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Development of the liver: Insights into organ and tissue morphogenesis

Published:January 12, 2018DOI:https://doi.org/10.1016/j.jhep.2018.01.005

      Summary

      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.

      Keywords

      Introduction

      In the last two decades most efforts to decipher the mechanisms of liver development were focussed on identifying regulators of cell fate specification and differentiation. As a result, a number of developmental disease mechanisms are now better understood and hepatocyte-like cells can be produced in vitro by recapitulating developmental processes.
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      However, the way in which the three-dimensional tissular architecture of the liver develops in the embryo remains much less explored. Yet, gaining new knowledge in this area has become key to enabling in vitro production of stem cell-derived hepatic tissue, in which hepatocyte cords, bile ducts and vasculature are reliably connected. This review focusses on new insights into the morphogenesis of the embryonic liver.

      Initiation of liver morphogenesis

      Origin of liver progenitor cells

      Three neighboring regions of the endoderm give rise to bipotent liver progenitors which eventually differentiate into hepatocytes and cholangiocytes.
      The liver is largely composed of hepatocytes and cholangiocytes, which differentiate from bipotent liver progenitors, the hepatoblasts. During development, hepatoblasts are specified in the ventral foregut endoderm by signals released from adjacent mesodermal tissues, including ligands of the Bone morphogenic protein (Bmp), Wnt and Fibroblast growth factor (Fgf) families, depending on the species.
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      At around embryonic day (E)8.5 in mice, hepatoblasts start expressing liver specific proteins, such as transcription factors Hhex and Prox1, followed soon after by α-fetoprotein (Afp), hepatocyte nuclear factor (Hnf)4α and albumin (Alb). Single-cell dye labelling has identified the origin of hepatoblasts within the foregut prior to the onset of fate specific gene expression.
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      Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues.
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      Bmp2 signaling regulates the hepatic vs. pancreatic fate decision.
      Liver progenitors arise from bilateral populations of lateral endoderm, which merge in the process of gut tube formation at the ventral midline (Fig. 1). A second smaller progenitor population, positioned slightly more anteriorly in chicken and mice, was identified in the ventral midline endodermal lip (VMEL), which moves caudally and contributes to large parts of the liver and other organ fates.
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      Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues.
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      The bilateral and VMEL progenitors initially respond to Fgf and Bmp respectively,
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      appearing to give rise to different parts of the liver. The latter was revealed when blocking early Fgf signalling elicited a differential survival response in the forming liver bud, with high apoptosis in the anterior bud, and low apoptosis posteriorly.
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      FGF signaling is required for anterior but not posterior specification of the murine liver bud.
      This indicates that the Fgf-responsive bilateral cells give rise to the posterior part of the liver bud, while the VMEL cells mostly contribute to the anterior population. It is unclear whether the two different progenitor sources correlate with distinct metabolic functions or spatial distribution within the adult liver and whether they exist in all vertebrates. Deleting Sfrp5 in frogs revealed that repression of non-canonical Wnt signalling is essential for epithelial integrity of the foregut endoderm prior to liver induction.
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      Figure thumbnail gr1
      Fig. 1Origin of hepatoblasts and early stages of liver budding. (A) Mouse and (B) zebrafish.

      Organ bud morphogenesis

      Newly specified hepatoblasts form the organ bud by undergoing a combination of morphogenetic processes, including cell shape changes, cell proliferation and migration. The first sign of liver bud morphogenesis is the thickening of the ventral foregut endoderm, which coincides with the start of hepatoblast gene expression and formation of the gut tube at around E8.5. Following inductive signalling from the adjacent mesoderm, early liver bud morphogenesis occurs via distinct steps: firstly, the cuboidal foregut epithelium changes into a thickened columnar epithelium of hepatoblasts, which subsequently transitions into a pseudostratified epithelium until it finally breaks down, at which point hepatoblasts delaminate and migrate into the adjacent mesenchyme. In the pseudostratified state, hepatoblasts undergo internuclear migration, with nuclei entering S-phase in the basal region and mitosis when positioned apically.
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      Hex homeobox gene controls the transition of the endoderm to a pseudostratified, cell emergent epithelium for liver bud development.
      The transcription factor Hhex is a critical regulator of the switch from columnar to pseudostratified epithelial morphology. Hhex mutants fail to form a thickened epithelium, while exhibiting impaired proliferation and a loss of hepatic gene expression by E10.5.
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      Hex homeobox gene controls the transition of the endoderm to a pseudostratified, cell emergent epithelium for liver bud development.
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      Liver organogenesis promoted by endothelial cells prior to vascular function.
      Ablation of endothelial cells by inactivation of Vascular endothelial growth factor receptor 2 (Vegfr2) blocks liver outgrowth at E9.5 and causes cessation of hepatic gene expression. In contrast, in zebrafish, blood vessels are only essential for supporting later stages of liver growth.
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      Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish.
      The onset of liver outgrowth coincides with a loss of contacts between hepatoblasts resulting from downregulation of E-cadherin. This allows hepatoblasts to migrate into the surrounding mesenchyme. Concomitantly, matrix metalloproteinases (MMPs) breakdown the laminin, collagen IV and fibronectin containing basal membrane, facilitating hepatoblast migration.
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      Immunolocalization of extracellular matrix components and integrins during mouse liver development.
      This extracellular matrix (ECM) remodelling is controlled most prominently by mesenchymally expressed Mmp2 and hepatoblast-expressed Mmp14.
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      Role of metalloproteinases at the onset of liver development.
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      Hand2 regulates extracellular matrix remodeling essential for gut-looping morphogenesis in zebrafish.
      Prox1 mutants exhibit impaired ECM degradation and maintain high E-cadherin expression, thereby impeding hepatoblast delamination and liver outgrowth.
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      Hepatocyte migration during liver development requires Prox1.
      In Prox1 mutants, a small liver bud forms, expressing Afp and Alb, but consisting largely of mesenchymal cells.
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      Hepatocyte migration during liver development requires Prox1.
      Prox1 expression in delaminating hepatoblasts is controlled at least in part by the transcriptional repressor Tbx3, which is required to maintain Prox1 expression. Liver outgrowth is defective in the Tbx3 knockout mice.
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      Tbx3 promotes liver bud expansion during mouse development by suppression of cholangiocyte differentiation.
      Other than in Prox1 mutants, the few hepatoblasts in Tbx3 mutants differentiate along the biliary lineage. The authors propose an alternative model, in which hampered hepatoblast delamination and emigration are secondary to the failure of hepatoblasts to differentiate into hepatocytes and not vice versa.
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      Tbx3 promotes liver bud expansion during mouse development by suppression of cholangiocyte differentiation.
      The Onecut transcription factors Hnf-6/Onecut1 and Onecut2 are additional regulators of hepatoblast migration, but unlike in Prox1 mutants, double mutant phenotypes are transient and liver outgrowth recovers, suggesting that other factors compensate.
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      The Onecut transcription factors HNF-6/OC-1 and OC-2 regulate early liver expansion by controlling hepatoblast migration.
      This work further identified key downstream genes involved in cell adhesion and migration.
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      The Onecut transcription factors HNF-6/OC-1 and OC-2 regulate early liver expansion by controlling hepatoblast migration.
      Tbx3, Prox1, Hnf6/Onecut1 and Onecut2, whose mutants exhibit similar phenotypes, form a regulatory gene network controlling hepatoblast migration, although the exact genetic hierarchy still needs to be determined.
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      Gene regulatory networks in differentiation and direct reprogramming of hepatic cells.
      Early hepatoblast proliferation and survival are interlinked with liver outgrowth, which has been described previously.
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      • Duncan S.A.
      Organogenesis and development of the liver.
      • Gordillo M.
      • Evans T.
      • Gouon-Evans V.
      Orchestrating liver development.
      • Wang S.
      • Miller S.R.
      • Ober E.A.
      • Sadler K.C.
      Making it new again: insight into liver development, regeneration, and disease from zebrafish research.

      Liver asymmetry

      A defining feature of the liver is its asymmetric position with respect to the long body axis, the incorrect alignment of the liver and other internal organs leads to a number of genetic disorders.
      A defining feature of the digestive organs, including the liver, is their asymmetric position with respect to the long body axis. Genetic disorders in humans, such as heterotaxy and situs inversus, exhibit an incorrect or reversed alignment of the majority of internal organs. Heterotaxy patients often have multiple organ defects including cardiovascular malformations and biliary atresia.
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      Biliary atresia: A comprehensive review.
      Such defects arise when the liver takes up its asymmetric position after hepatoblasts are initially specified at the embryonic midline. The first insight into how symmetry is broken came from studies in zebrafish, which suggested a repulsion-based mechanism (Fig. 1). Live-imaging showed that hepatoblasts are highly motile and that their directional migration to form the asymmetric liver bud is directed by repulsive signals from the adjacent right lateral plate mesoderm (LPM).
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      EphrinB1/EphB3b coordinate bidirectional epithelial-mesenchymal interactions controlling liver morphogenesis and laterality.
      These repulsive cell interactions are mediated by the transmembrane protein EphrinB1 (EfnB1) and its receptor EphB3b (EphB3b), expressed by hepatoblasts and the LPM, respectively. Disrupted EfnB1/EphB3b signalling leads to defects in asymmetric liver positioning and LPM morphogenesis, indicating that bidirectional interactions between hepatoblasts and adjacent mesoderm are crucial for the coordinated migration and morphogenesis of both tissues.
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      EphrinB1/EphB3b coordinate bidirectional epithelial-mesenchymal interactions controlling liver morphogenesis and laterality.
      A pioneering discovery in zebrafish identified the asymmetric movement of the bilateral LPM epithelia as essential for the early leftward placement of hepatoblasts and gut looping.
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      A cellular framework for gut-looping morphogenesis in zebrafish.
      During asymmetric liver positioning the left LPM moves dorsal to the endoderm and the right LPM moves ventrolateral. This asymmetric movement and the epithelial morphology of the LPM is disrupted in polarity gene mutants, including those with mutations in protein kinase C iota, pard6γb and actin modulators, who also exhibit a midline liver.
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      A cellular framework for gut-looping morphogenesis in zebrafish.
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      Mypt1-mediated spatial positioning of Bmp2-producing cells is essential for liver organogenesis.
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      Regulation of neurocoel morphogenesis by Pard6 gamma b.
      Similar LPM and liver defects in lamininb1a mutants and upon blocking MMPs showed that ECM remodelling, in particular temporally controlled laminin degradation, are essential for these morphogenetic movements.
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      Hand2 regulates extracellular matrix remodeling essential for gut-looping morphogenesis in zebrafish.
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      Laminin beta1a controls distinct steps during the establishment of digestive organ laterality.
      These results suggest that as the right LPM migrates leftwards it pushes hepatoblasts from the midline to their asymmetric position within the body (Fig. 1). The specific contributions of the LPM’s pushing force and active hepatoblast migration to hepatic left-right asymmetry remain to be determined. Chemical screening in zebrafish identified retinoic acid signalling via its receptor Rargb as an early regulator of organ asymmetry.
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      Rargb regulates organ laterality in a zebrafish model of right atrial isomerism.
      While asymmetric tissue morphogenesis of the left and right mesoderm govern asymmetric liver positioning in zebrafish and the mammalian intestine,
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      The chirality of gut rotation derives from left-right asymmetric changes in the architecture of the dorsal mesentery.
      the mechanism driving liver asymmetry in mammals is less well understood. It might be influenced by the major vessels, which are asymmetric and become enveloped by the liver, as suggested by abnormal liver lobe formation in laterality mutants.
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      Abnormal laterality and congenital cardiac anomalies. Relations of visceral and cardiac morphologies in the iv/iv mouse.
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      Lefty-1 is required for left-right determination as a regulator of lefty-2 and nodal.
      In chicken, NEURTURIN expression by the sinus venosus promotes the outgrowth of the liver bud, which expresses NEURTURIN GROWTH FACTOR RECEPTORα2, providing further support for such an interaction.
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      Liver and lobe growth during development

      In the adult organ the visceral ligaments divide the liver into multiple lobes, which are of a specific though unequal shape and size. This chararacteristic morphology of the hepatic lobes could be established in the embryo by differential hepatoblast proliferation or directional migration into specific domains of the nascent liver bud. While there is currently little evidence for coordinated hepatoblast rearrangement or active migration contributing to lobe formation, localised growth zones were implicated in hepatic lobe formation by examining proliferation patterns in chicken embryos. Increased cell division was detected at the periphery of the growing organ and in particular at the tips of the liver lobes, in contrast to few proliferation events in central regions of the liver.
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      Morphogenesis of chicken liver: identification of localized growth zones and the role of beta-catenin/Wnt in size regulation.
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      Characterization and functional analyses of hepatic mesothelial cells in mouse liver development.
      Conversely, hepatic differention, indicated by Alb expression and glycogen production, is most advanced in the centre of each lobe and declines towards the periphery and tips of the lobes, fitting with the notion that hepatocyte maturation is associated with progressively reduced proliferation. Functional studies showed that hepatoblast proliferation in these growth zones requires β-catenin.
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      In line with the idea that new hepatic precursors are added at the periphery of the growing liver primordium is the finding that mesothelial cells surrounding the liver secrete growth factors. Mesothelial cells are derived from the LPM and cover all visceral organs in a single layer, which is thought to have barrier-functions and thus act as a protective surface.
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      Moreover, lineage tracing of mesothelial progenitors showed their contribution to the mesothelium, as well as endothelial and hepatic stellate cells.
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      Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver.
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      The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebrafish: analysis of stellate cell entry into the developing liver.
      The functional importance of mesothelial cells for liver growth and lobulation was uncovered by inactivation of the highly expressed gene Wilms tumor1 (Wt1). Wt1 mutants exhibit small livers with incomplete lobulation at E13.5.
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      Wt1 and retinoic acid signaling are essential for stellate cell development and liver morphogenesis.
      Mutant and in vitro experiments showed Wt1 is important for mesothelial cell proliferation and differentiation, since loss of Wt1 function is associated with a significant reduction of pleiotrophin and midkine expression, two secreted proteins that promote hepatoblast proliferation. This mitogenic mesoderm-endoderm interaction was corroborated by in vitro co-culture of wild-type or Wt1 mutant mesothelial cells with wild-type hepatoblasts.
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      The size of the liver is tightly regulated in the adult, with a robust homeostatic and regenerative response ensuring its correct size.
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      Hippo pathway activity influences liver cell fate.
      A zebrafish study showed that Yap1 controls liver size by regulating nucleotide biosynthesis.
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      Although organ size is restored after surgical removal of large parts of the liver, its overall shape is signifcantly changed, because instead of restoration of the lost tissue by outgrowth from a blastema at the injury site, cells in the remnant liver proliferate, leading to an altered morphology. This suggests that the overall shape of the liver is less important for its metabolic functions than its volume and highly-specialised tissue architecture. The developing liver seems to have a similar potential to regulate organ size by adjusting the number of progenitor cells,
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      Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver.
      in a Hhex-dependent manner.
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      Hex homeobox gene controls the transition of the endoderm to a pseudostratified, cell emergent epithelium for liver bud development.

      Differentiation of the hepatic parenchyme

      The liver architecture is highly organised with polarised hepatocytes at the interface between endothelial sinusoids and bile canaliculi (Fig. 2). Establishment and maintenance of hepatocyte polarity requires the interplay of ECM interactions, cell adhesion and formation of cell junctions, cytoskeletal dynamics and intracellular trafficking.
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      Bipotent hepatoblasts are initially non-polarised and mixed with foetal haematopoietic stem cells. The latter only reside in the liver transiently and start declining between E17-21, leading to tissue maturation with increasing cellular adhesion of the hepatic parenchyme and formation of dense hepatocyte cords.
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      Zonula occludens-1 (ZO-1)/Tight junction protein 1 (Tjp1) staining of tight junctions shows that, initially, short bile canaliculi form around E17,
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      Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.
      including acini-like configurations, which likely form between clusters of hepatocytes and appear dilated.
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      The establishment of hepatocyte cell surface polarity during fetal liver development.
      Rapid extension and coalescense of these short canaliculi results in a canalicular network which is functionally connected to the intrahepatic bile ducts by E18
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      (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Differentiation of the hepatic parenchyme in mammals.
      The molecular mechanisms governing hepatocyte polarity have been extensively studied in in vitro systems, such as HepG2, MDCK and WIF-B cells or sandwich cultures of primary hepatocytes,
      • Gissen P.
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      Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.
      while there is a large gap in our understanding of how polarity is established within the tissue context during development.
      Haematopoietic progenitors, which are initially interspersed with hepatoblasts, release interleukin 6 family member Oncostatin M (OSM), which signals to hepatocytes through the receptor gp130 (IL6STP1). OSM can promote hepatocyte differentiation and formation of canalicular structures in culture,
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      Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer.
      but it is not required in the embryo, possibly because of compensation by Interleukin 6 itself.
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      • Miyajima A.
      Hepatocyte proliferation and tissue remodeling is impaired after liver injury in oncostatin M receptor knockout mice.
      In contrast, Hnf4α is a key transcriptional regulator of morphological and functional hepatocyte differentiation.
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      Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.
      Foetal inactivation of Hnf4α with Albumin-Afp:Cre specifically blocks the transition of round hepatoblasts into the characteristic mature polygonal shape, concomitant with loss of late hepatocyte gene expression. Hnf4α mutant hepatocytes show reduced cell contacts, disrupted expression of cell adhesion factors and localisation of junctional proteins, such as E-cadherin and ZO-1/Tjp1, respectively.
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      • et al.
      Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.
      Classic junctional and polarity complexes/proteins, as well as members of the planar cell polarity pathway are present in hepatocytes, though surprisingly little is known about their functions in setting up the unique hepatocyte polarity during development. Studying their early functions is of great clinical interest, given the strong correlation between hepatocellular carcinoma and disrupted expression of polarity proteins.

      Braiterman L. T., Hubbard A. L. Hepatocyte Surface Polarity: Its Dynamic Maintenance and Establishment. in The Liver: Biology and Pathobiology (ed. Arias, I.M.) (John Wiley & Sons Ltd, Chichester, UK, 2009).

      Moreover, disruption of cell polarity causes a redistribution of apical bile acid transporters and the formation of intracellular pseudocanaliculi.
      • Gissen P.
      • Arias I.M.
      Structural and functional hepatocyte polarity and liver disease.
      Membrane trafficking pathways, endocytic and recycling, sort polarised membrane proteins and junctional components to the correct domain of the plasma membrane.
      • Apodaca G.
      • Gallo L.I.
      • Bryant D.M.
      Role of membrane traffic in the generation of epithelial cell asymmetry.
      A key regulator in the liver is Liver kinase B1 (Lkb1), which controls intracellular trafficking and hepatocyte polarity via its effector AMP-activated protein kinase.
      • Gissen P.
      • Arias I.M.
      Structural and functional hepatocyte polarity and liver disease.
      Hepatocyte deletion of Lkb1 by Albumin:Cre, which often recombines genes at later stages of liver development compared to Albumin-Afp:Cre, results in fewer but longer and wider canalicular structures, delayed apical secretion and disrupted tight junctions.
      • Woods A.
      • Heslegrave A.J.
      • Muckett P.J.
      • Levene A.P.
      • Clements M.
      • Mobberley M.
      • et al.
      LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice.
      • Porat-Shliom N.
      • Tietgens A.J.
      • Van Itallie C.M.
      • Vitale-Cross L.
      • Jarnik M.
      • Harding O.J.
      • et al.
      Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo.
      Dye injections into the vascular and biliary network demonstrated that this leads to intermixing of blood and bile, and Lkb1 mutant mice develop cholestasis. Moreover, Rab5-mediated intracellular trafficking of early endosomes is necessary to maintain the localised protein distribution essential for liver function in the differentiated organ.
      • Zeigerer A.
      • Wuttke A.
      • Marsico G.
      • Seifert S.
      • Kalaidzidis Y.
      • Zerial M.
      Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.
      Conditional deletion of Vacuolar sorting protein 33b (Vps33b) or Vipar is associated with alterations in tight junction organisation and reduced apical microvilli, causing cholestasis in mice.
      • Cullinane A.R.
      • Straatman-Iwanowska A.
      • Zaucker A.
      • Wakabayashi Y.
      • Bruce C.K.
      • Luo G.
      • et al.
      Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization.
      • Hanley J.
      • Dhar D.K.
      • Mazzacuva F.
      • Fiadeiro R.
      • Burden J.J.
      • Lyne A.M.
      • et al.
      Vps33b is crucial for structural and functional hepatocyte polarity.
      • Matthews R.P.
      • Plumb-Rudewiez N.
      • Lorent K.
      • Gissen P.
      • Johnson C.A.
      • Lemaigre F.
      • et al.
      Zebrafish vps33b, an ortholog of the gene responsible for human arthrogryposis-renal dysfunction-cholestasis syndrome, regulates biliary development downstream of the onecut transcription factor hnf6.
      Similarly, lack of Claudin15 (Cldn15) in zebrafish leads to disrupted hepatocyte polarity and malformed canaliculi.
      • Cheung I.D.
      • Bagnat M.
      • Ma T.P.
      • Datta A.
      • Evason K.
      • Moore J.C.
      • et al.
      Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b.
      These phenotypes are transient in cldn15 mutants suggesting a robust regulatory network upstream of hepatocyte polarity.
      The characteristic cord-like hepatocyte arrangement could be generated by cell rearrangement or oriented cell division. Indeed, asymmetric cell division marked by inheritance of the entire apical membrane is mediated by Par1b and Leu-Gly-Asn repeat-enriched protein, which orient mitotic spindles away from the apical membrane.
      • Slim C.L.
      • Lazaro-Dieguez F.
      • Bijlard M.
      • Toussaint M.J.
      • de Bruin A.
      • Du Q.
      • et al.
      Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes.
      In contrast, symmetric divisions through the apical hepatocyte membrane, resulting in expanded canaliculi, have been observed in hepatocyte doublets in microniche cultures.
      • Li Q.
      • Zhang Y.
      • Pluchon P.
      • Robens J.
      • Herr K.
      • Mercade M.
      • et al.
      Extracellular matrix scaffolding guides lumen elongation by inducing anisotropic intercellular mechanical tension.
      Whether the latter contribute to liver development and tissue repair is unkown. Contact with the ECM, largely mediated by integrins, is necessary for epithelial differentiation. This is especially evident in two- or three-dimentional hepatocyte cultures or sandwich overlays, in which the acquisition of polarity is dependent on the correct ECM composition. However, unlike in most epithelial tissues, no basal lamina forms between the epithelial hepatocytes and endothelial cells, which together with a low-density ECM and fenestrated sinusoids facilitate the exchange between blood and hepatocytes. The ECM consists mainly of fibronectin, laminin and collagen type I and IV during midgestation stages, with fibronectin and laminin declining postnatally.
      • Baloch Z.
      • Klapper J.
      • Buchanan L.
      • Schwartz M.
      • Amenta P.S.
      Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study.
      Despite the unusual ECM configuration, its importance for hepatic differentiation is implicated by chimeric studies, in which Integrinβ1-deficient cells are found in almost all tissues, but do not contribute to the liver.
      • Fassler R.
      • Meyer M.
      Consequences of lack of beta 1 integrin gene expression in mice.
      Consistently, deletion of Integrin linked kinase 1 (Ilk1) in hepatoblasts by Albumin-Afp:Cre causes disorganised hepatic cords after birth, accompanied by higher hepatocyte and cholangiocyte proliferation and increased cytoplasmic and nuclear β-catenin
      • Gkretsi V.
      • Apte U.
      • Mars W.M.
      • Bowen W.C.
      • Luo J.H.
      • Yang Y.
      • et al.
      Liver-specific ablation of integrin-linked kinase in mice results in abnormal histology, enhanced cell proliferation, and hepatomegaly.
      resulting in twice the normal liver to body weight.
      • Weinstein M.
      • Monga S.P.
      • Liu Y.
      • Brodie S.G.
      • Tang Y.
      • Li C.
      • et al.
      Smad proteins and hepatocyte growth factor control parallel regulatory pathways that converge on beta1-integrin to promote normal liver development.
      ECM gene expression by hepatocytes and endothelial cells needs to be repressed to maintain liver functions. This is evident during chronic liver injury, wherein increased ECM deposition and formation of a basal lamina hamper physiologic exchange.
      The hepatic tissue architecture is further coordinated by bidirectional cell interactions between differentiating hepatoblasts and sinusoidal endothelial cells. Nascent endothelial cells migrate along the basal side of hepatocytes during the formation of the vascular network in zebrafish.
      • Sakaguchi T.F.
      • Sadler K.C.
      • Crosnier C.
      • Stainier D.Y.
      Endothelial signals modulate hepatocyte apicobasal polarization in zebrafish.
      Mutant analyses showed that the regular spacing of hepatocytes between the biliary and sinusoidal networks is regulated by endothelial expression of cerebral cavernous malformation 2 (ccm2) and EGF-like domain containing gene heart of glass (heg1).
      • Sakaguchi T.F.
      • Sadler K.C.
      • Crosnier C.
      • Stainier D.Y.
      Endothelial signals modulate hepatocyte apicobasal polarization in zebrafish.
      The division and alignment of hepatocytes along sinusoids also supports the recovery of functional tissue organisation in mammalian liver regeneration, as revealed by mathematical modelling.
      • Hoehme S.
      • Brulport M.
      • Bauer A.
      • Bedawy E.
      • Schormann W.
      • Hermes M.
      • et al.
      Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration.
      Conversely, Hnf4α-dependent differentiation of the polarised hepatic epithelium also influences the formation of a functional sinusoidal network in mice.
      • Parviz F.
      • Matullo C.
      • Garrison W.D.
      • Savatski L.
      • Adamson J.W.
      • Ning G.
      • et al.
      Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.

      Bile duct morphogenesis

      Development of intrahepatic ducts

      Bile duct development is initiated near the hilum of the liver, before progressing towards the lobes.
      It has been known for many years that bile duct development is initiated near the hilum of the liver before progressing towards the periphery of the lobes. However, our understanding of duct morphogenesis has recently improved with advanced three-dimensional imaging and computer-assisted analysis, and with retrograde ink injection studies enabling visualisation of bile duct lumina in the whole liver.
      • Tanimizu N.
      • Kaneko K.
      • Itoh T.
      • Ichinohe N.
      • Ishii M.
      • Mizuguchi T.
      • et al.
      Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.
      • Antoniou A.
      • Raynaud P.
      • Cordi S.
      • Zong Y.
      • Tronche F.
      • Stanger B.
      • et al.
      Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.
      • Takashima Y.
      • Terada M.
      • Kawabata M.
      • Suzuki A.
      Dynamic three-dimensional morphogenesis of intrahepatic bile ducts in mouse liver development.
      • Kaneko K.
      • Kamimoto K.
      • Miyajima A.
      • Itoh T.
      Adaptive remodeling of the biliary architecture underlies liver homeostasis.
      Yet such three-dimensional approaches have their limitations: ink injections cannot be implemented easily in early embryonic liver, and three-dimensional imaging has not yet captured the less mature structures. Indeed, duct morphogenesis was visualised by immunodetection of Osteopontin (Spp1) or Cytokeratin 19 (Krt19), two markers not detected at the onset of cholangiocyte differentiation, and nascent ductal structures would not be visualised with ink injections because they lack connectivity. An attempt to integrate the conclusions of classical two-dimensional histology and more recent three-dimensional analyses is shown (Fig. 3). Firstly, cholangiocytes line up around the periportal mesenchyme, forming a nearly continuous ductal plate, in which small lumina become rapidly detectable. These lumina are lined on the portal side by cholangiocytes and on the parenchymal side by hepatoblast-like cells,
      • Antoniou A.
      • Raynaud P.
      • Cordi S.
      • Zong Y.
      • Tronche F.
      • Stanger B.
      • et al.
      Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.
      • Raynaud P.
      • Tate J.
      • Callens C.
      • Cordi S.
      • Vandersmissen P.
      • Carpentier R.
      • et al.
      A classification of ductal plate malformations based on distinct pathogenic mechanisms of biliary dysmorphogenesis.
      • Vestentoft P.S.
      • Jelnes P.
      • Hopkinson B.M.
      • Vainer B.
      • Mollgard K.
      • Quistorff B.
      • et al.
      Three-dimensional reconstructions of intrahepatic bile duct tubulogenesis in human liver.
      but how these asymmetrically-lined lumina are formed is not well understood: hepatoblast-like cells might either derive directly from hepatoblasts that become committed to a ductal fate, or, as suggested by in vitro studies, they might originate from ductal plate cells which depolarise, dedifferentiate and migrate to fold up and delineate a lumen.
      • Antoniou A.
      • Raynaud P.
      • Cordi S.
      • Zong Y.
      • Tronche F.
      • Stanger B.
      • et al.
      Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.
      • Tanimizu N.
      • Miyajima A.
      • Mostov K.E.
      Liver progenitor cells fold up a cell monolayer into a double-layered structure during tubular morphogenesis.
      When the ducts mature, i.e. when they grow along the vein towards the periphery of the lobes, the hepatoblast-like cells differentiate to cholangiocytes, giving rise to bile ducts entirely lined by cholangiocytes. Three-dimensional analyses have revealed that duct growth is discontinuous. Along the axis of the future duct, the ductal plate forms small luminal spheres which progressively connect with each other, thereby elongating the duct. In parallel, a perivenous biliary plexus forms which eventually remodels into a dense network of small tubules connected to one or two main ducts. Lineage tracing suggests that ductal plate cells that are not involved in duct formation lose their molecular and morphological features and revert to a hepatocyte phenotype.
      • Carpentier R.
      • Suner R.E.
      • Van Hul N.
      • Kopp J.L.
      • Beaudry J.B.
      • Cordi S.
      • et al.
      Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells.
      Figure thumbnail gr3
      Fig. 3Intrahepatic bile duct morphogenesis in embryonic mouse liver. See text for details.

      Signalling pathways controlling bile duct morphogenesis

      Separating the molecular mechanisms that regulate the onset of ductal plate formation from those controlling differentiation of hepatoblasts to cholangiocytes remains difficult. The role of TGFβ, Notch, Wnt, Fgf, Laminin, Hippo and Epimorphin signalling in cholangiocyte differentiation has been reviewed elsewhere,
      • Gerard C.
      • Tys J.
      • Lemaigre F.P.
      Gene regulatory networks in differentiation and direct reprogramming of hepatic cells.
      • Raynaud P.
      • Carpentier R.
      • Antoniou A.
      • Lemaigre F.P.
      Biliary differentiation and bile duct morphogenesis in development and disease.
      • Zong Y.
      • Stanger B.Z.
      Molecular mechanisms of liver and bile duct development.
      • Jia Y.
      • Yao H.
      • Zhou J.
      • Chen L.
      • Zeng Q.
      • Yuan H.
      • et al.
      Role of epimorphin in bile duct formation of rat liver epithelial stem-like cells: involvement of small G protein RhoA and C/EBPbeta.
      but how these pathways control subsequent tubular network formation has yet to be discussed. TGFβ receptor II and TGFβ signalling are activated in hepatoblasts differentiating to ductal plate cholangiocytes, but they become repressed once the cholangiocytes delineate a lumen. Such repression is supported by direct and indirect observations. Indeed, inhibition of TGFβ represses formation of biliary-like cysts in an in vitro morphogenesis assay,
      • Tanimizu N.
      • Miyajima A.
      • Mostov K.E.
      Liver progenitor cells develop cholangiocyte-type epithelial polarity in three-dimensional culture.
      while persisting receptor expression is associated with delayed duct maturation in SRY-related HMG box transcription factor (Sox)4- and Sox9-deficient livers.
      • Antoniou A.
      • Raynaud P.
      • Cordi S.
      • Zong Y.
      • Tronche F.
      • Stanger B.
      • et al.
      Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.
      • Poncy A.
      • Antoniou A.
      • Cordi S.
      • Pierreux C.E.
      • Jacquemin P.
      • Lemaigre F.P.
      Transcription factors SOX4 and SOX9 cooperatively control development of bile ducts.
      Blocking Notch signalling by timed gene inactivation or pharmacological inhibition demonstrated that Jagged 1 and Notch 2, whose mutations cause bile duct paucity in patients affected by Alagille syndrome, are required for tube formation.
      • Tanimizu N.
      • Kaneko K.
      • Itoh T.
      • Ichinohe N.
      • Ishii M.
      • Mizuguchi T.
      • et al.
      Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.
      • Zong Y.
      • Panikkar A.
      • Xu J.
      • Antoniou A.
      • Raynaud P.
      • Lemaigre F.
      • et al.
      Notch signaling controls liver development by regulating biliary differentiation.
      • Lorent K.
      • Moore J.C.
      • Siekmann A.F.
      • Lawson N.
      • Pack M.
      Reiterative use of the notch signal during zebrafish intrahepatic biliary development.
      • Hofmann J.J.
      • Zovein A.C.
      • Koh H.
      • Radtke F.
      • Weinmaster G.
      • Iruela-Arispe M.L.
      Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development: insights into Alagille syndrome.
      Notch signalling initially induces differentiation of the single-layered ductal plate from hepatoblasts, following an interaction between the Jagged 1-positive portal mesenchyme and Notch 2 in hepatoblasts.
      • Hofmann J.J.
      • Zovein A.C.
      • Koh H.
      • Radtke F.
      • Weinmaster G.
      • Iruela-Arispe M.L.
      Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development: insights into Alagille syndrome.
      Notch then promotes tubulogenesis, most likely through Jagged-Notch interactions occurring between the cholangiocytes on the portal side and the hepatoblast-like cells located on the parenchymal side of the early luminal structures.
      • Zong Y.
      • Panikkar A.
      • Xu J.
      • Antoniou A.
      • Raynaud P.
      • Lemaigre F.
      • et al.
      Notch signaling controls liver development by regulating biliary differentiation.
      Therefore, in parallel to growth along the longitudinal axis of the ducts, differentiation proceeds radially. Genetic modifiers of Notch signalling are suspected to modulate the disease phenotype, but their identity remains elusive. However, a possible candidate is o-glycosyltransferase 1 (Poglut1, also called Rumi), which was recently discovered to post-translationally block Jagged 1 function.
      • Thakurdas S.M.
      • Lopez M.F.
      • Kakuda S.
      • Fernandez-Valdivia R.
      • Zarrin-Khameh N.
      • Haltiwanger R.S.
      • et al.
      Jagged1 heterozygosity in mice results in a congenital cholangiopathy which is reversed by concomitant deletion of one copy of Poglut1 (Rumi).
      POGLUT1 may thereby act as genetic modifier of Alagille syndrome, since its inactivation partially compensates for the duct paucity induced by heterozygous Jagged1 mutations. Other potential genetic modifiers are Hnf6 and the fringe family of glycosyltransferases.
      • Vanderpool C.
      • Sparks E.E.
      • Huppert K.A.
      • Gannon M.
      • Means A.L.
      • Huppert S.S.
      Genetic interactions between hepatocyte nuclear factor-6 and Notch signaling regulate mouse intrahepatic bile duct development in vivo.
      • Ryan M.J.
      • Bales C.
      • Nelson A.
      • Gonzalez D.M.
      • Underkoffler L.
      • Segalov M.
      • et al.
      Bile duct proliferation in Jag1/fringe heterozygous mice identifies candidate modifiers of the Alagille syndrome hepatic phenotype.
      Ductal plate-specific inactivation of β-catenin revealed that canonical Wnt signalling is dispensable for normal duct formation. Yet, the level of signalling must be tightly controlled, because β-catenin overexpression strongly perturbs ductal morphogenesis.
      • Cordi S.
      • Godard C.
      • Saandi T.
      • Jacquemin P.
      • Monga S.P.
      • Colnot S.
      • et al.
      Role of beta-catenin in development of bile ducts.
      Similarly, non-canonical Wnt5a signalling needs to be fine-tuned since it represses cholangiocyte differentiation in vivo and reduces tube size in in vitro experiments.
      • Kiyohashi K.
      • Kakinuma S.
      • Kamiya A.
      • Sakamoto N.
      • Nitta S.
      • Yamanaka H.
      • et al.
      Wnt5a signaling mediates biliary differentiation of fetal hepatic stem/progenitor cells in mice.
      Finally, studying ECM-cholangiocyte interactions highlighted the differentiation- and morphogenesis-promoting functions of collagens and of several laminins in vitro.
      • Tanimizu N.
      • Miyajima A.
      • Mostov K.E.
      Liver progenitor cells develop cholangiocyte-type epithelial polarity in three-dimensional culture.
      • Takayama K.
      • Mitani S.
      • Nagamoto Y.
      • Sakurai F.
      • Tachibana M.
      • Taniguchi Y.
      • et al.
      Laminin 411 and 511 promote the cholangiocyte differentiation of human induced pluripotent stem cells.
      • Dianat N.
      • Dubois-Pot-Schneider H.
      • Steichen C.
      • Desterke C.
      • Leclerc P.
      • Raveux A.
      • et al.
      Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells.
      • Ogawa M.
      • Ogawa S.
      • Bear C.E.
      • Ahmadi S.
      • Chin S.
      • Li B.
      • et al.
      Directed differentiation of cholangiocytes from human pluripotent stem cells.
      • Sampaziotis F.
      • Cardoso de Brito M.
      • Madrigal P.
      • Bertero A.
      • Saeb-Parsy K.
      • Soares F.A.
      • et al.
      Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.
      • De Assuncao T.M.
      • Sun Y.
      • Jalan-Sakrikar N.
      • Drinane M.C.
      • Huang B.Q.
      • Li Y.
      • et al.
      Development and characterization of human-induced pluripotent stem cell-derived cholangiocytes.
      However, less is known from in vivo functional studies, except that Integrinβ1 is required throughout differentiation and morphogenesis, and is activated by Laminin α5.
      • Tanimizu N.
      • Kikkawa Y.
      • Mitaka T.
      • Miyajima A.
      Alpha1- and alpha5-containing laminins regulate the development of bile ducts via beta1 integrin signals.
      Laminin α5 is necessary for biliary morphogenesis and is produced by the ducts, indicating that ducts activate a genetic programme sustaining their own morphogenesis.

      Cholangiocyte polarity and duct morphogenesis

      Polarisation is a hallmark of maturing cholangiocytes.
      • Tanimizu N.
      • Miyajima A.
      • Mostov K.E.
      Liver progenitor cells develop cholangiocyte-type epithelial polarity in three-dimensional culture.
      The earliest ductal plate cells are irregularly shaped but become cuboidal as soon as they line luminal structures. Apicobasal polarity then becomes evident as the primary cilia develop and Spp1 is expressed at the apical side; tight junctions separate the apicolateral and basolateral domains, the latter marked by high expression of E-cadherin. At the initial stage, i.e. when duct lumina are asymmetrically delineated by cholangiocytes and hepatoblast-like cells, the latter exhibit low E-cadherin levels while already expressing Spp1. This indicates that apicobasal polarisation is stimulated as soon as lumina are generated, even in cells lining the lumina which do not yet express typical cholangiocyte markers such as Sox9.
      • Antoniou A.
      • Raynaud P.
      • Cordi S.
      • Zong Y.
      • Tronche F.
      • Stanger B.
      • et al.
      Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.
      Some apical proteins such as ezrin display biliary-specific expression in developing livers and are not found in hepatocytes.
      • Claperon A.
      • Debray D.
      • Redon M.J.
      • Mergey M.
      • Ho-Bouldoires T.H.
      • Housset C.
      • et al.
      Immunohistochemical profile of ezrin and radixin in human liver epithelia during fetal development and pediatric cholestatic diseases.
      The morphogenic importance of tight junctions is underscored by the role of claudins, which are core constituents of tight junctions and whose mutations in humans cause sclerosing cholangitis.
      • Hadj-Rabia S.
      • Baala L.
      • Vabres P.
      • Hamel-Teillac D.
      • Jacquemin E.
      • Fabre M.
      • et al.
      Claudin-1 gene mutations in neonatal sclerosing cholangitis associated with ichthyosis: a tight junction disease.
      Analyses in developing zebrafish revealed that mutations in claudin 15-like b (cldn15lb) perturb bile duct remodelling, hepatocyte polarisation and bile canaliculi formation, suggesting that these three processes are tightly linked.
      • Gissen P.
      • Arias I.M.
      Structural and functional hepatocyte polarity and liver disease.
      • Cheung I.D.
      • Bagnat M.
      • Ma T.P.
      • Datta A.
      • Evason K.
      • Moore J.C.
      • et al.
      Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b.
      This potential link is further supported by the observation that cholestasis and abnormal duct development are caused by mutations in VPS33B, VIPAR, Vps18 and Atp6ap2, four genes coding for proteins controlling intracellular trafficking and hepatocyte polarisation.
      • Cullinane A.R.
      • Straatman-Iwanowska A.
      • Zaucker A.
      • Wakabayashi Y.
      • Bruce C.K.
      • Luo G.
      • et al.
      Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization.
      • Hanley J.
      • Dhar D.K.
      • Mazzacuva F.
      • Fiadeiro R.
      • Burden J.J.
      • Lyne A.M.
      • et al.
      Vps33b is crucial for structural and functional hepatocyte polarity.
      • Matthews R.P.
      • Plumb-Rudewiez N.
      • Lorent K.
      • Gissen P.
      • Johnson C.A.
      • Lemaigre F.
      • et al.
      Zebrafish vps33b, an ortholog of the gene responsible for human arthrogryposis-renal dysfunction-cholestasis syndrome, regulates biliary development downstream of the onecut transcription factor hnf6.
      • Gissen P.
      • Johnson C.A.
      • Morgan N.V.
      • Stapelbroek J.M.
      • Forshew T.
      • Cooper W.N.
      • et al.
      Mutations in VPS33B, encoding a regulator of SNARE-dependent membrane fusion, cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome.
      • EauClaire S.F.
      • Cui S.
      • Ma L.
      • Matous J.
      • Marlow F.L.
      • Gupta T.
      • et al.
      Mutations in vacuolar H+ -ATPase subunits lead to biliary developmental defects in zebrafish.
      • Sadler K.C.
      • Amsterdam A.
      • Soroka C.
      • Boyer J.
      • Hopkins N.
      A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease.
      Similarly, blocking canaliculi formation by inhibition of Mrp2 prevents remodelling of the ducts into a mature biliary plexus.
      • Tanimizu N.
      • Kaneko K.
      • Itoh T.
      • Ichinohe N.
      • Ishii M.
      • Mizuguchi T.
      • et al.
      Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.
      Along the same lines, duct tubulogenesis is inhibited in mice deficient in Lkb1, which is critical for normal canalicular structure and function.
      • Porat-Shliom N.
      • Tietgens A.J.
      • Van Itallie C.M.
      • Vitale-Cross L.
      • Jarnik M.
      • Harding O.J.
      • et al.
      Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo.
      • Just P.A.
      • Poncy A.
      • Charawi S.
      • Dahmani R.
      • Traore M.
      • Dumontet T.
      • et al.
      LKB1 and notch pathways interact and control biliary morphogenesis.
      Considering that initiation of bile flow through developing ducts coincides with duct remodelling, it is suggested that the influx of bile constitutes the actual trigger for bile duct remodelling.
      • Tanimizu N.
      • Kaneko K.
      • Itoh T.
      • Ichinohe N.
      • Ishii M.
      • Mizuguchi T.
      • et al.
      Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.
      Beyond tube formation and initiation of bile transport, epithelial maturation of cholangiocytes proceeds by strengthening the barrier function of tight junctions. This is controlled by the transcription factor Grainyhead-like 2, which stimulates expression of claudins and Rab25, with the latter promoting the localisation of Claudin 4 at the tight junction.
      • Senga K.
      • Mostov K.E.
      • Mitaka T.
      • Miyajima A.
      • Tanimizu N.
      Grainyhead-like 2 regulates epithelial morphogenesis by establishing functional tight junctions through the organization of a molecular network among claudin3, claudin4, and Rab25.
      • Tanimizu N.
      • Nakamura Y.
      • Ichinohe N.
      • Mizuguchi T.
      • Hirata K.
      • Mitaka T.
      Hepatic biliary epithelial cells acquire epithelial integrity but lose plasticity to differentiate into hepatocytes in vitro during development.
      E-cadherin expression is strongly enhanced at the basolateral side starting at the onset of cholangiocyte differentiation. Still, E-cadherin is required for adult biliary homeostasis, but not for morphogenesis.
      • Nakagawa H.
      • Hikiba Y.
      • Hirata Y.
      • Font-Burgada J.
      • Sakamoto K.
      • Hayakawa Y.
      • et al.
      Loss of liver E-cadherin induces sclerosing cholangitis and promotes carcinogenesis.
      Despite p120-catenin being well known for its E-cadherin-stabilising properties, it is dispensable for E-cadherin expression in cholangiocytes. Yet, its absence causes development of cholangiocytes displaying a hybrid hepatocyte-cholangiocyte phenotype, resulting in ductal plate malformation and lack of normal duct development.
      • van Hengel J.
      • Van den Broeke C.
      • Pieters T.
      • Libbrecht L.
      • Hofmann I.
      • van Roy F.
      Inactivation of p120 catenin in mice disturbs intrahepatic bile duct development and aggravates liver carcinogenesis.
      Planar cell polarity (PCP), which regulates directional cell behaviour, also controls tubulogenesis. The role of PCP in bile duct morphogenesis is supported by the observation that inhibition of genes typically involved in PCP, namely prickle-1a, vangl2 and ankrd6, reduces the number of ducts and duct interconnections in developing zebrafish liver.
      • Cui S.
      • Capecci L.M.
      • Matthews R.P.
      Disruption of planar cell polarity activity leads to developmental biliary defects.
      However, this is associated with defective vesicle transport in hepatocytes and abnormal bile formation, suggesting that perturbed bile flow may, again, contribute or even cause abnormal duct morphogenesis in these morphant livers. Still, the common bile duct, which belongs to the extrahepatic duct system, is shortened and shows fewer mucosal folds in mice with Prickle mutations, thereby extending the role of prickle to mammalian biliary tree development.
      • Gibbs B.C.
      • Damerla R.R.
      • Vladar E.K.
      • Chatterjee B.
      • Wan Y.
      • Liu X.
      • et al.
      Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects.
      Interestingly, this study revealed that a prickle mutation also causes defective development of primary cilia, which are connected both with PCP and apicobasal polarity.
      • Gibbs B.C.
      • Damerla R.R.
      • Vladar E.K.
      • Chatterjee B.
      • Wan Y.
      • Liu X.
      • et al.
      Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects.
      Primary cilia are at the origin of a wide range of diseases best known as ciliopathies, a number of which affect biliary development and cause aberrant cholangiocyte differentiation, ductal plate malformations, absence of bile ducts, and polycystic or dysplastic bile ducts.
      • Raynaud P.
      • Tate J.
      • Callens C.
      • Cordi S.
      • Vandersmissen P.
      • Carpentier R.
      • et al.
      A classification of ductal plate malformations based on distinct pathogenic mechanisms of biliary dysmorphogenesis.
      • Raynaud P.
      • Carpentier R.
      • Antoniou A.
      • Lemaigre F.P.
      Biliary differentiation and bile duct morphogenesis in development and disease.
      • Wills E.S.
      • Roepman R.
      • Drenth J.P.
      Polycystic liver disease: ductal plate malformation and the primary cilium.

      Bile duct morphogenesis in response to injury

      Following cholestatic injury bile duct morphology must adapt to deal with toxic accumulation of bile.
      Cholestatic injury forces the liver to cope with bile, the accumulation of which may eventually become toxic. Following bile duct ligation, the cholangiocytes lining terminal branches of interlobular ducts proliferate. Elongation and branching of ducts is stimulated, parallelled by a wrinkling of the epithelium (corrugation) leading to an increase in luminal surface. When injury persists over time, elongation and corrugation are amplified, and branched structures establish multiple connections; large bile ducts increase their diameter, unlike terminal ducts whose diameter remains unaffected.
      • Vartak N.
      • Damle-Vartak A.
      • Richter B.
      • Dirsch O.
      • Dahmen U.
      • Hammad S.
      • et al.
      Cholestasis-induced adaptive remodeling of interlobular bile ducts.
      Collectively, these morphological responses optimise bile absorption in the terminal branches and fluid drainage in larger ducts.
      The cholestasis-induced morphological response differs from toxin-mediated injury of the liver. Indeed, Itoh and coworkers studied bile duct remodelling triggered by a variety of hepatotoxic agents, extending earlier findings on ANIT-treated rats and elegantly illustrating, at high resolution, how bile duct anatomy adapts to the nature of the toxic injury in mice.
      • Kaneko K.
      • Kamimoto K.
      • Miyajima A.
      • Itoh T.
      Adaptive remodeling of the biliary architecture underlies liver homeostasis.
      • Masyuk T.V.
      • Ritman E.L.
      • LaRusso N.F.
      Quantitative assessment of the rat intrahepatic biliary system by three-dimensional reconstruction.
      Periportal hepatocyte damage induces a major expansion of the periportal biliary plexus, whereas pericentral hepatocyte death causes linear development of biliary branches towards the necrotic area. A combination of three-dimensional analysis, single-cell tracing and mathematical simulations has shown that cholangiocytes are heterogenous in terms of proliferative capacity: cholangiocytes lining peripheral ductules proliferate more upon injury than those lining larger ducts. Moreover, not all peripheral cholangiocytes proliferate and those that do generate clones of variable size, indicating that biliary remodelling results from a stochastic mode of response to injury rather than from a fixed pattern of stem cell-dependent tissue response.
      • Kamimoto K.
      • Kaneko K.
      • Kok C.Y.
      • Okada H.
      • Miyajima A.
      • Itoh T.
      Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling.
      The benefit for the liver of such biliary remodelling is not clear. However, the expanding ducts and plexus may constitute a source of growth factors for hepatocyte regeneration, or provide a frame for re-establishing contacts between hepatocytes and cholangiocytes.
      • Kaneko K.
      • Kamimoto K.
      • Miyajima A.
      • Itoh T.
      Adaptive remodeling of the biliary architecture underlies liver homeostasis.
      • Theise N.D.
      • Dolle L.
      • Kuwahara R.
      Low hepatocyte repopulation from stem cells: a matter of hepatobiliary linkage not massive production.
      The biliary response is expected to vary according to the nature of the hepatic injury, since, depending on the severity of hepatocyte damage biliary cells will or will not transdifferentiate towards hepatocytes to restore liver function, as shown in zebrafish and more recently in mice.
      • Lu W.Y.
      • Bird T.G.
      • Boulter L.
      • Tsuchiya A.
      • Cole A.M.
      • Hay T.
      • et al.
      Hepatic progenitor cells of biliary origin with liver repopulation capacity.
      • Lemaigre F.P.
      Determining the fate of hepatic cells by lineage tracing: facts and pitfalls.
      • Choi T.Y.
      • Ninov N.
      • Stainier D.Y.
      • Shin D.
      Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish.
      • Raven A.
      • Lu W.Y.
      • Man T.Y.
      • Ferreira-Gonzalez S.
      • O'Duibhir E.
      • Dwyer B.J.
      • et al.
      Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration.

      Development of the extrahepatic biliary tree

      The anatomy of the extrahepatic biliary tract, which comprises the gallbladder and the common, cystic and hepatic ducts, is similar in mice and humans,
      • Higashiyama H.
      • Sumitomo H.
      • Ozawa A.
      • Igarashi H.
      • Tsunekawa N.
      • Kurohmaru M.
      • et al.
      Anatomy of the murine hepatobiliary system: a whole-organ-level analysis using a transparency method.
      but its ontogeny did not attract as much attention as intrahepatic duct development. However, it is now well demonstrated that the extrahepatic ducts do not derive from the liver. Instead, they have a common origin with the ventral pancreas and develop as an elongated outgrowth of the ventral endoderm.
      • Spence J.R.
      • Lange A.W.
      • Lin S.C.
      • Kaestner K.H.
      • Lowy A.M.
      • Kim I.
      • et al.
      Sox17 regulates organ lineage segregation of ventral foregut progenitor cells.
      Tan and Moscoso showed that the extrahepatic ducts are located in the vicinity of the intrahepatic biliary ducts at the hilum of the developing human liver. Yet, how the intra- and extrahepatic ducts anastomose is still not clear.
      • Tan C.E.
      • Moscoso G.J.
      The developing human biliary system at the porta hepatis level between 11 and 25 weeks of gestation: a way to understanding biliary atresia. Part 2.
      The most distal parts of the extrahepatic ducts are likely to introgress slightly into the hilum of the liver and the largest hilar ducts of an adult liver may actually arise from the extrahepatic biliary tree. This is supported by our observation that inactivation of Sox4 and Sox9 in hepatoblasts preserves the hilar ducts while abolishing hepatoblast-derived peripheral duct formation.
      • Poncy A.
      • Antoniou A.
      • Cordi S.
      • Pierreux C.E.
      • Jacquemin P.
      • Lemaigre F.P.
      Transcription factors SOX4 and SOX9 cooperatively control development of bile ducts.
      Concordantly, the mutational spectrum associated with tumourigenesis is similar in hilar and extrahepatic cholangiocarcinoma, but different to that of intrahepatic cholangiocarcinoma, suggesting that the hilar and extrahepatic duct have a common gene expression background. This is expected to confer similar susceptibility to cancer and likely results from the hilar and extrahepatic ducts having a common embryonic origin.
      • Rizvi S.
      • Gores G.J.
      Liver capsule: Cholangiocarcinoma (CCA).
      Investigations into transcriptional regulation in extrahepatic ducts revealed that they originate from progenitors co-expressing the transcription factors Pdx1 and Sox17.
      • Spence J.R.
      • Lange A.W.
      • Lin S.C.
      • Kaestner K.H.
      • Lowy A.M.
      • Kim I.
      • et al.
      Sox17 regulates organ lineage segregation of ventral foregut progenitor cells.
      • Uemura M.
      • Hara K.
      • Shitara H.
      • Ishii R.
      • Tsunekawa N.
      • Miura Y.
      • et al.
      Expression and function of mouse Sox17 gene in the specification of gallbladder/bile-duct progenitors during early foregut morphogenesis.
      Sox17 is required to separate the prehepatic endodermal domain from the prepancreatic. In the absence of Sox17, the extrahepatic biliary tree fails to develop and is replaced by pancreatic tissue. Haploinsufficient Sox17 mice show hypoplastic gallbladder epithelium, and conversely, overexpression of Sox17 promotes ductal development.
      • Uemura M.
      • Ozawa A.
      • Nagata T.
      • Kurasawa K.
      • Tsunekawa N.
      • Nobuhisa I.
      • et al.
      Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice.
      Sox17 is involved in a feedback loop with hairy and enhancer of split 1 (Hes1), another factor whose absence causes gallbladder agenesis.
      • Fukuda A.
      • Kawaguchi Y.
      • Furuyama K.
      • Kodama S.
      • Horiguchi M.
      • Kuhara T.
      • et al.
      Ectopic pancreas formation in Hes1 -knockout mice reveals plasticity of endodermal progenitors of the gut, bile duct, and pancreas.
      • Sumazaki R.
      • Shiojiri N.
      • Isoyama S.
      • Masu M.
      • Keino-Masu K.
      • Osawa M.
      • et al.
      Conversion of biliary system to pancreatic tissue in Hes1-deficient mice.
      Sox17 is epistatic to Hhex, Hnf1β, Hnf6, and likely also to Forkhead box factor 1 (Foxf1) and Leucine-rich repeat-containing G-protein coupled receptor 4 (Lgr4), all genes whose loss-of-function causes various forms of extrahepatic biliary dysmorphogenesis, such as gallbladder agenesis, common bile duct enlargement, epithelial dysplasia or replacement by duodenal tissue.
      • Clotman F.
      • Lannoy V.J.
      • Reber M.
      • Cereghini S.
      • Cassiman D.
      • Jacquemin P.
      • et al.
      The onecut transcription factor HNF6 is required for normal development of the biliary tract.
      • Coffinier C.
      • Gresh L.
      • Fiette L.
      • Tronche F.
      • Schutz G.
      • Babinet C.
      • et al.
      Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta.
      • Hunter M.P.
      • Wilson C.M.
      • Jiang X.
      • Cong R.
      • Vasavada H.
      • Kaestner K.H.
      • et al.
      The homeobox gene Hhex is essential for proper hepatoblast differentiation and bile duct morphogenesis.
      • Kalinichenko V.V.
      • Zhou Y.
      • Bhattacharyya D.
      • Kim W.
      • Shin B.
      • Bambal K.
      • et al.
      Haploinsufficiency of the mouse Forkhead Box f1 gene causes defects in gall bladder development.
      • Yamashita R.
      • Takegawa Y.
      • Sakumoto M.
      • Nakahara M.
      • Kawazu H.
      • Hoshii T.
      • et al.
      Defective development of the gall bladder and cystic duct in Lgr4- hypomorphic mice.
      Congenital diseases of the extrahepatic ducts are rare and their aetiology is in most cases unknown. Yet their investigation is likely to uncover new mechanisms of extrahepatic duct development. Nf2 mutations in zebrafish are associated with choledochal cyst formation. In light of the repression exerted by Nf2 on the hippo effector Yap in intrahepatic ducts,
      • Sadler K.C.
      • Amsterdam A.
      • Soroka C.
      • Boyer J.
      • Hopkins N.
      A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease.
      • Zhang N.
      • Bai H.
      • David K.K.
      • Dong J.
      • Zheng Y.
      • Cai J.
      • et al.
      The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals.
      it is tempting to speculate that an Nf2-Hippo-Yap cascade modulates common bile duct growth. Clinically, the most important extrahepatic duct malformation is biliary atresia, a syndrome whose aetiology is still debated and for which several susceptibility genes have been proposed.
      • Lakshminarayanan B.
      • Davenport M.
      Biliary atresia: A comprehensive review.
      An in-depth description of this syndrome is out of the scope of the present review. However, its study revealed the role of Glypican 1 (Gpc1), a heparan sulfate proteoglycan that potentially binds FGF19 and Hedgehog ligands. GPC1 is indeed a biliary atresia susceptibility gene and its knock down in zebrafish results in intrahepatic duct paucity and gallbladder hypoplasia.
      • Cui S.
      • Leyva-Vega M.
      • Tsai E.A.
      • EauClaire S.F.
      • Glessner J.T.
      • Hakonarson H.
      • et al.
      Evidence from human and zebrafish that GPC1 is a biliary atresia susceptibility gene.
      Finally, the identification of biliatresone as a toxin that induces biliary atresia has revealed the importance of well-controlled redox homeostasis for normal extrahepatic bile duct development.
      • Zhao X.
      • Lorent K.
      • Wilkins B.J.
      • Marchione D.M.
      • Gillespie K.
      • Waisbourd-Zinman O.
      • et al.
      Glutathione antioxidant pathway activity and reserve determine toxicity and specificity of the biliary toxin biliatresone in zebrafish.

      Morphogenesis of the liver vasculature

      Origin of hepatic endothelial cells

      Hepatic endothelial cells derive from the mesoderm and endoderm, but all their sources have not yet been identified.
      The morphogenetic steps through which the hepatic venous system is patterned in human and mouse embryos have been extensively described.
      • Lassau J.P.
      • Bastian D.
      Organogenesis of the venous structures of the liver: a hemodynamic theory.
      • Collardeau-Frachon S.
      • Scoazec J.Y.
      Vascular development and differentiation during human liver organogenesis.
      • Crawford L.W.
      • Foley J.F.
      • Elmore S.A.
      Histology atlas of the developing mouse hepatobiliary system with emphasis on embryonic days 9.5-18.5.
      • Cast A.E.
      • Walter T.J.
      • Huppert S.S.
      Vascular patterning sets the stage for macro and micro hepatic architecture.
      According to the “vestigial” theory, the umbilical and vitelline veins give rise to the hepatic venous system. However, this theory was questioned in humans since the vitelline veins are not functional and only the left umbilical vein is present at the stage of liver organogenesis.
      • Lassau J.P.
      • Bastian D.
      Organogenesis of the venous structures of the liver: a hemodynamic theory.
      This suggests that the venous system is essentially shaped by the blood flow from the left umbilical vein.
      Several sources of sinusoidal endothelial cells have been considered. Lineage tracing in mouse embryos did not provide evidence for septum transversum-derived vasculature.
      • Asahina K.
      • Zhou B.
      • Pu W.T.
      • Tsukamoto H.
      Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver.
      However, in the avian embryo, there is evidence that mesothelial cells invade the liver and contribute in part to liver sinusoidal endothelial cells (LSEC).
      • Perez-Pomares J.M.
      • Carmona R.
      • Gonzalez-Iriarte M.
      • Macias D.
      • Guadix J.A.
      • Munoz-Chapuli R.
      Contribution of mesothelium-derived cells to liver sinusoids in avian embryos.
      More recent studies in mice have found that the endocardium of the sinus venosus, which is located near the liver diverticulum and hepatic bud at E 8.0-8.5, contributes no less than a third of the endothelium lining the sinusoids, the portal and the central veins.
      • Zhang H.
      • Pu W.
      • Tian X.
      • Huang X.
      • He L.
      • Liu Q.
      • et al.
      Genetic lineage tracing identifies endocardial origin of liver vasculature.
      The sinus venosus flanks the liver at the budding stage. The sinus venosus-derived cells are attracted to the early liver by Vegf secreted by hepatoblasts, and they persist into adulthood where they participate in liver regeneration following injury.
      • Matsumoto K.
      • Yoshitomi H.
      • Rossant J.
      • Zaret K.S.
      Liver organogenesis promoted by endothelial cells prior to vascular function.
      • Zhang H.
      • Pu W.
      • Tian X.
      • Huang X.
      • He L.
      • Liu Q.
      • et al.
      Genetic lineage tracing identifies endocardial origin of liver vasculature.
      In parallel, lineage tracing using Foxa2:T2AiCre or Foxa2:CreER mice, which enables labelling of endoderm-derived cells, suggests that the endoderm also contains progenitors for a subset of endothelial cells.
      • Goldman O.
      • Han S.
      • Hamou W.
      • Jodon de Villeroche V.
      • Uzan G.
      • Lickert H.
      • et al.
      Endoderm generates endothelial cells during liver development.
      The percentage of endoderm-derived endothelial cells was estimated to be around 15%, using Foxa2:T2AiCre mice. This number must be considered with caution since Foxa2:T2AiCre also labels cardiac cells located close to the developing liver,
      • Horn S.
      • Kobberup S.
      • Jorgensen M.C.
      • Kalisz M.
      • Klein T.
      • Kageyama R.
      • et al.
      Mind bomb 1 is required for pancreatic beta-cell formation.
      and whether Foxa2:T2AiCre-labelled cells overlap with sinus venosus cells has not been verified. Moreover, when human or mouse embryonic stem cells are programmed in vitro towards an endoderm fate and subsequently grow in hepatic-inducing medium, a population of endothelial cells emerges with residual Hnf4 expression, a marker of endoderm-derived hepatoblasts. Similarly, sections of human foetal liver specimens also show cells double positive for HNF4 and endothelial marker CD31.
      • Goldman O.
      • Han S.
      • Sourrisseau M.
      • Dziedzic N.
      • Hamou W.
      • Corneo B.
      • et al.
      KDR identifies a conserved human and murine hepatic progenitor and instructs early liver development.
      Together, these data indicate that part of the liver endothelial cells are derived from the sinus venosus and endoderm, and possibly mesothelium. Vitelline veins are located in the vicinity of the liver and share a common marker with sinusoidal cells (Stabilin 2), suggesting that they can also contribute to liver endothelial cell development.
      • Shiojiri N.
      • Niwa T.
      • Sugiyama Y.
      • Koike T.
      Preferential expression of connexin37 and connexin40 in the endothelium of the portal veins during mouse liver development.
      • Nonaka H.
      • Tanaka M.
      • Suzuki K.
      • Miyajima A.
      Development of murine hepatic sinusoidal endothelial cells characterized by the expression of hyaluronan receptors.
      In zebrafish, elegant lineage tracing studies demonstrated that the liver vasculature is essentially of venous origin.
      • Hen G.
      • Nicenboim J.
      • Mayseless O.
      • Asaf L.
      • Shin M.
      • Busolin G.
      • et al.
      Venous-derived angioblasts generate organ-specific vessels during zebrafish embryonic development.

      Morphogenesis of veins, arteries and sinusoids

      The spatial organisation of blood flow in adult liver lobules is well known and recent improvements in casting, imaging and modelling provide additional quantitative information regarding the three-dimensional architecture of the liver vasculature.
      • Morales-Navarrete H.
      • Segovia-Miranda F.
      • Klukowski P.
      • Meyer K.
      • Nonaka H.
      • Marsico G.
      • et al.
      A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture.
      • Peeters G.
      • Debbaut C.
      • Laleman W.
      • Monbaliu D.
      • Vander Elst I.
      • Detrez J.R.
      • et al.
      A multilevel framework to reconstruct anatomical 3D models of the hepatic vasculature in rat livers.
      • Walter T.J.
      • Sparks E.E.
      • Huppert S.S.
      3-dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system.
      However, it remains unclear how the branching pattern is established during embryogenesis and whether vessel morphogenesis results from angiogenesis or vasculogenesis. The portal vein is the first to organise a branched structure, constituting a frame for development of the biliary tree, hepatic artery branching and haematopoietic stem cell expansion. The portal mesenchyme instructs ductal plate development (see earlier), and biliary cells then promote hepatic artery morphogenesis.
      • Clotman F.
      • Libbrecht L.
      • Gresh L.
      • Yaniv M.
      • Roskams T.
      • Rousseau G.G.
      • et al.
      Hepatic artery malformations associated with a primary defect in intrahepatic bile duct development.
      • Fabris L.
      • Cadamuro M.
      • Libbrecht L.
      • Raynaud P.
      • Spirli C.
      • Fiorotto R.
      • et al.
      Epithelial expression of angiogenic growth factors modulate arterial vasculogenesis in human liver development.
      Expression data suggest that artery development depends on VEGF and Angiopoietin according to a sequence of events summarised (Fig. 4). VEGF-A is expressed widely in developing liver and most strongly in ductal plate cells.
      • Fabris L.
      • Cadamuro M.
      • Libbrecht L.
      • Raynaud P.
      • Spirli C.
      • Fiorotto R.
      • et al.
      Epithelial expression of angiogenic growth factors modulate arterial vasculogenesis in human liver development.
      • Gouysse G.
      • Couvelard A.
      • Frachon S.
      • Bouvier R.
      • Nejjari M.
      • Dauge M.C.
      • et al.
      Relationship between vascular development and vascular differentiation during liver organogenesis in humans.
      It recruits VEGFR-2-positive endothelial cells to the portal mesenchyme near the ductal plate, where they aggregate as VEGFR-1-positive endothelial cells. The latter assemble into vascular structures with myofibroblast-derived mural cells expressing TIE-2, a receptor for Angiopoietin-1 secreted by hepatoblasts. Arterial remodelling is then likely promoted by autocrine secretion of Angiopoietin-2. In parallel, a vascular peribiliary plexus develops, but its origin remains debated as it may result from branching of the hepatic artery or from local vasculogenesis.
      • Gouysse G.
      • Couvelard A.
      • Frachon S.
      • Bouvier R.
      • Nejjari M.
      • Dauge M.C.
      • et al.
      Relationship between vascular development and vascular differentiation during liver organogenesis in humans.
      • Terada T.
      • Nakanuma Y.
      Development of human peribiliary capillary plexus: a lectin-histochemical and immunohistochemical study.
      In line with this model, inactivation of VEGF-A in mid-gestational hepatocytes and cholangiocytes causes a reduction in the number of hepatic endothelial cells.
      • Walter T.J.
      • Cast A.E.
      • Huppert K.A.
      • Huppert S.S.
      Epithelial VEGF signaling is required in the mouse liver for proper sinusoid endothelial cell identity and hepatocyte zonation in vivo.
      The interaction between developing bile ducts and hepatic arteries may differ slightly in humans and mice, since arterial morphogenesis in humans occurs along the ductal plate, whereas in mice, arteries only form postnatally and alongside mature bile ducts.
      • Clotman F.
      • Libbrecht L.
      • Gresh L.
      • Yaniv M.
      • Roskams T.
      • Rousseau G.G.
      • et al.
      Hepatic artery malformations associated with a primary defect in intrahepatic bile duct development.
      Figure thumbnail gr4
      Fig. 4Model for development of hepatic artery branches in humans.
      Developing portal vessels also constitute a frame for haematopoietic stem cell development, and the work of Khan and coworkers establishes a nice parallel between morphogenesis and function.
      • Khan J.A.
      • Mendelson A.
      • Kunisaki Y.
      • Birbrair A.
      • Kou Y.
      • Arnal-Estape A.
      • et al.
      Fetal liver hematopoietic stem cell niches associate with portal vessels.
      The periportal mesenchyme contains Nestin+ cells that form a niche for the haematopoietic stem cell population, which expands proportionately to the growth of the portal vessel surface. Interestingly, at birth, the portal vein switches from an arterial-like EfnB2+/EphB4 phenotype to a venous EfnB2/EphB4+ state, which is associated with disappearance of the Nestin+ niche and egression of haematopoietic cells.
      It has been proposed that sinusoidal development follows a three-phase process.
      • Collardeau-Frachon S.
      • Scoazec J.Y.
      Vascular development and differentiation during human liver organogenesis.
      • Poisson J.
      • Lemoinne S.
      • Boulanger C.
      • Durand F.
      • Moreau R.
      • Valla D.
      • et al.
      Liver sinusoidal endothelial cells: Physiology and role in liver diseases.
      The earliest LSECs are non-fenestrated and lined by a laminin-rich basal membrane; they express non-specific endothelial markers such as CD31, CD34 and IF10. Subsequently, LSECs follow a stereotypic differentiation process: they become fenestrated, the perisinusoidal matrix develops a laminin-poor and tenascin-rich composition, and CD34/CD31/IF10 expression is repressed while other markers more specific for adult sinusoids, such as CD4, CD32 and ICAM-1 are induced. Hepatoblasts promote this differentiation by VEGF secretion and repression of TGFβ;
      • Walter T.J.
      • Cast A.E.
      • Huppert K.A.
      • Huppert S.S.
      Epithelial VEGF signaling is required in the mouse liver for proper sinusoid endothelial cell identity and hepatocyte zonation in vivo.
      • Yoshida M.
      • Nishikawa Y.
      • Omori Y.
      • Yoshioka T.
      • Tokairin T.
      • McCourt P.
      • et al.
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      Sinusoid development and morphogenesis may be stimulated by VEGF-Flk-1 signaling during fetal mouse liver development.
      it occurs around the 12th week of gestation in humans and around E14-15 in mouse embryos, coinciding with the acquisition of haematopoietic properties. In this context, sinusoidal morphology was recently shown to be critical for tissular distribution of macrophages: the diaphragm of sinusoidal fenestrae contains Plasmalemma vesicle-associated protein (Plvap) facilitating egression of foetal monocytes from the embryonic liver. It likely traps chemoattractants which, in the absence of Plvap, aberrantly maintain the monocytes within the liver.
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      Isolation of periportal, midlobular, and centrilobular rat liver sinusoidal endothelial cells enables study of zonated drug toxicity.
      In normal adult liver, LSECs are mostly quiescent and self-renewal of LSECs is ensured by resident LSEC progenitors. In chronic liver disease, LSECs undergo capillarisation, i.e. loss of fenestrae and basal membrane synthesis. Bone marrow cells are then recruited by hepatic VEGF secretion, and contribute to liver angiogenesis (reviewed in
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      ).

      Conclusions

      In conclusion, the use of distinct model organisms combined with the availability of improved three-dimensional imaging approaches has uncovered a series of new morphogenetic processes in developing and adult liver. Still, molecular mechanisms driving morphogenesis are only beginning to emerge, and very little is known about how hepatocyte cords, bile ducts and vessels interact or connect with each other in an organised fashion during liver development. In-depth characterisation of both differentiation and morphogenesis is a prerequisite for understanding the pathophysiology of developmental diseases and for in vitro reconstitution of liver tissue for regenerative medicine.

      Financial support

      EAO was supported by the Novo Nordisk Foundation ( NNF17CC0027852 ) and Danish National Research Foundation ( DNRF116 ). FL was supported by the Interuniversity Attraction Pole Programme (Belgian Science Policy, PVII-47), the D.G. Higher Education and Scientific Research of the French Community of Belgium (ARC 15/20-065), and the F.R.S.-FNRS (Belgium: Grants T.007214 and J.0058.15).

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contribution

      EAO and FPL wrote the manuscript.

      Supplementary data

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