Characterization of Zebrafish Mutants to Model Human Genetic Defects in the LRRC56 Gene.

Title: Characterization of Zebrafish Mutants to Model Human Genetic Defects in the LRRC56 Gene.
Authors: Ivare, Joshua
Date: 2020-02-12
Abstract: In humans, defects of motile cilia are characterized by respiratory infections, in some cases, congenital heart disease, and in about 50% of patients, laterality defects where the proper placement of internal organs is disrupted. Recently, two autopsied human fetuses identified as carrying a homozygous missense variant of the Leucine-rich repeat (LRR)-containing gene 56 (LRRC56) gene showed mirror image placement of thoracic and abdominal structures as well as complex cardiac anomalies. Furthermore, LRRC56 was recently reported to be a homolog of the Outer Dynein Arm 8 gene (oda8), a gene necessary for the cytoplasmic maturation of the outer dynein arms (ODA) in the flagella of algae. Therefore, to confirm that the observed phenotypes of the human fetuses are the result of an inactive LRRC56 protein, we created a zebrafish lrrc56 knockout with a 4bp deletion in the first exon of the lrrc56 gene (lrrc56-/-) using the CRISPR/Cas9 genome editing technology. We hypothesize that if the missense mutation of the LRRC56 gene in the human fetuses indeed renders the LRRC56 protein inactive, a knockout of the zebrafish lrrc56 gene might produce in zebrafish similar phenotypes to those observed in the mutant human fetuses. We show that knocking out the zebrafish lrrc56 gene leads to spinal defects in the lrrc56-/- zebrafish. By performing gene expression analyses on the lrrc56-/- mutants, we observed defects in visceral organ left/right asymmetry. Injection of the wild type lrrc56 mRNA rescued the phenotypes of spinal and visceral organ asymmetry defects in the lrrc56-/- mutants while the injection of a lrrc56 mRNA possessing the same missense mutation observed in the two autopsied fetuses failed to rescue these phenotypes. When we performed IHC with an α-tubulin antibody to detect cilia in the zebrafish Kupffer’s Vesicle (KV), we observed that the cilia present in the KV of the lrrc56-/- mutants were longer than those in WT zebrafish. Together, our results verify that the observed phenotypes of the fetuses are due to the homozygous missense LRRC56 variant they possessed.
CollectionThèses, 2011 - // Theses, 2011 -