Introduction: Cystic fibrosis (CF) patients and CF mouse models have an increased risk for gastrointestinal tumors1,2. CF mice show augmented intestinal proliferation of unknown etiology, which together with an altered intestinal environment may predispose to neoplastic development. Here, we examined the involvement of canonical Wnt/β-catenin signaling in proliferation of the intestinal stem cell (ISC) population of the CF mouse intestine. Directed cell migration, cell polarity and tight junction (TJ) remodeling must keep pace with increased epithelial proliferation. CF patients and CF mouse models also exhibit increased intestinal barrier permeability which likely contributes to other manifestations of CF disease3,4. Therefore, we also examined the involvement of non-canonical Wnt signaling, in particular components of the planar cell polarity (PCP) pathway, in TJ remodeling in the CF mouse intestine. A unifying hypothesis to altered Wnt signaling in the CF crypt epithelium is based on our previous finding that CF crypts have sustained increases in intracellular pH (pHi) due to a coincident intracellular retention of Cl- and HCO3- resulting from loss of the CF transmembrane conductance regulator CFTR anion conductance. An alkaline pHi increases the plasma membrane association of disheveled (Dvl), a mutual transducer of both canonical and non-canonical Wnt signaling, thereby facilitating initial Wnt signaling cascades. Methods: In these studies we employed transgenic mouse models crossed to wild-type (WT) and Cftr knockout (CF) sex-matched littermate mice to estimate membrane association of Dvl2-dGFP in cultured organoids by confocal fluorescence microscopy. All animal experiments were performed on an approved IACUC protocol. Tight junction permeability was assessed by 3K fluorescent dextran in spherical intestinal enteroids (2-3 days post-passage). Immunofluorescence (IF) was used to investigate localization of the Rho GTPase Cdc42 in WT and CF enteroids. Matched Caco2 cells with WT CFTR and CFTR knocked out (KO) by gene-editing, which show a hyperproliferative phenotype, were investigated for changes in GTP-bound (active) form of Cdc42. Results: Increased intestinal proliferation in CF mice in vivo was recapitulated in CF enteroids relative to WT. Studies further showed increased proliferation of Lgr5+ ISCs in CF mice crossed to Lgr5-eGFP reporter mice as compared to WT. CF enteroids expressing Dvl2-eGFP in a Dvl2 KO background and membrane dTomato showed increased membrane association of Dvl2 beginning in crypt-base columnar cells (CBC) and extending as well as amplifying in the apical region of crypt cells of the transit-amplifying zone (TAZ) as compared to WT. The increased membrane association of Dvl2-eGFP in CF enteroid crypts was reversed by treatment with sphingosine, a cation membrane lipid, and by two-day exposure to pH 6.4 medium, which also decreased active b-catenin, relative to untreated CF enteroids. IF showed expression of the PCP Wnt receptor Frizzled 6 and increased TJ localization of the PCP pathway effector Cdc42. Western blots indicated reduced expression of the TJ scaffold protein ZO-1 and unchanged to slightly increased protein expression of Cdc42. Tight junction transcriptome measured by RT-qPCR Taqman array plates showed decreases in ZO-1, partitioning defective polarity proteins Par 3 and Par6b and, paradoxically, decreased Cdc42 mRNA relative to WT. Spherical CF enteroids showed increased “leak” permeability to 3K-dextran, as compared to WT. ELISA based studies of CF Caco2 cells indicating increased GTP-bound Cdc42 and a proliferative phenotype by exhibiting multiple filopodia, a hallmark of Cdc42 activity. Conclusions: The above findings support the hypothesis that loss of CFTR in the proliferative compartment of intestinal crypts results in an alkaline pHi and increased membrane association of the Wnt transducer Dvl. The consequences may include a) facilitation of Wnt/βb-catenin signaling and a contribution to ISC hyperproliferation in the CF intestine, thereby increasing the potential for DNA replication errors; and b) facilitation of non-canonical Wnt PCP signaling involving increased activation of the Rho GTPase Cdc42 and increased “leak” permeability of the CF intestine, thereby providing a propensity for barrier dysfunction in the CF intestine. Together, altered Wnt signaling resulting from dysregulated pHi in the proliferative crypt compartment may contribute to the increased risk of intestinal cancer and impaired intestinal barrier permeability in CF patients.