Loss of a Candidate Biliary Atresia Susceptibility Gene, add3a, Causes Biliary Developmental Defects in Zebrafish

Abstract

Objectives Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the bile ducts of neonates. Although BA is the leading indication for pediatric liver transplantation, the etiology remains elusive. Adducin 3 (ADD3) and X-prolyl aminopeptidase 1 (XPNPEP1) are two genes previously identified in genome-wide association studies (GWAS) as potential BA susceptibility genes. Using zebrafish, we investigated the importance of ADD3 and XPNPEP1 in functional studies. Methods To determine whether loss of either gene leads to biliary defects, we performed morpholino antisense oligonucleotide (MO) knockdown studies targeting add3a and xpnpep1 in zebrafish. Individuals were assessed for decreases in biliary function and the presence of biliary defects. Quantitative PCR (qPCR) was performed on pooled 5 days post fertilization (dpf) larvae to assess variations in transcriptional expression of genes of interest. Results While both xpnpep1 and add3a are expressed in the developing zebrafish liver, only knockdown of add3a produced intrahepatic defects and decreased biliary function. Similar results were observed in homozygous add3a mutants. Morpholino antisense oligonucleotide-mediated knockdown of add3a also showed higher mRNA expression of Hedgehog (Hh) targets. Inhibition of Hh signaling rescued biliary defects caused by add3a knockdown. Combined knockdown of add3a and glypican-1 (gpc1), another mediator of Hh activity that is also a BA susceptibility gene, resulted in more severe biliary defects than knockdown of either alone. Conclusions Our results support previous studies identifying ADD3 as a putative genetic risk factor for BA susceptibility. Our results also provide evidence that add3a may be affecting the Hedgehog pathway, an important factor in BA pathogenesis.

Three-dimensional structural analysis reveals a Cdk5-mediated kinase cascade regulating hepatic biliary network branching in zebrafish

“…Biliary atresia is not generally thought of as an inherited disease, although there are some lines of evidence to support the hypothesis that genetic factors contribute to disease susceptibility (Garcia-Barceló et al, 2010;Tang et al, 2016;Tsai et al, 2014). Our study has indicated that inhibiting the Cdk5-mediated pathway can generate phenotypes similar to those seen in biliary atresia patients.…”

Embryonic cholecystitis and defective gallbladder contraction in the Sox17-haploinsufficient mouse model of biliary atresia

“…It has also been speculated that the SHH signaling pathway regulates the epithelial-mesenchymal transition of cholangiocytes (Omenetti et al, 2008;Jung et al, 2015). The effects of excess SHH signaling encompass defective hepatobiliary ducts in a zebrafish model (Cui et al, 2013;Tang et al, 2016), suggesting that excess SHH signaling can have detrimental effects on the proper development and maintenance of the biliary duct. The current phenotype of reduced SHH signaling in the defective gallbladder might be explained by the distinct roles of SHH signaling during developmental and early pathogenic stages (i.e.…”

Using Zebrafish to Model Liver Diseases-Where Do We Stand?

“…They turned to the zebrafish model to validate the biological function of the candidate genes by using the morpholino oligonucleotide knockdown technique. Matthews’ group revealed that knocking down glypican 1 ( gpc1 ), which encodes a cell-surface heparan sulfate proteoglycan, and adducin 3 ( add3 ), which encodes a membrane skeletal protein, both resulted in decreased PED6 uptake in the gallbladder and a less complex intrahepatic biliary network [28, 29]. gpc1 knockdown also led to a smaller gallbladder [28].…”

“…The main challenge for the follow-up studies of patient GWAS is to determine which variants are indeed causative. Four groups have used zebrafish to validate GWAS variants in BA patients, as well as in patients with elevated liver enzyme levels [28, 98, 31, 29]. Furthermore, CRISPR/Cas9-mediated targeted mutagenesis can now be applied in zebrafish not only to generate null mutations, but also to precisely recapitulate the same mutations found in patients.…”