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

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

Coordinated development of the mouse extrahepatic bile duct: Implications for neonatal susceptibility to biliary injury.

BACKGROUND & AIMS: The extrahepatic bile duct is the primary tissue initially affected by biliary atresia. Biliary atresia is a cholangiopathy which exclusively affects neonates. Current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts. METHODS: We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes, as well as isolated extrahepatic bile ducts, and a collagen reporter mouse. The methods used included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix. RESULTS: The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling of the matrix suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. CONCLUSION: We identified 4 key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis. LAY SUMMARY: Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction, resulting in liver injury. We identify 4 key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.

Distal bile duct carcinomas and pancreatic ductal adenocarcinomas: postulating a common tumor entity.

The set definition of distal cholangiocarcinomas and adenocarcinomas of the pancreatic head is challenged by their close anatomical relation, similar growth pattern, and corresponding therapeutic outcome. They show a mutual development during embryologic organ formation and share phenotypic characteristics. This review will highlight the similarities with regard to the common origin of their primary organs, histopathological similarities, and modern clinical management. Thus, we propose to subsume those entities under a common superfamily.

Development of extrahepatic bile duct excluding gall bladder in human fetuses: histological, histochemical, and immunohistochemical analysis.

BACKGROUND: The fetal development of extrahepatic bile ducts (EBD) is unkown. MATERIALS AND METHODS: Development of EBD was examined by immunohistochemistry in 16 fetuses of 7-40 gestational week (GW). Gall bladder (GB) was not investigated. RESULTS: At seven GW, a hepato-pancreatic bud (HPB) was seen near the hepatic hilus. At eight GW, embryonic EBD, GB and pacreas developed from HPB. Portal veins (PV) and hepatic arteries (HAs) were present in EBD at eight GW. Liver parenchyma was already present in seven GW. At eight GW, EBD at porta hepatis (PH) was already established; PH EBD was derived from ductal plate (DP). The distal and middle EBD gradually develeped and took shape of EBD at nine GW. In PH, cystic and hepatic ducts developed from DP at eight GW. EBD developed further, accompanying many nerve fibers (NF) at PH and distal and middle EBD. Apparent PV and HA were seen around 12 GW. Around 20 GW, HA and capillaries proliferated, giving rise to peribiliary capillary plexus (PCP) in all parts of EBD. EBD grew gradually further, and around 30 GW extrahepatic peribiliary glands (EPG) emerged from EBD but not from cystic duct. Around 36 GW, exocrine pancreatic acinar cells emerged from remodeled DP at PH. At term (40 GW), EBD was established but was as yet immature. Numerous NF were present around EBD. Histochemically, EBD epithelium had no mucins at 7-12 GW but contained neutral and acidic mucins at 23-40 GW. EPG had abundant neutral and acidic mucins. Immunohistochemically, alpha-fetoprotein (AFP) was consistently positive in the epithelial and mesenychyma. The NF and muscles of HPB present at seven GW were positive for neural cell adhesion molecule (NCAM), neuron-specific enolase (NSE), platelet-derived growth factor receptor-α (PDGFRA), and KIT, but they disappeared in nine GW. Expressions of cytokeratin (CK) seven and CK19 in EBD and EPG were slight or none, while expression of CK8 was moderate, and that of CK18 was strong. NF were positive for NCAM, NSE, synaptophysin, and chromogranin, and PDGFRA. MUC1 and MUC6 apomucins were noted in EBD and EPG. EPG contained numerous endocrine cells positive for chromogranin, synaptophysin, NCAM and NSE. A few endocrine cells positive for these antigens were seen in EBD. Numeous KIT-positive stem cells (SC) were seen in PH, EBD, PV, HA, PCP, and EPG. NCAM-positive and bcl-2-positive SC were also located in these structures. Epithelial cells of EBD and EPG showed expressions of MET, PDGFRA, CA19-9, MUC1, MUC2, MUC6, KIT, bcl-2, and ErbB2. No expressions of HepPar1, carcinoembryonic antigen (CEA), and epithelial membrane antigen (EMA) were noted. CONCLUSIONS: Although the findings have limitatios because this study of humans are descriptive one, the present data suggest that the processes of the development and differentiation of EBD system may be associated with EBD SC, CK prolifes, SFC/KIT signaling, HGF/MET signaling, PDGRa/PDGFRA signaling, fibroblast growth factor/ErbB2 signaling, neuroendocrine lineage, NF differentiation, pancreatic aninar cell differentiation, PCP differentiation, MUC apomucins differentiation, and expressions of AFP and CA19-9. HepPar1, EMA and CEA were not involved in them.

Molecular mechanisms of bile duct development.

The mammalian biliary system, consisting of the intrahepatic and extrahepatic bile ducts, is responsible for transporting bile from the liver to the intestine. Bile duct dysfunction, as is seen in some congenital biliary diseases such as Alagille syndrome and biliary atresia, can lead to the accumulation of bile in the liver, preventing the excretion of detoxification products and ultimately leading to liver damage. Bile duct formation requires coordinated cell-cell interactions, resulting in the regulation of cell differentiation and morphogenesis. Multiple signaling molecules and transcription factors have been identified as important regulators of bile duct development. This review summarizes recent progress in the field. Insights gained from studies of the molecular mechanisms of bile duct development have the potential to reveal novel mechanisms of differentiation and morphogenesis in addition to potential targets for therapy of bile duct disorders.

Multipotent stem cells in the biliary tree.

The biliary tree system consists of two divisions: intrahepatic bile ducts and extrahepatic bile ducts. The development of the biliary tree, and secondarily the liver, shares a common origin with ventral pancreas. A common progenitor for liver, biliary duct system, and ventral pancreas exists at early stages of development, when the anterior definitive endoderm is forming the foregut. Several studies indicate that the biliary tree contains stem cell compartments for liver, pancreas and the bile duct system and persisting into adulthood. These stem cell compartments are present in the peribiliary glands and possibly give rise to committed progenitors in gallbladder that does not have peribiliary glands. The biliary tree stem/progenitors represent a new source of cells that can be used as tools for regenerative medicine of liver, bile duct and pancreas.

Tbx3 promotes liver bud expansion during mouse development by suppression of cholangiocyte differentiation.

UNLABELLED: After specification of the hepatic endoderm, mammalian liver organogenesis progresses through a series of morphological stages that culminate in the migration of hepatocytes into the underlying mesenchyme to populate the hepatic lobes. Here, we show that in the mouse the transcriptional repressor Tbx3, a member of the T-box protein family, is required for the transition from a hepatic diverticulum with a pseudo-stratified epithelium to a cell-emergent liver bud. In Tbx3-deficient embryos, proliferation in the hepatic epithelium is severely reduced, hepatoblasts fail to delaminate, and cholangiocyte rather than hepatocyte differentiation occurs. Molecular analyses suggest that the primary function of Tbx3 is to maintain expression of hepatocyte transcription factors, including hepatic nuclear factor 4a (Hnf4a) and CCAAT/enhancer binding protein (C/EBP), alpha (Cebpa), and to repress expression of cholangiocyte transcription factors such as Onecut1 (Hnf6) and Hnf1b. CONCLUSION: Tbx3 controls liver bud expansion by suppressing cholangiocyte and favoring hepatocyte differentiation in the liver bud.

Embryology of extra- and intrahepatic bile ducts, the ductal plate.

In the human embryo, the first anlage of the bile ducts and the liver is the hepatic diverticulum or liver bud. For up to 8 weeks of gestation, the extrahepatic biliary tree develops through lengthening of the caudal part of the hepatic diverticulum. This structure is patent from the beginning and remains patent and in continuity with the developing liver at all stages. The hepatic duct (ductus hepaticus) develops from the cranial part (pars hepatica) of the hepatic diverticulum. The distal portions of the right and left hepatic ducts develop from the extrahepatic ducts and are clearly defined tubular structures by 12 weeks of gestation. The proximal portions of the main hilar ducts derive from the first intrahepatic ductal plates. The extrahepatic bile ducts and the developing intrahepatic biliary tree maintain luminal continuity from the very start of organogenesis throughout further development, contradicting a previous study in the mouse suggesting that the extrahepatic bile duct system develops independently from the intrahepatic biliary tree and that the systems are initially discontinuous but join up later. The normal development of intrahepatic bile ducts requires finely timed and precisely tuned epithelial-mesenchymal interactions, which proceed from the hilum of the liver toward its periphery along the branches of the developing portal vein. Lack of remodeling of the ductal plate results in the persistence of an excess of embryonic bile duct structures remaining in their primitive ductal plate configuration. This abnormality has been termed the ductal plate malformation.

The homeobox gene Hhex is essential for proper hepatoblast differentiation and bile duct morphogenesis.

Hhex is required for early development of the liver. A null mutation of Hhex results in a failure to form the liver bud and embryonic lethality. Therefore, Hhex null mice are not informative as to whether this gene is required during later stages of hepatobiliary morphogenesis. To address this question, we derived Hhex conditional null mice using the Cre-loxP system and two different Cre transgenics (Foxa3-Cre and Alfp-Cre). Deletion of Hhex in the hepatic diverticulum (Foxa3-Cre;Hhex(d2,3/-)) led to embryonic lethality and resulted in a small and cystic liver with loss of Hnf4alpha and Hnf6 expression in early hepatoblasts. In addition, the gall bladder was absent and the extrahepatic bile duct could not be identified. Loss of Hhex in the embryonic liver (Alfp-Cre;Hhex(d2,3/-)) caused irregular development of intrahepatic bile ducts and an absence of Hnf1beta in many (cystic) biliary epithelial cells, which resulted in a slow, progressive form of polycystic liver disease in adult mice. Thus, we have shown that Hhex is required during multiple stages of hepatobiliary development. The altered expression of Hnf4alpha, Hnf6 and Hnf1beta in Hhex conditional null mice suggests that Hhex is an essential component of the genetic networks regulating hepatoblast differentiation and intrahepatic bile duct morphogenesis.

Pathogenesis of extrahepatic bile duct atresia (EHBA): comprehension from a surgical point of view.

INTRODUCTION: The aim of the study is to establish a complete comprehension of the pathogenesis of Biliary Atresia, and to explain both the variable and redundant pathomorphological, as well as, histological findings. MATERIALS AND METHODS: The pathomorphological and histological findings in 223 patients with histologically evident EHBA were recorded retrospectively (72 patients) or prospectively (151 patients), according to a projected ascending study. These findings were compared with histological findings in human and rat embryos. RESULTS: 1) The pathomorphological findings recorded in patients with EHBA were also found in stages of normal embryogenesis of the bile duct system in human and rat embryos. 2) Each histological finding in Biliary Atresia corresponds to a finding in an interrupted stage of the normal development in human and rat embryos. 3) The findings in patients and embryos can be explained completely by a disturbed intrinsic epithelium/mesoderm interaction. 4) Some findings in Biliary Atresia cannot be explained easily by the assumption of an extrinsic factor. CONCLUSION: There is no finding in Biliary Atresia which cannot be completely explained as the result of an intrinsic developmental error, probably due to disturbances or interruption of epithelium/mesoderm interaction during embryogenesis.

Immunolocalization of extracellular matrix components and integrins during mouse liver development.

Intrahepatic biliary cell differentiation takes place in periportal hepatoblasts under the influence of the subjacent connective tissue, the mechanism of which is still unclear. This study was undertaken to analyze the immunolocalization of extracellular matrix components and their cellular receptors during mouse liver development, with special attention given to biliary differentiation and vascular development. In young fetal mouse liver, primitive structures of sinusoids were developed between hepatic cords associated with hematopoietic cells demonstrated by immunohistochemistry of basal laminar components, the alpha6 integrin subunit, and PECAM-1. Portal veins and hepatic veins showed different staining intensities of alpha2, alpha3, and alpha6 integrin subunits from early stages of development. Anti-beta4 integrin subunit antibodies reacted with portal veins, but not with hepatic veins after perinatal stages. Their different phenotypes may be related to the preferential differentiation of periportal bile ducts. In intrahepatic bile duct development, periportal hepatoblasts adjacent to the connective tissue were immunostained for each basal laminar component on the basal side at almost the same time; alpha3, alpha5, alpha6, and beta4 integrin subunits were immunohistochemically detectable later than the basal laminar components. These staining patterns of intrahepatic bile duct cells clearly differed from those of extrahepatic bile duct cells from the beginning of their development, suggesting that these ducts are of different origins. In conclusion, the vascular structures, including sinusoids, portal veins, and hepatic veins, develop from early stages of liver development, and the extracellular matrix components may play important roles in biliary differentiation and vascular development. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

The onecut transcription factor HNF6 is required for normal development of the biliary tract.

During liver development, hepatoblasts differentiate into hepatocytes or biliary epithelial cells (BEC). The BEC delineate the intrahepatic and extrahepatic bile ducts, and the gallbladder. The transcription factors that control the development of the biliary tract are unknown. Previous work has shown that the onecut transcription factor HNF6 is expressed in hepatoblasts and in the gallbladder primordium. We now show that HNF6 is also expressed in the BEC of the developing intrahepatic bile ducts, and investigate its involvement in biliary tract development by analyzing the phenotype of Hnf6(-/-) mice. In these mice, the gallbladder was absent, the extrahepatic bile ducts were abnormal and the development of the intrahepatic bile ducts was perturbed in the prenatal period. The morphology of the intrahepatic bile ducts was identical to that seen in mice whose Hnf1beta gene has been conditionally inactivated in the liver. HNF1beta expression was downregulated in the intrahepatic bile ducts of Hnf6(-/-) mice during development. Furthermore, we found that HNF6 can stimulate the Hnf1beta promoter. We conclude that HNF6 is essential for differentiation and morphogenesis of the biliary tract and that intrahepatic bile duct development is controlled by a HNF6-->HNF1beta cascade.

New clues for the developing human biliary system at the porta hepatis.

The human biliary system is formed from the hepatic diverticulum, a structure which develops from the embryonic foregut in the fourth week of gestation. The cephalic portion of the hepatic diverticulum lies within the septum transversum, and gives rise to entodermal cells which become the primitive hepatocytes. The caudal part of the hepatic diverticulum is molded by mesenchyme to form the gallbladder, cystic duct, and extrahepatic bile duct. The gallbladder is initially tubular in shape, and undergoes morphological changes to become saccular during the 11th week of gestation. The extrahepatic bile duct elongates and widens as gestation progresses, and intramural mucus glands develop. There is no solid stage during the development of the extrahepatic bile duct. The extrahepatic bile duct is a well-defined tubular structure by the 6th week of gestation, whereas the intrahepatic biliary system during this period of gestation is represented by the primitive ductal plate. The ductal plate undergoes structural changes from the 11th week of gestation, beginning at the porta hepatis and progressing through gestation to the periphery of the liver. This remodeling process shapes the ductal plate from a flat sheath of biliary epithelium surrounding the portal vein branches into a network of interconnecting tubular structures. Mesenchyme plays an important role in ductal plate remodeling. The intrahepatic biliary system is in luminal continuity with the extrahepatic bile duct throughout gestation at the porta hepatis. The major bile ducts at the porta hepatis are fully formed by the 16th week of gestation.

Cytokeratin subtypes in biliary atresia: immunohistochemical study.

The etiology of biliary atresia (BA) remains unknown, but ductal-plate malformation and insufficient ductal-plate remodeling have been suggested to play important roles, so it is beneficial to examine the maturation and differentiation of bile ducts in BA. Different epithelial types are characterized by the expression of specific cytokeratin (CK) subtypes. CK can therefore serve as a 'lineage marker' of epithelial cells. CK subtypes have not been previously examined in BA. In this study, we examined the maturation of bile-duct cells in BA (n = 45) using immunohistochemistry of CK subtypes, with mouse monoclonal antibodies to CAM5.2, and CK subtypes 7, 8, 13, 14, 17, 19 and 20. We then compared these findings with pediatric non-BA (n = 11) and fetal (n = 21) liver. We semiquantitatively evaluated the findings using a H score method. In the fetal liver, immunoreactivity for CAM5.2, CK-7, CK-8 and CK-19 was detected in bile-duct cells, and CAM5.2 and CK-8 immunoreactivity was also detected in hepatocytes. The distribution of these CK subtypes was the same in fetal, pediatric non-BA and BA liver. However, CK-7 immunoreactivity was markedly weaker in bile ducts of fetal (H scores: ductal plate 0 +/- 0; remodeling 9.5 +/- 40.3; remodeled 37.3 +/- 60.8) and BA (H score: 200.9 +/- 55.3) liver compared to non-BA liver (H score: 251.1 +/- 33.5). In addition, CK-20 was detected in the bile ducts of the fetal and BA liver, but not in non-BA liver. These findings suggest that the expression patterns of CK subtypes in bile-duct cells in BA are similar to that in developing bile-duct cells, which is indicative of bile-duct cell immaturity.

Induction of bile ducts in embryonic liver by mesenchyme: a new perspective for the treatment of biliary atresia?

Presently only those forms of Extrahepatic Biliary Atresia (EHBA) with minimal or no intrahepatic manifestations can be treated successfully by extensive hepatoportoenterostomy. Intraoperative macro- and microscopic observations show that the typical pathogenetic manifestations in EHBA are most prominent at the porta hepatis. We therefore postulate that EHBA is the result of a defective embryonic development of the porta hepatis. In rat embryos hepatic bile duct formation is initiated at the porta hepatis and in this context mesenchyme from the periportal region seems to play a major inductive role. In order to demonstrate the role of invading periportal mesenchyme for the process of bile duct rudiment formation we established an organ culture model of the embryonic porta hepatis by recombining periportal mesenchyme with peripheral liver fragments from 15 days old rat embryos (Carnegie Stage 21). The degree of mesenchyme invasion as well as the formation of mesenchyme-surrounded liver cell clusters, rosettes or vesicles (bile duct rudiments) were assessed. Mesenchyme from the porta hepatis invaded the peripheral liver fragments and induced the formation of mesenchyme-surrounded liver cell clusters and rosettes with the beginning of lumen formation. Kidney mesenchyme recombined with liver fragments as a mesenchymal alternative showed almost the same effect, lung mesenchyme showed only a very weak inductive effect. To assess the effect of a diffusible factor versus direct cell contact, a millipore filter with and without paraffin coating was interposed between mesenchyme containing tissue and peripheral liver tissue fragments. Without direct cell contact to mesenchyme no hepatoblast cluster or rosette formation could be observed. Comparing this result to the normal development of the liver in rats our investigations suggest that the embryogenesis of the porta hepatis is probably defined by the following two developmental steps: First, differentiation of the intrahepatic bile duct system which is induced by invading mesenchyme originating from the extrahepatic periportal region and realized by epithelium mesenchyme interaction. Second, fusion of extra- and intrahepatic bile duct systems at the level of the later porta hepatis. Disturbances of this complex process can possibly lead to biliary atresia. Further investigations regarding details of the role of the mesenchyme, its inductive factors and the kidney mesenchyme's inductive potential in liver development may provide a new perspective for future treatment of biliary atresia.

Embryology, anatomy, and surgical applications of the extrahepatic biliary system.

As technology has improved and the ability to apply this technology in the surgical arena has grown, surgeons have been able to perform more sophisticated operative procedures. Hepatobiliary surgeons are now able to use laparoscopy, immunosuppressive drugs, and technical advances in cryosurgery to accomplish magnificent results. The success and safety of laparoscopic cholecystectomy, orthotopic liver transplantation, and trisegmentectomy for hepatic tumors depend on a high regard for and an accurate knowledge of the anatomy and some of the common embryologic anomalies of the biliary tree. The blood supply, ductal variations, and gallbladder anatomy of this area are often the source of major challenge to unprepared and unaware surgeons. The authors have attempted to stimulate an interest in, a respect for, and perhaps some desire to learn more about the important and fascinating anatomy of this region.

Developing human biliary system in three dimensions.

BACKGROUND: In the development of the human biliary system, although the extrahepatic bile ducts develop from the embryonic hepatic diverticulum, there is increasing evidence to suggest that the intrahepatic bile ducts originate within the liver from the ductal plate. The ductal plate develops as a sheath of primitive biliary epithelium in the mesenchyme along the portal vein branches. Through an orderly process of selection and deletion, the ductal plate is remodelled into the adult system of anastomosing tubular bile ducts. The ductal plate remodelling process occurs at the porta hepatis between 11 and 13 weeks of gestation and progresses towards the periphery of the liver. METHODS: In this project, for the first time, we have used computerised three-dimensional reconstruction techniques to visualise the developing human biliary system. Paraffin-embedded tissue from eight human embryos or fetuses between 5.5 and 16 weeks of gestation were serially sectioned, and their images were aligned, digitised, and used for three-dimensional reconstruction. RESULTS AND CONCLUSIONS: Three-dimensional images of the extrahepatic and the intrahepatic biliary systems were obtained, and the following conclusions were drawn. (1) The intrahepatic biliary system, both at the porta hepatis and within the liver, developed from the ductal plate through a consistent pattern of remodelling. (2) Prior to the remodelling process, the ductal plate was of similar morphology irrespective of site and gestation. (3) The extrahepatic biliary system was in direct luminal continuity with the developing intrahepatic biliary system throughout gestation and did not show the presence of a "solid stage" in any of the embryos or fetuses studied.

The developing human biliary system at the porta hepatis level between 29 days and 8 weeks of gestation: a way to understanding biliary atresia. Part 1.

The developing biliary system in normal human embryos from 29 days to 8 weeks post-fertilization was studied. The primitive extrahepatic bile duct that originates from the embryonic hepatic foregut diverticulum is in contact with the hepatic anlage from the start of organogenesis and remains so throughout the gestational ages examined. The primitive extrahepatic bile duct maintains continuity with the ductal plate from which intrahepatic bile ducts are eventually formed. Contrary to long-held concepts of biliary development, no 'solid stage' of entodermal occlusion of the common bile duct lumen was found at any stage of gestation in the material investigated. Therefore, biliary atresia is not caused by incomplete vacuolization of the 'solid stage'.

The developing human biliary system at the porta hepatis level between 11 and 25 weeks of gestation: a way to understanding biliary atresia. Part 2.

In biliary atresia, inflammation and destruction of extrahepatic and intrahepatic bile ducts with eventual fibrous obliteration occurs, causing neonatal obstructive jaundice. The onset of the disorder may start antenatally and progress after birth, and the porta hepatis is a constant site of involvement. To date, little is known about the intrauterine development of the bile ducts at the porta hepatis. The present work gives an account of the developmental pattern of bile ducts at the level of the porta hepatis in the normal human fetus from the 11th to the 25th weeks of gestation. It has been observed that the proximal portion of the hilar bile ducts derives from the intrahepatic biliary ductal plate. This occurs following a predictable remodeling sequence by which, from many ductal plate-derived ductules, those destined to become definitive bile ducts are enveloped in a concentric cuff of mesenchyma. Those which are not are deleted. The distal portions of the right and left main hepatic ducts develop from the extrahepatic bile duct. There was no gestational period in which the extrahepatic bile duct and the intrahepatic biliary system were separated. Furthermore, the developing intrahepatic bile ducts maintain luminal continuity with the common bile duct from the start of organogenesis. Biliary atresia may result from: (i) failure to establish a definitive type of bile duct; (ii) leakage of bile from primitive bile ducts resulting in an interstitial inflammatory reaction in the adjacent mesenchyma; and (iii) continuous proliferation of primitive bile ducts at the level of the porta hepatis beyond the 25th week of gestation, as a failed compensatory mechanism.