Identification of Epidermal Pdx1 Expression Discloses Different Roles of Notch1 and Notch2 in Murine G12D Kras -Induced Skin Carcinogenesis In Vivo Pawel K. Mazur1, Barbara M. Gru¨ ner1, Hassan Nakhai2, Bence Sipos3, Ursula Zimber-Strobl4, Lothar J. Strobl4, Freddy Radtke5, Roland M. Schmid1, Jens T. Siveke1* 1 2nd Department of Internal Medicine, Klinikum Rechts der Isar University Hospital, Technical University of Munich, Munich, Germany, 2 Department of Biological Sciences, University of Warwick, Coventry, United Kingdom, 3 Institute of Pathology, University Hospital, University of Tu¨ bingen, Tu¨ bingen, Germany, 4 Department of Gene Vectors, Helmholtz Zentrum, German Research Center for Environmental Health, Munich, Germany, 5 ISREC, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL SV ISREC), Lausanne, Switzerland Abstract Background: The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic KrasG12D mice with ablation of Notch1 and/or Notch2. Methodology/Principal Findings: Surprisingly, mice with activated KrasG12D and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and b- catenin signaling inhibition capabilities. Conclusions/Significance: Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors. Citation: Mazur PK, Gru¨ ner BM, Nakhai H, Sipos B, Zimber-Strobl U, et al. (2010) Identification of Epidermal Pdx1 Expression Discloses Different Roles of Notch1 and Notch2 in Murine KrasG12D-Induced Skin Carcinogenesis In Vivo. PLoS ONE 5(10): e13578. doi:10.1371/journal.pone.0013578 Editor: Joseph Alan Bauer, Bauer Research Foundation, United States of America Received February 6, 2010; Accepted September 22, 2010; Published October 22, 2010 Copyright: 2010 Mazur et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the German Cancer Aid (#107195), The Lustgarten Foundation (RFP05-14 and 06-12) and the German Research Foundation (SI 1549/1-1; all to J.T.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jens.siveke@lrz.tum.de Introduction Conditional tissue-specific modulation of genes using Cre/loxP recombination in genetically engineered mice provides an enormous leap forward to study gene function in detail yet requires detailed knowledge of gene regulation and expression patterns. For pancreatic targeting of genes, Pdx1-Cre mice are commonly used [1–3], in which Cre-recombinase is expressed under a 4.5 to 5.5 kb fragment of the Pdx1 promoter. The transcription factor Pdx1 (pancreas and duodenum homeobox gene 1) directs pancreatic cell formation, maintenance and function. Pdx1 is expressed in the region of the endoderm that ultimately gives rise to stomach, pancreas and duodenum and its function is critical for posterior foregut development [4]. Postnatally, Pdx1 is mainly expressed in insulin-producing endocrine cells of the pancreas. Ablation of Pdx1 results in defects of different cell types including malformations of the pylorus and duodenum, absence of Brunner’s glands and reduced numbers of specific enteroendocrine cell types in the stomach and intestine. Loss of Pdx1 function results in pancreatic agenesis, while heterozygous expression leads to defects in glucose homeostasis. Pdx1-deficient mice survive up to 6.5 days after birth, are severely dehydrated, have no fur and a delicate, cracking skin. [5–8]. Here, we report epidermal PDX1 expression observed due to an unexpected skin tumor formation in Pdx1-Cre mice with activation of oncogenic KrasG12D and loss of Notch1 but not Notch2. Notch proteins are evolutionarily conserved large transmem- brane receptors, which upon ligand binding undergo proteolytic cleavage mediated by the c-secretase-presenilin complex releasing the intracellular fragment (NIC). NIC is translocated to the nucleus where it binds and activates the mammalian repressor RBP-Jk thereby regulating fetal and postnatal cell fate decisions PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13578