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.     

Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta

The inactivation of the Hnf1beta gene identified an essential role in epithelial differentiation of the visceral endoderm and resulted in early embryonic death. In the present study, we have specifically inactivated this gene in hepatocytes and bile duct cells using the Cre/loxP system. Mutant animals exhibited severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD). The paucity of small IHBD was linked to a failure in the organization of duct structures during liver organogenesis, suggesting an essential function of Hnf1b in bile duct morphogenesis. Mutant mice also lacked interlobular arteries. As HNF1beta is not expressed in these cells, it further emphasizes the link between arterial and biliary formation. Hepatocyte metabolism was also affected and we identified hepatocyte-specific HNF1beta target genes involved in bile acids sensing and in fatty acid oxidation.     

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established^1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.     

The functions of bile ducts in sheep

Pressure changes in the gallbladder and the bile flow and pressure changes in the common bile duct were determined in sheep. The experiments were conducted on animals with external junction of choleslochus and cholecystostomy performed previously. The experiments demonstrated pressure in the sheep of the functional sphincter of Mirizzi at the boundary between the intrahepatic and extrahepatic bile ducts. A correlation was demonstrated also between the function of this sphincter and that of Oddi's sphincter. The conditions for bile filling of the extrahepatic bile ducts and gallbladder were determined. The process of bile excretion into the duodenum and the role of bile duct sphincters in this process are discussed. Attention is called to the relationship between the pressure in the gallbladder and the tonus of bile duct sphinters.     

Histochemical analyses of hepatic architecture of the hagfish with special attention to periportal biliary structures

The hagfish liver was histochemically examined with special attention to biliary structures around the portal veins. Hepatocytes were organized into tubular structures surrounded by sinusoids. Biliary ductule structures, which resemble the ductal plates transiently appearing in mammalian liver development, were observed around the portal veins, but they did not appear around central veins. Thus, the hagfish liver demonstrates the same basic structure as the mammalian liver; that is, a vascular system from portal to central veins via sinusoids, and portal triad structures consisting of portal veins, hepatic arteries, and intrahepatic bile ducts. The epithelial cells of the ductal platelike structures strongly expressed cytokeratin, had some lectin-binding sites, and were delineated by the basal lamina, which was reactive for periodic acid-Schiff (PAS) staining and Iectin histochemistry. The lumina of the ductal plate-like structures were comparatively small and heterogeneous in diameter around the portal veins, suggesting that the biliary structures may not be efficient for bile secretion. The epithelial cells of the gall bladder had a simple columnar shape and were a PAS-positive cytoplasm. Those of bile ducts near the hilus, including extrahepatic and hepatic ducts, were simple columnar or cuboidal cells, and had large lumina. The cytoplasm in these cells was PAS-positive. These phenotypes with the expression of lectin-binding sites were clearly different from those of the ductal plate-like structures in the liver proper, suggesting that the extrahepatic and intrahepatic biliary structures may have different developmental origins.     

Expression of neural cell adhesion molecule in human liver development and in congenital and acquired liver diseases

In the liver, neural cell adhesion molecule (NCAM) is a marker of immature cells committed to the biliary lineage and is expressed by reactive bile ductules in human liver diseases. We investigated the possible role of NCAM in the development of intrahepatic bile ducts and aimed at determining whether immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases. Therefore, we performed immunohistochemistry for NCAM and bile duct cell markers cytokeratin 7 and cytokeratin 19 on frozen sections of 85 liver specimens taken from 14 fetuses, 10 donor livers, 18 patients with congenital liver diseases characterized by ductal plate malformations (DPMs), and 43 cirrhotic explant livers. Duplicated ductal plates and incorporating bile ducts during development showed a patchy immunoreactivity for NCAM, while DPMs were continuously positive for NCAM. Bile ducts showing complete or patchy immunoreactivity for NCAM were found in cirrhotic livers, with higher frequency in biliary than in posthepatitic cirrhosis. Our results suggest that NCAM may have a function in the development of the intrahepatic bile ducts and that NCAM-positive immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases.     

Histopathology findings in common marmosets (<Emphasis Type="Italic">Callithrix jacchus</Emphasis> Linnaeus, 1758) with chronic weight loss associated with bile tract obstruction by infestation with <Emphasis Type="Italic">Platynosomum</Emphasis> (Loos, 1907)

Chronic weight loss in marmosets is often associated with wasting marmoset syndrome (WMS), an important disease that occurs in callitrichid colonies around the world. Even though its etiology is very difficult to determine, particular variables, such as weight loss, diarrhea and alopecia, associated or not with infestation in the pancreatic ducts with Trichospirura leptossoma (Nematoda: Thelazioidea), seem to be linked with the syndrome. This study investigated the histopathology of the lungs, duodenum, liver, gallbladder, extrahepatic bile ducts and pancreatic ducts of six common marmosets (Callithrix jacchus) suffering from severe non-diarrheic weight loss. Three individuals died naturally and the other three were euthanized. Microscopic findings showed the presence of adult flukes (Platynosomum) in the liver. These flukes, which provoke common infection in cats, were also observed inside the gallbladder as well as in the intra and extrahepatic bile ducts in common marmosets. Portal fibrosis was observed in two animals, which developed chronic fibrosing hepatopathy (biliary pattern, grade 3). The disease progresses without diarrhea and without pancreatic lesions or infestation. With the progression, the animals presented with ascending cholangitis, cholestasis and portal fibrosis, sometimes culminating in secondary biliary cirrhosis. Therefore, this infirmity, associated with chronic weight loss in common marmosets, could be another etiological factor linked with WMS.