The Role of ARF6 in Biliary Atresia

Abstract

Background & aims: Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).

Methods: To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).

Results: Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3' flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10-7), ERK/MAPK and CREB canonical pathways (p<1 x10-34), and functional networks for cellular development and proliferation (p<1 x10-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

Conclusions: The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.

Fig 1. Eigenstrat Principal Components Analysis.

(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 ARF6 gene (UCSC genome browser evaluation show enriched histone marks H3K4me1 overlayed with H3K27Ac is an indicative of enhancer region). (C) i-iv show ARF6 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.

Fig 2. arf6 knockdown results in developmental biliary defects in zebrafish.

(A, B) Whole-mount in situ hybridization showing arf6a and arf6b expression in developing embryos/larvae. Arrows point to the liver; arrowheads the pancreas; brackets mark the intestinal bulb. (C) The Tg(Tp1:GFP) and Tg(fabp10a:dsRed) 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 arf6-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.

Fig 3. arf6 regulates intrahepatic biliary morphogenesis.

(A-C) Confocal images showing the development of the intrahepatic biliary network. Tg(Tp1:GFP) embryos/larvae were processed for immunostaining with anti-GFP (green). For 40 and 48 hpf, anti-Prox1 staining (red) was also used. Brackets delineate the length of BEC filopodia at 62, 72, and 96 hpf, and the length of interconnecting bile preductules at 5 dpf (A); graphs show their quantitation (B, C). (D) Confocal images showing the location of BEC nuclei in the entire liver. The Tg(Tp1:H2B-mCherry) line reveals BEC nuclei. Dashed lines outline clusters with four or more BECs. Graph shows the percentage of BECs present as single cells, doublets, triplets, or in clusters of four or more cells. (E) Confocal images of the liver immunostained for GFP (green) and Abcb11 (red). The Tg(Tp1:GFP) line and anti-Abcb11 reveal the intrahepatic biliary structure and bile canaliculi, respectively. All dotted lines outline the liver. Asterisks, statistical significance (* p<0.0001); error bars, ± SEM; scale bars, 25 μm.

Fig 4. EGFR signaling regulates biliary morphogenesis.

(A) Whole-mount in situ hybridization image showing egfra expression in the liver at 72 hpf. (B) Confocal images of the liver showing the intrahepatic biliary structure, as revealed by Tp1:GFP expression. Tg(Tp1:GFP) 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 Tp1: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 Tp1: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.

Fig 5. EGFR signaling and Arf6 act in the same pathway in the regulation of intrahepatic biliary morphogenesis.

Instead of 4 μM AG1478 and 2 ng of arf6-ATG MO, 1 μM AG1478 and 0.5 ng of arf6-ATG MO were used. (A) Epifluorescence images showing the expression of Tp1:GFP and fabp10a: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.

Fig 6. Quantitative RT-PCR analysis of EGFR pathway genes in liver tissue from zebrafish.

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

Fig 7. A proposed mechanism for poor bile duct development in BA.

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 ARF6 originating from the regulatory regions defined by rs3126184 and rs10140366 in human BA, and arf6 knockdown or EGFR inhibition with AG1478 in zebrafish embryos lead to poor bile duct development.