receptor was abrogated (Fig. 5D and 6A). Additionally, we provide in vitro evidence of different inhibition capacities between both receptors (Fig. 6C) further supporting the postulate of distinct molecular functions of Notch1 and Notch2. In line with the non-redundant roles of Notch1 and Notch2 in keratinocytes is the accelerated papilloma formation in double Notch1/2-deficient mice (Fig. 1A and B), suggesting that Notch2 cannot fully compensate for Notch1 loss. Besides different roles in regulation of p21 and b-catenin, Notch expression dosage may play a role as was recently shown [17]. In this study Notch1 loss promoted skin tumorigenesis in a non-cell autonomous manner by impairing skin-barrier integrity and creating a wound-like microenvironment in the epidermis. Of note, Notch2 ablation alone had no such capabilities unless combined with a Notch3 knockout, suggesting that a certain threshold of Notch signaling is essential for skin homeostasis. In conclusion, our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function as well as distinctive roles of Notch1 and Notch2 in skin tumori- genesis potentially via different p21 and b-catenin pathway modulation. Materials and Methods Mouse strains Kras+/LSL-G12D, Notch1fl/fl, Notch2fl/fl, Pdx1-Cre and Keratin5-Cre transgenic mice have been described before [2,21,22,27,35]. Mice were interbred to obtain Pdx1-Cre;Kras+/LSL-G12D (Pdx1-Cre;Kras), Pdx1-Cre;Kras+/LSL-G12D;Notch1fl/fl (Pdx1-Cre;Kras;N1ko), Pdx1-Cre;K- ras+/LSL-G12D;Notch2fl/fl (Pdx1-Cre;Kras;N1ko), Keratin5-Cre;Notch1fl/fl (K5N1ko) and Keratin5-Cre;Notch2fl/fl (K5N2ko) mice. Previously described reporter strains LSL-ROSA26R-LacZ, Notch1-GFP and Notch2lacZ [23,38,39,55], were used as indicated in the text. All animals were of mixed C57BL/6J;129SV background. Animal care and experimental protocols were conducted in accordance with German animal protection laws and approved by the Institutional Animal Care and Use Committee at the Technical University of Munich. Statistical Analyses Kaplan-Meier curves were calculated using the tumor free survival time for each mouse from all littermate groups. The log- rank test was used to test for significant differences between the four groups. For gene expression analysis the unpaired two-tailed Student’s t-test was used. For P values the following scale was used: * p,0.05, ** p,0.01, *** p,0.001. Histology and Immunohistology For morphologic, immunohistochemical, and immunofluores- cence studies specimens were fixed in 4% buffered formalin then processed as described previously [56] and embedded in paraffin. Tissues were sectioned 4 mm and stained with hematoxylin and eosin (HE) or used for immunohistochemical studies with antibodies: CDK4 (Santa Cruz Biotechnology), K14, K10, K6, Loricrin (Covance), Notch1 (Abcam), Notch2 (The Developmental Studies Hybridoma Bank), pERK, (Cell Signaling), p63, CyclinD1 (BD), active-b-catenin (Upstate), PDX1 (gift of C.V. Wright). X- Gal staining of cryosections (10 mm) was carried out according to standard protocol, counterstained with nuclear fast red. Immuno- fluorescence was performed using Alexa 488 and 555 (Invitrogen). Nuclei were stained with DAPI. Pictures were taken using an Axiovert 200 M fluorescence inverse microscope equipped with the Axiovision software (Zeiss). Pdx1 Is Expressed in the Skin Histopathological Evaluation HE stained sections were evaluated by a pathologist (B.S.) with expertise in human and mouse cancer pathology. The pathologist, where needed, also reviewed immunohistochemical stainings. Western Blot Analysis Protein extracts from freshly isolated primary keratinocyte cells were obtained using RIPA buffer containing proteinase inhibitors - Complete (Roche). Lysates were separated on standard SDS- PAGE electrophoresis, transferred to PDVF membranes as described previously [56] and incubated with antibodies: b-actin (Sigma), Notch1 (BD Pharmigen), Notch2 (The Developmental Studies Hybridoma Bank), p21 (LabVison), active b-catenin (Upstate). Antibody binding was visualized using horseradish peroxidase-labeled secondary antibodies and ECL reagent (Amersham). Primary Keratinocytes Culture Keratinocytes were isolated from 3 to 4 week old mice as described previously [57]. Briefly, mice in anlagen phase were sacrificed, trunk skin was removed disinfected and enzymatically treated to allow separation of epidermis from dermis. Detaching keratinocytes were collected, filtered through Teflon mesh (100 mm), washed and plated on Petri dish previously coated with collagen and fibronectin. Cells were maintained in DMEM Spiner modification media (Sigma) with addition of 8% FCS treated with Chelex (BioRad), 10 mg/ml Transferrin, 5 mg/ml Insulin, 10 mM Phosphoethyloamine, 10 mM Ethyloamine, 0.05 nM CaCl2 (Sig- ma), 10 ng EGF, 0.36 mg/ml Hydrocortisone (Chemicon), 1% Glutathion, 1% Pen/Strep (Invitrogen). Keratinocytes were plated and cultured for 3 to 5 days before use in luciferase and differentiation assays. Growth medium was changed every day. Induction of keratinocyte differentiation was achieved by addition of CaCl2 to final concentration of 1.2–2 mM. Fluorescent Activated Cell Sorting for Cre-mediated recombination analysis in Keratinocytes Total isolated keratinocytes were stained with K14 or K10 antibodies (Covance) for 1 h at 4uC. Cells were washed in PBS +1% BSA and stained with the secondary antibody Alexa 488 (Invitrogen). Keratinocytes were washed and stained with propidium iodide followed by sorting using a FACS Aria 2 (BD Bioscience). DNA was isolated from the sorted cells utilizing DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s instructions. Recombination of genomic DNA was quantified by qPCR using the following program: 95uC for 10 min, 35 cycles of 95uC for 10 sec, 62uC for 10 sec and 72uC for 30 sec on a LightCycler 480 (Roche). All samples were analyzed in triplicate. b-globin genomic fragment was used for normalization. The following primers were used: b-globin-F 59-CCAATCTGCTCACACAGGATAGAGAGG- GCAGG-39 b-globin-R 59-CCTTGAGGCTGTCCAAGTGATTCAGGC- CATCG-39 Del Notch1-F 59-TGT GCT TTC ACA CTG GCA CAG-39 Del Notch1-R 59-CCA CTT AGA AGG AAT TCC ACC-39 Luciferase assay A luciferase reporter assay was performed with a pair of luciferase reporter constructs TOPFLASH, containing three copies of the TCF/LEF binding sites and FOP-FLASH, containing mutated binding sites (Upstate Biotechnology). Primary keratinocytes were cultured in 6-well plates and transiently PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13578