As mentioned above, the extrahepatic biliary tree is derived from the caudal part of the hepatic diverticulum as the latter elongates and widens. This portion of the endoderm is closely associated with the ventral pancreatic bud, and gene expression maps have identified a number of transcription factors that are common to the epithelial cells lining the developing ducts and to the pancreas and duodenum (Pdx-1, Prox-1, HNF-6). Mice deficient in these factors exhibit abnormalities of the biliary tree and gallbladder with abnormal differentiation toward a pancreatic phenotype (Lemaigre 2009; Strazzabosco and Fabris 2012). The morphogenesis of the biliary tree depends on close interaction with the surrounding mesenchyme. By the 4th week post-fertilization, the cystic duct and gallbladder are visible as a bud on the hepatic diverticulum (Fig. 9.1a). By the 5th week, the gallbladder, cystic duct, hepatic ducts, common bile duct, and pancreatic duct are well demarcated. The gallbladder elongates but remains tubular until the 11th week when it dilates into a saccular shape (Tan and Vijayan 2001). The distal common bile duct enters the duodenum where it is joined by the duct from the ventral pancreas. This junction initially lies outside the duodenum until the 8th week of gestation at which time it migrates into the duodenal wall. Failure of this process may result in an abnormally long choledocho-pancreatic channel associated with choledochal cyst formation. The major bile ducts at the porta hepatis are fully formed by the 11th week of gestation. Initially believed to be a solid cord of cells, more recent studies, such as those by Tan (Adeva et al. 2006), suggest that the anlage of the extrahepatic biliary tree has a continuous patent lumen, which is visible, according to these authors, as early as 29 days post-fertilization. The most cranial portions of the hepatic ducts would appear to be in direct continuity with converging ductules at the hepatic hilum, which in turn are continuous with the ductal plate and the developing intrahepatic ducts (Nakanuma et al. 1997). These observations would preclude the possibility that biliary atresia results either from a failure of “canalization” or from nonunion of separately developing cranial and caudal portions of the biliary tree. Although much less is known about the molecular mechanisms responsible for the development of the extrahepatic than the intrahepatic biliary tree, murine models deficient in Pdx-1 or Hes-1 (a Notch-dependent transcription factor), HNF6, HNF-1b, or Foxf1 (a transcription factor target for the sonic Hedgehog signaling) have an altered development of the gallbladder and of the common bile duct (Strazzabosco and Fabris 2012).

The development of the intrahepatic bile ducts (IHBDs) begins between the 5th and 9th week post-fertilization. The biliary epithelium is believed to arise from precursor bipotential hepatoblasts which can differentiate into either hepatocytes or biliary epithelial cells, also referred to as cholangiocytes (Spagnoli et al. 1998). Beginning around the 6th week, the hepatoblasts adjacent to the developing portal tract mesenchyme flatten and become a continuous layer of biliary-type cuboidal cells, the ductal plate (Fig. 9.1b). At the hilum, the ductal plate is in direct continuity with the developing hepatic duct, and its remodeling leads to the formation of the intrahepatic portion of the right and left branches of the hepatic duct (Tan, 1994 #2649). The differentiation of the hepatoblasts into cells destined for the biliary tree and cells destined for the hepatic parenchyma appears to be determined at the time of ductal plate formation. Immunohistochemical studies have illustrated the development of the intrahepatic bile ducts, exploiting the differences in cytokeratin (CK) subtype expression between hepatocytes and biliary epithelial cells: precursor hepatoblasts express CK 8, 18, and 19; mature hepatocytes express CK 8 and 18 but not 19, whereas biliary epithelium expresses CK 8,18, 19, and 20. CK7, in the authors’ experience, is not as robustly expressed as CK19 in biliary epithelium until the third trimester. Around weeks 6–7 a prominent rim of CK19+ cells forms along the outer boundary of the developing portal tracts, forming the so-called ductal plate (Desmet 1998; Van Eyken et al. 1988). During the next few weeks, the rim becomes a continuous double-layered saccular sleeve around each portal tract (Fig. 9.4). Remodeling of the ductal plate, starting around week 12, is a dynamic process weaving together loss of continuity of the epithelial sleeve with the formation of discrete tubular spaces, the incorporation of the primitive tubules more centrally into the portal tract mesenchyme, and the elimination of excess epithelial elements. This tubulogenesis is transiently asymmetrical, driven by cholangiocyte-like cells on the portal tract side, and by hepatocyte-like cells on the parenchymal side of the ductal plate (Strazzabosco and Fabris 2012). The ductal structures gradually assume a symmetrical arrangement as the hepatoblasts are replaced by cholangiocytes. This process is believed to proceed from the hepatic hilum and extend centrifugally toward the periphery of the liver, resulting in a continuous development of IHBDs throughout fetal life, and appears to continue into the first month of life ex utero (Crawford 2002). Thus, in later stages, histologic examination of the fetal liver reveals more mature ducts in the hilar region, while the ducts in the periphery are still in ductal plate configuration (Desmet 1998) (Fig. 9.5). The development of the intrahepatic biliary tree is under the control of numerous transcription factors, including HNF6 and HNF1β (Strazzabosco and Fabris 2012). In addition, Notch-2 and JAG1 appear to also play key roles in biliary development, exemplified by paucity of bile ducts seen in Alagille syndrome resulting from mutations in JAG1. Studies in mice have shown that Jagged-1 is expressed by portal mesenchymal cells and interacts with Notch-2 expressed in hepatoblasts at the ductal plate. Inactivation of Jagged-1 in mesenchymal cells results in defective maturation of bile ducts in mice (Hofmann et al. 2010). Studies using resin casts in mouse models have shown that the three-dimensional density of the intrahepatic biliary tree appears to be regulated by Notch gene dosage during development (Sparks et al. 2010).

The intrahepatic biliary system is not mature at the time of birth, with expression of canalicular transporters present by mid-gestation, but with a different immmunohistochemical pattern than in adults (Chen et al. 2005). Development of the intrahepatic bile ducts also continues after birth, and remodeling of the ductal plate in the most peripheral portal tracts is probably not complete until 6 weeks after birth (Van Eyken et al. 1988); this has implications for the diagnosis of bile duct paucity on a liver biopsy in a premature infant. The adult liver, weighing approximately 1,400 g, is more than 10 times the weight of the newborn liver, but information on the development and growth of the liver after birth is scanty. Landing and Wells (1991) proposed that the postnatal human liver grows by an increase in the number of peripheral lobules with associated branching and elongation of the portal tracts. The intrahepatic tree can be divided into large bile ducts (300–800 μm in humans), which include hepatic, segmental, and area ducts, and small bile ducts (<300 μm), which include septal bile ducts (100–300 μm), interlobular bile ducts (15–100 μm), and bile ductules (diameter <15 μm) (Strazzabosco and Fabris 2008). The number of orders of intrahepatic branches in the fetus or newborn is not yet known (Crawford 2002), while according to Ludwig (Beuers and Sackmann 1998), 10–12 orders of branches are present in the adult liver. The large ducts have associated mucous peribiliary glands and can be grossly identified, while the small ducts are not associated with peribiliary glands and are identifiable only by microscopy. The interlobular (or terminal) bile ducts branch into bile ductules, which seem to have both an intraportal and periportal segment and are entirely lined by cholangiocytes and invested by a thin layer of mesenchyme. These ductules collect bile from the canals of Hering, which are lined in part by hepatocytes and in part by cholangiocytes and represent the link between the hepatocyte canaliculi and the biliary tree. Canals of Hering appear as small clusters of cells positively stained by cholangiocyte immunomarkers (cytokeratin 7 or 19), located at the periphery of the portal tracts and periportal region (Roskams et al. 2004) (Fig. 9.6). Canals of Hering and bile ductules appear to house the progenitor cells that will proliferate and result in the ductular reaction of chronic liver disease. Cholangiocytes possess primary cilia, which play an important role in regulating their biological activities. Cholangiocyte cilia also contain proteins such as polycystin and fibrocystin, defects of which are involved in biliary dysgenesis (Strazzabosco and Fabris 2008). Cholangiocytes exhibit functional heterogeneity depending on their location in the biliary tree: those in the larger bile ducts play a role in bile production via both absorptive and secretory functions, whereas cholangiocytes in the smaller branches, such as bile ductules and canals of Hering, have the ability to participate in inflammatory processes and to act as progenitor cells (Strazzabosco and Fabris 2008).

The peribiliary glands are histologically divided into intramural and extramural structures. The former consist of simple tubular glands with much mucin and are sparsely and irregularly distributed within the ductal wall. The latter are characterized by the presence of excretory units that consist of seromucinous acini and a conducting system in the periductal tissue. Pancreatic exocrine acini are occasionally admixed with extramural glands. These peribiliary glands appear in the late fetal period and complete their development about 15 years after birth along with the peribiliary capillary plexus. Extramural and intramural glands secrete neutral and acid mucin into the ductal lumen. Extramural glands contain several enzymes for digestion of protein and lipids. Neural and vascular supply of these glands may be related to the regulation of their secretion. Specific and nonspecific immune responses within this glandular system may also be essential in the sterility of bile (Nakanuma et al. 1994).