Retinal sensitivity to light is controlled both by the level of illumination (light adaptation at the photoreceptor and network levels) and by an endogenous circadian clock. Considerable evidence suggests that broad and coordinated programs of gene expression underlie the changes in sensitivity driven by light and by the circadian clock, respectively, and that these programs overlap or converge in common pathways. Although there are sporadic examples of retinal transcripts regulated by light, a circadian clock, or both, there is as yet no comprehensive view of retinal gene expression in a natural light-dark cycle or in constant darkness. Microarray technology, developed for massively parallel analysis of gene expression, now permits detailed investigation of biological processes too complex for piecemeal approaches. Described here is a proposal to use cDNA microarrays to analyze gene expression in the mouse retina on a genome-wide scale. Two kinds of microarrays will be used for the analysis. The first contains 40,000 unique, fully sequenced mouse cDNAs, representing on the order of 50% of all mouse genes. The second is a custom microarray which will contain a few-thousand unique retinal cDNAs that encode secreted and transmembrane proteins, to be obtained in a large-scale """"""""secretion trap"""""""" screen. This cDNA set will be highly enriched for molecules involved in intercellular communication, such as neuropeptides, receptors, and ion channels, known and unknown, likely to play a role in the control of retinal sensitivity. Retinal gene expression will be monitored at 4-hour intervals over two days, both from mice kept in a 24 hour light dark cycle and from mice kept in constant darkness. The incorporation of both conditions into a single experiment is a key-feature of the proposal. This experiment should provide comprehensive, parallel views of the dynamics of (i) diurnal gene expression over the course of a natural light-dark cycle and (ii) circadian clock-regulated gene expression. The design will permit transcripts regulated by light to be distinguished from those regulated by a circadian clock, and it should provide insight into how the natural temporal pattern in a light-dark cycle arises from the interaction of a light-driven transcriptional program with a clock-driven one. Overlap between the two programs will likely highlight transcripts involved in changes in sensitivity of the retina to light. It is likely that a comprehensive view of natural retinal gene expression programs in light and darkness will provide deep insights into retinal physiology and open new avenues of inquiry regarding the coordinated responses of the retina to light. Aberrant transcriptional events are known to underlie light-induced photoreceptor degeneration, and knowledge of retinal gene expression programs will almost certainly stimulate progress in understanding retinal diseases such as age-related macular degeneration and retinitis pigmentosa.
