Using Xenopus genetics to understand vertebrate development and disease
National Institute for Medical Research
The University of New Mexico
Albuquerque, New Mexico
Harvesting medical benefits from the human genome depends on understanding the tasks specific genes perform in living organisms. Our group uses genetics to describe gene roles important for initial formation and function of tissues and organs, by comparing normal embryogenesis to mutations with disruptions in individual genes. Since all vertebrates (including humans) share most gene functions, we use an aquatic african frog, Xenopus tropicalis, as a genetic model because of its simple chromosomal structure and easily-studied eggs. As a platform for high-throughput functional genomic studies of vertebrate development and organogenesis, X. tropicalis is uniquely flexible. Like its well-studied relative Xenopus laevis, all developmental processes from fertilization onwards occur externally, where they can be observed and manipulated experimentally. Embryos are abundant (up to 5,000 or more from a single mating in X. tropicalis), and can be easily injected with synthetic mRNAs at early stages for gain-of-function assays. The fatemap of the early embryo is stable, with little cell mixing, so injected gene products can be targeted to regions that form specific organs. Tissue explants are robust and can differentiate in simple salt solutions, or can be grafted onto other embryos. Genomic resources include a high quality draft genomic sequence assembly and more than one million ESTs. Unlike the genomes of X. laevis and teleosts such as zebrafish, the X. tropicalis genome has not undergone any additional duplications since the early vertebrate common ancestor, and the genome shows a high degree of synteny (conservation of gene order along the chromosomes) with amniotes. This simpler diploid genomic structure facilitates loss-of-function genetic studies. X. tropicalis also reaches sexual maturity in as little as 3 months. We are developing procedures for rapidly mapping and cloning X. tropicalis mutations, taking advantage of the abundant meioses (up to 10,000) provided by a single mating and the ease of gynogenesis and haploid genetics. In the first cloning of an X. tropicalis mutation (Abu-Daya et al 2009), we showed that a defect in the myh6 gene (which can lead to atrial-septal defects in human) causes the non-contractile hearts in the frog muzak phenotype. This mutation ablates both myh6 protein and heartbeat, so the roles played by myosin thick filaments and contractile activity in myofibrillogenesis and cardiogenesis can be studied, helping model human disease states in both heart and skeletal muscle.
Cardiac development in the absence of heartbeat (see Figure). 3-D models of wild type (top) and muzak mutant (bottom) X. tropicalis tadpole hearts. Muzak mutant hearts, which never beat, develop with elongated ventricles and constricted endocardium, and fail to form valves and trabeculae. Atrium, green; ventricle, red; outflow tract, blue; endothelial layer (right panels) orange.