Van Gogh-like 2 is essential for the architectural patterning of the mammalian biliary tree.

BACKGROUND & AIMS: In the developing liver, bipotent epithelial progenitor cells undergo lineage segregation to form hepatocytes, which constitute the bulk of the liver parenchyma, and biliary epithelial cells (cholangiocytes), which comprise the bile duct (a complex tubular network that is critical for normal liver function). Notch and TGFß signalling promote the formation of a sheet of biliary epithelial cells, the ductal plate, that organises into discontinuous tubular structures. How these structures elongate and connect to form a continuous duct remains undefined. We aimed to define the mechanisms by which the ductal plate transitions from a simple sheet of epithelial cells into a complex and connected bile duct. METHODS: By combining single-cell RNA sequencing of embryonic mouse livers with genetic tools and organoid models we functionally dissected the role of planar cell polarity in duct patterning. RESULTS: We show that the planar cell polarity protein VANGL2 is expressed late in intrahepatic bile duct development and patterns the formation of cell-cell contacts between biliary cells. The patterning of these cell contacts regulates the normal polarisation of the actin cytoskeleton within biliary cells and loss of Vangl2 function results in the abnormal distribution of cortical actin remodelling, leading to the failure of bile duct formation. CONCLUSIONS: Planar cell polarity is a critical step in the post-specification sculpture of the bile duct and is essential for establishing normal tissue architecture. IMPACT AND IMPLICATIONS: Like other branched tissues, such as the lung and kidney, the bile ducts use planar cell polarity signalling to coordinate cell movements; however, how these biochemical signals are linked to ductular patterning remains unclear. Here we show that the core planar cell polarity protein VANGL2 patterns how cell-cell contacts form in the mammalian bile duct and how ductular cells transmit confluent mechanical changes along the length of a duct. This work sheds light on how biological tubes are patterned across mammalian tissues (including within the liver) and will be important in how we promote ductular growth in patients where the duct is mis-patterned or poorly formed.

Oestradiol promotes the intrahepatic bile duct development of C57BL/6CrSlc mice during embryonic period via Notch signalling pathway.

Oestradiol (E2) is a critical factor for multiple systems' development during the embryonic period. Here, we aimed to investigate the effects of oestradiol on intrahepatic bile duct development, which may allow a better understanding of congenital bile duct dysplasia. DLK+ hepatoblasts were extracted from the C57BL/6CrSlc foetal mice and randomly divided into control group, oestradiol groups (1, 10, 100 nM) and oestradiol (10 nM) + DAPT (inhibitor of Notch signalling; 40 µM) group for in vitro experiments. For in vivo analysis, pregnant mice were divided into control group, oestradiol (intraperitoneal injection of 0.6 mg/kg/day) ± DAPT (subcutaneous injection of 10 mg/kg/day) groups and tamoxifen (gavage administration of 0.4 mg/kg/day) group. The results showed that oestradiol promoted hepatoblast differentiation into cholangiocytes and intrahepatic bile duct development during the embryonic period. Tamoxifen, an antioestrogenic drug, inhibited the above processes. Moreover, oestradiol promoted the expression of Notch signalling pathway-associated proteins and genes both in vitro and in vivo. Notably, DAPT addition inhibited the oestradiol-mediated effects. In conclusion, oestradiol can promote hepatoblast differentiation into cholangiocytes and intrahepatic bile duct development of C57BL/6CrSlc mice during embryonic period via the Notch signalling pathway.

NCK-associated protein 1 like (nckap1l) minor splice variant regulates intrahepatic biliary network morphogenesis.

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.

Development of the Intrahepatic and Extrahepatic Biliary Tract: A Framework for Understanding Congenital Diseases.

The involvement of the biliary tract in the pathophysiology of liver diseases and the increased attention paid to bile ducts in the bioconstruction of liver tissue for regenerative therapy have fueled intense research into the fundamental mechanisms of biliary development. Here, I review the molecular, cellular and tissular mechanisms driving differentiation and morphogenesis of the intrahepatic and extrahepatic bile ducts. This review focuses on the dynamics of the transcriptional and signaling modules that promote biliary development in human and mouse liver and discusses studies in which the use of zebrafish uncovered unexplored processes in mammalian biliary development. The review concludes by providing a framework for interpreting the mechanisms that may help us understand the origin of congenital biliary diseases.

Von Meyenburg Complex: Current Concepts and Imaging Misconceptions.

Von Meyenburg complexes, also known as biliary hamartomas, are ductal plate malformations that are usually diagnosed on imaging studies when there are multiple as small hepatic cysts. However, because of variations in histology, they actually have a wide spectrum of imaging appearances ranging from solid, to mixed solid and cystic, to cystic lesions. The objective of the review is to provide up-to-date information about the embryopathogenesis of von Meyenburg complexes, their imaging appearances, diagnostic pitfalls, and clinical significance.

Molecular regulation of mammalian hepatic architecture.

The essential liver exocrine and endocrine functions require a precise spatial arrangement of the hepatic lobule consisting of the central vein, portal vein, hepatic artery, intrahepatic bile duct system, and hepatocyte zonation. This allows blood to be carried through the liver parenchyma sampled by all hepatocytes and bile produced by the hepatocytes to be carried out of the liver through the intrahepatic bile duct system composed of cholangiocytes. The molecular orchestration of multiple signaling pathways and epigenetic factors is required to set up lineage restriction of the bipotential hepatoblast progenitor into the hepatocyte and cholangiocyte cell lineages, and to further refine cell fate heterogeneity within each cell lineage reflected in the functional heterogeneity of hepatocytes and cholangiocytes. In addition to the complex molecular regulation, there is a complicated morphogenetic choreography observed in building the refined hepatic epithelial architecture. Given the multifaceted molecular and cellular regulation, it is not surprising that impairment of any of these processes can result in acute and chronic hepatobiliary diseases. To enlighten the development of potential molecular and cellular targets for therapeutic options, an understanding of how the intricate hepatic molecular and cellular interactions are regulated is imperative. Here, we review the signaling pathways and epigenetic factors regulating hepatic cell lineages, fates, and epithelial architecture.

Generation of Hepatic Organoids with Biliary Structures.

Incorporation of bile drainage system into engineered liver tissue is an important issue to advance liver regenerative medicine. Our group reported that three-dimensional (3D) coculture of fetal liver cells (FLCs) and adult rat biliary epithelial cells (BECs) allows reconstruction of hepatic spheroids that possess bile ductular structures. In this chapter, we describe the detailed protocol to isolate FLCs and BECs and to generate the spheroids with bile drainage system using these two types of primary cells.

Proliferation-independent role of NF2 (merlin) in limiting biliary morphogenesis.

The architecture of individual cells and cell collectives enables functional specification, a prominent example being the formation of epithelial tubes that transport fluid or gas in many organs. The intrahepatic bile ducts (IHBDs) form a tubular network within the liver parenchyma that transports bile to the intestine. Aberrant biliary 'neoductulogenesis' is also a feature of several liver pathologies including tumorigenesis. However, the mechanism of biliary tube morphogenesis in development or disease is not known. Elimination of the neurofibromatosis type 2 protein (NF2; also known as merlin or neurofibromin 2) causes hepatomegaly due to massive biliary neoductulogenesis in the mouse liver. We show that this phenotype reflects unlimited biliary morphogenesis rather than proliferative expansion. Our studies suggest that NF2 normally limits biliary morphogenesis by coordinating lumen expansion and cell architecture. This work provides fundamental insight into how biliary fate and tubulogenesis are coordinated during development and will guide analyses of disease-associated and experimentally induced biliary pathologies.

Intrahepatic bile ducts guide establishment of the intrahepatic nerve network in developing and regenerating mouse liver.

Epithelial organs consist of multiple tissue structures, such as epithelial sheets, blood vessels and nerves, which are spatially organized to achieve optimal physiological functions. The hepatic nervous system has been implicated in physiological functions and regeneration of the liver. However, the processes of development and reconstruction of the intrahepatic nerve network and its underlying mechanisms remain unknown. Here, we demonstrate that neural class III ß-tubulin (TUBB3)+ nerve fibers are not distributed in intrahepatic tissue at embryonic day 17.5; instead, they gradually extend along the periportal tissue, including intrahepatic bile ducts (IHBDs), after birth. Nerve growth factor (Ngf) expression increased in biliary epithelial cells (BECs) and mesenchymal cells next to BECs before nerve fiber extension, and Ngf was upregulated by hairy enhancer of slit 1 (Hes family bHLH transcription factor 1; Hes1). Ectopic NGF expression in mature hepatocytes induced nerve fiber extension into the parenchymal region, from where these fibers are normally excluded. Furthermore, after BECs were damaged by the administration of 4,4-diaminodiphenylmethane, the nerve network appeared shrunken; however, it was reconstructed after IHBD regeneration, which depended on the NGF signal. These results suggest that IHBDs guide the extension of nerve fibers by secreting NGF during nerve fiber development and regeneration.

Development of the liver: Insights into organ and tissue morphogenesis.

Recent development of improved tools and methods to analyse tissues at the three-dimensional level has expanded our capacity to investigate morphogenesis of foetal liver. Here, we review the key morphogenetic steps during liver development, from the prehepatic endoderm stage to the postnatal period, and consider several model organisms while focussing on the mammalian liver. We first discuss how the liver buds out of the endoderm and gives rise to an asymmetric liver. We next outline the mechanisms driving liver and lobe growth, and review morphogenesis of the intra- and extrahepatic bile ducts; morphogenetic responses of the biliary tract to liver injury are discussed. Finally, we describe the mechanisms driving formation of the vasculature, namely venous and arterial vessels, as well as sinusoids.

TGFß signaling controls intrahepatic bile duct development may through regulating the Jagged1-Notch-Sox9 signaling axis.

Due to the inherent limitations of the mouse models, the molecular mechanism of TGFß signaling involved in the development of intrahepatic bile ducts (IHBDs) has been investigated little. Here, we investigated the role of TGFß signaling and its regulatory mechanism in IHBDs development. We demonstrate that TGFß signaling pathway activity is essential for IHBDs development. When blocking TGFß signaling at E10.5, the number of bile ducts in hilum was reduced more than two fold and number of CK19 positive chlangiocytes in periphery was reduced more than 3.5-fold compared with controls. We also show that alpha-smooth muscle actin (α-SMA)-immunoreactive cells are located in the portal vein mesenchyme (PVM) adjacent to the bile ducts during IHBDs development and identify the α-SMA positive cells expressing the Notch ligand Jagged1 in the periportal area. Importantly, after blocking TGFß signaling, the expression of Jagged1 was selectively decreased in the PVM but not in biliary epithelial cells (BECs), which is associated with the transformation of portal mesenchyme cells (PMCs) into portal myofibroblasts (PMFs). In addition, Sox9, which is downstream of Notch, is decreased after blocking the TGFß signaling pathway in the liver. Our findings uncover a novel mechanism of TGFß signaling in controlling the development of IHBDs may through regulating the Jagged1-Notch-Sox9 signaling axis.

Endoderm Jagged induces liver and pancreas duct lineage in zebrafish.

Liver duct paucity is characteristic of children born with Alagille Syndrome (ALGS), a disease associated with JAGGED1 mutations. Here, we report that zebrafish embryos with compound homozygous mutations in two Notch ligand genes, jagged1b (jag1b) and jagged2b (jag2b) exhibit a complete loss of canonical Notch activity and duct cells within the liver and exocrine pancreas, whereas hepatocyte and acinar pancreas development is not affected. Further, animal chimera studies demonstrate that wild-type endoderm cells within the liver and pancreas can rescue Notch activity and duct lineage specification in adjacent cells lacking jag1b and jag2b expression. We conclude that these two Notch ligands are directly and solely responsible for all duct lineage specification in these organs in zebrafish. Our study uncovers genes required for lineage specification of the intrahepatopancreatic duct cells, challenges the role of duct cells as progenitors, and suggests a genetic mechanism for ALGS ductal paucity.The hepatopancreatic duct cells connect liver hepatocytes and pancreatic acinar cells to the intestine, but the mechanism for their lineage specification is unclear. Here, the authors reveal that Notch ligands Jagged1b and Jagged2b induce duct cell lineage in the liver and pancreas of the zebrafish.

Human ductal plate and its derivatives express antigens of cholangiocellular, hepatocellular, hepatic stellate/progenitor cell, stem cell, and neuroendocrine lineages, and proliferative antigens.

Molecular mechanisms of human ductal plate (DP) development and differentiation (DD) are unclear. The author immunohistochemically investigated expressions of cholangiocellular antigens (CEA, CA19-9, EMA, MUC1, MUC2, MUC5AC, MUC6, mucins, CK7, and CK19), hepatocellular antigens (HepPar1, AFP, CK8, and CK18), hepatic stellate/progenitor cell (HSC) antigens or stem cell (SC) antigens (C-erbB2, CD56, chromogranin, synaptophysin, bcl2, NSE, NCAM, KIT, and PDGFRA), and proliferating antigen (Ki67) in 32 human fetal livers (HFL). The DD of human intrahepatic bile duct (IBD) could be categorized into four stages: DP, remodeling DP, remodeled DP, and immature IBD. All the molecules examined were expressed in the DP and DP derivatives. These results suggest that human DP or DP derivatives have capacities to differentiate into cholangiocellular, hepatocellular, HSC, SC, and neuroendocrine lineages. The data also suggest that NCAM, KIT/SC factor-signaling, NSE, HGF/MET signaling, PDGFa/PDGFRA signaling, chromogranin, synaptophysin, and CD56 play important roles in DD of DP and biliary cells of HFL. DP, DP derivatives, and IBD in HFL have proliferative capacity.

Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.

UNLABELLED: The intrahepatic bile duct (IHBD) is a highly organized tubular structure consisting of cholangiocytes, biliary epithelial cells, which drains bile produced by hepatocytes into the duodenum. Although several models have been proposed, it remains unclear how the three-dimensional (3D) IHBD network develops during liver organogenesis. Using 3D imaging techniques, we demonstrate that the continuous luminal network of IHBDs is established by 1 week after birth. Beyond this stage, the IHBD network consists of large ducts running along portal veins (PVs) and small ductules forming a mesh-like network around PVs. By analyzing embryonic and neonatal livers, we found that newly differentiated cholangiocytes progressively form a continuous and homogeneous luminal network. Elongation of this continuous network toward the liver periphery was attenuated by a potent Notch-signaling inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. Subsequent to this first step, the fine homogenous network is reorganized into the mature hierarchical network consisting of large ducts and small ductules. Between E17 and E18, when the homogenous network is radically reorganized into the mature hierarchical network, bile canaliculi rapidly extend and bile flow into IHBDs may increase. When formation of bile canaliculi was blocked between E16 and E18 by a multidrug resistance protein 2 inhibitor (benzbromarone), the structural rearrangement of IHBDs was significantly suppressed. CONCLUSION: Establishment of the mature IHBD network consists of two sequential events: (1) formation of the continuous luminal network regulated by the Notch-signaling pathway and (2) dynamic rearrangement of the homogeneous network into the hierarchical network induced by increased bile flow resulting from the establishment of hepatobiliary connections. (Hepatology 2016;64:175-188).

Hsp90 and hepatobiliary transformation during sea lamprey metamorphosis.

BACKGROUND: Biliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics. RESULTS: We hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection. CONCLUSIONS: HSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.

Compound heterozygous PKHD1 variants cause a wide spectrum of ductal plate malformations.

Ductal plate malformations (DPM) present with a wide phenotypic spectrum comprising Von Meyenburg complexes (VMC), Caroli disease (CD), Caroli syndrome (CS), and autosomal recessive polycystic kidney disease (ARPKD). Variants in PKHD1 are responsible for ARPKD and CS with a high inter- and intra-familial phenotypic variability. Rare familial cases of CD had been reported and exceptional cases of CD are associated with PKHD1 variants. In a family of three siblings presenting with a wide spectrum of severity of DPM, we performed whole exome sequencing and identified two PKHD1 compound heterozygous variants (c.10444G>A; p.Arg3482Cys and c.5521C>T; p.Glu1841Lys), segregating with the symptoms. Two compound heterozygous PKHD1 variants, including one hypomorphic variant, were identified in two other familial cases of DPM with at least one patient presenting with CD. This report widens the phenotypic variability of PKHD1 variants to VMC, and others hepatic bile ducts malformations with inconstant renal phenotype in adults and highlights the important intra-familial phenotypic variability. It also showed that PKHD1 might be a major gene for CD. This work adds an example of the contribution of exome sequencing, not only in the discovery of new genes but also in expanding the phenotypic spectrum of well-known disease-associated genes, using reverse phenotyping.

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.

Id2a is required for hepatic outgrowth during liver development in zebrafish.

During development, inhibitor of DNA binding (Id) proteins, a subclass of the helix-loop-helix family of proteins, regulate cellular proliferation, differentiation, and apoptosis in various organs. However, a functional role of Id2a in liver development has not yet been reported. Here, using zebrafish as a model organism, we provide in vivo evidence that Id2a regulates hepatoblast proliferation and cell death during liver development. Initially, in the liver, id2a is expressed in hepatoblasts and after their differentiation, id2a expression is restricted to biliary epithelial cells. id2a knockdown in zebrafish embryos had no effect on hepatoblast specification or hepatocyte differentiation. However, liver size was greatly reduced in id2a morpholino-injected embryos, indicative of a hepatic outgrowth defect attributable to the significant decrease in proliferating hepatoblasts concomitant with the significant increase in hepatoblast cell death. Altogether, these data support the role of Id2a as an important regulator of hepatic outgrowth via modulation of hepatoblast proliferation and survival during liver development in zebrafish.

Dynamic three-dimensional morphogenesis of intrahepatic bile ducts in mouse liver development.

UNLABELLED: During liver development, biliary epithelial cells differentiated from bipotential hepatic progenitor cells (hepatoblasts) form a cell layer, called the ductal plate surrounding portal veins (PVs), and develop into intrahepatic bile ducts (IBDs) following developmental programs. Because IBDs make duct structures in the liver, it is necessary to perform sequential and three-dimensional (3D) analyses from the early stages of liver development to address the process of morphogenesis in detail. However, to date, the development of IBDs has mainly been investigated using tissue sections in two-dimensional planes, and examinations of the 3D morphogenesis and quantitative analyses based on morphometrics have not been performed. Therefore, in this study, we simulated the solid structures of IBDs from mouse embryos to adults in silico, analyzed the subjects for the length and number of developing duct structures, number of predicted connections, and discrete distance from the PV, and examined the developmental process of the IBD in detail in a quantitative manner. CONCLUSIONS: Through quantitative analyses with spatiotemporal observations using a 3D structural reconstruction model and morphometrics, we succeeded in constructing a 3D dynamic model of bile duct formation. Because the 3D reconstruction technique used in this study is available for analyzing solid structures in tissues that are difficult to approach, it shows promise for wide use in the fields of biology and medicine.

Dyskerin expression in human fetal, adult and neoplastic intrahepatic bile ducts: correlations with cholangiocarcinoma aggressiveness.

AIMS: To investigate the immunohistochemical expression of dyskerin, a biomarker involved in ribosome production and telomere maintenance, in human fetal, adult and neoplastic bile ducts, and possible correlations with cholangiocarcinoma aggressiveness. METHODS AND RESULTS: Sixty consecutive intrahepatic cholangiocarcinomas were collected and used for tissue microarray construction (total: 176 cores); clinical data and follow-up were also collected. Five fetal and 10 normal adult livers were included as controls. Automated immunohistochemistry for dyskerin, p53, and Ki67, and nucleolar silver staining, were performed. In normal livers, dyskerin expression was negative in smaller bile ducts (mean 44.8 µm) and positive in bile ducts of larger diameter (mean 116.1 µm; P < 0.001). Expression was positive in 56.7% of cholangiocarcinomas, and correlated with p53 mutation (P = 0.008) and a higher proliferative (Ki67) index (P = 0.003), which were included as markers of tumour aggressiveness. Finally, dyskerin-positive cholangiocarcinomas showed a negative trend in disease-free survival (P = 0.078) on univariate analysis. CONCLUSIONS: The non-neoplastic biliary tree seems to progressively lose dyskerin expression from the major branches to the peripheral portal bile ducts. Similarly, intrahepatic cholangiocarcinomas showed two patterns of dyskerin expression, and the dyskerin-positive phenotype seemed to characterize more aggressive cholangiocarcinomas.