Advertisement

Development of the bile ducts: Essentials for the clinical hepatologist

  • Mario Strazzabosco
    Correspondence
    Corresponding author. Addresses: Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, 333 Cedar Street, LMP 1080, New Haven, CT 06520, USA. Tel.: +1 203 785 7281; fax: +1 203 785 7273, Department of Clinical Medicine and Prevention, Università di Milano-Bicocca, Via Cadore 48, 20052 Monza (Milano), Italy. Tel.: +39 02 6448 8052; fax: +39 02 6448 8363.
    Affiliations
    Section of Digestive Diseases, Yale University, New Haven, CT, USA

    Department of Clinical Medicine, University of Milan-Bicocca, Milan, Italy
    Search for articles by this author
  • Luca Fabris
    Affiliations
    Department of Clinical Medicine, University of Milan-Bicocca, Milan, Italy

    Department of Surgical and Gastroenterological Sciences, University of Padova, Italy
    Search for articles by this author
Open AccessPublished:January 16, 2012DOI:https://doi.org/10.1016/j.jhep.2011.09.022

      Summary

      Several cholangiopathies result from a perturbation of developmental processes. Most of these cholangiopathies are characterised by the persistence of biliary structures with foetal configuration. Developmental processes are also relevant in acquired liver diseases, as liver repair mechanisms exploit a range of autocrine and paracrine signals transiently expressed in embryonic life. We briefly review the ontogenesis of the intra- and extrahepatic biliary tree, highlighting the morphogens, growth factors, and transcription factors that regulate biliary development, and the relationships between developing bile ducts and other branching biliary structures. Then, we discuss the ontogenetic mechanisms involved in liver repair, and how these mechanisms are recapitulated in ductular reaction, a common reparative response to many forms of biliary and hepatocellular damage. Finally, we discuss the pathogenic aspects of the most important primary cholangiopathies related to altered biliary development, i.e. polycystic and fibropolycystic liver diseases, Alagille syndrome.

      Abbreviations:

      GW (week of gestation), Sox9 (SRY-related HMG box transcription factor 9), K (cytokeratin), HNF (hepatocyte nuclear factor), TGF (transforming growth factor), TβRII (transforming growth factor receptor type II), PCP (planar cell polarity), IL (interleukin), Hh (Hedgehog), Shh (sonic Hedgehog), DPM (ductal plate malformations), VEGF (vascular endothelial growth factor), PBP (peribiliary plexus), RBP-JK (recombination signal binding protein for immunoglobulin kappa J), AGS (Alagille syndrome), Dvl (Dishevelled), IGF1 (insulin-like growth factor-1), CTGF (connective tissue growth factor), SDF-1 (stromal cell-derived factor 1), TNFα (tumour necrosis factor-α), NCAM (neural cell adhesion molecule), HPC (hepatic progenitor cells), IHBC (intermediate hepato-biliary cells), RDC (reactive ductular cells), MKS (Meckel syndrome), ARPKD (autosomal recessive polycystic kidney disease), CHF (congenital hepatic fibrosis), CD (Caroli’s disease), FPC (fibrocystin), ADPKD (autosomal dominant polycystic kidney disease), PLD (polycystic liver disease), PC (polycystin), PKA (protein kinase A), ER (endoplasmic reticulum), CCA (cholangiocarcinoma), MCP-1 (monocyte chemotactic protein-1), CINC (cytokine-induced neutrophil chemoattractant), ET-1 (endothelin-1)

      Introduction

      Development of the biliary system is a unique process that has been thoroughly reviewed in several recent papers [
      • Lemaigre F.P.
      Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies.
      ,
      • Raynaud P.
      • Carpentier R.
      • Antoniou A.
      • Lemaigre F.P.
      Biliary differentiation and bile duct morphogenesis in development and disease.
      ]. Here, we will focus on those concepts of biliary development that are “essential” to understand congenital and acquired cholangiopathies.
      Cholangiopathies are an heterogeneous group of liver diseases, caused by congenital, immune-mediated, toxic, infectious or idiopathic insults to the biliary tree [
      • Lazaridis K.N.
      • Strazzabosco M.
      • Larusso N.F.
      The cholangiopathies: disorders of biliary epithelia.
      ,
      • Strazzabosco M.
      • Fabris L.
      • Spirli C.
      Pathophysiology of cholangiopathies.
      ]. In addition to being responsible for significant morbidity and mortality, cholangiopathies account for the majority of liver transplants in paediatrics and a significant percentage of liver transplants in young adults. Many cholangiopathies are congenital, resulting initially from an altered development of the biliary tree, eventually accompanied by necro-inflammatory processes [
      • Desmet V.J.
      Congenital diseases of intrahepatic bile ducts: variations on the theme “ductal plate malformation”.
      ,
      • Desmet V.J.
      Ludwig symposium on biliary disorders: Part I. Pathogenesis of ductal plate abnormalities.
      ]. For the clinical hepatologist, this means a good working knowledge of the mechanisms of liver development is necessary for the care of these patients.
      We will briefly review the general aspects of bile duct development and morphogenesis and the main molecular mechanisms involved in bile duct ontogenesis. Then we will highlight the role of these mechanisms in liver repair. Lastly, we will discuss the cholangiopathies related to altered development, with a special emphasis on those caused by a single genetic defect.

      General aspects of bile duct morphogenesis during liver development

      The liver develops as a tissue bud deriving from a diverticulum of the ventral foregut endoderm, which extends into the septum transversum, a structure located between the pericardial and peritoneal cavities. The ventral foregut endoderm develops two protrusions: the cranial part leads to the formation of the intrahepatic bile ducts, while the caudal part generates the extrahepatic biliary tree [
      • Lemaigre F.P.
      Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies.
      ,
      • Roskams T.
      • Desmet V.
      Embryology of extra- and intrahepatic bile ducts, the ductal plate.
      ,
      • Tan J.
      • Hytiroglou P.
      • Wieczorek R.
      • Park Y.N.
      • Thung S.N.
      • Arias B.
      • et al.
      Immunohistochemical evidence for hepatic progenitor cells in liver diseases.
      ].

      Intrahepatic biliary tree

      The development of the intrahepatic biliary epithelium begins around the 8th week of gestation (GW), and proceeds centrifugally from the hilum to the periphery of the liver following the portal vein system [
      • Desmet V.J.
      Congenital diseases of intrahepatic bile ducts: variations on the theme “ductal plate malformation”.
      ,
      • Desmet V.J.
      Ludwig symposium on biliary disorders: Part I. Pathogenesis of ductal plate abnormalities.
      ,
      • Van Eyken P.
      • Sciot R.
      • Callea F.
      • Van der Steen K.
      • Moerman P.
      • Desmet V.J.
      The development of the intrahepatic bile ducts in man: a keratin-immunohistochemical study.
      ]. At birth, the intrahepatic biliary epithelium is still immature, and its maturation is completed during the first years of life [
      • Van Eyken P.
      • Sciot R.
      • Callea F.
      • Van der Steen K.
      • Moerman P.
      • Desmet V.J.
      The development of the intrahepatic bile ducts in man: a keratin-immunohistochemical study.
      ]. The sequence of events leading to the development of the ductal plate and the intrahepatic bile ducts are shown in Fig. 1 [
      • Nakanuma Y.
      • Hoso M.
      • Sanzen T.
      • Sasaki M.
      Microstructure and development of the normal and pathologic biliary tract in humans, including blood supply.
      ,
      • Libbrecht L.
      • Cassiman D.
      • Desmet V.
      • Roskams T.
      Expression of neural cell adhesion molecule in human liver development and in congenital and acquired liver diseases.
      ,
      • Fabris L.
      • Cadamuro M.
      • Libbrecht L.
      • Raynaud P.
      • Spirlì C.
      • Fiorotto R.
      • et al.
      Angiogenic growth factors secreted by liver epithelial cells modulate arterial vasculogenesis during human liver development.
      ]. Whether or not segments of the ductal plate, that are not incorporated into the nascent bile ducts, are gradually deleted by apoptosis is a matter of controversy. Recent data from Lemaigre’s group [
      • Carpentier R.
      • Suñer 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.
      ] suggest that ductal plate remodelling does not occur by proliferation and apoptosis. Rather, portions of the ductal plate appear to differentiate into periportal hepatocytes and adult hepatic progenitor cells [
      • Carpentier R.
      • Suñer 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.
      ]. The role of ductal plate cells as potential stem cells has been recently addressed by Furuyama et al. [
      • Furuyama K.
      • Kawaguchi Y.
      • Akiyama H.
      • Horiguchi M.
      • Kodama S.
      • Kuhara T.
      • et al.
      Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.
      ]. A crucial event in the development of the intrahepatic biliary system, as well as in liver repair, is tubulogenesis. Biliary tubule formation depends upon a unique process of transient asymmetry. Careful studies performed in mouse embryos have shown that nascent tubules are formed by ductal plate cells resembling cholangiocytes (positive for the SRY-related HMG box transcription factor 9 [Sox9] and cytokeratin-19 [K19]) on the side facing the portal tract, and by ductal plate cells resembling hepatoblasts (positive for the hepatocyte nuclear factor 4 [HNF4] and the transforming growth factor receptor type II [TβRII]) on the side facing the parenchyma. The “portal” layer shows higher levels of E-cadherin and is in contact with laminin, while the “parenchymal” layer is characterised by the apical expression of osteopontin [
      • Antoniou A.
      • Raynaud P.
      • Cordi S.
      • Zong Y.
      • Tronche F.
      • Stanger B.Z.
      • et al.
      Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.
      ]. After the formation of a lumen, the nascent bile duct becomes symmetrical as “hepatoblasts” are replaced by “cholangiocytes”, and the ductal structure matures along a cross-sectional axis and a cranio-caudal axis, extending from the hilum to the periphery. Thus, the progressive elongation of the duct requires a mechanism able to orient cell mitoses along the aforementioned axes in a coordinated manner. Through this mechanism, called “planar cell polarity” (PCP), the epithelial cells are uniformly oriented within the ductal plane to maintain the tubular architecture. PCP is a process that is controlled by the non-canonical Wnt pathway, and is defective in fibropolycystic liver diseases (see below) [
      • Fischer E.
      • Legue E.
      • Doyen A.
      • Nato F.
      • Nicolas J.F.
      • Torres V.
      • et al.
      Defective planar cell polarity in polycystic kidney disease.
      ].
      Figure thumbnail gr1
      Fig. 1Embryological stages of intrahepatic bile duct development. The development of intrahepatic bile ducts starts when the periportal hepatoblasts, in close contact with the portal mesenchyme surrounding a portal vein branch, begin to organise into a single layered sheath of small flat epithelial cells, called ductal plate (“ductal plate stage”, A). In the following weeks, discreet portions of the ductal plates are duplicated by a second layer of cells (double layered ductal plate, B), which then dilate to form tubular structures in the process of being incorporated into the mesenchyma of the nascent portal space (incorporating bile duct, “migratory stage”, C). Once incorporated into the portal space, the immature tubules are remodelled into individualised bile ducts (incorporated bile duct, “bile duct stage”, D).
      The mechanisms that regulate the termination of biliary development are not well known. Recent work from Kaestner’s laboratory suggests that by inhibiting NF-κB-dependent cytokine expression (specifically IL-6), the transcription factors Foxa1/2, may act as a termination signal in bile duct development. Mice with liver-specific deletion of both Foxa1 and Foxa2 showed an increased amount of dysmorphic bile ducts [
      • Li Z.
      • White P.
      • Tuteja G.
      • Rubins N.
      • Sackett S.
      • Kaestner K.H.
      Foxa1 and Foxa2 regulate bile duct development in mice.
      ]. On the other hand, a decrease in Notch signalling could change the fate of the non-duplicated ductal plate segments [
      • Zong Y.
      • Panikkar A.
      • Xu J.
      • Antoniou A.
      • Raynaud P.
      • Lemaigre F.
      • et al.
      Notch signaling controls liver development by regulating biliary differentiation.
      ] and promote their differentiation towards alternative pathways.

      Extrahepatic biliary tree

      Cholangiocytes lining the extrahepatic bile ducts derive from the caudal part of the ventral foregut endoderm located between the liver and the pancreatic buds, a region that expresses a combination of transcription factors common to the pancreas and duodenum (Pdx-1, Prox-1, HNF-6). The extrahepatic part of the biliary tree develops before the intrahepatic part; the two systems merge at the level of the hepatic duct/hilum. Molecular mechanisms regulating the development of the extrahepatic bile ducts are less well known than those regulating the development of the intrahepatic bile ducts. Mice deficient in Pdx-1 [
      • Fukuda A.
      • Kawaguchi Y.
      • Furuyama K.
      • Kodama S.
      • Kuhara T.
      • Horiguchi M.
      • et al.
      Loss of the major duodenal papilla results in brown pigment biliary stone formation in pdx1 null mice.
      ] or Hes1 (a Notch-dependent transcription factor), HNF6, HNF-1β, or Foxf1 (a transcription factor target for the sonic Hedgehog signalling) result in an altered development of the gallbladder and of the common bile duct [
      • 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 HNF1β.
      ,
      • 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.
      ].

      Relationships between biliary and arterial development

      Branches of the hepatic artery develop in close proximity to ductal plates. On one side, the biliary epithelium guides arterial development, on the other, the developing intrahepatic bile ducts are nourished by the peribiliary plexus (PBP), a network of capillaries emerging from the finest branches of the hepatic artery at the periphery of the liver lobule. PBP is crucial in maintaining the integrity and function of the biliary epithelium [
      • Gaudio E.
      • Pannarale L.
      • Franchitto A.
      • Onori P.
      • Marinozzi G.
      Hepatic microcirculation as a morpho-functional basis for the metabolic zonation in normal and pathological rat liver.
      ,
      • Gaudio E.
      • Onori P.
      • Franchitto A.
      • Pannarale L.
      • Alpini G.
      • Alvaro D.
      Hepatic microcirculation and cholangiocyte physiopathology.
      ,
      • Gaudio E.
      • Franchitto A.
      • Pannarale L.
      • Carpino G.
      • Alpini G.
      • Francis H.
      • et al.
      Cholangiocytes and blood supply.
      ].
      The patterning of the intrahepatic biliary tree develops in strict harmony with hepatic arteriogenesis. For example, inactivation of Hnf6 or Hnf1ß, transcription factors involved in intrahepatic bile duct epithelium development, resulted in anomalies of hepatic artery branches paralleling bile duct abnormalities [
      • Lemaigre F.P.
      Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies.
      ,
      • Fabris L.
      • Cadamuro M.
      • Libbrecht L.
      • Raynaud P.
      • Spirlì C.
      • Fiorotto R.
      • et al.
      Angiogenic growth factors secreted by liver epithelial cells modulate arterial vasculogenesis during human liver development.
      ]. One of the signals linking ductal and arterial development in the liver is the vascular endothelial growth factor (VEGF), which cooperates with angiopoietin-1 (Ang-1). The developing bile ducts produce VEGF-A that in turn acts on endothelial cells and their precursors, which both express the receptor VEGFR-2, to promote arterial and PBP vasculogenesis. At the same time, Ang-1 produced by hepatoblasts is likely to induce the maturation of hepatic artery terminations by recruiting mural pericytes to the nascent endothelial layer [
      • Fabris L.
      • Cadamuro M.
      • Libbrecht L.
      • Raynaud P.
      • Spirlì C.
      • Fiorotto R.
      • et al.
      Angiogenic growth factors secreted by liver epithelial cells modulate arterial vasculogenesis during human liver development.
      ]. In ductal plate malformations (DPM), the dysmorphic bile ducts are surrounded by an increased number of vascular structures [
      • Desmet V.J.
      Congenital diseases of intrahepatic bile ducts: variations on the theme “ductal plate malformation”.
      ,
      • Fabris L.
      • Cadamuro M.
      • Fiorotto R.
      • Roskams T.
      • Spirlì C.
      • Melero S.
      • et al.
      Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.
      ,
      • Spirli C.
      • Okolicsanyi S.
      • Fiorotto R.
      • Fabris L.
      • Cadamuro M.
      • Lecchi S.
      • et al.
      Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice.
      ]. For example, in cystic cholangiopathies, the epithelium retains an immature phenotype typical of the embryonic ductal plate coupled with the production of VEGF and angiopoietins [
      • Fabris L.
      • Cadamuro M.
      • Fiorotto R.
      • Roskams T.
      • Spirlì C.
      • Melero S.
      • et al.
      Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.
      ]. Production of angiogenic factor by immature cholangiocytes promotes an abundant pericystic vascularisation, thus providing the vascular supply around the growing liver cysts [
      • Spirli C.
      • Okolicsanyi S.
      • Fiorotto R.
      • Fabris L.
      • Cadamuro M.
      • Lecchi S.
      • et al.
      Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice.
      ] (see below).
      The anatomical and functional association between bile ductules and arterial vascularisation is maintained also in adult life and during liver repair. A malfunction of this system may cause ductopenia, as observed in chronic allograft rejection and in other chronic cholangiopathies caused by an ischaemic damage [
      • Deltenre P.
      • Valla D.C.
      Ischemic cholangiopathy.
      ]. Ductular reaction is a common histopathological response to many forms of liver damage (see below); the increase in bile ductules at the portal tract interface is paralleled by an increased number of hepatic arterioles and capillaries [
      • Gaudio E.
      • Onori P.
      • Franchitto A.
      • Pannarale L.
      • Alpini G.
      • Alvaro D.
      Hepatic microcirculation and cholangiocyte physiopathology.
      ,
      • Gaudio E.
      • Franchitto A.
      • Pannarale L.
      • Carpino G.
      • Alpini G.
      • Francis H.
      • et al.
      Cholangiocytes and blood supply.
      ]. These features are reproduced in an experimental rat model of selective cholangiocyte proliferation (α-naphthylisothiocyanate treatment), where an extensive neovascularisation of the arterial bed develops in strict conjunction with increased cholangiocyte mass [
      • Masyuk T.V.
      • Ritman E.L.
      • LaRusso N.F.
      Hepatic artery and portal vein remodeling in rat liver: vascular response to selective cholangiocyte proliferation.
      ]. Furthermore, expansion of the biliary tree after bile duct ligation in rat is also followed by substantial adaptive modifications of the PBP [
      • Gaudio E.
      • Franchitto A.
      • Pannarale L.
      • Carpino G.
      • Alpini G.
      • Francis H.
      • et al.
      Cholangiocytes and blood supply.
      ,
      • 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.
      ].

      Transcription factors, growth factors and morphogens involved in the ontogenesis of the biliary epithelium

      At the time of liver specification, i.e. when the endoderm becomes committed towards a liver cell fate, liver development is driven by several transcription factors including hepatocyte nuclear factor 1β (HNF1β) [
      • 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 HNF1β.
      ,
      • Lemaigre F.
      • Zaret K.S.
      Liver development update. New embryo models, cell lineage control, and morphogenesis.
      ], Foxa1 and Foxa2 [
      • Li Z.
      • White P.
      • Tuteja G.
      • Rubins N.
      • Sackett S.
      • Kaestner K.H.
      Foxa1 and Foxa2 regulate bile duct development in mice.
      ], and GATA-4 [
      • Bossard P.
      • Zaret K.S.
      GATA transcription factors as potentiators of gut endoderm differentiation.
      ,
      • Rojas A.
      • De Val S.
      • Heidt A.B.
      • Xu S.M.
      • Bristow J.
      • Black B.L.
      Gata4 expression in lateral mesoderm is downstream of BMP4 and is activated directly by Forkhead and GATA transcription factors through a distal enhancer element.
      ,
      • Haworth K.E.
      • Kotecha S.
      • Mohun T.J.
      • Latinkic B.V.
      GATA4 and GATA5 are essential for heart and liver development in Xenopus embryos.
      ]. Extracellular signals, like fibroblast growth factor (FGF) secreted by the cardiogenic mesoderm [
      • Rossi J.M.
      • Dunn N.R.
      • Hogan B.L.
      • Zaret K.S.
      Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm.
      ], bone morphogenetic protein-4 (BMP-4) and extracellular matrix constituents [
      • Rojas A.
      • De Val S.
      • Heidt A.B.
      • Xu S.M.
      • Bristow J.
      • Black B.L.
      Gata4 expression in lateral mesoderm is downstream of BMP4 and is activated directly by Forkhead and GATA transcription factors through a distal enhancer element.
      ,
      • Rossi J.M.
      • Dunn N.R.
      • Hogan B.L.
      • Zaret K.S.
      Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm.
      ], signal to competent cells and appear also to be involved in biliary differentiation.
      The differentiation of the intrahepatic biliary epithelium and its tubular morphogenesis are finely regulated by signals exchanged between epithelial cells and a variety of other non-parenchymal cells. These signals encompass a series of morphogens and growth factors that regulate the commitment of hepatoblasts to the biliary lineage, or tubule formation by cholangiocytes, or more often, both processes. The portal mesenchyme generates a portal to parenchymal gradient of transforming growth factor-β (TGF-β2 and TGF-β3). TGF-β stimulates hepatoblasts to undergo a switch towards a biliary phenotype [
      • Clotman F.
      • Lemaigre F.P.
      Control of hepatic differentiation by activin/TGFbeta signaling.
      ]. The developing ductal plates transitorily express the TGF-β-receptor type II (TβRII); expression of TβRII is repressed in differentiated cholangiocytes.
      HNF6 and HNF1β are transcription factors able to regulate multiple steps of biliary development and morphogenesis. Their expression is upregulated in cholangiocytes and in progenitor cells committed to the cholangiocyte lineage. In mice, their absence is associated with cystic dysgenesis of the biliary tree [
      • 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 HNF1β.
      ,
      • Igarashi P.
      • Shao X.
      • McNally B.T.
      • Hiesberger T.
      Roles of HNF-1beta in kidney development and congenital cystic diseases.
      ]. HNF6 and HNF1β regulate distinct stages of bile duct morphogenesis. Whereas the absence of HNF6 caused an early defect in biliary cell differentiation, which can be somehow repaired, a defect in HNF1β appears to be associated with a defect in the maturation of the primitive ductal structure [
      • 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.
      ]. Furthermore, HNF6 stimulates expression of Pkhd1 while a deletion of HNF1β causes an aberrant cystic development and defects in PCP, a feature associated with fibropolycystic liver diseases [
      • Fischer E.
      • Legue E.
      • Doyen A.
      • Nato F.
      • Nicolas J.F.
      • Torres V.
      • et al.
      Defective planar cell polarity in polycystic kidney disease.
      ]. It is noteworthy that HNF1β expression appears to be under the control of Notch signalling.
      Notch signalling is a fundamental mechanism that confers cell fate instructions during the development of various tissues. Several studies using genetic mouse models and zebrafish demonstrated that Notch signalling is required at different stages during biliary tree development: from the formation of the ductal plate to ductal plate remodelling and tubule formation [
      • Lorent K.
      • Yeo S.Y.
      • Oda T.
      • Chandrasekharappa S.
      • Chitnis A.
      • Matthews R.P.
      • et al.
      Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy.
      ,
      • Lozier J.
      • McCright B.
      • Gridley T.
      Notch signaling regulates bile duct morphogenesis in mice.
      ]. The Notch genes encode four transmembrane receptors (Notch 1, 2, 3, and 4), which can interact with a number of ligands (Jagged-1, Jagged-2, Delta-like 1, 3, and 4). Notch signalling requires the establishment of cell–cell contacts. Through this interaction among neighbouring cells, Notch receptors expressed by “receiving” cells are activated by the binding of ligands expressed on the surface of “transmitting” cells. Notch may stimulate cells to undergo a phenotypic switch through a process of “lateral induction”. Alternatively, Notch may promote the maintenance of the original phenotype through “lateral inhibition” [
      • Cornell R.A.
      • Eisen J.S.
      Notch in the pathway: the roles of Notch signaling in neural crest development.
      ]. In liver development, Notch signalling appears to control the ability of hepatoblasts and mature hepatocytes to differentiate into cholangiocytes by altering the expression of liver-enriched transcription factors, and to regulate the formation of biliary tubules [
      • Zong Y.
      • Panikkar A.
      • Xu J.
      • Antoniou A.
      • Raynaud P.
      • Lemaigre F.
      • et al.
      Notch signaling controls liver development by regulating biliary differentiation.
      ]. Notch-dependent signalling mechanisms are summarised in Fig. 2. Notch drives the activation of Notch effector genes such as Hairy and Enhancer of Split homologues (Hes1 and Hey-1), via the transcription factor recombination signal binding protein for immunoglobulin kappa J (RBP-Jk), which in turn activates transcription factors specifically expressed by cholangiocytes, including HNF1β and Sox9 [
      • Tchorz J.S.
      • Kinter J.
      • Müller M.
      • Tornillo L.
      • Heim M.H.
      • Bettler B.
      Notch2 signaling promotes biliary epithelial cell fate specification and tubulogenesis during bile duct development in mice.
      ].
      Figure thumbnail gr2
      Fig. 2Notch signalling. Jagged binding to a Notch receptor leads to the proteolytic processing and subsequent translocation into the nucleus of the Notch intracellular domain (NICD) of the receptor. Cleavage of NICD is an essential step in this process and is mediated by a γ-secretase enzyme in the cytoplasm. Once delivered into the nucleus, NICD forms a complex with its DNA-binding partner, the recombination signal binding protein for immunoglobulin kappa J (RBP-Jk). The formation of this complex leads to the upregulation of cholangiocyte-specific transcription factors, such as HNF1β and the SRY-related HGM box transcription factor 9 (Sox9), and to the downregulation of hepatocyte-specific transcription factors such as HNF1α and HNF4. Sox9 in particular, is the most specific and earliest marker of biliary cells in the developing liver, as it controls the timing and maturation of primitive ductal structures in tubulogenesis.
      Studies in mice have shown that Jagged-1 is expressed by periportal mesenchymal cells and interacts with Notch-2 expressed by hepatoblasts favouring their differentiation into ductal plate cells. Perturbations in Jagged-1/Notch-2 interactions cause Alagille syndrome (AGS), a genetic cholangiopathy characterised by ductopenia and defective peripheral branching of the biliary tree [
      • Fabris L.
      • Cadamuro M.
      • Guido M.
      • Spirli C.
      • Fiorotto R.
      • Colledan M.
      • et al.
      Analysis of liver repair mechanisms in Alagille syndrome and biliary atresia reveals a role for notch signaling.
      ]. In fact, Jagged-1 inactivation in the portal vein mesenchymal cells, but not in the endothelial cells, results in a defective development of the bile ducts that do not mature beyond the initial formation of the ductal plate [
      • 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.
      ].
      A major effect of Notch signalling is to modulate tubule formation, a property that is necessary to effectively repair the biliary tree. The effects of Notch receptors can be modified by other ligands such as the glycosyltransferases encoded by Fringe genes, and by the dosage of the Jagged-1 gene [
      • Loomes K.M.
      • Russo P.
      • Ryan M.
      • Nelson A.
      • Underkoffler L.
      • Glover C.
      • et al.
      Bile duct proliferation in liver-specific Jag1 conditional knockout mice: effects of gene dosage.
      ]. The impact of Notch signalling on the intrahepatic bile duct branching is highlighted in a recent study by Sparks et al. [
      • Sparks E.E.
      • Huppert K.A.
      • Brown M.A.
      • Washington M.K.
      • Huppert S.S.
      Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.
      ], which demonstrated that the density of three-dimensional peripheral intrahepatic bile duct architecture during liver development depends on Notch gene dosage.
      Canonical Wingless (Wnt)/β-catenin participates in several stages of bile duct development. Specific Wnt ligands, such as Wnt3a, induce biliary differentiation, characterised by the appearance of K19 positivity and generation of duct-like structures in mouse embryonic liver cell cultures. Wnt/β-catenin appears to play a key role in biliary commitment, by repressing hepatocyte differentiation and promoting ductal plate remodelling [
      • Lemaigre F.P.
      Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies.
      ,
      • Raynaud P.
      • Carpentier R.
      • Antoniou A.
      • Lemaigre F.P.
      Biliary differentiation and bile duct morphogenesis in development and disease.
      ]. The deletion of β-catenin in the developing hepatoblasts in transgenic mice leads to a paucity of bile ducts and to multiple defects in hepatoblast maturation, expansion, and survival [
      • Tan X.
      • Yuan Y.
      • Zeng G.
      • Apte U.
      • Thompson M.D.
      • Cieply B.
      • et al.
      Beta-catenin deletion in hepatoblasts disrupts hepatic morphogenesis and survival during mouse development.
      ]. Wnt can also signal through β-catenin-independent pathways. Non-canonical Wnt pathways seem to be crucial in the regulation of PCP [
      • Montcouquiol M.
      • Crenshaw 3rd, E.B.
      • Kelley M.W.
      Noncanonical Wnt signaling and neural polarity.
      ], which is lost when Pkhd1, the gene encoding for fibrocystin (see below), is defective [
      • Fischer E.
      • Legue E.
      • Doyen A.
      • Nato F.
      • Nicolas J.F.
      • Torres V.
      • et al.
      Defective planar cell polarity in polycystic kidney disease.
      ]. Notably, inversin, a cilium-associated protein regulating the left–right symmetry, modulates non-canonical Wnt signalling by interacting directly with Dvl [
      • Simons M.
      • Gloy J.
      • Ganner A.
      • Bullerkotte A.
      • Bashkurov M.
      • Krönig C.
      • et al.
      Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways.
      ] and if defective, causes aberrant development of intrahepatic bile ducts and cyst formation [
      • Fischer E.
      • Legue E.
      • Doyen A.
      • Nato F.
      • Nicolas J.F.
      • Torres V.
      • et al.
      Defective planar cell polarity in polycystic kidney disease.
      ]. Canonical and non-canonical Wnt pathways are illustrated in Fig. 3.
      Figure thumbnail gr3
      Fig. 3Wnt signalling (canonical and non-canonical). Wnt signals in two different ways depending upon the activation (canonical) or the non-activation (non canonical) of β-catenin. In the canonical Wnt pathway (A), the binding of Wnt to Frizzled (Fzd) receptors activates Dishevelled (Dvl), which prevents the phosphorylation and the following ubiquitination of β-catenin. If β-catenin is not phosphorylated, it can thus accumulate in the cytoplasm and then translocate to the nucleus, where it activates Wnt target genes by interacting with the TCF/LEF family of transcription factors. In the non-canonical Wnt pathway (B), the binding of specific Wnt isoforms (Wnt 4, 5a, 11) to Fzd can activate Dvl but the downstream signal pathways involve small GTPases and the C-Jun N-terminal kinase (JNK) instead of β-catenin. Once activated, Dvl leads to increased intracellular Ca2+ levels that activate a number of proteins, including PKA, CaMK and NFAT. Acting as transcription factors, these proteins may activate downstream effectors that are crucial regulators of different cellular responses, such as planar cell polarity and cytoskeletal rearrangement.
      Similar to Notch and Wnt, Hedgehog signalling (Hh) is a morphogenetic pathway involved in liver development. Signalling mechanisms of the Hh pathway are shown in Fig. 4. On the contrary to stromal and progenitor cells, mature epithelial cells do not retain the ability to respond to Hh signals. During development, sonic Hh is expressed in the ventral foregut endoderm, but its expression is then downregulated when the liver bud is formed [
      • Hirose Y.
      • Itoh T.
      • Miyajima A.
      Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells.
      ]. In the foetal liver, Hh and Gli1, transcription factors, downstream of Hh signalling, are expressed in liver progenitor cells but their expression disappears as soon as the development proceeds. Therefore, activation of Hh signals is requested in a temporally restricted manner to promote the early hepatoblast proliferation, but then this pathway needs to be switched off to allow hepatoblasts to differentiate normally [
      • Hirose Y.
      • Itoh T.
      • Miyajima A.
      Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells.
      ].
      Figure thumbnail gr4
      Fig. 4Sonic Hedgehog (Shh) signalling. In physiological conditions (A), Patched (Ptc) receptor binds to and consequently suppresses the function of its co-receptor Smoothened (Smo). This maintains the bonding of the downstream regulator Glioblastoma-3 (Gli3) to a tetramer complex encompassing its suppressor factors, Suppressor Fused (Su(Fu)), Costal2 (Cos2), and Fused (Fu). In this complex, Gli3 is proteolytically cleaved by protein kinase A (PKA), and converted into the repressor form (Gli3R), which exerts an inhibitory effect on nuclear transcription factors regulating Hh-responsive genes (Ptc, Gli1, Gli2). When Shh interacts with Ptc (B), it prevents its inhibitory action on Smo. Following Smo activation, the tetramer complex is disassembled so that Su(Fu) inhibitory effect is restricted to Cos2 and PKA, thereby preventing the proteolytic cleavage of Gli3. Gli3 can thus enter the nucleus in the activated state (Gli3A) to promote activation of Hh target genes.
      In addition to differentiation and tissue remodelling, biliary tubulogenesis requires cell proliferation. While the process of remodelling depends on complex interactions with mesenchymal and endothelial cells, tubule elongation is stimulated by a number of paracrine or autocrine factors, including oestrogens [
      • Alvaro D.
      • Alpini G.
      • Onori P.
      • Perego L.
      • Svegliati Baroni G.
      • Franchitto A.
      • et al.
      Estrogens stimulate proliferation of intrahepatic biliary epithelium in rats.
      ], insulin-like growth factor-1 (IGF1) [
      • Alvaro D.
      • Macarri G.
      • Mancino M.G.
      • Marzioni M.
      • Bragazzi M.
      • Onori P.
      • et al.
      Serum and biliary insulin-like growth factor I and vascular endothelial growth factor in determining the cause of obstructive cholestasis.
      ,
      • Onori P.
      • Alvaro D.
      • Floreani A.R.
      • Mancino M.G.
      • Franchitto A.
      • Guido M.
      • et al.
      Activation of the IGF1 system characterizes cholangiocyte survival during progression of primary biliary cirrhosis.
      ], interleukin-6 (IL-6) [
      • Park J.
      • Gores G.J.
      • Patel T.
      Lipopolysaccharide induces cholangiocyte proliferation via an interleukin-6-mediated activation of p44/p42 mitogen-activated protein kinase.
      ], VEGF [
      • Fabris L.
      • Cadamuro M.
      • Fiorotto R.
      • Roskams T.
      • Spirlì C.
      • Melero S.
      • et al.
      Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.
      ], which are able to stimulate cholangiocyte proliferation.
      When tubule formation finishes, cholangiocytes become quiescent. Expression of ciliary proteins and control of cytokine secretion are important mechanisms involved in the termination of the developmental process. Foxa1 and Foxa2 are liver-specific transcription factors, which play a crucial role in establishing the developmental competence of the foregut endoderm and liver specification [
      • Li Z.
      • White P.
      • Tuteja G.
      • Rubins N.
      • Sackett S.
      • Kaestner K.H.
      Foxa1 and Foxa2 regulate bile duct development in mice.
      ]. Interestingly, mice with liver-specific deletion of Foxa1/2 develop dysmorphic and dilated bile ducts surrounded by increased portal fibrosis. These changes are, in part, caused by the persistent expression of IL-6 by the biliary epithelium. In fact, the IL-6 promoter is negatively regulated by Foxa1/2 through binding to the glucocorticoid receptor. In absence of Foxa1/2, IL-6 expression is not suppressed leading to both autocrine effects on cholangiocytes (proliferation) and to paracrine effects on inflammatory cells and myofibroblasts (peribiliary fibrosis) [
      • Li Z.
      • White P.
      • Tuteja G.
      • Rubins N.
      • Sackett S.
      • Kaestner K.H.
      Foxa1 and Foxa2 regulate bile duct development in mice.
      ]. This observation suggests that Foxa1/2 are among the transcription factors involved in the termination of bile duct development. Given the strong ability of reactive cholangiocytes to secrete large amounts of IL-6, this mechanism can be relevant also in liver repair (see below).

      Ductular reaction, a reparative response to biliary and hepatocellular damage, is characterised by features reminiscent of biliary ontogenesis

      Mechanisms of liver repair recapitulating liver developmental processes are an emerging concept. In fact, many molecular factors (growth factors, transcription factors, morphogens), transiently engaged in the development of the biliary epithelium during embryonic life, are reactivated in adulthood in response to acute or chronic liver damage [
      • Fabris L.
      • Cadamuro M.
      • Libbrecht L.
      • Raynaud P.
      • Spirlì C.
      • Fiorotto R.
      • et al.
      Angiogenic growth factors secreted by liver epithelial cells modulate arterial vasculogenesis during human liver development.
      ,
      • Fabris L.
      • Strazzabosco M.
      • Crosby H.A.
      • Ballardini G.
      • Hubscher S.G.
      • Kelly D.A.
      • et al.
      Characterization and isolation of ductular cells coexpressing neural cell adhesion molecule and Bcl-2 from primary cholangiopathies and ductal plate malformations.
      ].
      In the adult liver, the division of mature epithelial cells, i.e. hepatocytes and/or cholangiocytes, drives normal tissue homeostasis and regeneration after acute and transient hepatocellular or biliary damage. On the other hand, in chronic liver diseases, liver repair relies on the activation of hepatic progenitor cells (HPC). HPC are bipotent cells located in close proximity to the terminal cholangioles at their interface with the canals of Hering. HPC are able to amplify and differentiate into cells committed to hepatocellular or biliary lineages [
      • Sell S.
      Is there a liver stem cell?.
      ,
      • Sell S.
      Liver stem cells.
      ,
      • Crosby H.A.
      • Hubscher S.
      • Fabris L.
      • Joplin R.
      • Sell S.
      • Kelly D.
      • et al.
      Immunolocalization of putative human liver progenitor cells in livers from patients with end-stage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6.
      ,
      • Roskams T.
      • Theise N.D.
      • Balabaud C.
      • Bhagat G.
      • Bhathal P.S.
      • Bioulac-Sage P.
      • et al.
      Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.
      ]. In humans, differentiation towards hepatocytes occurs via intermediate hepato-biliary cells (IHBC), whereas differentiation towards the biliary lineage leads to the formation of reactive ductules (RDC) [
      • Roskams T.
      • Theise N.D.
      • Balabaud C.
      • Bhagat G.
      • Bhathal P.S.
      • Bioulac-Sage P.
      • et al.
      Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.
      ]. These cellular elements encompass the “hepatic reparative complex” and can be recognised from their expression of cytokeratin-7 (K7) and/or -19 (K19), cytoskeletal proteins present in the biliary lineage, but not in hepatocytes (Fig. 5) [
      • Van Eyken P.
      • Sciot R.
      • Callea F.
      • Van der Steen K.
      • Moerman P.
      • Desmet V.J.
      The development of the intrahepatic bile ducts in man: a keratin-immunohistochemical study.
      ,
      • Fabris L.
      • Strazzabosco M.
      • Crosby H.A.
      • Ballardini G.
      • Hubscher S.G.
      • Kelly D.A.
      • et al.
      Characterization and isolation of ductular cells coexpressing neural cell adhesion molecule and Bcl-2 from primary cholangiopathies and ductal plate malformations.
      ]. Although transdifferentiation from hepatocytes (formerly recognised as “ductular metaplasia” of hepatocytes) may occur under certain circumstances, reactive ductules are believed to derive mostly from the progenitor cell compartment.
      Figure thumbnail gr5
      Fig. 5Epithelial phenotypes involved in liver repair driven by the activation of hepatic progenitor cells (“Hepatic Reparative Complex”). In acute and chronic liver diseases, especially in fulminant hepatic failure, liver repair is driven by the activation of hepatic progenitor cells (HPC) to differentiate into hepatocytes and/or cholangiocytes. However, hepatocyte and cholangiocyte proliferation can be directly stimulated without exploiting HPC activation in experimental models, such as partial hepatectomy and acute biliary obstruction, respectively. HPC are small epithelial cells with an oval nucleus and scant cytoplasm, similar to oval cells in rodents treated with carcinogens. HPC originate from a niche located in the smaller branches of the biliary tree and in the canals of Hering. HPC behave as a bipotent, transit amplifying compartment. Differentiation of HPC towards hepatocytes occurs via intermediate hepatobiliary cells (IHBC), while differentiation towards the biliary lineage leads to the formation of reactive ductular cells (RDC). HPC, IHBC and RDC constitute the “hepatic reparative complex”, and can be distinguished by morphology and pattern of K7 expression. Whereas Wnt signalling is a key regulator of proliferation of HPC, Notch and Hh signalling are mostly involved in biliary differentiation through RDC generation, along with other cytokines released from the inflammatory microenvironment (TNF-α, TWEAK, TGF-β, HGF, VEGF, IL-6) (see text for details).
      Initially, RDC organise in clusters and do not encircle a lumen. As RDC’s participation in tissue remodelling develops, they eventually reorganise into a richly anastomosing tubular network. Tubule formation during biliary repair is a fundamental process aimed at generating a compensatory increase of the ductal mass to prevent the development of extensive liver necrosis due to the leakage of bile into the parenchyma. It is important to recognise that activation and proliferation of HPC is not sufficient to repair biliary damage unless progenitor cells and reactive cholangiocytes acquire the ability to form new branching tubular structures, thus restoring the ductal mass [
      • Fausto N.
      • Campbell J.S.
      • Riehle K.J.
      Liver regeneration.
      ].
      In addition to replacement of damaged cells and development of branching tubules, liver repair requires the generation of a fibro-vascular stroma able to sustain and feed the remodelling ductal structures. As a consequence, RDC de novo express a variety of cytokines, chemokines, growth factors, angiogenic factors and adhesion molecules, along with their receptors. This property enables RDC to establish an extensive crosstalk with other liver cell types, including hepatocytes, stellate cells and endothelial cells (Fig. 6) [
      • Strazzabosco M.
      • Spirli C.
      • Okolicsanyi L.
      Pathophysiology of the intrahepatic biliary epithelium.
      ]. When the biliary tree is damaged, these interactions become functionally relevant leading to a significant expansion of the reactive cholangiocyte compartment. Most of the molecular signatures expressed by reactive cholangiocytes, including VEGF [
      • Fabris L.
      • Cadamuro M.
      • Libbrecht L.
      • Raynaud P.
      • Spirlì C.
      • Fiorotto R.
      • et al.
      Angiogenic growth factors secreted by liver epithelial cells modulate arterial vasculogenesis during human liver development.
      ], TGF-β2 [
      • Dünker N.
      • Krieglstein K.
      Tgfbeta2 −/− Tgfbeta3 −/− double knockout mice display severe midline fusion defects and early embryonic lethality.
      ], connective tissue growth factor (CTGF) [
      • Surveyor G.A.
      • Brigstock D.R.
      Immunohistochemical localization of connective tissue growth factor (CTGF) in the mouse embryo between days 7.5 and 14.5 of gestation.
      ], stromal cell-derived factor 1 (SDF-1) [
      • Coulomb-L’Hermin A.
      • Amara A.
      • Schiff C.
      • Durand-Gasselin I.
      • Foussat A.
      • Delaunay T.
      • et al.
      Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells.
      ], IL-6 [
      • Jia C.
      Advances in the regulation of liver regeneration.
      ], tumour necrosis factor-α (TNFα) [
      • Jia C.
      Advances in the regulation of liver regeneration.
      ], neural cell adhesion molecule (NCAM) and Bcl-2 [
      • Fabris L.
      • Strazzabosco M.
      • Crosby H.A.
      • Ballardini G.
      • Hubscher S.G.
      • Kelly D.A.
      • et al.
      Characterization and isolation of ductular cells coexpressing neural cell adhesion molecule and Bcl-2 from primary cholangiopathies and ductal plate malformations.
      ], are transiently expressed by ductal plate cells in foetal life, consistent with the concept that ductular reaction recapitulates liver ontogenesis [
      • Fabris L.
      • Strazzabosco M.
      • Crosby H.A.
      • Ballardini G.
      • Hubscher S.G.
      • Kelly D.A.
      • et al.
      Characterization and isolation of ductular cells coexpressing neural cell adhesion molecule and Bcl-2 from primary cholangiopathies and ductal plate malformations.
      ,
      • LeSage G.
      • Alvaro D.
      • Benedetti A.
      • Glaser S.
      • Marucci L.
      • Baiocchi L.
      • et al.
      Cholinergic system modulates growth, apoptosis, and secretion of cholangiocytes from bile duct-ligated rats.
      ,
      • Marzioni M.
      • Glaser S.
      • Francis H.
      • Marucci L.
      • Benedetti A.
      • Alvaro D.
      • et al.
      Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.
      ,
      • Gigliozzi A.
      • Alpini G.
      • Baroni G.S.
      • Marucci L.
      • Metalli V.D.
      • Glaser S.S.
      • et al.
      Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis.
      ] (Fig. 7).
      Figure thumbnail gr6
      Fig. 6Reactive ductular cells acquire the ability to exchange a range of paracrine signals with mesenchymal, vascular and inflammatory cells. Owing to the de novo expression of a variety of cytokines, chemokines, growth factors, angiogenic factors, together with a rich expression of many of the respective cognate receptors, reactive ductular cells can establish an extensive crosstalk with other liver cell types, particularly with stellate cells, endothelial cells and inflammatory cells. In response to biliary damage, these interactions become functionally relevant leading to the generation of a fibro-vascular stroma able to sustain and feed the ductular reaction, and to the recruitment of a peribiliary inflammatory infiltrate which further enhances the bile duct damage.
      Figure thumbnail gr7
      Fig. 7Phenotypic changes of ductular reactive cells shared with ductal plate cells. An important feature of reactive cholangiocytes is the foetal reminiscence of their phenotype. Reactive ductular cells express neuroendocrine features, adhesion molecules, cytokines and chemokines, receptors and other metabolically active molecules, which are transiently expressed by ductal plate cells during embryonic development.
      The molecular mechanisms that activate ductular reaction require a finely coordinated process that also shares a number of similarities with biliary embryogenesis. Ductular reaction is elicited by inflammatory signals released from the local microenvironment. TNF-α [
      • Yasoshima M.
      • Kono N.
      • Sugawara H.
      • Katayanagi K.
      • Harada K.
      • Nakanuma Y.
      Increased expression of interleukin-6 and tumour necrosis factor-alpha in pathologic biliary epithelial cells: in situ and culture study.
      ], TWEAK [
      • Jakubowski A.
      • Ambrose C.
      • Parr M.
      • Lincecum J.M.
      • Wang M.Z.
      • Zheng T.S.
      • et al.
      TWEAK induces liver progenitor cell proliferation.
      ], TGF-β [
      • Napoli J.
      • Prentice D.
      • Niinami C.
      • Bishop G.A.
      • Desmond P.
      • McCaughan G.W.
      Sequential increases in the intrahepatic expression of epidermal growth factor, basic fibroblast growth factor, and transforming growth factor beta in a bile duct ligated rat model of cirrhosis.
      ], HGF [
      • Napoli J.
      • Prentice D.
      • Niinami C.
      • Bishop G.A.
      • Desmond P.
      • McCaughan G.W.
      Sequential increases in the intrahepatic expression of epidermal growth factor, basic fibroblast growth factor, and transforming growth factor beta in a bile duct ligated rat model of cirrhosis.
      ], VEGF [
      • Fabris L.
      • Cadamuro M.
      • Fiorotto R.
      • Roskams T.
      • Spirlì C.
      • Melero S.
      • et al.
      Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.
      ,
      • Gouw A.S.
      • van den Heuvel M.C.
      • Boot M.
      • Slooff M.J.
      • Poppema S.
      • de Jong K.P.
      Dynamics of the vascular profile of the finer branches of the biliary tree in normal and diseased human livers.
      ], sonic Hedgehog (shh) [
      • Omenetti A.
      • Diehl A.M.
      The adventures of sonic hedgehog in development and repair: II. Sonic hedgehog and liver development, inflammation, and cancer.
      ], and Wnt/β-catenin [
      • Sackett S.D.
      • Gao Y.
      • Shin S.
      • Esterson Y.B.
      • Tsingalia A.
      • Hurtt R.S.
      • et al.
      Foxl1 promotes liver repair following cholestatic injury in mice.
      ] signalling are among the key signalling pathways. As mentioned above, proliferation of reactive cholangiocytes may be achieved by switching off transcription factors (Foxa1/2) used to terminate development. Unfortunately, activation/deactivation of developmental mechanisms in the context of non-resolving inflammation, leads to the development of portal fibrosis in cholangiopathies. The ability of “reactive” cholangiocytes to recruit inflammatory, vascular and mesenchymal cells, with which they exchange a variety of paracrine signals, subsequently leads to excessive collagen deposition and stimulation of angiogenesis, and ultimately to cirrhosis [
      • Fabris L.
      • Strazzabosco M.
      Epithelial–mesenchymal interactions in biliary diseases.
      ].
      Several morphogens involved in biliary development, such as Wnt, Hedgehog, and Notch, also play a pivotal role in liver repair. Recent findings suggest that the Wnt/β-catenin pathway is strongly involved in the normal activation and proliferation of adult HPC in both acute and chronic human liver diseases [
      • Spee B.
      • Carpino G.
      • Schotanus B.A.
      • Katoonizadeh A.
      • Vander Borght S.
      • Gaudio E.
      • et al.
      Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling.
      ]. Oval cell activation in β-catenin conditional knockout mice was dramatically reduced, after chemical treatment to induce acute liver damage [
      • Apte U.
      • Thompson M.D.
      • Cui S.
      • Liu B.
      • Cieply B.
      • Monga S.P.
      Wnt/beta-catenin signaling mediates oval cell response in rodents.
      ].
      Whereas Wnt signalling is a key regulator of proliferation of HPC, Notch signalling is mostly involved in biliary differentiation. The role of Notch is underscored by the peculiar liver phenotype observed in AGS. AGS is characterised by a marked reduction in RDC and HPC, in sharp contrast with biliary atresia, which is a congenital cholangiopathy with similar levels of homeostasis but much faster evolution to biliary cirrhosis [
      • Fabris L.
      • Cadamuro M.
      • Guido M.
      • Spirli C.
      • Fiorotto R.
      • Colledan M.
      • et al.
      Analysis of liver repair mechanisms in Alagille syndrome and biliary atresia reveals a role for notch signaling.
      ]. This difference is likely related to a Notch-dependent block in cell fate determination upstream of HNF1β. Recent data from our group show that following treatment with cholestatic agents, HPC activation and tubule formation are dramatically impaired in mice with a liver-specific defect in RBP-Jk [
      • Fiorotto R.
      • Spirli C.
      • Scirpo R.
      • Fabris L.
      • Huppert S.
      • Torsello B.
      • et al.
      Defective progenitor cells activation and biliary tubule formation in liver conditional RBP-jk-knock out mice exposed to cholestatic injuries reveals a key role for Notch signaling in liver repair.
      ].
      In addition to Notch, Hh signalling is also relevant in congenital cholangiopathies. Perturbations in the Hh signalling have been associated with DPM in Meckel syndrome (MKS), a rare autosomal recessive disease caused by a defect in MKS1 gene encoding a protein associated with the base of cilia. MKS causes perinatal lethality and is characterised by a complex syndrome including polycystic kidneys, occipital meningoencephalocele, postaxial polydactyly in addition to DPM [
      • Weatherbee S.D.
      • Niswander L.A.
      • Anderson K.V.
      A mouse model for Meckel syndrome reveals Mks1 is required for ciliogenesis and Hedgehog signaling.
      ]. In liver repair, activation of the Hh signalling promotes the expansion of a subset of immature ductular cells that co-express mesenchymal markers and may be profibrogenic [
      • Choi S.S.
      • Omenetti A.
      • Syn W.K.
      • Diehl A.M.
      The role of Hedgehog signaling in fibrogenic liver repair.
      ]. This mechanism is relevant in biliary atresia, where excessive activation of Hh signalling halts bile duct morphogenesis and promotes accumulation of immature ductular cells with a mesenchymal phenotype, which in turn enhance fibrogenesis [
      • Omenetti A.
      • Bass L.M.
      • Anders R.A.
      • Clemente M.G.
      • Francis H.
      • Guy C.D.
      • et al.
      Hedgehog activity, epithelial–mesenchymal transitions, and biliary dysmorphogenesis in biliary atresia.
      ]. These data suggest that aberrant activation of Hh signalling may be responsible for biliary fibrosis featuring developmental cholangiopathies.

      Primary cholangiopathies related to an altered development of the biliary epithelium

      Cholangiopathies related to altered biliary development [
      • Desmet V.J.
      Congenital diseases of intrahepatic bile ducts: variations on the theme “ductal plate malformation”.
      ,
      • Jørgensen M.J.
      The ductal plate malformation.
      ] (Table 1) represent important disease models; understanding their pathogenetic mechanisms offers important clues on how specific genes regulating developmental processes are involved in biliary repair and reaction to damage. Recently, based on the phenotype of several mouse models with specific defects of biliary morphogenesis, DPM have been classified into three groups, with: (a) defective differentiation of biliary precursors cells (HNF6 deficiency), (b) defective maturation of primitive ductal structure (HNF1β deficiency) or (c) defective duct expansion during development with preserved biliary differentiation (cystin-1 deficiency) [
      • 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.
      ]. Most DPM are embryonically lethal or are part of complex syndromic diseases. Herein, we will outline the most recent advances in the pathogenesis of congenital cholangiopathies that may be clinically relevant for both paediatric and adult clinical hepatologists. The most common DPM are the Von Meyenburg complexes, also known as biliary microhamartomas (Fig. 8B). These are benign lesions characterised by irregularly shaped and dilated biliary structures, embedded in a dense fibrous stroma. Their distribution is generally focal, but when diffuse they can be associated with cystic lesions, as seen in congenital hepatic fibrosis (see below) [
      • Desmet V.J.
      Ludwig symposium on biliary disorders: Part I. Pathogenesis of ductal plate abnormalities.
      ]. Von Meyenburg complexes have also been found in higher numbers in the liver of patients with autosomal dominant polycystic kidney disease [
      • Redston M.S.
      • Wanless I.R.
      The hepatic von Meyenburg complex: prevalence and association with hepatic and renal cysts among 2843 autopsies.
      ,
      • Tsui W.M.
      How many types of biliary hamartomas and adenomas are there?.
      ]. The association of Von Meyenburg complexes to cholangiocarcinoma is uncommon, but it has been sporadically described [
      • Jain D.
      • Sarode V.R.
      • Abdul-Karim F.W.
      • Homer R.
      • Robert M.E.
      Evidence for the neoplastic transformation of Von Meyenburg complexes.
      ,
      • Song J.S.
      • Lee Y.J.
      • Kim K.W.
      • Huh J.
      • Jang S.J.
      • Yu E.
      Cholangiocarcinoma arising in Von Meyenburg complexes: report of four cases.
      ,
      • Xu A.M.
      • Xian Z.H.
      • Zhang S.H.
      • Chen X.F.
      Intrahepatic cholangiocarcinoma arising in multiple bile duct hamartomas: report of two cases and review of the literature.
      ].
      Table 1Primary cholangiopathies related to altered biliary development.
      AGS, Alagille syndrome; ADPKD, autosomal dominant polycystic kidney disease; PLD, polycystic liver disease; ARPKD, autosomal recessive polycystic kidney disease; CHF, congenital hepatic fibrosis; CD, Caroli’s disease.
      Figure thumbnail gr8
      Fig. 8Main differences in liver phenotype between ARPKD and ADPKD. On magnetic resonance imaging, liver cysts appear as focal lesions with regular margins, which are of small size and in continuity with the biliary tree in ARPKD (A), whereas they are large, of different size and scattered throughout the hepatic parenchyma leading to extensive cyst substitution in ADPKD (D). At histological examination, irregularly shaped biliary structures (microhamartomas) surrounded by an extensive deposition of fibrotic tissue, are present in the portal tracts in ARPKD (B, H&E; magnification: 100×), while in ADPKD, biliary cysts appear as large, circular biliary structures, lined by cuboidal or flattened epithelium, with negligible amount of peribiliary fibrosis (E, H&E, magnification: 100×). The pathogenetic mechanism underlying cyst formation is characterised by progressive segmental dilation of biliary structures which maintain their connection to the biliary tree in ARPKD (C), whereas biliary cysts detach from the bile duct and then progressively increase in size in ADPKD (F). Alternatively, based on previous histopathological studies [
      • Karhunen P.J.
      Adult polycystic liver disease and biliary microhamartomas (von Meyenburg’s complexes).
      ,
      • Grimm P.C.
      • Crocker J.F.
      • Malatjalian D.A.
      • Ogborn M.R.
      The microanatomy of the intrahepatic bile duct in polycystic disease: comparison of the cpk mouse and human.
      ,
      • Ramos A.
      • Torres V.E.
      • Holley K.E.
      • Offord K.P.
      • Rakela J.
      • Ludwig J.
      The liver in autosomal dominant polycystic kidney disease. Implications for pathogenesis.
      ], liver cysts in ADPKD may also derive from dilatation of components of Von Meyenburg complexes.

      Autosomal recessive polycystic kidney disease (ARPKD), congenital hepatic fibrosis (CHF) and Caroli’s disease (CD)

      The presence of ductal plate remnants, Von Meyenburg complexes, and biliary cysts variably associated with an intense peribiliary fibrosis, are the main features of fibropolycystic diseases [
      • Desmet V.J.
      Ludwig symposium on biliary disorders: Part I. Pathogenesis of ductal plate abnormalities.
      ]. These include ARPKD, and its hepatic variants, CHF and CD, and a variety of congenital syndromes (Meckel and Joubert syndromes), which are often embryonically lethal. In CHF and CD, biliary malformations are associated with progressive portal fibrosis leading to portal hypertension, without progressing to frank cirrhosis. In patients with CD, the risk of developing cholangiocarcinoma is substantially increased (about 10%).
      CHF and CD are caused by mutations in PKHD1, a gene encoding for fibrocystin (FPC). FPC is a large membrane, receptor-like protein expressed by the basal body of cilia, subcellular organelles that sense the direction of ductal bile flow, and by centromeres of renal tubular and bile duct epithelial cells [
      • Ward C.J.
      • Hogan M.C.
      • Rossetti S.
      • Walker D.
      • Sneddon T.
      • Wang X.
      • et al.
      The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.
      ,
      • Zhang M.Z.
      • Mai W.
      • Li C.
      • Cho S.Y.
      • Hao C.
      • Moeckel G.
      • et al.
      PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells.
      ,
      • Harris P.C.
      • Torres V.E.
      Polycystic kidney disease.
      ]. Although FPC functions are largely unknown, some of its properties have been recently outlined [
      • Banales J.M.
      • Masyuk T.V.
      • Gradilone S.A.
      • Masyuk A.I.
      • Medina J.F.
      • LaRusso N.F.
      The cAMP effectors Epac and protein kinase a (PKA) are involved in the hepatic cystogenesis of an animal model of autosomal recessive polycystic kidney disease (ARPKD).
      ]. FPC is thought to be involved in a variety of functions, from proliferation to secretion, terminal differentiation, tubulogenesis, and interactions with the extracellular matrix. Silencing Pkhd1 in cultured mouse renal tubular cells altered cytoskeletal organisation and impaired cell–cell and cell–matrix contacts [
      • Mai W.
      • Chen D.
      • Ding T.
      • Kim I.
      • Park S.
      • Cho S.Y.
      • et al.
      Inhibition of Pkhd1 impairs tubulomorphogenesis of cultured IMCD cells.
      ]. Recent evidences indicate that FPC is also involved in planar cell polarity. In the Pck rat, a model orthologue of ARPKD, FPC deficiency is strongly correlated with the loss of PCP at the renal level. This leads to a perturbed mitotic alignment on circumferential tubular cell number expansion, which is ultimately responsible for renal tubular enlargement and cyst formation [
      • Fischer E.
      • Legue E.
      • Doyen A.
      • Nato F.
      • Nicolas J.F.
      • Torres V.
      • et al.
      Defective planar cell polarity in polycystic kidney disease.
      ]. The mechanistic relationships between biliary dysgenesis and portal fibrosis are not understood and this is an area of current investigation.

      Autosomal dominant polycystic kidney disease (ADPKD) and polycystic liver disease (PLD)

      Autosomal dominant polycystic kidney disease (ADPKD) and polycystic liver disease (PLD) are inherited with an autosomal dominant transmission. These conditions do not cause significant liver fibrosis, but rather an enormous increase in liver mass. The formation and progressive enlargement of multiple cysts scattered throughout the liver parenchyma characterise both ADPKD and PLD [
      • Tahvanainen E.
      • Tahvanainen P.
      • Kääriäinen H.
      • Höckerstedt K.
      Polycystic liver and kidney diseases.
      ]. Despite extensive cyst substitution of the hepatic parenchyma, liver function is generally well preserved and portal hypertension is rare.
      The patients are usually asymptomatic, unless acute and chronic complications (including cyst infections or bleeding and mass effect) develop. ADPKD is caused by mutations in the PKD1 or PKD2 genes [
      • Igarashi P.
      • Somlo S.
      Genetics and pathogenesis of polycystic kidney disease.
      ,
      • Wilson P.D.
      Polycystic kidney disease: new understanding in the pathogenesis.
      ]. The transcribed products of these two genes, polycystin-1 (PC1) and polycystin-2 (PC2), are membrane proteins located in the renal tubular and biliary epithelia. PC1 and PC2 regulate signalling pathways that are involved in epithelial cell morphogenesis, differentiation and proliferation. In conditional, liver specific mice defective for PC-1 or PC-2, polycystic liver disease develops even if PC1 and PC2 are deleted after birth, indicating that PC expression maintain a fundamental morphogenetic role also during adult life [
      • Spirli C.
      • Okolicsanyi S.
      • Fiorotto R.
      • Fabris L.
      • Cadamuro M.
      • Lecchi S.
      • et al.
      Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice.
      ,
      • Spirli C.
      • Okolicsanyi S.
      • Fiorotto R.
      • Fabris L.
      • Cadamuro M.
      • Lecchi S.
      • et al.
      ERK1/2-dependent vascular endothelial growth factor signaling sustains cyst growth in polycystin-2 defective mice.
      ]. Thus, altered PC function may cause a lack of differentiating signals favouring the maintenance of an immature and proliferative phenotype by biliary epithelial cells ultimately responsible for cyst formation. In fact, cholangiocytes lining the liver cysts present strong phenotypic and functional similarities with ductal plate/reactive ductules [
      • Fabris L.
      • Cadamuro M.
      • Fiorotto R.
      • Roskams T.
      • Spirlì C.
      • Melero S.
      • et al.
      Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.
      ]. Among them are an aberrant secretion of several cytokines and chemokines, including IL-6 [
      • Nichols M.T.
      • Gidey E.
      • Matzakos T.
      • Dahl R.
      • Stiegmann G.
      • Shah R.J.
      • et al.
      Secretion of cytokines and growth factors into autosomal dominant polycystic kidney disease liver cyst fluid.
      ], IL-8 [
      • Nichols M.T.
      • Gidey E.
      • Matzakos T.
      • Dahl R.
      • Stiegmann G.
      • Shah R.J.
      • et al.
      Secretion of cytokines and growth factors into autosomal dominant polycystic kidney disease liver cyst fluid.
      ], and CXCR2 [
      • Amura C.R.
      • Brodsky K.S.
      • Gitomer B.
      • McFann K.
      • Lazennec G.
      • Nichols M.T.
      • et al.
      CXCR2 agonists in ADPKD liver cyst fluids promote cell proliferation.
      ], a marked overexpression of oestrogen receptors, IGF1, the IGF1 and growth hormone receptors as well as VEGF and its cognate receptor, VEGFR-2 [
      • Fabris L.
      • Cadamuro M.
      • Fiorotto R.
      • Roskams T.
      • Spirlì C.
      • Melero S.
      • et al.
      Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.
      ]. In particular, VEGF potently stimulates the progression of liver cysts in ADPKD via autocrine stimulation of cholangiocyte proliferation and paracrine promotion of pericystic angiogenesis. We have shown that in cystic cholangiocytes from Pkd2-defective mice a MEK/ERK1/2/mTOR pathway is overactive and is responsible for increased hypoxia-inducible factor 1α-dependent VEGF production and increased VEGFR-2-mediated autocrine stimulation of cyst growth. This mechanism represents a potential target for therapy, as the blockade of angiogenic signalling using a competitive inhibitor of VEGFR-2, or the administration of mTOR inhibitors block the growth of liver cysts in Pkd2KO mice, and reduces the proliferative activity of the cystic epithelium [
      • Spirli C.
      • Okolicsanyi S.
      • Fiorotto R.
      • Fabris L.
      • Cadamuro M.
      • Lecchi S.
      • et al.
      ERK1/2-dependent vascular endothelial growth factor signaling sustains cyst growth in polycystin-2 defective mice.
      ]. Overall, these data indicate that polycystic liver diseases should be considered as congenital diseases of cholangiocyte signalling [
      • Strazzabosco M.
      • Somlo S.
      Polycystic liver diseases: congenital disorders of cholangiocyte signaling.
      ]. Phenotypic changes in cyst cholangiocytes with functional relevance for cyst formation and progression are summarised in Table 2. Each pathway is also therapeutically relevant as a potential target amenable of pharmacological interference aimed at reducing disease progression. Future studies will clarify whether the use of agents interfering with VEGF or mTOR signalling can be clinically useful and if their use can be extended to other cholangiopathies [
      • Spirli C.
      • Okolicsanyi S.
      • Fiorotto R.
      • Fabris L.
      • Cadamuro M.
      • Lecchi S.
      • et al.
      Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice.
      ].
      Table 2Phenotypic changes in cyst cholangiocytes.
      In PLD, genetic defects do not involve ciliary proteins. PLD is caused by mutations in PRKCSH, a gene encoding for protein kinase C substrate 80K-H also called hepatocystin [
      • Drenth J.P.
      • te Morsche R.H.
      • Smink R.
      • Bonifacino J.S.
      • Jansen J.B.
      Germline mutations in PRKCSH are associated with autosomal dominant polycystic liver disease.
      ], or in the SEC63 gene [
      • Davila S.
      • Furu L.
      • Gharavi A.G.
      • Tian X.
      • Onoe T.
      • Qian Q.
      • et al.
      Mutations in SEC63 cause autosomal dominant polycystic liver disease.
      ]. SEC63 encodes a component of the molecular machinery regulating translocation and folding of newly synthesised membrane glycoproteins. Hepatocystin and SEC63 are expressed on the endoplasmic reticulum (ER) [
      • Janssen M.J.
      • Waanders E.
      • Woudenberg J.
      • Lefeber D.J.
      • Drenth J.P.
      Congenital disorders of glycosylation in hepatology: the example of polycystic liver disease.
      ], and defects in other enzymatic activities associated with the ER, such as xylosyl transferase 2 responsible for initiating heparin sulphate and chondroitin sulphate biosynthesis, have been linked to the development of renal and liver cysts [
      • Condac E.
      • Silasi-Mansat R.
      • Kosanke S.
      • Schoeb T.
      • Towner R.
      • Lupu F.
      • et al.
      Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis.
      ]. PLD serves to highlight the important concept that cystic liver disease does not necessarily derive from ciliary dysfunction, but that the defective proteins are expressed in multiple cellular locations, including the ER.

      Alagille syndrome (AGS)

      Alagille syndrome (AGS) is a complex, multisystemic disorder inherited as an autosomal dominant trait and characterised by ductopenia. AGS exhibits a wide range of extrahepatic manifestations (cardiac, vascular, skeletal, ocular, facial, renal, central nervous system), hence the term of “syndromic bile duct paucity” [
      • Krantz I.D.
      • Piccoli D.A.
      • Spinner N.B.
      Clinical and molecular genetics of Alagille syndrome.
      ,
      • Piccoli D.A.
      • Spinner N.B.
      Alagille syndrome and the Jagged1 gene.
      ]. The hepatic phenotype is recognised by variable degrees of cholestasis, jaundice and pruritus. Fibrosis is not a prominent feature of AGS and frank evolution to cirrhosis is rare, however, rare cases lead to liver transplantation [
      • Emerick K.M.
      • Rand E.B.
      • Goldmuntz E.
      • Krantz I.D.
      • Spinner N.B.
      • Piccoli D.A.
      Features of Alagille syndrome in 92 patients: frequency and relation to prognosis.
      ]. In nearly 80% of AGS patients, a mutation in the genes JAGGED1 [
      • Oda T.
      • Elkahloun A.G.
      • Pike B.L.
      • Okajima K.
      • Krantz I.D.
      • Genin A.
      • et al.
      Mutations in the human Jagged1 gene are responsible for Alagille syndrome.
      ,
      • Li L.
      • Krantz I.D.
      • Deng Y.
      • Genin A.
      • Banta A.B.
      • Collins C.C.
      • et al.
      Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.
      ], or less frequently, NOTCH2 can be identified [
      • McDaniell R.
      • Warthen D.M.
      • Sanchez-Lara P.A.
      • Pai A.
      • Krantz I.D.
      • Piccoli D.A.
      • et al.
      NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway.
      ]. AGS differs from other cholestatic cholangiopathies in the extent and severity of ductular reaction. The near absence of HPC and RDC in AGS (Fig. 5) is conversely coupled with an extensive accumulation of IHBC. IHBC do not express the biliary specific transcription factor HNF1β, whose expression is controlled by Notch signalling [
      • Fabris L.
      • Cadamuro M.
      • Guido M.
      • Spirli C.
      • Fiorotto R.
      • Colledan M.
      • et al.
      Analysis of liver repair mechanisms in Alagille syndrome and biliary atresia reveals a role for notch signaling.
      ]. This imbalance in the cellular elements of the “hepatic reparative complex”, which is a peculiar histopathological feature of AGS, supports the concept that Notch signalling plays an essential role in liver repair by regulating the generation of biliary committed precursors as well as the branching tubularisation [
      • Fiorotto R.
      • Spirli C.
      • Scirpo R.
      • Fabris L.
      • Huppert S.
      • Torsello B.
      • et al.
      Defective progenitor cells activation and biliary tubule formation in liver conditional RBP-jk-knock out mice exposed to cholestatic injuries reveals a key role for Notch signaling in liver repair.
      ].

      Conclusions

      Several cholangiopathies are caused by a malfunction of developmental mechanisms. In these diseases, cholangiocyte dysfunction is often caused by a specific genetic defect relevant to morphogenesis. Also, cholangiocytes express a range of phenotypic features reminiscent of foetal behaviour. In different forms of acquired liver damage, liver repair exploits several developmental mechanisms, in a sort of “recapitulation of ontogenesis”. Ductular reactive cells generated in response to liver damage express several of the autocrine and paracrine signals transitorily expressed by ductal plate cells during liver development. Therefore, understanding the molecular mechanisms regulating development of the biliary tree may help solve several challenging issues facing modern Hepatology. These may range from the search of novel treatments for patients with biliary diseases, to the need of limit/prevent pathologic repair in chronic liver diseases, to the design of bioartificial liver support devices, to strategies for liver regenerative medicine.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Financial support:

      Supported by NIH DK079005 , Yale University Liver Center (NIH DK34989 ) and by a grant from “PSC partners for a care” to M.S. Supported by Telethon ( GGP09189 ), Associazione Scientifica Gastroenterologica di Treviso (ASGET, “associazione di promozione sociale senza scopo di lucro”) and by Progetto di Ricerca Ateneo 2008 ( CPDA083217 ) to L.F.

      Acknowledgements

      The authors wish to thank Massimiliano Cadamuro, Ph.D. (Department of Surgical and Gastroenterological Sciences, University of Padua) for the micrographs of histology and assistance in the preparation of the graphics in the manuscript, and Dr. Roberto Agazzi, MD (Department of Radiology, Ospedali Riuniti di Bergamo) for providing the radiological images of Caroli’s disease and polycystic liver.

      References

        • Lemaigre F.P.
        Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies.
        Gastroenterology. 2009; 137: 62-79
        • Raynaud P.
        • Carpentier R.
        • Antoniou A.
        • Lemaigre F.P.
        Biliary differentiation and bile duct morphogenesis in development and disease.
        Int J Biochem Cell Biol. 2011; 43: 245-256
        • Lazaridis K.N.
        • Strazzabosco M.
        • Larusso N.F.
        The cholangiopathies: disorders of biliary epithelia.
        Gastroenterology. 2004; 127: 1565-1577
        • Strazzabosco M.
        • Fabris L.
        • Spirli C.
        Pathophysiology of cholangiopathies.
        J Clin Gastroenterol. 2005; 39: S90-S102
        • Desmet V.J.
        Congenital diseases of intrahepatic bile ducts: variations on the theme “ductal plate malformation”.
        Hepatology. 1992; 16: 1069-1083
        • Desmet V.J.
        Ludwig symposium on biliary disorders: Part I. Pathogenesis of ductal plate abnormalities.
        Mayo Clin Proc. 1998; 73: 80-89
        • Roskams T.
        • Desmet V.
        Embryology of extra- and intrahepatic bile ducts, the ductal plate.
        Anat Rec. 2008; 291: 628-635
        • Tan J.
        • Hytiroglou P.
        • Wieczorek R.
        • Park Y.N.
        • Thung S.N.
        • Arias B.
        • et al.
        Immunohistochemical evidence for hepatic progenitor cells in liver diseases.
        Liver. 2002; 22: 365-373
        • Van Eyken P.
        • Sciot R.
        • Callea F.
        • Van der Steen K.
        • Moerman P.
        • Desmet V.J.
        The development of the intrahepatic bile ducts in man: a keratin-immunohistochemical study.
        Hepatology. 1988; 8: 1586-1595
        • Nakanuma Y.
        • Hoso M.
        • Sanzen T.
        • Sasaki M.
        Microstructure and development of the normal and pathologic biliary tract in humans, including blood supply.
        Microsc Res Tech. 1997; 38: 552-570
        • Libbrecht L.
        • Cassiman D.
        • Desmet V.
        • Roskams T.
        Expression of neural cell adhesion molecule in human liver development and in congenital and acquired liver diseases.
        Histochem Cell Biol. 2001; 116: 233-239
        • Fabris L.
        • Cadamuro M.
        • Libbrecht L.
        • Raynaud P.
        • Spirlì C.
        • Fiorotto R.
        • et al.
        Angiogenic growth factors secreted by liver epithelial cells modulate arterial vasculogenesis during human liver development.
        Hepatology. 2008; 47: 719-728
        • Carpentier R.
        • Suñer 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.
        Gastroenterology. 2011; 141: 1432-1438
        • Furuyama K.
        • Kawaguchi Y.
        • Akiyama H.
        • Horiguchi M.
        • Kodama S.
        • Kuhara T.
        • et al.
        Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.
        Nat Genet. 2011; 43: 34-41
        • Antoniou A.
        • Raynaud P.
        • Cordi S.
        • Zong Y.
        • Tronche F.
        • Stanger B.Z.
        • et al.
        Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.
        Gastroenterology. 2009; 136: 2325-2333
        • Fischer E.
        • Legue E.
        • Doyen A.
        • Nato F.
        • Nicolas J.F.
        • Torres V.
        • et al.
        Defective planar cell polarity in polycystic kidney disease.
        Nat Genet. 2006; 38: 21-23
        • Li Z.
        • White P.
        • Tuteja G.
        • Rubins N.
        • Sackett S.
        • Kaestner K.H.
        Foxa1 and Foxa2 regulate bile duct development in mice.
        J Clin Invest. 2009; 119: 1537-1545
        • Zong Y.
        • Panikkar A.
        • Xu J.
        • Antoniou A.
        • Raynaud P.
        • Lemaigre F.
        • et al.
        Notch signaling controls liver development by regulating biliary differentiation.
        Development. 2009; 136: 1727-1739
        • Fukuda A.
        • Kawaguchi Y.
        • Furuyama K.
        • Kodama S.
        • Kuhara T.
        • Horiguchi M.
        • et al.
        Loss of the major duodenal papilla results in brown pigment biliary stone formation in pdx1 null mice.
        Gastroenterology. 2006; 130: 855-867
        • 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.
        Development. 2002; 129: 1819-1828
        • 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 HNF1β.
        Development. 2002; 129: 1829-1838
        • 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.
        J Biol Chem. 2002; 277: 12369-12374
        • Gaudio E.
        • Pannarale L.
        • Franchitto A.
        • Onori P.
        • Marinozzi G.
        Hepatic microcirculation as a morpho-functional basis for the metabolic zonation in normal and pathological rat liver.
        Ital J Anat Embryol. 1995; 100: 419-428
        • Gaudio E.
        • Onori P.
        • Franchitto A.
        • Pannarale L.
        • Alpini G.
        • Alvaro D.
        Hepatic microcirculation and cholangiocyte physiopathology.
        Ital J Anat Embryol. 2005; 110: 71-75
        • Gaudio E.
        • Franchitto A.
        • Pannarale L.
        • Carpino G.
        • Alpini G.
        • Francis H.
        • et al.
        Cholangiocytes and blood supply.
        World J Gastroenterol. 2006; 12: 3546-3552
        • Fabris L.
        • Cadamuro M.
        • Fiorotto R.
        • Roskams T.
        • Spirlì C.
        • Melero S.
        • et al.
        Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.
        Hepatology. 2006; 43: 1001-1012
        • Spirli C.
        • Okolicsanyi S.
        • Fiorotto R.
        • Fabris L.
        • Cadamuro M.
        • Lecchi S.
        • et al.
        Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice.
        Hepatology. 2010; 51: 1778-1788
        • Deltenre P.
        • Valla D.C.
        Ischemic cholangiopathy.
        Semin Liver Dis. 2008; 28: 235-246
        • Masyuk T.V.
        • Ritman E.L.
        • LaRusso N.F.
        Hepatic artery and portal vein remodeling in rat liver: vascular response to selective cholangiocyte proliferation.
        Am J Pathol. 2003; 162: 1175-1182
        • 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.
        J Hepatol. 2003; 39: 686-692
        • Lemaigre F.
        • Zaret K.S.
        Liver development update. New embryo models, cell lineage control, and morphogenesis.
        Curr Opin Genet Dev. 2004; 14: 582-590
        • Bossard P.
        • Zaret K.S.
        GATA transcription factors as potentiators of gut endoderm differentiation.
        Development. 1998; 125: 4909-4917
        • Rojas A.
        • De Val S.
        • Heidt A.B.
        • Xu S.M.
        • Bristow J.
        • Black B.L.
        Gata4 expression in lateral mesoderm is downstream of BMP4 and is activated directly by Forkhead and GATA transcription factors through a distal enhancer element.
        Development. 2005; 132: 3405-3417
        • Haworth K.E.
        • Kotecha S.
        • Mohun T.J.
        • Latinkic B.V.
        GATA4 and GATA5 are essential for heart and liver development in Xenopus embryos.
        BMC Dev Biol. 2008; 8: 74
        • Rossi J.M.
        • Dunn N.R.
        • Hogan B.L.
        • Zaret K.S.
        Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm.
        Genes Dev. 2001; 15: 1998-2009
        • Clotman F.
        • Lemaigre F.P.
        Control of hepatic differentiation by activin/TGFbeta signaling.
        Cell Cycle. 2006; 5: 168-171
        • Igarashi P.
        • Shao X.
        • McNally B.T.
        • Hiesberger T.
        Roles of HNF-1beta in kidney development and congenital cystic diseases.
        Kidney Int. 2005; 68: 1944-1947
        • 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.
        Hepatology. 2011; 53: 1959-1966
        • Lorent K.
        • Yeo S.Y.
        • Oda T.
        • Chandrasekharappa S.
        • Chitnis A.
        • Matthews R.P.
        • et al.
        Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy.
        Development. 2004; 131: 5753-5766
        • Lozier J.
        • McCright B.
        • Gridley T.
        Notch signaling regulates bile duct morphogenesis in mice.
        PLoS One. 2008; 3: e1851
        • Cornell R.A.
        • Eisen J.S.
        Notch in the pathway: the roles of Notch signaling in neural crest development.
        Semin Cell Dev Biol. 2005; 16: 663-672
        • Tchorz J.S.
        • Kinter J.
        • Müller M.
        • Tornillo L.
        • Heim M.H.
        • Bettler B.
        Notch2 signaling promotes biliary epithelial cell fate specification and tubulogenesis during bile duct development in mice.
        Hepatology. 2009; 50: 871-879
        • Fabris L.
        • Cadamuro M.
        • Guido M.
        • Spirli C.
        • Fiorotto R.
        • Colledan M.
        • et al.
        Analysis of liver repair mechanisms in Alagille syndrome and biliary atresia reveals a role for notch signaling.
        Am J Pathol. 2007; 171: 641-653
        • 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.
        Development. 2010; 137: 4061-4072
        • Loomes K.M.
        • Russo P.
        • Ryan M.
        • Nelson A.
        • Underkoffler L.
        • Glover C.
        • et al.
        Bile duct proliferation in liver-specific Jag1 conditional knockout mice: effects of gene dosage.
        Hepatology. 2007; 45: 323-330
        • Sparks E.E.
        • Huppert K.A.
        • Brown M.A.
        • Washington M.K.
        • Huppert S.S.
        Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.
        Hepatology. 2010; 51: 1391-1400
        • Tan X.
        • Yuan Y.
        • Zeng G.
        • Apte U.
        • Thompson M.D.
        • Cieply B.
        • et al.
        Beta-catenin deletion in hepatoblasts disrupts hepatic morphogenesis and survival during mouse development.
        Hepatology. 2008; 47: 1667-1679
        • Montcouquiol M.
        • Crenshaw 3rd, E.B.
        • Kelley M.W.
        Noncanonical Wnt signaling and neural polarity.
        Annu Rev Neurosci. 2006; 29: 363-386
        • Simons M.
        • Gloy J.
        • Ganner A.
        • Bullerkotte A.
        • Bashkurov M.
        • Krönig C.
        • et al.
        Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways.
        Nat Genet. 2005; 37: 537-543
        • Hirose Y.
        • Itoh T.
        • Miyajima A.
        Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells.
        Exp Cell Res. 2009; 315: 2648-2657
        • Alvaro D.
        • Alpini G.
        • Onori P.
        • Perego L.
        • Svegliati Baroni G.
        • Franchitto A.
        • et al.
        Estrogens stimulate proliferation of intrahepatic biliary epithelium in rats.
        Gastroenterology. 2000; 119: 1681-1691
        • Alvaro D.
        • Macarri G.
        • Mancino M.G.
        • Marzioni M.
        • Bragazzi M.
        • Onori P.
        • et al.
        Serum and biliary insulin-like growth factor I and vascular endothelial growth factor in determining the cause of obstructive cholestasis.
        Ann Intern Med. 2007; 147: 451-459
        • Onori P.
        • Alvaro D.
        • Floreani A.R.
        • Mancino M.G.
        • Franchitto A.
        • Guido M.
        • et al.
        Activation of the IGF1 system characterizes cholangiocyte survival during progression of primary biliary cirrhosis.
        J Histochem Cytochem. 2007; 55: 327-334
        • Park J.
        • Gores G.J.
        • Patel T.
        Lipopolysaccharide induces cholangiocyte proliferation via an interleukin-6-mediated activation of p44/p42 mitogen-activated protein kinase.
        Hepatology. 1999; 29: 1037-1043
        • Fabris L.
        • Strazzabosco M.
        • Crosby H.A.
        • Ballardini G.
        • Hubscher S.G.
        • Kelly D.A.
        • et al.
        Characterization and isolation of ductular cells coexpressing neural cell adhesion molecule and Bcl-2 from primary cholangiopathies and ductal plate malformations.
        Am J Pathol. 2000; 156: 1599-1612
        • Sell S.
        Is there a liver stem cell?.
        Cancer Res. 1990; 50: 3811-3815
        • Sell S.
        Liver stem cells.
        Mod Pathol. 1994; 7: 105-112
        • Crosby H.A.
        • Hubscher S.
        • Fabris L.
        • Joplin R.
        • Sell S.
        • Kelly D.
        • et al.
        Immunolocalization of putative human liver progenitor cells in livers from patients with end-stage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6.
        Am J Pathol. 1998; 152: 771-779
        • Roskams T.
        • Theise N.D.
        • Balabaud C.
        • Bhagat G.
        • Bhathal P.S.
        • Bioulac-Sage P.
        • et al.
        Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.
        Hepatology. 2004; 39: 1739-1745
        • Fausto N.
        • Campbell J.S.
        • Riehle K.J.
        Liver regeneration.
        Hepatology. 2006; 43: S45-S53
        • Strazzabosco M.
        • Spirli C.
        • Okolicsanyi L.
        Pathophysiology of the intrahepatic biliary epithelium.
        J Gastroenterol Hepatol. 2000; 15: 244-253
        • Dünker N.
        • Krieglstein K.
        Tgfbeta2 −/− Tgfbeta3 −/− double knockout mice display severe midline fusion defects and early embryonic lethality.
        Anat Embryol (Berl). 2002; 206: 73-83
        • Surveyor G.A.
        • Brigstock D.R.
        Immunohistochemical localization of connective tissue growth factor (CTGF) in the mouse embryo between days 7.5 and 14.5 of gestation.
        Growth Factors. 1999; 17: 115-124
        • Coulomb-L’Hermin A.
        • Amara A.
        • Schiff C.
        • Durand-Gasselin I.
        • Foussat A.
        • Delaunay T.
        • et al.
        Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells.
        Proc Natl Acad Sci USA. 1999; 96: 8585-8590
        • Jia C.
        Advances in the regulation of liver regeneration.
        Expert Rev Gastroenterol Hepatol. 2011; 5: 105-121
        • LeSage G.
        • Alvaro D.
        • Benedetti A.
        • Glaser S.
        • Marucci L.
        • Baiocchi L.
        • et al.
        Cholinergic system modulates growth, apoptosis, and secretion of cholangiocytes from bile duct-ligated rats.
        Gastroenterology. 1999; 117: 191-199
        • Marzioni M.
        • Glaser S.
        • Francis H.
        • Marucci L.
        • Benedetti A.
        • Alvaro D.
        • et al.
        Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin.
        Gastroenterology. 2005; 128: 121-137
        • Gigliozzi A.
        • Alpini G.
        • Baroni G.S.
        • Marucci L.
        • Metalli V.D.
        • Glaser S.S.
        • et al.
        Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis.
        Gastroenterology. 2004; 127: 1198-1209
        • Yasoshima M.
        • Kono N.
        • Sugawara H.
        • Katayanagi K.
        • Harada K.
        • Nakanuma Y.
        Increased expression of interleukin-6 and tumour necrosis factor-alpha in pathologic biliary epithelial cells: in situ and culture study.
        Lab Invest. 1998; 78: 89-100
        • Jakubowski A.
        • Ambrose C.
        • Parr M.
        • Lincecum J.M.
        • Wang M.Z.
        • Zheng T.S.
        • et al.
        TWEAK induces liver progenitor cell proliferation.
        J Clin Invest. 2005; 115: 2330-2340
        • Napoli J.
        • Prentice D.
        • Niinami C.
        • Bishop G.A.
        • Desmond P.
        • McCaughan G.W.
        Sequential increases in the intrahepatic expression of epidermal growth factor, basic fibroblast growth factor, and transforming growth factor beta in a bile duct ligated rat model of cirrhosis.
        Hepatology. 1997; 26: 624-633
        • Gouw A.S.
        • van den Heuvel M.C.
        • Boot M.
        • Slooff M.J.
        • Poppema S.
        • de Jong K.P.
        Dynamics of the vascular profile of the finer branches of the biliary tree in normal and diseased human livers.
        J Hepatol. 2006; 45: 393-400
        • Omenetti A.
        • Diehl A.M.
        The adventures of sonic hedgehog in development and repair: II. Sonic hedgehog and liver development, inflammation, and cancer.
        Am J Physiol Gastrointest Liver Physiol. 2008; 294: G595-G598
        • Sackett S.D.
        • Gao Y.
        • Shin S.
        • Esterson Y.B.
        • Tsingalia A.
        • Hurtt R.S.
        • et al.
        Foxl1 promotes liver repair following cholestatic injury in mice.
        Lab Invest. 2009; 89: 1387-1396
        • Fabris L.
        • Strazzabosco M.
        Epithelial–mesenchymal interactions in biliary diseases.
        Semin Liver Dis. 2011; 31: 11-32
        • Spee B.
        • Carpino G.
        • Schotanus B.A.
        • Katoonizadeh A.
        • Vander Borght S.
        • Gaudio E.
        • et al.
        Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling.
        Gut. 2010; 59: 247-257
        • Apte U.
        • Thompson M.D.
        • Cui S.
        • Liu B.
        • Cieply B.
        • Monga S.P.
        Wnt/beta-catenin signaling mediates oval cell response in rodents.
        Hepatology. 2008; 47: 288-295
        • Fiorotto R.
        • Spirli C.
        • Scirpo R.
        • Fabris L.
        • Huppert S.
        • Torsello B.
        • et al.
        Defective progenitor cells activation and biliary tubule formation in liver conditional RBP-jk-knock out mice exposed to cholestatic injuries reveals a key role for Notch signaling in liver repair.
        Hepatology. 2010; 52: 406A
        • Weatherbee S.D.
        • Niswander L.A.
        • Anderson K.V.
        A mouse model for Meckel syndrome reveals Mks1 is required for ciliogenesis and Hedgehog signaling.
        Hum Mol Genet. 2009; 18: 4565-4575
        • Choi S.S.
        • Omenetti A.
        • Syn W.K.
        • Diehl A.M.
        The role of Hedgehog signaling in fibrogenic liver repair.
        Int J Biochem Cell Biol. 2011; 43: 238-244
        • Omenetti A.
        • Bass L.M.
        • Anders R.A.
        • Clemente M.G.
        • Francis H.
        • Guy C.D.
        • et al.
        Hedgehog activity, epithelial–mesenchymal transitions, and biliary dysmorphogenesis in biliary atresia.
        Hepatology. 2011; 53: 1246-1258
        • Jørgensen M.J.
        The ductal plate malformation.
        Acta Pathol Microbiol Scand Suppl. 1977; 257: 1-87
        • Redston M.S.
        • Wanless I.R.
        The hepatic von Meyenburg complex: prevalence and association with hepatic and renal cysts among 2843 autopsies.
        Mod Pathol. 1996; 9: 233-237
        • Tsui W.M.
        How many types of biliary hamartomas and adenomas are there?.
        Adv Anat Pathol. 1998; 5: 16-20
        • Jain D.
        • Sarode V.R.
        • Abdul-Karim F.W.
        • Homer R.
        • Robert M.E.
        Evidence for the neoplastic transformation of Von Meyenburg complexes.
        Am J Surg Pathol. 2000; 24: 1131-1139
        • Song J.S.
        • Lee Y.J.
        • Kim K.W.
        • Huh J.
        • Jang S.J.
        • Yu E.
        Cholangiocarcinoma arising in Von Meyenburg complexes: report of four cases.
        Pathol Int. 2008; 58: 503-512
        • Xu A.M.
        • Xian Z.H.
        • Zhang S.H.
        • Chen X.F.
        Intrahepatic cholangiocarcinoma arising in multiple bile duct hamartomas: report of two cases and review of the literature.
        Eur J Gastroenterol Hepatol. 2009; 21: 580-584
        • Ward C.J.
        • Hogan M.C.
        • Rossetti S.
        • Walker D.
        • Sneddon T.
        • Wang X.
        • et al.
        The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.
        Nat Genet. 2002; 30: 259-269
        • Zhang M.Z.
        • Mai W.
        • Li C.
        • Cho S.Y.
        • Hao C.
        • Moeckel G.
        • et al.
        PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells.
        Proc Natl Acad Sci USA. 2004; 101: 2311-2316
        • Harris P.C.
        • Torres V.E.
        Polycystic kidney disease.
        Annu Rev Med. 2009; 60: 321-337
        • Banales J.M.
        • Masyuk T.V.
        • Gradilone S.A.
        • Masyuk A.I.
        • Medina J.F.
        • LaRusso N.F.
        The cAMP effectors Epac and protein kinase a (PKA) are involved in the hepatic cystogenesis of an animal model of autosomal recessive polycystic kidney disease (ARPKD).
        Hepatology. 2009; 49: 160-174
        • Mai W.
        • Chen D.
        • Ding T.
        • Kim I.
        • Park S.
        • Cho S.Y.
        • et al.
        Inhibition of Pkhd1 impairs tubulomorphogenesis of cultured IMCD cells.
        Mol Biol Cell. 2005; 16: 4398-4409
        • Tahvanainen E.
        • Tahvanainen P.
        • Kääriäinen H.
        • Höckerstedt K.
        Polycystic liver and kidney diseases.
        Ann Med. 2005; 37: 546-555
        • Igarashi P.
        • Somlo S.
        Genetics and pathogenesis of polycystic kidney disease.
        J Am Soc Nephrol. 2002; 13: 2384-2398
        • Wilson P.D.
        Polycystic kidney disease: new understanding in the pathogenesis.
        Int J Biochem Cell Biol. 2004; 36: 1868-1873
        • Spirli C.
        • Okolicsanyi S.
        • Fiorotto R.
        • Fabris L.
        • Cadamuro M.
        • Lecchi S.
        • et al.
        ERK1/2-dependent vascular endothelial growth factor signaling sustains cyst growth in polycystin-2 defective mice.
        Gastroenterology. 2010; 138: 360-371
        • Nichols M.T.
        • Gidey E.
        • Matzakos T.
        • Dahl R.
        • Stiegmann G.
        • Shah R.J.
        • et al.
        Secretion of cytokines and growth factors into autosomal dominant polycystic kidney disease liver cyst fluid.
        Hepatology. 2004; 40: 836-846
        • Amura C.R.
        • Brodsky K.S.
        • Gitomer B.
        • McFann K.
        • Lazennec G.
        • Nichols M.T.
        • et al.
        CXCR2 agonists in ADPKD liver cyst fluids promote cell proliferation.
        Am J Physiol Cell Physiol. 2008; 294: C786-C796
        • Strazzabosco M.
        • Somlo S.
        Polycystic liver diseases: congenital disorders of cholangiocyte signaling.
        Gastroenterology. 2011; 140: 1855-1859
        • Drenth J.P.
        • te Morsche R.H.
        • Smink R.
        • Bonifacino J.S.
        • Jansen J.B.
        Germline mutations in PRKCSH are associated with autosomal dominant polycystic liver disease.
        Nat Genet. 2003; 33: 345-347
        • Davila S.
        • Furu L.
        • Gharavi A.G.
        • Tian X.
        • Onoe T.
        • Qian Q.
        • et al.
        Mutations in SEC63 cause autosomal dominant polycystic liver disease.
        Nat Genet. 2004; 36: 575-577
        • Janssen M.J.
        • Waanders E.
        • Woudenberg J.
        • Lefeber D.J.
        • Drenth J.P.
        Congenital disorders of glycosylation in hepatology: the example of polycystic liver disease.
        J Hepatol. 2010; 52: 432-440
        • Condac E.
        • Silasi-Mansat R.
        • Kosanke S.
        • Schoeb T.
        • Towner R.
        • Lupu F.
        • et al.
        Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis.
        Proc Natl Acad Sci USA. 2007; 104: 9416-9421
        • Krantz I.D.
        • Piccoli D.A.
        • Spinner N.B.
        Clinical and molecular genetics of Alagille syndrome.
        Curr Opin Pediatr. 1999; 11: 558-564
        • Piccoli D.A.
        • Spinner N.B.
        Alagille syndrome and the Jagged1 gene.
        Semin Liver Dis. 2001; 21: 525-534
        • Emerick K.M.
        • Rand E.B.
        • Goldmuntz E.
        • Krantz I.D.
        • Spinner N.B.
        • Piccoli D.A.
        Features of Alagille syndrome in 92 patients: frequency and relation to prognosis.
        Hepatology. 1999; 29: 822-829
        • Oda T.
        • Elkahloun A.G.
        • Pike B.L.
        • Okajima K.
        • Krantz I.D.
        • Genin A.
        • et al.
        Mutations in the human Jagged1 gene are responsible for Alagille syndrome.
        Nat Genet. 1997; 16: 235-242
        • Li L.
        • Krantz I.D.
        • Deng Y.
        • Genin A.
        • Banta A.B.
        • Collins C.C.
        • et al.
        Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.
        Nat Genet. 1997; 16: 243-251
        • McDaniell R.
        • Warthen D.M.
        • Sanchez-Lara P.A.
        • Pai A.
        • Krantz I.D.
        • Piccoli D.A.
        • et al.
        NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway.
        Am J Hum Genet. 2006; 79: 169-173
        • Karhunen P.J.
        Adult polycystic liver disease and biliary microhamartomas (von Meyenburg’s complexes).
        Acta Pathol Microbiol Immunol Scand A. 1986; 94: 397-400
        • Grimm P.C.
        • Crocker J.F.
        • Malatjalian D.A.
        • Ogborn M.R.
        The microanatomy of the intrahepatic bile duct in polycystic disease: comparison of the cpk mouse and human.
        J Exp Pathol. 1990; 71: 119-131
        • Ramos A.
        • Torres V.E.
        • Holley K.E.
        • Offord K.P.
        • Rakela J.
        • Ludwig J.
        The liver in autosomal dominant polycystic kidney disease. Implications for pathogenesis.
        Arch Pathol Lab Med. 1990; 114: 180-184