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Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.

ABSTRACT: 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 bile ducts. Here, we investigated duct development in mouse liver and characterized the role of the SRY-related HMG box transcription factor 9 (SOX9).We identified SOX9 as a new biliary marker and used it in immunostaining experiments to characterize bile duct morphogenesis. The expression of growth factors was determined by in situ hybridization and immunostaining, and their role was studied on cultured hepatoblasts. SOX9 function was investigated by phenotyping mice with a liver-specific inactivation of Sox9.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-beta 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 alpha and Homolog of Hairy/Enhancer of Split-1, as well as of the transforming growth factor-beta receptor type II, which are regulators of biliary development.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.

SUBMITTER: Antoniou A

PROVIDER: S-EPMC2743481 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.

Antoniou Aline A Raynaud Peggy P Cordi Sabine S Zong Yiwei Y Tronche François F Stanger Ben Z BZ Jacquemin Patrick P Pierreux Christophe E CE Clotman Frederic F Lemaigre Frederic P FP

Gastroenterology 20090221 7

<h4>Background & aims</h4>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 bile ducts. Here, we investigated duct development in mouse liver and characterized the role of the SRY-related HMG box transcription factor 9 (SOX9).<h4>Methods</h4>We identified SOX9 as a new biliary marker and used it in immunostaining experiments to characterize bile duct morphogenesis. The ...[more]

PMID: 19403103

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Project description:BackgroundDuring liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis.ResultsProtocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15½ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1).Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis.ConclusionsThe developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.

Project description:Formation of intrahepatic bile ducts (IHBDs) proceeds in accordance with their microenvironment. Particularly, mesenchymal cells around portal veins regulate the differentiation and ductular morphogenesis of cholangiocytes in the developing liver; however, further studies are needed to fully understand the arrangement of IHBDs into a continuous hierarchical network. This study aims to clarify the interaction between biliary and liver mesenchymal cells during IHBD formation. To identify candidate factors contributing to this cell-cell interaction, mesenchymal cells were isolated from embryonic day 16.5 matrix metalloproteinase 14 (MMP14)-deficient (knockout [KO]) mice livers, in which IHBD formation is retarded, and compared with those of the wild type (WT). WT mesenchymal cells significantly facilitated the formation of luminal structures comprised of hepatoblast-derived cholangiocytes (cholangiocytic cysts), whereas MMP14-KO mesenchymal cells failed to promote cyst formation. Comprehensive analysis revealed that expression of vasoactive intestinal peptide (VIP) was significantly suppressed in MMP14-KO mesenchymal cells. VIP and VIP receptor 1 (VIPR1) were mainly expressed in periportal mesenchymal cells and cholangiocytic progenitors during IHBD development, respectively, in vivo. VIP/VIPR1 signaling significantly encouraged cholangiocytic cyst formation and up-regulated tight junction protein 1, cystic fibrosis transmembrane conductance regulator, and aquaporin 1, in vitro. VIP antagonist significantly suppressed the tight junction assembly and the up-regulation of ion/water transporters during IHBD development in vivo. In a cholestatic injury model of adult mice, exogenous VIP administration promoted the restoration of damaged tight junctions in bile ducts and improved hyperbilirubinemia. Conclusion: VIP is produced by periportal mesenchymal cells during the perinatal stage. It supports bile duct development by establishing tight junctions and up-regulating ion/water transporters in cholangiocytes. VIP contributes to prompt recovery from cholestatic damage through the establishment of tight junctions in the bile ducts.

Project description:Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

Dataset Information

Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.

Publications

Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.

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