exquisitely sensitive to the time of day. This ability to temporally regulate physiology is not due simply to changes in light and dark, but is controlled by a circadian clock, an intrinsic component of vertebrate retinas. Many aspects of physiology including retinomotor movements, neuromodulator synthesis, and gene expression are under circadian control, but the molecular mechanism of this regulation is not known. However, a common theme to clock function seems to be regulation at the level of gene expression. The overall goal of this research plan is to define the molecular mechanisms of circadian clock regulation of gene expression in the vertebrate retina. The applicant has chosen to focus this research on the clock that was recently demonstrated in retinal photoreceptors in the African clawed frog, Xenopus laevis. The Xenopus retina is an outstanding system in which to study vertebrate clock mechanisms because the clock is functional and entrainable in cultured preparations of Xenopus retinas or photoreceptor layers. This proposal focuses on two genes that have recently been shown to be regulated by this retinal clock: tryptophan hydroxylase (TPH) and rhythmic messenger 1 (RM1). The first specific aim addresses the newly identified photoreceptor-specific message, RM1, which exhibits extremely high amplitude rhythmic expression. These studies will include basic characterization of RM1, including sequence and gene structure, localization, and other studies aimed at an understanding of RM1 function in the retina.
The second aim i s to examine possible effectors involved in acute regulation of TPH and RM1 message levels in the retina, including such things as light and dopamine. Development of a transfectable culture system with an intact circadian clock is the goal of specific aim 3. This is followed by the fourth specific aim, which is to define promotor elements and factors involved in the transcriptional regulation of gene expression by the clock. These studies will provide a better understanding of the molecular mechanisms of the retinal circadian clock which will have profound impact on retinal physiology as a whole.
