer (K14+) and the spinous layer (K10+) of the epidermis we tested freshly isolated keratinocytes from Pdx1-Cre;N1ko mice. Cells were fractioned for K14 and K10 expression respectively using fluorescent activated cell sorting (FACS). Cre-mediated recombi- nation was measured using quantitative PCR amplifying the recombined allele of floxed Notch1 that was normalized to input and then compared to fully recombined DNA. We found that only 5% of DNA isolated from total keratinocytes underwent recombination in Pdx-Cre;N1ko mice and most of them were found in the suprabasal layer. We sporadically (below 0.5%) found K14+ cells with recombined Notch1 loci hypothesizing that these cells could be the cell-of-origin for papilloma development (Fig. 3B). As papilloma development in Pdx1-Cre mice usually occurred in regions susceptible to grooming, scratching and wounding, we speculated that PDX1 expression may be induced in wounded skin triggering Cre-mediated KrasG12D activation and Notch1 ablation. PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13578