Figure 1. Constitutive activation of the PI3K-signaling pathway induces premalignant biliary intraepithelial neoplasia (BilIN).

(A) Left panel: Genetic strategy and recombination scheme to analyze the patterns of Pdx1-Cre transgene expression using a conditional tdTomato reporter allele (LSL-R26^tdTomato/+ ). Right panel: Macroscopic fluorescence and white light images of Pdx1-Cre;LSL-R26^tdTomato/+ reporter mouse. Visualization of tdTomato reveals reporter gene expression (red) in the pancreas, duodenum, gallbladder and common bile duct (CBD). Scale bars, 1 cm. (B) Left panel: Genetic strategy and recombination scheme to analyze the patterns of Pdx1-Cre transgene expression using a switchable floxed double color fluorescent tdTomato-EGFP Cre reporter line (R26^mT-mG ). Right panel, left image: Representative confocal microscopic image of tdTomato (red color, non Cre-recombined cells) and Cre-induced EGFP (green color) expression in the common bile duct. Note: Cre-mediated EGFP expression in the biliary epithelium and peribiliary glands, but not stromal cells. Nuclei were counterstained with TOPRO-3 (blue). Right image: Representative immunofluorescence staining of CK19 (red color) of Pdx1-Cre;R26^mT-mGI+ animal. Note: Blue color shows expression of tdTomato in unrecombined cells. Green color labels Cre-recombined cells that express EGFP. Co-localization of CK19 immunofluorescence staining (red) and EGFP expression (green) results in yellow color. Scale bars, 50 μm. (C-E) Genetic strategy used to express oncogenic Pik3ca^H1047R (C), Kras^G12D (D), or only Cre as control (E) in the common bile duct (upper panels). H&E staining and immunohistochemical analysis of PI3K/AKT pathway activation in the biliary epithelium of the common bile duct and different grades of dysplasia in biliary intraepithelial neoplasia (BilIN) (lower panels). Scale bars, 50 μm for micrographs and 20 μm for insets.