In the retina, many aspects of physiology and function are controlled by circadian (24-h) clocks, and clock malfunction impinges on information processing and cell viability. However, there is a fundamental gap in understanding how clocks control functional pathways in both healthy and diseased retinal tissue. Continued existence of this gap represents an important problem because, until it is filled, understanding of the mechanisms that link circadian clock malfunction and retinal disorders will remain largely incomprehensible. Our long-term goal is to better understand how circadian clocks control development and maintenance of visual processing in the retina. The objective of this project is to determine how circadian clocks within photoreceptors (i.e. rods, cones and opn4/melanopsin-expressing retinal ganglion cells or ipRGCs) control the development, maintenance and/or function of these cells. Our central hypothesis is that circadian clocks are present in most retinal cell types and each cell type's clock controls specific aspects of retinal development and function through a restricted clock pathway. Our central hypothesis has been formulated on the basis of our own preliminary data and recent publications in the field. The rationale for the proposed research is that by genetically silencing the clock mechanism specifically in a photoreceptor cell type, we will be able to link this cell type's clock to distinct clock pathways associated with retinal development and function. We have developed new genetically modified mouse lines and generated strong preliminary data. We will pursue two Specific Aims: 1) Characterize retina-specific and photoreceptor cell type-specific clock-deficient mouse models; and 2) Identify candidate genes and signaling pathways under the control of the photoreceptor clocks. Under the first aim, morphological analysis of retinal tissue and a variety of electrophysiological and behavioral approaches will be used in the conditional clock-deficient mouse lines already created. Under the second aim, we will combine Fluorescence-Assisted single-Cell Sorting (FACS) of cones, rods or ipRGCs, RNA sequencing and analysis, Fluorescence RNA In Situ Hybridization (FISH), and immuno-cytochemistry. In addition, we have established a plan to prioritize RNAseq data analysis to a few specific biological processes of interest. The proposed research is significant because it is expected to vertically advance and expand understanding of how circadian clocks control retinal development and function and will provide critical missing information about the clock pathways involved. Ultimately, such knowledge has the potential to increase our understanding of the general rules governing the maintenance of photoreceptors and of the events leading to their malfunction in degenerative diseases such as age-related macular degeneration.
The goal of this project is to start to establish a mechanistic model of the control of photoreceptor development, maintenance and function by circadian clocks in the mammalian retina. Dysfunction of circadian signaling in the retina has been linked to morphological defects and functional deficits, including loss of photoreceptor cells. Therefore this study may provide new insights into the mechanisms that underlie some type of retinal degeneration, such as age-related macular degeneration, so as to suggest novel treatments for these diseases.
