Pdx1 Is Expressed in the Skin Figure 2. Pdx1 is physiologically expressed in the adult mouse epidermis. A: Immunohistochemical PDX1 staining of normal wildtype epidermis (i, ii) reveals that PDX1 is expressed in suprabasal keratinocytes (black arrowheads) and only rarely in basal cells (black arrows). Pdx1- Cre;Kras;N1ko papilloma (iii) is strongly positive for PDX1. Inclusion (iii) shows positive staining of pancreatic islet cells. Nuclei were contrastained with methyl green (i, ii) or hematoxilin (iii). B: Immunofluorescent PDX1 staining (i) indicates positive keratinocytes in the suprabasal (white arrowheads) and the basal (arrow) layer of the skin. Signal strength is comparable to that in duodenum cells (ii, arrowheads) and weaker than in pancreatic islet cells (ii, inclusion). Double immunofluorescence (iii) demonstrates that the majority of PDX1+ cells co-localize with a suprabasal marker Keratin10 (arrowheads) however, a small subset of PDX1+ cells can be found in the basal layer of the epidermis (arrow). Asterisks indicate unspecific staining of stratum corneum. C: Pdx1 expression in cultured keratinocytes is increased during Ca++-induced differentiation. Quantitative RT-PCR of Pdx1, Keratin10, Loricrin and p63 transcripts in induced primary keratinocytes in vitro. D: Schematic representation of PDX1 expression in the epidermal layers: (SC) Stratum Corneum, (GL) Granular Layer, (SL) Spinous Layer, (BL) Basal Layer, (BM) Basement Membrane, (D) Dermis and their markers: Loricrin, K1/10, K5/14. The scale bars represent 50 mm. doi:10.1371/journal.pone.0013578.g002 To test this hypothesis, wounds were induced on the back skin of wild type mice. Six days after wound formation mice were sacrificed and sections of scared skin were dissected and analyzed. Increased PDX1 expression was found in the scar tissue and in the transition zone between normal and wounded epidermis (Fig. 3C). PDX1 staining pattern was nuclear and partially cytoplasmic as previously described [29–32]. Quantitative RT-PCR indicated a three-fold induction of Pdx1 and highly increased Keratin6 transcript levels in wounded compared to normal epidermis (Fig. 3D) supporting PDX1 expression in wounded skin. In summary these results denote (i) physiological Pdx1 expression in the skin, (ii) restricted to differentiated keratinocytes but sporad- ically present in K14+ basal cells, (iii) mosaic Pdx1-Cre epidermal expression, and (iv) Pdx1 induction in wounded skin. Histopathology of skin tumors developing in Pdx1- Cre;Kras;N1ko mice Histological investigations revealed that the papillomas and hyperplastic epithelium cover thin expansions of a fibroblastic stroma often with mild chronic inflammatory infiltrates. Local hyperplasia and squamous papillomas were well differentiated, rarely demonstrating focal dysplasia (Fig. 1E). Sections of typical papillomas were analyzed by immunofluorescence for differentiation markers including Keratin 14, 10 and Loricrin. In the papillomas all three keratins were expressed in a manner similar to normal skin, except that there was a delay in the onset of K10 expression consistent with an expansion of the proliferative compartment expressing K14 and CyclinD1 (Fig. 4). In line with the hyperplastic character was the expression of K6, a keratin usually expressed in hair follicles or in pathological conditions resulting in hyperplasia (Fig. 4). The observed keratin expression pattern is characteristic of well-differentiated squamous papillomas. Older mice developed hyperproliferative lesions that exhibited cellular atypia, increased mitosis and an invasive growth pattern with characteristic keratin ‘pearls’ formation and a high degree of keratinization that are diagnostic of well-differentiated SCC. Of note, no basal cell carcinomas (BCC) were observed in Pdx1-Cre;Kras;N1ko mice and no signs of a metastatic disease were observed. Immunohistochemical characterization of papillomas revealed strong activation of Ras-dependent phospho-ERK consistent with previous studies [33] as well as robust MYC expression associated with skin neoplastic transformation [34]. Interestingly, robust p63 expres- sion throughout the papilloma tissue was noted. Normally, the presence of p63 is restricted to the thin layer of basal keratinocytes due to inhibition by Notch1. Expression of p63 is characteristic for progenitor and multiplying cells of the epidermis. Expanded and strong CyclinD1 staining supports this conclusion (Fig. 4). This expression pattern is common and characteristic for cutaneous neoplasia. Notch1 but not Notch2 is a tumor suppressor in the skin Although the role of Notch receptors in the skin has already been intensively studied [12–17], we aimed to characterize epidermal Notch1 and Notch2 deficiency in our model. To do so, Notch1fl/fl [21] and Notch2fl/fl [22] mice were crossed with basal keratinocyte-specific Keratin5-Cre mice [35] (named K5;N1ko and K5;N2ko respectively). These mice were born at the expected Mendelian ratio (Fig. 5B) and successful recombination of the floxed loci was confirmed in isolated primary keratinocytes by immunoblot (Fig. 6A). Consistent with previous studies, K5;N1ko mice did not develop proper hair follicles showing a ‘naked’ phenotype. Additionally, the epidermis was thinner, easily cracking and prone to injury (Fig. 5B, D and E). Such a phenotype has been attributed to a role of Notch1 in the stimulation of keratinocyte differentiation [19,36,37]. Before the age of 9 months, K5;N1ko mice developed extensive hyperplasia and keratinization of the corneal epithelium, which resulted in opaque plague formation and blindness (Fig. 5B and C) [14]. All analyzed mice (n = 4) developed skin neoplasia at 9 to 12 months of age and additionally BCC, SCC and papillomas were noticed (Fig. 5B and C). By contrast, K5;N2ko mice featured a non-pathological skin and hair follicle formation (Fig. 5B and D) with normal growth cycles. However, impairment of hair growth direction that manifested in more upwards-ruffle appearance of fur was observed (Fig. 5B). Mice followed up to 12 months of age (n = 4) did not show any sign of tumorigenesis. Taken together, our findings confer that Notch1, but not Notch2 is a tumor suppressor and plays a crucial role in proper skin development and differentiation. Since expression in different compartments may explain distinct Notch1 and Notch2 functions, we analyzed the expression pattern of these receptors using immunohistochemical staining as well as transgenic Notch1-GFP [38] and Notch2lacZ knockin [39] reporter mice. We found Notch2 and X-Gal as a surrogate for Notch2 expression in spinous and granular layers of the epidermis (Fig. 5A). Notch1 and GFP expression in Notch1-GFP mice was found throughout the epidermal layers as previously described [37], including the basal layer of keratinocytes formed by stem cells and highly proliferative transit amplifying cells (Fig. 5A). Besides these differences in expression, different and context- specific functions of Notch1 and Notch2 have been described. We thus isolated and cultured primary keratinocytes from K5;N1ko and K5;N2ko mice, which showed no protein expression of the respective Notch receptor (Fig. 6A) and significantly downregu- lated levels of Hes1 transcripts (Fig. 6B) Notch1 signaling is essential for proper skin differentiation through induction of p21 (WAF1/Cip1) [37,40]. We speculated that Notch2 signaling might not be required for this process since it is expressed mainly by differentiated keratinocytes. p21 is a cyclin-dependent kinase inhibitor that induces cell cycle arrest [41], predictably its loss is commonly associated with skin malignancies, particularly in an active Ras context [34]. We found that p21 expression was highly reduced in Notch1 ablated cells whereas no significant differences were noted in Notch2 deficient keratinocytes both on mRNA and protein level (Fig. 6A, PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13578