Sox9 links biliary maturation to branching morphogenesis

During liver development, bipotential epithelial progenitor cells, termed hepatoblasts, give rise to hepatocytes and biliary epithelial cells (BECs). BECs line the intrahepatic bile ducts which are essential for liver function. The adult intrahepatic bile duct (IHBD) network is hierarchical in structure with smaller, peripheral “ductules” draining into larger “ducts” that eventually exit the liver. Despite proposed models from histological and genetic studies, mechanisms differentiating local “duct” or “ductule” hierarchies and driving the global IHBD architecture are poorly understood. Previous studies of IHBD development have focused on mechanisms of BEC specification/ de novo bile duct formation by tubulogenesis. However, the contribution of pre-existing BECs to IHBD expansion and morphogenesis is unclear. Previous studies on liver specific Sox9 knockout (Sox9cKO) mice showed that Sox9 is important for timing of BEC specification during development and prevents cystogenesis in aged mice. We hypothesize that SOX9 is an important regulator of IHBD local and global architecture during development by promoting IHBD branching morphogenesis. Using whole-lobe 3D imaging and iDISCO+, we find that adult, embryonic, and postnatal Sox9cKO mice demonstrate ductal paucity with bias towards ductule, rather than duct, loss. Bulk RNA-sequencing reveals that Sox9cKO BECs are transcriptionally “immature” and have gene expression signatures consistent with a failure to fully acquire BEC identity. scRNA-sequencing identifies ductule-like cells in a subset of Sox9cKO BECs correlating with elevated Inhba, the gene precursor of Activin A. Sox9cKO biliary organoids exhibit non-cystic morphology that is reproduced in Activin A treated WT organoids. In vitro and in vivo, Activin A signaling inhibition experiments partially rescue BEC morphology. Together, our data demonstrate that SOX9 drives BEC maturation and promotes ductule formation, in part through inhibiting Activin A. Our data support a model for IHBD development involving distinct mechanisms governing Sox9-independent large duct tubulogenesis versus Sox9-dependent small ductule branching morphogenesis. F31DK134199; PI: Hannah Hrncir T34GM149511; PI: Anita Corbett, PhD; R01DK132653; PI: Adam Gracz, PhD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.