Ethan D. Buhr, PhD - US grants

The retina is not a static tissue, but a dynamic population of cells whose function is modulated through the day. The circadian regulation of the retina is evident from the level of gene transcription to the connectivity of cell networks. The circadian regulation of gene transcription includes genes involved in cellular metabolism and synaptic transduction. This daily rhythmicity of retinal physiology persists in the absence of light and in many cases even when the retina is cultured in isolation. This demonstrates that the circadian rhythms of the retina are controlled by local circadian clocks. Intriguingly, these retinal circadian clocks are also able to synchronize, or entrain, to light:dark cycles when cultured in a dish. These are the only known mammalian circadian oscillators which exhibit this light sensitivity. The identity of the cell type or types which exhibits robust circadian gene expression and susceptibility to light cycles is still unknown. Using a bioluminescent reporter of the circadian clock gene, Per2, the phase, amplitude and period of the circadian clocks present in many mouse tissues can be measured in organotypic tissue culture as a representation of the animal's endogenous rhythms prior to culture of the tissue. This technique has confirmed that the cells in the mouse retina which express the circadian clock are viable in culture and offers a unique opportunity to determine their identity. Using immunohistochemistry, the retinal cells which are strongly expressing PER2 as synchronized by a light:dark cycle will be identified by co-labeling with markers for retinal cell types. Also, mutations in genes which reduce or remove the function of key retinal cell types will be tested for their influence on retinal circadian rhythms. Because the cultures are still light entrainable, this also offers an opportunity to determine the wavelength of light to which the rhythms are most sensitive, which provides insight into the photoreceptive molecule. In addition, tissue culture allows for the use of pharmacology to test the cellular pathways involved in photo transduction. Inhibitors of known visual second messenger pathways and other circadian regulatory pathways will be used in the presence of light:dark cycles to test their influence on synchronization to light. This will be done with complementary genetic mutations of known retinal cell types as described above. Finally, the ontogeny of retinal circadian clocks and their light sensitivity will be assessed in mouse pups in order to describe the onset of circadian rhythmicity in the developing retina. These experiments will provide answers to fundamental questions in regular retinal anatomy and physiology. They will also open up future avenues of research and potential therapeutic regulation of retinal function.

Project Summary/Abstract The 24 hour solar cycle is one of the most predictable, yet potentially damaging, aspects of life on Earth. Because of this, most animals utilize molecular circadian clocks which anticipate the rising sun and are prepared when the light is at its most UV-enriched. There are various ways in which this is achieved at the behavioral, anatomic, and physiologic levels. OPN5 is an opsin which is maximally sensitive to short- wavelength light. Recently, we found that OPN5 is a critical element in the mammalian retina?s ability to synchronize its local circadian rhythms to light cycles. The skin and the cornea are also daily exposed to sunlight in most animals, and both tissues contain robust, autonomous circadian clocks. We and others have observed the expression of OPN5 in both of these tissues. This proposal aims to characterize the role of these extraretinal OPN5 photoreceptors on the circadian rhythms of these tissues. Specifically, the aim of this project is to identify the specific cells in the skin and cornea that express OPN5, characterize their photic response, characterize the mechanism these cells use to disseminate photic information, and characterize the circadian nature of the light response that these tissues display. Using a mouse line from which a circadian luciferase reporter can be measured as a representation of clock gene expression, we have demonstrated that clocks in both skin and corneas can be synchronized by light and that this circadian photoreception is regulated by OPN5. This circadian reporter will be used to monitor tissues in culture, and a mouse line which includes a fluorescent protein only in OPN5 expressing cells will allow us to ascertain the precise identity of the extraretinal photoreceptor cells. Lastly, this proposal seeks to link the expression of extraocular photoreception to potential health risks. Skin, in particular, has been shown to have precise times of day when it is most vulnerable to UV light for development of skin cancer and erythema. This proposal will examine the role OPN5 plays in controlling timing of cell division, acute responses to UV light, the sensitivity during development, and the global transcriptional landscape of the skin and cornea. By analyzing the molecular state of tissues exposed to specific light conditions at various times of day we will gain a better understanding of the ways mammalian cells cope with high energy light, and we will begin to understand the way they use OPN5 to regulate circadian clocks to anticipate photic changes in their environment.