Intrahepatic Bile Ducts Develop According to a New Mode of Tubulogenesis Regulated by the Transcription Factor SOX9

BACKGROUND & AIMS: A number of diseases are characterized by defective formation of the intrahepatic bile ducts. In the embryo, hepatoblasts differentiate to cholangiocytes, which give rise to the intrahepatic bile ducts. Here, we investigated how these ducts develop in mouse liver and characterized the role of the transcription factor SRY-related HMG box transcription factor 9 (SOX9). METHODS: We identified SOX9 as a new biliary marker and used it with other cell markers in immunostaining experiments to characterize the process of bile duct morphogenesis. The expression of growth factors was determined by in situ hybridization and immunostaining, and their role was studied on cultured embryonic hepatoblasts. SOX9 function was investigated by phenotyping mice with a liver-specific inactivation of Sox9. RESULTS: Biliary tubulogenesis started with formation of asymmetrical ductal structures, lined on the portal side by cholangiocytes and on the parenchymal side by hepatoblasts. When the ducts grew from the hilum to the periphery, the hepatoblasts lining the asymmetrical structures differentiated to cholangiocytes, thereby allowing formation of symmetrical ducts lined only by cholangiocytes. We also provide evidence that transforming growth factor-? promotes differentiation of the hepatoblasts lining the asymmetrical structures. In the absence of SOX9, the maturation of asymmetrical structures into symmetrical ducts was delayed. This was associated with abnormal expression of CCAAT/Enhancer Binding Protein ? and Homolog of Hairy/Enhancer of Split-1, as well as of the transforming growth factor-? receptor type II, which are regulators of biliary development. CONCLUSIONS: Our results suggest that biliary development proceeds according to a new mode of tubulogenesis characterized by transient asymmetry and whose timing is controlled by SOX9