INSERTIONAL MUTAGENESIS IN ZEBRAFISH IDENTIFIES 2 NOVEL GENES, PESCADILLO AND DEAD EYE, ESSENTIAL FOR EMBRYONIC-DEVELOPMENT

Recently our laboratory described an efficient method for generating r etroviral provirus insertions in the zebrafish germ line, and we showe d that provirus insertions induce embryonic mutations at a frequency o f roughly one mutant per 70 insertions. To date we have isolated four insertional mutants and, using the proviruses as a molecular tag, have cloned the genes disrupted in three of them. The proviruses in all th ree mutants lie within or just 5' of the first coding exon, point in t he opposite transcriptional orientation from the gene, and disrupt tra nscription. Here we present a molecular characterization of two genes identified by this method and describe the associated mutant phenotype s. The pescadillo (pes) gene is predicted to encode a protein of 582 a mino acids with no recognizable functional motifs, which is highly con served from yeast to humans. pes mRNA is expressed widely and dynamica lly during the first 3 days of embryogenesis. Prominent sites of expre ssion are the eyes and optic tectum on day 1, the fin buds, liver prim ordium, and gut on day 2, and the branchial arches on day 3. Beginning at day 3 of embryogenesis, pes mutant embryos exhibit small eyes, a r educed brain and visceral skeleton, shortened fins, and a lack of expa nsion of the liver and gut, and then die on the sixth day of developme nt. The dead eye (dye) gene encodes a protein of 820 amino acids that is homologous to genes of unknown function in human, mouse, and Xenopu s, and that has weak homology with the yeast NIC96 (nucleoporin-intera cting component) gene. dye: mutants can be recognized on day 2 of embr yogenesis by the presence of necrotic cells in the tectum and eyes. dy e mutants die on day 5 of development. These results demonstrate the p ower of insertional mutagenesis in zebrafish for rapidly finding and c haracterizing novel genes essential for embryonic development. Using o ur current methodology, we estimate that our laboratory could screen s imilar to 25,000 insertions in 2-3 years, identifying perhaps 250-350 embryonic lethal genes. Assuming that all genes are accessible to prov iral insertion, the wider application of this approach could lead to t he rapid identification of the majority of genes that are required for embryonic development of this vertebrate.