Circadian clocks control many processes important for normal functioning of living organisms, including behavior, physiology and biochemistry. These clocks are endogenous timekeeping devices and have been shown to be present in organisms ranging from bacteria to humans. Recent work has resulted in the identification of a number of genes involved in the central circadian clock and it has become clear that many of these genes are conserved in the animal kingdom. Studies of the Drosophila clock have yielded a model for the central clock mechanisms, but significant differences between the Drosophila and vertebrate clocks exist making it difficult to extrapolate from the Drosophila model to vertebrates. In this proposal, experiments are described to study the molecular mechanism of the vertebrate circadian clock within the retina of Xenopus laevis. The Xenopus retina contains many well described cellular and biochemical rhythms that can be manipulated in vitro. Furthermore, new methods for generating transgenic Xenopus embryos allow precise manipulation of gene expression within the intact retina, making this an extremely tractable system for studies of clock mechanisms.
The first aim of this application will focus on cloning and characterization of Xenopus homologs of the known clock genes.
The second aim will test the roles of Clock and bmal1 gene in the central clock mechanism by introduction of mutant versions of these genes and by altering expression levels of the endogenous clock genes in transgenic Xenopus embryos. In the third aim, several promoters that will drive rhythmic gene expression will be cloned and tested. Lines of Xenopus that will express the reporter gene luciferase under the control of these rhythmic promoters will be generated for use as a convenient measurement of rhythms in vivo.
