During liver development, hepatocytes and biliary cells differentiate from common progenitors called hepatoblasts. The factors that control hepatoblast fate decision are unknown. Here we report that a gradient of activin/ TGF␤ signaling controls hepatoblast differentiation. High activin/TGF␤ signaling is required near the portal vein for differentiation of biliary cells. The Onecut transcription factors HNF-6 and OC-2 inhibit activin/TGF␤ signaling in the parenchyma, and this allows normal hepatocyte differentiation. In the absence of Onecut factors, the shape of the activin/TGF␤ gradient is perturbed and the hepatoblasts differentiate into hybrid cells that display characteristics of both hepatocytes and biliary cells. Thus, a gradient of activin/TGF␤ signaling modulated by Onecut factors is required to segregate the hepatocytic and the biliary lineages.Supplemental material is available at http://www.genesdev.org.

SummaryZebrafish vps33b, an ortholog of the gene responsible for human arthrogryposis-renal dysfunction-cholestasis syndrome, regulates biliary development downstream of the onecut transcription factor hnf6

During liver development, hepatocytes undergo a maturation process that leads to the fully differentiated state. This relies at least in part on the coordinated action of liver-enriched transcription factors (LETFs), but little is known about the dynamics of this coordination. In this context we investigate here the role of the LETF hepatocyte nuclear factor 6 (HNF-6; also called Onecut-1) during hepatocyte differentiation. We show that HNF-6 knockout mouse fetuses have delayed expression of glucose-6-phosphatase (g6pc), which catalyzes the final step of gluconeogenesis and is a late marker of hepatocyte maturation. Using a combination of in vivo and in vitro gain-and loss-of-function approaches, we demonstrate that HNF-6 stimulates endogenous g6pc gene expression directly via a synergistic and interdependent action with HNF-4 and that it involves coordinate recruitment of the coactivator PGC-1␣. The expression of HNF-6, HNF-4, and PGC-1␣ rises steadily during liver development and precedes that of g6pc. We provide evidence that threshold levels of HNF-6 are required to allow synergism between HNF-6, HNF-4, and PGC-1␣ to induce time-specific expression of g6pc. Our observations on the regulation of g6pc by HNF-6 provide a model whereby synergism, interdependency, and threshold concentrations of LETFs and coactivators determine time-specific expression of genes during liver development.

A network of liver-enriched transcription factors controls differentiation and morphogenesis of the liver. These factors interact via direct, feedback, and autoregulatory loops. Previous work has suggested that hepatocyte nuclear factor (HNF)-6/OC-1 and HNF-3␣/FoxA1 participate coordinately in this hepatic network. We investigated how HNF-6 controls the expression of Foxa1. We observed that Foxa1 expression was upregulated in the liver of Hnf6 ؊/؊ mouse embryos and in bipotential mouse embryonic liver (BMEL) cell lines derived from embryonic Hnf6 ؊/؊ liver, suggesting that HNF-6 inhibits the expression of Foxa1. Because no evidence for a direct repression of Foxa1 by HNF-6 was found, we postulated the existence of an indirect mechanism. We found that the expression of a mediator and targets of the transforming growth factor beta (TGF-␤) signaling was increased both in Hnf6 ؊/؊ liver and in Hnf6 ؊/؊ BMEL cell lines. Using these cell lines, we demonstrated that TGF-␤ signaling was increased in the absence of HNF-6, and that this resulted from upregulation of TGF-␤ receptor II expression. We also found that TGF-␤ can stimulate the expression of A network of liver-enriched transcription factors (LETFs) controls differentiation and morphogenesis of the liver. 1-3 It comprises factors from several families that have been identified through biochemical and genetic studies. These include the CCAAT/enhancer binding proteins, the proline acidrich factors, the homeodomain proteins of the hepatocyte nuclear factor (HNF)-1 family, winged helix proteins (HNF-3/FoxA), nuclear receptors (HNF-4, LXR, FXR and FTF/hB1F/CPF families), and the Onecut (OC) factors (HNF-6/OC-1, OC-2 and OC-3). These LETFs regulate the expression of genes whose products control the development of the liver and its physiological functions. Evidence for a network of LETFs stemmed from studies based on transfection of hepatoma lines, which showed that HNF-1␣ and HNF-4 can control each other. 4 -7 Other groups extended this notion by showing that a combination of LETFs acting in synergy can directly regulate the expression of a transcription factor

SummaryZebrafish vps33b, an ortholog of the gene responsible for human arthrogryposis-renal dysfunction-cholestasis syndrome, regulates biliary development downstream of the onecut transcription factor hnf6