Eigenstrat Principal Components Analysis.

<p>(A) Boxed region shows well stratified cases and controls carried forward in analysis. (B) The BA susceptibility locus defined by rs3126184 and rs10140366 lies in an enhancer region in the 3’ flanking region of the <i>ARF6</i> gene (UCSC genome browser evaluation show enriched histone marks H3K4me1 overlayed with H3K27Ac is an indicative of enhancer region). (C) i-iv show <i>ARF6</i> immunostaining in liver explants from normal children with intact bile ducts (BD) (i), children with BA with bile duct paucity in portal tracts (PT) (ii) or cirrhosis (iii), and a child with hepatocellular carcinoma (iv). T = tumor cells, L = lobule.</p

EGFR signaling regulates biliary morphogenesis.

<p>(A) Whole-mount in situ hybridization image showing <i>egfra</i> expression in the liver at 72 hpf. (B) Confocal images of the liver showing the intrahepatic biliary structure, as revealed by <i>Tp1</i>:GFP expression. <i>Tg(Tp1</i>:<i>GFP)</i> embryos were treated with DMSO or 4 μM AG1478 from 48 to 96 hpf, and processed for whole-mount immunostaining with anti-GFP antibody. The length of BEC filopodia was quantified as shown in a graph. Brackets delineate the length of BEC filopodia. (C) Confocal images of the liver showing the expression of <i>Tp1</i>:GFP (green) and Abcb11 (red) for biliary structure and bile canaliculi, respectively. (D) Confocal images of the liver showing the location of BEC nuclei in the entire liver, as assessed by <i>Tp1</i>:H2B-mCherry expression. Dashed lines outline clusters with four or more BECs. Graph showing the percentage of BECs present as single cells, doublets, triplets, or in clusters of four or more cells. (E) Epifluorescence images showing PED-6 accumulation in the gallbladder in DMSO- or AG1478-treated larvae at 5 dpf. Graph showing the percentage of larvae exhibiting different levels of PED-6 accumulation in the gallbladder. Arrows point to the gallbladder. All dotted lines outline the liver. n indicates the number of larvae examined; asterisks indicate statistical significance (* p<0.0001). Error bars, ± SEM; scale bars, 25 μm.</p

EGFR signaling and Arf6 act in the same pathway in the regulation of intrahepatic biliary morphogenesis.

<p>Instead of 4 μM AG1478 and 2 ng of <i>arf6</i>-ATG MO, 1 μM AG1478 and 0.5 ng of <i>arf6</i>-ATG MO were used. (A) Epifluorescence images showing the expression of <i>Tp1</i>:GFP and <i>fabp10a</i>:dsRed revealed a severe defect in the intrahepatic biliary structure only when the MO injection was combined with the AG1478 treatment. Based on the severity of the biliary defect, larvae were divided into three groups: normal, intermediate, and severe. Arrows point to the liver and dotted lines outline the liver. Scale bars, 100 μm. (B) Graph showing the percentage of larvae in each group shown in A. (C) Graph showing the percentage of larvae exhibiting different levels of PED-6 accumulation in the gallbladder at 5 dpf. n indicates the number of larvae examined.</p

<i>arf6</i> knockdown results in developmental biliary defects in zebrafish.

<p>(A, B) Whole-mount <i>in situ</i> hybridization showing <i>arf6a</i> and <i>arf6b</i> expression in developing embryos/larvae. Arrows point to the liver; arrowheads the pancreas; brackets mark the intestinal bulb. (C) The <i>Tg(Tp1</i>:<i>GFP)</i> and <i>Tg(fabp10a</i>:<i>dsRed)</i> lines reveal the intrahepatic biliary structure and liver size, respectively. Epifluorescence images showing the expression of these transgenes revealed a defect in the intrahepatic biliary structure in <i>arf6</i>-ATG MO-injected larvae. Based on the severity of the biliary defect, larvae were divided into three groups: normal, intermediate, and severe. Graph shows the percentage of larvae in each group. Arrows point to the liver; dotted lines outline the liver (A-C). (D) Epifluorescence images showing PED-6 accumulation in the gallbladder (arrows). Based on PED-6 levels in the gallbladder, larvae were divided into three groups: absent, small/faint, and normal. Graph shows the percentage of larvae in each group. n, the number of larvae examined; scale bars, 100 μm.</p

A proposed mechanism for poor bile duct development in BA.

<p>Ligation of activated EGFR to GEP100 initiates sequential activation of ARF6, ERK/MAPK and CREB signaling proteins resulting in cellular development and proliferation. Negative regulation of <i>ARF6</i> originating from the regulatory regions defined by rs3126184 and rs10140366 in human BA, and <i>arf6</i> knockdown or EGFR inhibition with AG1478 in zebrafish embryos lead to poor bile duct development.</p

Quantitative RT-PCR analysis of EGFR pathway genes in liver tissue from zebrafish.

<p>The relative expression level of genes was shown in fold change in <i>arf6</i> morphants over un-injected controls. Error bars shown, ± SEM (average of three independent experiments).</p