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.
Highlights
Genetic studies have arguably contributed more to our understanding of animal development than any other approach
Others, such as the cyd vicious phenotype, do not appear to resemble known mutations, and confirm that X. tropicalis forward genetics can be a highly effective tool for discovery of novel gene functions. While this pilot screen clearly demonstrates the feasibility of the approaches we have taken, there are a number of factors to consider for future screens
Gynogenetic Forward Screen Limitations In our screen strategy, in vitro mutagenesis followed by gynogenesis and morphological inspection was employed to perform rapid surveillance of postneurulation phenotypes
Summary
Genetic studies have arguably contributed more to our understanding of animal development than any other approach. Invertebrate genetic models have helped identify the transcriptional control networks underpinning the basic animal body plan [1,2]; among vertebrates, the mouse has been an especially powerful tool for genetic studies since the development of gene targeting [3,4], but forward screens for embryonic mutations in this system are challenging due to the intrauterine mode of development. Where duplicated genes have not diverged functionally, they may be inaccessible to forward genetic screens. While it is not clear whether an increased redundancy has been retained relative to other vertebrates, subfunctionalization and neofunctionalization in teleosts have resulted in a significant degree of reorganization of genetic roles [11]. Since teleosts are the most evolutionarily diverse vertebrates, systematic comparison with canonical tetrapod genomes is essential for understanding gene function in vertebrate development
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