Cancer susceptibility in zebrafish loss of heterozygosity mutants

Abstract

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We have performed a genetic screen in the zebrafish, Danio rerio, to identify genes that are critical in the maintenance, or stability, of the vertebrate genome. Loss of heterozygosity (LOH) can occur by a variety of mechanisms and has been shown to be a key step in carcinogenesis. Germline genomic instability (gin) mutations were generated in zebrafish by ENU mutagenesis, and then identified in a screen for somatic loss of heterozygosity, using the golden (gol) pigmentation gene as a readily scorable “target”. Loss of heterozygosity can be scored as mosaic eye pigmentation in 48 to 72-hour old gol/+ zebrafish embryos, due to LOH at the golden locus. The long-term objective of this research is to understand the processes that maintain genomic stability, and how aberrations in these processes lead to cancer. Twelve gin mutations have been recovered from this screen and are currently being characterized. They are recessive, homozygous lethal mutations that may represent as many as 12 different genes. Complementation analysis indicates that 7 gin mutations interact through a combination of maternal and zygotic activity. Microarray experiments are underway to identify the changes in gene expression that correlate with these mutations. The discovery of tumors in heterozygous adults of all gin lines is consistent with the expectation that genomic instability mutations are critical in cancer. Tumors have been found in a variety of tissues with multiple tumors in some fish. Molecular analysis of tumors in heterozygous adults of the gin-10 mutation has shown somatic loss of heterozygosity for an SSR marker near the gin-10 locus. Tumor incidence was compared between gin-10 carriers and age matched wildtype fish. The results of this tumor survey clearly demonstrate that the tumor incidence in gin heterozygote adults is as high as 10X that of wildtype fish. Characterization of the gin mutations, their interactions, pathobiology, the molecular analysis of gin tumors and the advantages of zebrafish as a model organism for cancer will be discussed.