Intrahepatic cholangiocyte regeneration from an Fgf-dependent extrahepatic progenitor niche in a zebrafish model of Alagille Syndrome.

BACKGROUND AND AIMS: Alagille Syndrome (ALGS) is a congenital disorder caused by mutations in the Notch ligand gene JAGGED1, leading to neonatal loss of intrahepatic duct (IHD) cells and cholestasis. Cholestasis can resolve in certain patients with ALGS, suggesting regeneration of IHD cells. However, the mechanisms driving IHD cell regeneration following Jagged loss remains unclear. Here, we show that cholestasis due to developmental loss of IHD cells can be consistently phenocopied in zebrafish with compound jagged1b and jagged2b mutations or knockdown. APPROACH AND RESULTS: Leveraging the transience of jagged knockdown in juvenile zebrafish, we find that resumption of Jagged expression leads to robust regeneration of IHD cells through a Notch-dependent mechanism. Combining multiple lineage tracing strategies with whole-liver three-dimensional imaging, we demonstrate that the extrahepatic duct (EHD) is the primary source of multipotent progenitors that contribute to the regeneration, but not to the development, of IHD cells. Hepatocyte-to-IHD cell transdifferentiation is possible but rarely detected. Progenitors in the EHD proliferate and migrate into the liver with Notch signaling loss and differentiate into IHD cells if Notch signaling increases. Tissue-specific mosaic analysis with an inducible dominant-negative Fgf receptor suggests that Fgf signaling from the surrounding mesenchymal cells maintains this extrahepatic niche by directly preventing premature differentiation and allocation of EHD progenitors to the liver. Indeed, transcriptional profiling and functional analysis of adult mouse EHD organoids uncover their distinct differentiation and proliferative potential relative to IHD organoids. CONCLUSIONS: Our data show that IHD cells regenerate upon resumption of Jagged/Notch signaling, from multipotent progenitors originating from an Fgf-dependent extrahepatic stem cell niche. We posit that if Jagged/Notch signaling is augmented, through normal stochastic variation, gene therapy, or a Notch agonist, regeneration of IHD cells in patients with ALGS may be enhanced.

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.

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.

Biogenic amine actions on cholangiocyte function.

Biogenic amines, such as serotonin, histamine, dopamine, and the catecholamines epinephrine and norepinephrine, regulate a multitude of cellular responses. A great deal of effort has been invested into understanding the effects of these molecules and their corresponding receptor systems on cholangiocyte secretion, apoptosis, and growth. This review summarizes the results of these efforts and highlights the importance of these regulatory molecules on the physiology and pathophysiology of cholangiocytes.

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.

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).

Effect of aging on the adult extrahepatic bile duct: a sonographic study.

OBJECTIVE: To determine whether the size of the extrahepatic bile duct increases with age in adults. METHODS: We prospectively collected data on 251 patients aged 20 years or older who underwent abdominal sonography. None of the patients had a history of liver, gallbladder, biliary, or pancreatic disease or surgery. The extrahepatic bile duct was measured at 3 locations: in the porta hepatis, in the most distal aspect of the head of the pancreas, and midway between these points. Least squares linear regression was used to correlate patient age and the size of the extrahepatic bile duct. RESULTS: There were 126 men and 125 women aged 20 to 94 years (mean +/- SD, 52.5 +/- 17.63 years). Twelve percent of the study population were younger than 30 years, and 12% were older than 80 years. The mean diameters of the common bile duct in the 3 locations were as follows: proximal, 3.39 +/- 1.14 mm; middle, 3.72 +/- 1.28 mm; and distal, 4.28 +/- 1.18 mm. The overall mean for all measures was 3.66 +/- 1.15 mm. The width of the common bile duct ranged from 1.0 to 8.6 mm. There was a significant correlation between common bile duct size and age (r = 0.535; P < .001). Mean common bile duct sizes were 3.128 +/- 0.862 mm in the patients younger than 50 years and 4.19 +/- 1.15 mm in the patients older than 50 years (P < .001 by independent t test for equality of means). We have found that the duct gradually dilated 0.04 mm/y. CONCLUSIONS: This study revealed an age-dependent change in the diameter of the extrahepatic bile duct. We suggest that the upper normal limit of the duct in elderly persons be set at 8.5 mm.

Is age associated with size of adult extrahepatic bile duct: sonographic study.

PURPOSE: To determine if the size of the extrahepatic bile duct increases with age in adults. MATERIALS AND METHODS: A total of 258 consecutive patients 18 years and older, without known biliary or pancreatic disease, who were fasting to undergo routine abdominal sonography were examined. The transverse and anteroposterior dimensions of the extrahepatic bile duct were measured proximally at the porta hepatis, at the middle above the head of the pancreas, and distally at the head of the pancreas. Simple linear regression of the average of these measurements against age tested the hypothesis of a slope of 1.0 mm per decade. RESULTS: The sample included a wide variety of ages: 55 years +/- 16 (mean +/- SD), with a range of 20-92 years, including 151 men and 107 women. One-tenth of the cohort were younger than 35 years old and one-tenth were older than 77 years old. The six measurements were proximal-transverse 3.5 mm +/- 1.0, proximal-anteroposterior 2.9 mm +/- 1.1, middle-transverse 3.9 mm +/- 1.2, middle-anteroposterior 3.4 mm +/- 1.2, distal-transverse 4.1 mm +/- 1.2, distal-anteroposterior 3.5 mm +/- 1.2. Least squares regression slope differed significantly from 0.1 mm per year (95% CI; -0.000703, +0.00110) and in fact contained zero. CONCLUSION: Findings were not able to help confirm an association between age and size of the extrahepatic bile duct in an asymptomatic adult population.

Development of the adult endocrine pancreas during metamorphosis in the sea lamprey, Petromyzon marinus L. II. Electron microscopy and immunocytochemistry.

The development of the adult endocrine pancreas was followed throughout metamorphosis in the sea lamprey using electron microscopy and immunocytochemistry. It was discovered that the caudal pancreas develops from the larval extrahepatic common bile duct through the process of transdifferentiation (dedifferentiation/redifferentiation). Early in metamorphosis the bile duct epithelial cells possess large vacuoles, resembling autophagic vacuoles, containing recognizable cell material. There is a loss of the large bundles of intermediate filaments characteristic of the larval bile duct epithelium. These same cells are then seen to contain granules immunoreactive for insulin. Pancreatic islets develop within the base of the bile duct epithelium from these transdifferentiated cells and migrate into the surrounding connective tissue to form the caudal pancreas. The cranial pancreas was found to develop from the epithelia lining the developing adult diverticulum and anterior intestine in a similar fashion as those in the larva. The second cell type to appear in either portion of the developing pancreas is similar to the third cell type of the adult: cells immunoreactive for somatostatin do not appear until late in metamorphosis in either region.