The long-term goal of this research project is to understand cellular and molecular mechanisms of the circadian system in vertebrate retinal photoreceptors. Circadian rhythms are observed in a variety of biological processes, and disruption of the circadian rhythm is related to physiological diseases and psychological disorders. Since a number of retinal processes show circadian rhythms, fulfillment of this research project will also contribute to prevention of retinal diseases. Several putative molecular components of the circadian system have been identified recently in vertebrates, and the functional roles of those components are of fundamental interest in circadian research.
The specific aim of this proposed research is to develop an experimental system for rapid analysis of photoreceptor gene functions and to apply it to circadian research. The isolated Xenopus retinal photoreceptor layer will be used because this photoreceptor preparation eliminates complex interactions from other cell types. This enables us to investigate effects of experimental treatments and output measurements from a nearly pure population photoreceptor cells. In addition, cellular mechanisms of photic entrainment of the circadian oscillator can be investigated because this system is directly photosensitive. We will investigate whether morpholino antisense oligonucleotide techniques are useful for the study of photoreceptor gene functions. Morpholino oligos have overcome major problems encountered in other antisense techniques developed earlier and have been proven to be a powerful tool to study gene functions in research area such as developmental biology. In addition, a recently developed carrier molecule, ethoxylated polyethylenimine (EPEI), has enabled application of the morpholino technique to several cultured cell lines and primary cultured cells. We will employ western blotting analysis to examine specificities and functional effect of morpholino oligos on translation of a target mRNA and fluorescence confocal microscopy analysis to examine the delivery efficiency of morpholino oligos into photoreceptor cells. We will also examine the strength of the morpholino technique in circadian research by investigating functional roles of CLOCK and CRYPTOCHROMES in the regulation of a photoreceptor circadian rhythm. Since various genome projects have provided tremendous amount of gene sequence data, the next step is identification of functions of those genes. Successful completion of this proposed research will dramatically accelerate functional genomics research in vertebrate photoreceptors.
