MicroRNA-337-3p controls hepatobiliary gene expression and transcriptional dynamics during hepatic cell differentiation.

Transcriptional networks control the differentiation of the hepatocyte and cholangiocyte lineages from embryonic liver progenitor cells and their subsequent maturation to the adult phenotype. However, how relative levels of hepatocyte and cholangiocyte gene expression are determined during differentiation remains poorly understood. Here, we identify microRNA (miR)-337-3p as a regulator of liver development. miR-337-3p stimulates expression of cholangiocyte genes and represses hepatocyte genes in undifferentiated progenitor cells in vitro and in embryonic mouse livers. Beyond the stage of lineage segregation, miR-337-3p controls the transcriptional network dynamics of developing hepatocytes and balances both cholangiocyte populations that constitute the ductal plate. miR-337-3p requires Notch and transforming growth factor-ß signaling and exerts a biphasic control on the hepatocyte transcription factor hepatocyte nuclear factor 4α by modulating its activation and repression. With the help of an experimentally validated mathematical model, we show that this biphasic control results from an incoherent feedforward loop between miR-337-3p and hepatocyte nuclear factor 4α. CONCLUSION: Our results identify miR-337-3p as a regulator of liver development and highlight how tight quantitative control of hepatic cell differentiation is exerted through specific gene regulatory network motifs. (Hepatology 2018;67:313-327).

LKB1 and Notch Pathways Interact and Control Biliary Morphogenesis.

BACKGROUND: LKB1 is an evolutionary conserved kinase implicated in a wide range of cellular functions including inhibition of cell proliferation, regulation of cell polarity and metabolism. When Lkb1 is inactivated in the liver, glucose homeostasis is perturbed, cellular polarity is affected and cholestasis develops. Cholestasis occurs as a result from deficient bile duct development, yet how LKB1 impacts on biliary morphogenesis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We characterized the phenotype of mice in which deletion of the Lkb1 gene has been specifically targeted to the hepatoblasts. Our results confirmed that lack of LKB1 in the liver results in bile duct paucity leading to cholestasis. Immunostaining analysis at a prenatal stage showed that LKB1 is not required for differentiation of hepatoblasts to cholangiocyte precursors but promotes maturation of the primitive ductal structures to mature bile ducts. This phenotype is similar to that obtained upon inactivation of Notch signaling in the liver. We tested the hypothesis of a functional overlap between the LKB1 and Notch pathways by gene expression profiling of livers deficient in Lkb1 or in the Notch mediator RbpJκ and identified a mutual cross-talk between LKB1 and Notch signaling. In vitro experiments confirmed that Notch activity was deficient upon LKB1 loss. CONCLUSION: LKB1 and Notch share a common genetic program in the liver, and regulate bile duct morphogenesis.

Transcription factors SOX4 and SOX9 cooperatively control development of bile ducts.

In developing liver, cholangiocytes derive from the hepatoblasts and organize to form the bile ducts. Earlier work has shown that the SRY-related High Mobility Group box transcription factor 9 (SOX9) is transiently required for bile duct development, raising the question of the potential involvement of other SOX family members in biliary morphogenesis. Here we identify SOX4 as a new regulator of cholangiocyte development. Liver-specific inactivation of SOX4, combined or not with inactivation of SOX9, affects cholangiocyte differentiation, apico-basal polarity and bile duct formation. Both factors cooperate to control the expression of mediators of the Transforming Growth Factor-ß, Notch, and Hippo-Yap signaling pathways, which are required for normal development of the bile ducts. In addition, SOX4 and SOX9 control formation of primary cilia, which are known signaling regulators. The two factors also stimulate secretion of laminin α5, an extracellular matrix component promoting bile duct maturation. We conclude that SOX4 is a new regulator of liver development and that it exerts a pleiotropic control on bile duct development in cooperation with SOX9.

Hepatic Notch2 deficiency leads to bile duct agenesis perinatally and secondary bile duct formation after weaning.

UNLABELLED: Notch signaling plays an acknowledged role in bile-duct development, but its involvement in cholangiocyte-fate determination remains incompletely understood. We investigated the effects of early Notch2 deletion in Notch2(fl/fl)/Alfp-Cre(tg/-) ("Notch2-cKO") and Notch2(fl/fl)/Alfp-Cre(-/-) ("control") mice. Fetal and neonatal Notch2-cKO livers were devoid of cytokeratin19 (CK19)-, Dolichos-biflorus agglutinin (DBA)-, and SOX9-positive ductal structures, demonstrating absence of prenatal cholangiocyte differentiation. Despite extensive cholestatic hepatocyte necrosis and growth retardation, mortality was only ~15%. Unexpectedly, a slow process of secondary cholangiocyte differentiation and bile-duct formation was initiated around weaning that histologically resembled the ductular reaction. Newly formed ducts varied from rare and non-connected, to multiple, disorganized tubular structures that connected to the extrahepatic bile ducts. Jaundice had disappeared in ~30% of Notch2-cKO mice by 6 months. The absence of NOTCH2 protein in postnatally differentiating cholangiocyte nuclei of Notch2-cKO mice showed that these cells had not originated from non-recombined precursor cells. Notch2 and Hnf6 mRNA levels were permanently decreased in Notch2-cKO livers. Perinatally, Foxa1, Foxa2, Hhex, Hnf1ß, Cebpα and Sox9 mRNA levels were all significantly lower in Notch2-cKO than control mice, but all except Foxa2 returned to normal or increased levels after weaning, coincident with the observed secondary bile-duct formation. Interestingly, Hhex and Sox9 mRNA levels remained elevated in icteric 6 months old Notch2-cKOs, but decreased to control levels in non-icteric Notch2-cKOs, implying a key role in secondary bile-duct formation. CONCLUSION: Cholangiocyte differentiation becomes progressively less dependent on NOTCH2 signaling with age, suggesting that ductal-plate formation is dependent on NOTCH2, but subsequent cholangiocyte differentiation is not.