Genetic screens for p53 pathway genes in zebrafish: the identification and characterization of novel gene wdr43

Zebrafish are vertebrate organisms offering many distinct advantages to study both genetics and development. The body of this dissertation describes research establishing and using zebrafish to model the p53 pathway.;The gene p53 is implicated in the vast majority of human tumors. It is known as, "the Guardian of the Genome" because of its functions of monitoring and maintaining the fidelity of the genome. The first resulting manuscript from our research centered on developing a zebrafish model for the human syndrome Li-Fraumeni. Dysfunction of the p53 pathway has been shown to be the primary cause of their propensity toward tumorigenesis. We first showed the p53 pathway is conserved in zebrafish. An F3 screen was devised and carried out using a reproducible apoptotic phenotype seen in p53 deficient fish. This led to the discovery of a viable zebrafish with no functional p53. These fish develop tumors that closely mimic Li-Fraumeni patients providing a zebrafish model to study human disease.;The next resulting manuscript describes a highly efficient method of genotyping zebrafish using high resolution melting analysis (HRMA). The sheer number of zebrafish used in laboratories necessitates a technique that is efficient, cost effective and accurate. HRMA was used to genotype zebrafish mutant embryos with 100 percent accuracy, removing the need for restriction enzymes and agarose gel electrophoresis. This technique worked consistently well with three different types of mutations: point mutations, deletions, and insertions and is applicable to other model organisms.;The third manuscript results from a secondary screen of a known collection of insertional mutant zebrafish. A novel gene wdr43 was recovered in the screen using a phenotype of p53 pathway activation. This gene was shown to activate downstream targets of p53. The mutant exhibited specific p53 dependent phenotypes, including apoptosis. Mutants displayed aberrant mitotic spindle formation which appeared to result in chromosomal instability.;The wdr43 mutant also displayed p53 independent developmental phenotypes. Specifically the gut and eyes appeared to halt differentiation at particular stages of development, while other organs of the fish continued to develop appropriately. This ongoing project helps to elucidate the p53 independent role of wdr43 in development.