In vivo Studies of Vertebrate Circadian Clock Genes

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DESCRIPTION (provided by applicant): 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. In humans, disruptions of these clocks occur during jet lag, shift work and in some sleep disorders. 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 within the animal kingdom. Although a general molecular clock model has been proposed, many of the steps within this model are still not well understood. 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 mechanism in vivo. The first and second aims of this proposal will focus on in vitro studies of two aspects of cryptochrome function. These proteins are critical components of the negative feedback loop of the clock and we will analyze how the cryptochromes move from the cytoplasm to the nucleus (aim 1) and how they cause repression of the transcriptional apparatus once they are in the nucleus (aim 2). The third aim will test the function of the cryptochromes in vivo by introduction of mutant versions and/or by altering expression levels of these genes in transgenic Xenopus embryos. In the fourth aim, we will make specific "molecular lesions" that disrupt the clock in specific cell types within the retina. This will be done by overexpressing mutant clock genes under the control of several different cell-specific promoters in order to address how individual clocks in the different cell types orchestrate tissue-level rhythmicity. These experiments take advantage of the strengths of the Xenopus system which allow mechanistic studies to be done that are difficult to do in other vertebrate systems. Because these clocks are conserved, information gained from these studies will provide insight into vertebrate clocks in general, including those in humans.