Randolph P. Matthews

Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy

The Alagille Syndrome (AGS) is a heritable disorder affecting the liver and other organs. Causative dominant mutations in human Jagged 1 have been identified in most AGS patients. Related organ defects occur in mice that carry jagged 1 and notch 2 mutations. Multiple jagged and notch genes are expressed in the developing zebrafish liver. Compound jagged and notch gene knockdowns alter zebrafish biliary, kidney, pancreatic, cardiac and craniofacial development in a manner compatible with an AGS phenocopy. These data confirm an evolutionarily conserved role for Notch signaling in vertebrate liver development, and support the zebrafish as a model system for diseases of the human biliary system.

The microRNA-30 family is required for vertebrate hepatobiliary development

BACKGROUND & AIMS The function of microRNA (miRNA) in liver development is unknown. To address this issue, we characterized miRNA expression in the embryonic mouse liver, performed functional miRNA analysis in zebrafish larvae, and identified novel hepatic miRNA targets. METHODS Hepatic RNA isolated from mice at embryonic days 15.5, 18.5, and postnatal day 2 was hybridized to a mouse miRNA microarray. The microarray results were confirmed by Northern blot hybridization and quantitative reverse-transcription polymerase chain reaction. The spatial distribution of selected miRNAs was determined by in situ hybridization. Functional analysis of miR-30a was performed in zebrafish using antisense-mediated miRNA knockdown. Targets of miR-30a were identified by microarray analysis of gene expression following knockdown in cultured cells. RESULTS A set of 38 differentially expressed fetal hepatic miRNAs was identified. Several of these miRNAs were found to exhibit distinct temporal and spatial patterns of expression in hepatocytes, cholangiocytes, and nonepithelial cells within the liver. Two (miR-30a and miR-30c) are the first examples of ductal plate and bile duct-specific hepatic miRNAs. Knockdown of miR-30a in the zebrafish larva results in defective biliary morphogenesis. Several newly identified targets of miR-30a are known regulators of liver development and function. CONCLUSIONS We have identified miRNAs whose spatial and temporal patterns of expression are suggestive of functional roles in hepatic development and/or function. One of these, the biliary miRNA miR-30a, is required for biliary development in zebrafish. This is the first demonstration of a functional role for miRNA in hepatic organogenesis.

Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization

Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC) is a multisystem disorder associated with abnormalities in polarized liver and kidney cells. Mutations in VPS33B account for most cases of ARC. We identified mutations in VIPAR (also called C14ORF133) in individuals with ARC without VPS33B defects. We show that VIPAR forms a functional complex with VPS33B that interacts with RAB11A. Knockdown of vipar in zebrafish resulted in biliary excretion and E-cadherin defects similar to those in individuals with ARC. Vipar- and Vps33b-deficient mouse inner medullary collecting duct (mIMDC-3) cells expressed membrane proteins abnormally and had structural and functional tight junction defects. Abnormal Ceacam5 expression was due to mis-sorting toward lysosomal degradation, but reduced E-cadherin levels were associated with transcriptional downregulation. The VPS33B-VIPAR complex thus has diverse functions in the pathways regulating apical-basolateral polarity in the liver and kidney.

TNFα-dependent hepatic steatosis and liver degeneration caused by mutation of zebrafish s-adenosylhomocysteine hydrolase

Hepatic steatosis and liver degeneration are prominent features of the zebrafish ducttrip (dtp) mutant phenotype. Positional cloning identified a causative mutation in the gene encoding S-adenosylhomocysteine hydrolase (Ahcy). Reduced Ahcy activity in dtp mutants led to elevated levels of S-adenosylhomocysteine (SAH) and, to a lesser degree, of its metabolic precursor S-adenosylmethionine (SAM). Elevated SAH in dtp larvae was associated with mitochondrial defects and increased expression of tnfa and pparg, an ortholog of the mammalian lipogenic gene. Antisense knockdown of tnfa rescued hepatic steatosis and liver degeneration in dtp larvae, whereas the overexpression of tnfa and the hepatic phenotype were unchanged in dtp larvae reared under germ-free conditions. These data identify an essential role for tnfa in the mutant phenotype and suggest a direct link between SAH-induced methylation defects and TNF expression in human liver disorders associated with elevated TNFα. Although heterozygous dtp larvae had no discernible phenotype, hepatic steatosis was present in heterozygous adult dtp fish and in wild-type adult fish treated with an Ahcy inhibitor. These data argue that AHCY polymorphisms and AHCY inhibitors, which have shown promise in treating autoimmunity and other disorders, may be a risk factor for steatosis, particularly in patients with diabetes, obesity and liver disorders such as hepatitis C infection. Supporting this idea, hepatic injury and steatosis have been noted in patients with recently discovered AHCY mutations.

Zebrafish vps33b, an ortholog of the gene responsible for human arthrogryposis-renal dysfunction-cholestasis syndrome, regulates biliary development downstream of the onecut transcription factor hnf6

Arthrogryposis-renal dysfunction-cholestasis syndrome (ARC) is a rare cause of cholestasis in infants. Causative mutations in VPS33B, a gene that encodes a Class C vacuolar sorting protein, have recently been reported in individuals with ARC. We have identified a zebrafish vps33b-ortholog that is expressed in developing liver and intestine. Knockdown of vps33b causes bile duct paucity and impairs intestinal lipid absorption, thus phenocopying digestive defects characteristic of ARC. By contrast, neither motor axon nor kidney epithelial defects typically seen in ARC could be identified in vps33b-deficient larvae. Biliary defects in vps33b-deficient zebrafish larvae closely resemble the bile duct paucity associated with knockdown of the onecut transcription factor hnf6. Consistent with this, reduced vps33b expression was evident in hnf6-deficient larvae and in larvae with mutation of vhnf1, a downstream target of hnf6. Zebrafish vhnf1, but not hnf6, increases vps33b expression in zebrafish embryos and in mammalian liver cells. Electrophoretic mobility shift assays suggest that this regulation occurs through direct binding of vHnf1 to the vps33b promoter. These findings identify vps33b as a novel downstream target gene of the hnf6/vhnf1 pathway that regulates bile duct development in zebrafish. Furthermore, they show that tissue-specific roles for genes that regulate trafficking of intracellular proteins have been modified during vertebrate evolution.

Evidence From Human and Zebrafish That GPC1 Is a Biliary Atresia Susceptibility Gene

BACKGROUND & AIMS Biliary atresia (BA) is a progressive fibroinflammatory disorder of infants involving the extrahepatic and intrahepatic biliary tree. Its etiology is unclear but is believed to involve exposure of a genetically susceptible individual to certain environmental factors. BA occurs exclusively in the neonatal liver, so variants of genes expressed during hepatobiliary development could affect susceptibility. Genome-wide association studies previously identified a potential region of interest at 2q37. We continued these studies to narrow the region and identify BA susceptibility genes. METHODS We searched for copy number variants that were increased among patients with BA (n = 61) compared with healthy individuals (controls; n = 5088). After identifying a candidate gene, we investigated expression patterns of orthologues in zebrafish liver and the effects of reducing expression, with morpholino antisense oligonucleotides, on biliary development, gene expression, and signal transduction. RESULTS We observed a statistically significant increase in deletions at 2q37.3 in patients with BA that resulted in deletion of one copy of GPC1, which encodes glypican 1, a heparan sulfate proteoglycan that regulates Hedgehog signaling and inflammation. Knockdown of gpc1 in zebrafish led to developmental biliary defects. Exposure of the gpc1 morphants to cyclopamine, a Hedgehog antagonist, partially rescued the gpc1-knockdown phenotype. Injection of zebrafish with recombinant Sonic Hedgehog led to biliary defects similar to those of the gpc1 morphants. Liver samples from patients with BA had reduced levels of apical GPC1 in cholangiocytes compared with samples from controls. CONCLUSIONS Based on genetic analysis of patients with BA and zebrafish, GPC1 appears to be a BA susceptibility gene. These findings also support a role for Hedgehog signaling in the pathogenesis of BA.

DNA hypomethylation causes bile duct defects in zebrafish and is a distinguishing feature of infantile biliary atresia

Infantile cholestatic disorders arise in the context of progressively developing intrahepatic bile ducts. Biliary atresia (BA), a progressive fibroinflammatory disorder of extra‐ and intrahepatic bile ducts, is the most common identifiable cause of infantile cholestasis and the leading indication for liver transplantation in children. The etiology of BA is unclear, and although there is some evidence for viral, toxic, and complex genetic causes, the exclusive occurrence of BA during a period of biliary growth and remodeling suggests an importance of developmental factors. Interestingly, interferon‐γ (IFN‐γ) signaling is activated in patients and in the frequently utilized rhesus rotavirus mouse model of BA, and is thought to play a key mechanistic role. Here we demonstrate intrahepatic biliary defects and up‐regulated hepatic expression of IFN‐γ pathway genes caused by genetic or pharmacological inhibition of DNA methylation in zebrafish larvae. Biliary defects elicited by inhibition of DNA methylation were reversed by treatment with glucocorticoid, suggesting that the activation of inflammatory pathways was critical. DNA methylation was significantly reduced in bile duct cells from BA patients compared to patients with other infantile cholestatic disorders, thereby establishing a possible etiologic link between decreased DNA methylation, activation of IFN‐γ signaling, and biliary defects in patients. Conclusion: Inhibition of DNA methylation leads to biliary defects and activation of IFN‐γ‐responsive genes, thus sharing features with BA, which we determine to be associated with DNA hypomethylation. We propose epigenetic activation of IFN‐γ signaling as a common etiologic mechanism of intrahepatic bile duct defects in BA. (HEPATOLOGY 2011;)

Fructose Leads to Hepatic Steatosis in Zebrafish That Is Reversed by Mechanistic Target of Rapamycin (mTOR) Inhibition

Nonalcoholic fatty liver disease (NAFLD), the accumulation of lipid within hepatocytes, is increasing in prevalence. Increasing fructose consumption correlates with this increased prevalence, and rodent studies directly support fructose leading to NAFLD. The mechanisms of NAFLD and in particular fructose‐induced lipid accumulation remain unclear, although there is evidence for a role for endoplasmic reticulum (ER) stress and oxidative stress. We have evidence that NAFLD models demonstrate activation of the target of rapamycin complex 1 (Torc1) pathway. We set out to assess the contribution of ER stress, oxidative stress, and Torc1 up‐regulation in the development of steatohepatitis in fructose‐treated larval zebrafish. Zebrafish were treated with fructose or glucose as a calorie‐matched control. We also treated larvae with rapamycin, tunicamycin (ER stress), or valinomycin (oxidative stress). Fish were stained with oil red O to assess hepatic lipid accumulation, and we also performed quantitative polymerase chain reaction (qPCR)and western blot analysis. We performed immunostaining on samples from patients with NAFLD and nonalcoholic steatohepatitis (NASH). Treatment with fructose induced hepatic lipid accumulation, mitochondrial abnormalities, and ER defects. In addition, fructose‐treated fish showed activation of inflammatory and lipogenic genes. Treatment with tunicamycin or valinomycin also induced hepatic lipid accumulation. Expression microarray studies of zebrafish NAFLD models showed an elevation of genes downstream of Torc1 signaling. Rapamycin treatment of fructose‐treated fish prevented development of hepatic steatosis, as did treatment of tunicamycin‐ or valinomycin‐treated fish. Examination of liver samples from patients with hepatic steatosis demonstrated activation of Torc1 signaling. Conclusion: Fructose treatment of larval zebrafish induces hepatic lipid accumulation, inflammation, and oxidative stress. Our results indicate that Torc1 activation is required for hepatic lipid accumulation across models of NAFLD, and in patients. (Hepatology 2014;60:1581–1592)

Transcription factor onecut3 regulates intrahepatic biliary development in zebrafish

Members of the onecut family of transcription factors play important roles in the development of the liver and pancreas. We have shown previously that onecut1 (hnf6) is important during the terminal stages of intrahepatic biliary development in zebrafish. Here we report the characterization of a third zebrafish onecut gene, onecut3 (oc3), and assay its expression during development and its role in biliary duct formation using morpholino antisense oligonucleotide‐mediated knockdown. These experiments reveal an important role for oc3 during the earliest stages of zebrafish biliary development, and suggest that zebrafish oc3 is the functional ortholog of mammalian hnf6, a gene that directs biliary differentiation from bipotential progenitor cells. Consistent with this, zebrafish hnf6 expression was significantly reduced in oc3‐deficient larvae. Knockdown of hnf6 in wild‐type zebrafish larvae also significantly reduced oc3 expression, suggesting a complex interaction between onecut family member proteins during the latter stages of zebrafish biliary development. Developmental Dynamics 237:124–131, 2008.

p53-Mediated Biliary Defects Caused by Knockdown of cirh1a, the Zebrafish Homolog of the Gene Responsible for North American Indian Childhood Cirrhosis

North American Indian Childhood Cirrhosis (NAIC) is a rare, autosomal recessive, progressive cholestatic disease of infancy affecting the Cree-Ojibway first Nations of Quebec. All NAIC patients are homozygous for a missense mutation (R565W) in CIRH1A, the human homolog of the yeast nucleolar protein Utp4. Utp4 is part of the t-Utp subcomplex of the small subunit (SSU) processome, a ribonucleoprotein complex required for ribosomal RNA processing and small subunit assembly. NAIC has thus been proposed to be a primary ribosomal disorder (ribosomopathy); however, investigation of the pathophysiologic mechanism of this disease has been hindered by lack of an animal model. Here, using a morpholino oligonucleotide (MO)-based loss-of-function strategy, we have generated a model of NAIC in the zebrafish, Danio rerio. Zebrafish Cirhin shows substantial homology to the human homolog, and cirh1a mRNA is expressed in developing hepatocytes and biliary epithelial cells. Injection of two independent MOs directed against cirh1a at the one-cell stage causes defects in canalicular and biliary morphology in 5 dpf larvae. In addition, 5 dpf Cirhin-deficient larvae have dose-dependent defects in hepatobiliary function, as assayed by the metabolism of an ingested fluorescent lipid reporter. Previous yeast and in vitro studies have shown that defects in ribosome biogenesis cause stabilization and nuclear accumulation of p53, which in turn causes p53-mediated cell cycle arrest and/or apoptosis. Thus, the nucleolus appears to function as a cellular stress sensor in some cell types. In accordance with this hypothesis, transcriptional targets of p53 are upregulated in Cirhin-deficient zebrafish embryos, and defects in biliary function seen in Cirhin-deficient larvae are completely abrogated by mutation of tp53. Our data provide the first in vivo evidence of a role for Cirhin in biliary development, and support the hypothesis that congenital defects affecting ribosome biogenesis can activate a cellular stress response mediated by p53.