PROJECT SUMMARY The liver exhibits remarkable capacity for regeneration, but chronic injury or severe acute damage can overwhelm regenerative mechanisms and lead to end-stage liver disease. The lack of effective medical therapies combined with insufficient donor organ availability necessitates the development of new regenerative medicine- based therapies and a deeper understanding of the basic mechanisms of liver repair. The transcription factor Sox9 is required for stem/progenitor cell function in a number of epithelial tissues and has been shown to establish cellular identity. In the liver, Sox9 is required for timing of biliary epithelial cell (BEC) specification in development and is broadly expressed in adult BECs. Sox9 deficiency worsens cholangiopathy in mouse models of Alagille Syndrome. My preliminary data demonstrate ductal paucity in Sox9 knockout mice, suggesting a central role for Sox9 in BEC specification. This proposal seeks to define functional regulation of intrahepatic BEC networks by Sox9, during development and injury-associated ductular reaction (DR). DR is a common characteristic of liver disease and is defined by the proliferative expansion of BECs. Furthermore, based on damage type, timing, and/or extent of damage, DR can involve lineage conversion between mature hepatocytes and BECs that contributes to tissue regeneration. The genetic regulation of DR remains poorly understood. The central hypothesis of this proposal is that Sox9 is required to establish proper intrahepatic BEC networks in development and ductular reaction. I will test this hypothesis with the following specific aims: Aim 1A will establish the developmental requirement of Sox9 to form functional intrahepatic bile ducts in mice through the use of histology, bile acid assays, organoid assays, and 3D tissue imaging. Aim 1B will investigate the role of Sox9 in specifying BEC subpopulations through scRNA-seq. Aim 2 will determine how Sox9 maintains adult BEC populations during DR using inducible Sox9 knockout mice. BEC function will be assayed by liver serum biochemistry, histology, and lineage tracing. This project will determine how Sox9 establishes and maintains BEC populations and provide me with training towards my goal of obtaining a research-focused faculty position studying GI tissue homeostasis and regeneration. This work is significant because the findings will help develop an understanding of basic mechanisms of liver repair that can be used to identify novel targets for therapeutics to restore liver function in end stage liver disease.