Having a well-annotated genome and numerous, transparent progeny, zebrafish afford a powerful vertebrate model to development and human disease employing forward and reverse genetic approaches. Our preliminary forward genetic screen for chemically induced mutations have uncovered numerous recessive and dominant adult mutants with scoliotic features. We propose to continue this productive screen towards establishing a comprehensive list of the genes required for normal spine development in zebrafish. We will identify the molecular nature of isolated mutants and characterize the defects at embryonic and tissue levels. The genes identified in zebrafish will become candidates in human genetic and genomic analyses.
To validate candidate loci associated in human adolescent idiopathic scoliosis (AIS) patients in Project 1 (Human Genetics) and Project 3 (Genomics) of this program, we will take advantage of the recent progress in reverse genetic methods in zebrafish. Using significant experience we developed in TALEN and CRISPR-Cas9-mediated gene disruption, and improved methods of gene editing we recently established, we will engineer null mutations or humanized no-synonymous mutations in the zebrafish. We will employ these robust methods for testing candidate loci of human AIS patients and provide models of AIS in a genetically tractable vertebrate zebrafish model.
By combining the complementary expertise of the participating research groups, advances in human genetics and genomics with the strengths of zebrafish as a robust animal model, this program will provide the first atlas of genes critical to proper spine development in general and to AIS specifically.