Two pilot genetic screens using the diploid amphibian Xenopus tropicalis are proposed. The major objective of the proposed research is to examine the feasibility of both a traditional chemical mutagenesis screen and a new anti-sense morpholino oligonucleotide based screen in Xenopus tropicalis. Previously, large-scale mutagenesis screens in zebrafish successfully identified a number of genes important for vertebrate embryogenesis. Despite this success, there is evidence that many genes and pathways were not identified due to the genomic complexity of teleost fish. While zebrafish, Danio rerio, are clearly diploid organisms, an ancient genome-wide duplication event is manifested in the functional overlap of genes critical to early embryogenesis. Hence, systematic genetic screens in other amenable vertebrates are warranted to uncover phenotypes hidden by genomic redundancy in the fish. The amphibian Xenopus tropicalis, by virtue of its diploid genome, relatively short generation time, and tetrapod evolutionary status, has been identified as a good candidate for mutagenesis screens. The reliable transgenic technique for X. tropicalis can also contribute in several ways. While the low efficiency of transgenesis currently limits its utility for insertional mutagenesis, use of stable transgenic reporter lines can effectively streamline functional analysis of injected gene products or anti-sense oligonucleotides. Two pilot genetic screens are proposed here. The first is a forward genetic approach employing in vitro chemical mutagenesis of sperm, in vitro fertilization and identification of carriers of zygotic-effect recessive-lethal mutations. The second screen takes advantage of ongoing expressed sequence tag projects and utilizes anti-sense morpholino oligonucleotides to disrupt gene-products of zygotic as well as some maternally expressed genes during embryogenesis. The forward and reverse screens proposed here also complement ongoing X. tropicalis infrastructure development. Functional characterization of EST sequences will help integrate genetic and physical mapping efforts already underway.
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