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

Tang, V., Cofer, Z.C., Cui, S., Sapp, V., Loomes, K.M., Matthews, R.P.
Journal of pediatric gastroenterology and nutrition   63(5): 524-530 (Journal)
Registered Authors
Cui, Shuang Alice, Matthews, Randy
MeSH Terms
  • Aminopeptidases/genetics*
  • Animals
  • Biliary Atresia/genetics*
  • Calmodulin-Binding Proteins/genetics*
  • Fluorescent Antibody Technique
  • Gene Knockdown Techniques
  • Genetic Predisposition to Disease
  • Real-Time Polymerase Chain Reaction
  • Zebrafish/genetics*
27526058 Full text @ J. Pediatr. Gastroenterol. Nutr.
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.
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.
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.
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.This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Genes / Markers
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Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes