Genetic Screens for Mutations Affecting Development of Xenopus tropicalis

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Abstract
We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics. Amphibian embryos can be used to understand how all vertebrates, including mammals, develop from fertilized single-celled eggs to establish a body plan and form different cell types and functional organs. Genetic methods are used to analyze what goes wrong in embryos lacking working versions of individual genes, and help to understand those genes' specific functions. However, genetic analysis of previously studied amphibians has been difficult because of these species' long generation time and complex genetic structure. The authors have established methods for systematically studying disrupted genes in the frog Xenopus tropicalis, which has a relatively short generation time, simple genetic structure, and an easily studied externally-developing embryo. They describe their methods for creating and characterizing X. tropicalis mutations, using both forward genetics (where a mutation's effects on the embryo are first characterized, then the DNA defect is later identified) and reverse genetics (where animals carrying mutations in a known DNA sequence are first identified, and the effects of that mutation are characterized subsequently). Studies of amphibian development using tissue culture, transplantation, and molecular tools have been fundamental to understanding vertebrate early development. These studies will be greatly enriched by the addition of forward and reverse genetics to complement emerging genomic tools.