Organization and development of the S-cone system

National Eye Institute

Investigators

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Abstract

Short-wavelength-sensitive cones (S-cones) convey essential visual information for color, circadian rhythm, and even mood and cognitive functions, thus are critical for survival. We have long been interested in understanding how S-cones develop, connect selectively with downstream neurons, and form circuits that encode short-wavelength signals. S-cones are the minority (<10%) in most species, and therefore, they are under-studied for technical reasons. We exploit unique features of different animal models to investigate the S-cone system in the retina. We leveraged the cone-dominance of the ground squirrel (GS) retina to genetically profile S- and M-cones. High throughput single cell RNA-seq is a great tool to rapidly investigate thousands of cells for gene expression, while a Smart-Seq approach (sequencing fewer cells but with more reads/cell) may offer a higher gene detection rate. We used both techniques to profile S- and M-cones. In addition to 10X single cell sequencing (10Xseq), we developed a protocol to dissociate and isolate live S- and M-cone photoreceptors with an anti-S-opsin antibody (live labeling) to distinguish S- and M-cones after tissue dissociation. Cells were then collected and pooled using a micromanipulator followed by RNA-extraction, reverse transcription, and library preparation. With this approach we detected 44 DEGs (21 in M- and 23 in S-cones, adj. p-value < 0.05). Many of these genes are verified in 10Xseq data, in which a S-cone cluster can easily be identified. The fact that we could identify S-cones at all demonstrates the advantage of using the cone-dominant GS: recent papers that sequenced retinal single cells in other species failed to identify S-cone clusters or were only able to identify a few DEGs. We verified some of the DEGs with fluorescent in situ hybridization (FISH). This library of DEGs likely contain genes critical for S- and M-cone development, maintenance, and connectivity. We verified some of these genes by fluorescent in situ hybridization (FISH). To avoid missing any genes expressed transiently during development, we also performed Smart-Seq of developing GS S- and M-cones (P6,7,9,14,21). This experiment not only verified the results from the adult GS, but also provided a rich dataset for mining genes that regulate S- and M-cone development and synaptic formation. We will further investigate the function of these cone subtype specific genes.