The biliary tree is a hierarchically organized system of tubes that drain bile from the liver into the intestine. Integral to the development of this structure is NOTCH signaling, which directs biliary differentiation and tubulogenesis in embryonic liver development1-3. Disruption of this signaling pathway results in a paucity of intrahepatic bile ducts, as observed in the human disease Alagille syndrome (ALGS)4-6. By extinguishing all NOTCH signaling in the hepatic progenitor cells of mice, we observe complete biliary agenesis and extensive cholestatic liver damage similar to human cases of ALGS. Uniquely, this mouse is able to generate a new biliary system postnatally, derived entirely from transdifferentiated hepatocytes and dependent on TGF? signaling7. We observe that as cholestatic injury increases in the NOTCH-deficient liver, resident hepatocytes acquire biliary characteristics and proliferate around and between portal veins to form areas of dense cellular processes called ductular reactions (DRs). DRs appear as vascularized, homogenous ductular structures at least partially contiguous with the common bile duct and involve a complex assortment of cell types. Overtime, DRs will resolve into a hierarchical biliary tree equivalent in size and function to that of a wild-type mouse7 ? how this occurs is unclear, but involves a demonstrable reduction in ductule number, suggesting a heterogeneous commitment among hepatocyte-derived ductular cells to a mature cholangiocyte fate. Our preliminary evidence in combination with recently published studies suggests there are multiple signaling modules, including TGF?, WNT, and YAP, that operate in a dynamic fashion to orchestrate the transdifferentiation, expansion, and reorganization of hepatocyte-derived ductular cells into the finalized biliary network7-12. Understanding the transcriptional changes occurring in these cells as the hepatocyte-derived biliary system progressively matures would provide direct insight into the signaling pathways driving both transdifferentiation and morphogenesis, as well as reveal the resident cell types directing these processes.
I aim to define the lineage trajectory of hepatocyte-derived ductular cells that successfully transdifferentiate into cholangiocytes, the signals directing their assembly into a functional biliary tree, and the cell populations in the DR microenvironment that are responsible. To accomplish this, I will first single-cell sequence pure populations of hepatocyte-derived ductular cells from progressive stages of biliary tree maturation. Second, I will single-cell sequence ductular and non-ductular cells isolated from the DR together to elucidate the ligand-receptor relationships driving hepatocyte fate conversion and subsequent tubulogenesis. Understanding the blueprint for the successful genesis of the hepatocyte-derived biliary tree, independent of NOTCH signaling, has direct clinical ramifications for the treatment of diseases involving biliary paucity, malformation, and disappearance, as well as the engineering of appropriately complex and faithful tissues ex vivo for transplantation or drug discovery.
Cholestatic liver diseases, stemming from defects in the specification and morphogenesis of the biliary tree, are responsible for a plurality of pediatric liver transplants in the United States13, and biliary disorders frequently recur following transplant intervention in adults14. Studying the mechanisms directing the growth and maturation of the hepatocyte-derived biliary tree will reveal specific cellular targets for inducing the expansion of the native biliary system in vivo, thereby reducing the requirement for cell or organ transplantation, which is limited in feasibility and accessibility. Furthermore, by identifying the signaling mechanisms and cells of origin which drive the generation of a hierarchical ductular structure within an extant organ, my work will advance the biological accuracy of ex vivo tissues as they are engineered for transplantation or drug discovery.
