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.

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.

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