Forward genetics combined with unsupervised classifications identified zebrafish mutants affecting biliary system formation.

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

NCK-associated protein 1 like (nckap1l) minor splice variant regulates intrahepatic biliary network morphogenesis.

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.

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

The intrahepatic biliary network is a highly branched three-dimensional network lined by biliary epithelial cells, but how its branching patterns are precisely established is not clear. We designed a new computer-based algorithm that quantitatively computes the structural differences of the three-dimensional networks. Utilizing the algorithm, we showed that inhibition of Cyclin-dependent kinase 5 (Cdk5) led to reduced branching in the intrahepatic biliary network in zebrafish. Further, we identified a previously unappreciated downstream kinase cascade regulated by Cdk5. Pharmacological manipulations of this downstream kinase cascade produced a crowded branching defect in the intrahepatic biliary network and influenced actin dynamics in biliary epithelial cells. We generated larvae carrying a mutation in cdk5 regulatory subunit 1a (cdk5r1a), an essential activator of Cdk5. cdk5r1a mutant larvae show similar branching defects as those observed in Cdk5 inhibitor-treated larvae. A small-molecule compound that interferes with the downstream kinase cascade rescued the mutant phenotype. These results provide new insights into branching morphogenesis of the intrahepatic biliary network.