Project summary Currently, there is no cure for intrahepatic bile duct (IHBD) paucity except for liver transplantation. IHBD paucity causes accumulation of pathologic bile within the liver resulting in bile stasis and over time can lead to chronic cholestasis. Diseases such as Alagille syndrome (ALGS), biliary atresia, progressive familial intrahepatic cholestasis, alpha1-antitrypsin deficiency, primary biliary cirrhosis, and primary sclerosing cholangitis are associated with IHBD paucity. The standard of care for some of these diseases includes reducing bile acids through surgical biliary diversion, inhibition of the ileal bile acid transporter (i.e., IBAT) or by altering the composition of the bile acid pool (using bile acids salts or FXR ligand agonists) to interrupt and modify enterohepatic circulation. Currently, there are no clinical approaches to augment IHBD architecture or bile duct number in patients with these diseases. Therefore, in the absence of new approaches, patients with IHBD paucity will remain dependent on liver transplantation as definitive therapy for the foreseeable future. We have published that hepatocyte-to-cholangiocyte transdifferentiation in the absence of epithelial Notch activity is competent through a Tgfbr2-dependent mechanism. However, hepatocyte-to-cholangiocyte transdifferentiation is inefficient in patients with ALGS, due to global JAGGED1 (JAG1) haploinsufficiency with a significant number of patients requiring liver transplantation. Recent lineage tracing and single-cell RNA sequencing studies inform us that even though arising from a common mesenchymal progenitor, the identity of periportal mesenchyme is divergent from hepatic stellate cells and a critical knowledge gap in all aspects of IHBD development. Preliminary work strongly supports a role for periportal mesenchyme in this process, but the underlying role of periportal mesenchyme in IHBD paucity is superficial at best, especially regarding the mechanisms regulating biliary epithelial-periportal mesenchymal crosstalk. Our long-term goal is to understand the mechanisms regulating epithelial-mesenchymal crosstalk during IHBD specification, morphogenesis, maintenance, and response to injury/disease. Based on published and unpublished results we propose to address the central hypothesis that Jag1 haploinsufficiency impacts the periportal mesenchyme indirectly by reducing Notch activity in the hepatic epithelium hindering the implementation of the full cholangiocyte transcriptional program and thereby influencing the periportal mesenchyme by disrupting optimum crosstalk between the epithelium and mesenchyme. Our aims are to: 1) define the role Gli1 periportal mesenchyme plays to regulate epithelial IHBD development, and 2) define the role epithelial Ihh plays to regulate epithelial-periportal mesenchymal crosstalk. Long-term, the improved understanding of epithelial-periportal mesenchymal crosstalk will enable the development of targeted therapies capable ...