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Andrew Smith, Conor Daly, Ross F Collery, Vijender Chaitankar, Matthew Brooks, Tiziana Cogliati, Anand Swaroop, Breandan N Kennedy; Identification and Characterisation of Novel Cone Photoreceptor-Enriched Factors Conserved in Zebrafish, Mouse and Human. Invest. Ophthalmol. Vis. Sci. 2016;57(12):572.
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© ARVO (1962-2015); The Authors (2016-present)
The inability of the retina to detect/transmit light-triggered signals due to dysfunction or death of photoreceptor cells is manifested in incurable blinding conditions. The purpose of this research is to identify novel cone-enriched factors conserved in zebrafish, mouse and human macula and characterise their involvement in cone development, function and survival.
Microarray analysis of cone photoreceptors of TG(3.2TαCP:EGFP) zebrafish and RNAseq data of photoreceptors of the cone dominant Nrl -/- mouse and rod dominant Nrl-GFP mouse were compared to identify conserved cone-enriched factors. Human retina and macular RNAseq data was analysed to confirm evolutionary conservation. Genes were ranked on cone enrichment. PCR was performed in wild type (Tü) zebrafish eyes at developmental stages: 3, 4 & 5 days post-fertilization (dpf). Fluorescent in situ hybridisation (FISH) of high-ranking factors was performed on developing and adult TG(3.2TαCP:EGFP) zebrafish. Gene knockdown of clul1, one of these high-ranking genes was performed using morpholino technology. Morphants visual behaviour was assessed using the optokinetic response, and retinal integrity examined using light microscopy. We are currently developing CRISPR-Cas9 mediated gene knockout for our 5 highest-ranking genes.
Upon ranking based on enrichment in zebrafish and mice, twentyseven novel, conserved, cone-enriched genes were selected for further analysis. These factors were conserved and enriched in human retina. High-ranking genes were confirmed to be present during development at 3, 4 and 5 dpf in wildtype (Tü) zebrafish, with the genes clul1 and es1 demonstrating an incremental increase in expression. FISH revealed the gene clul1 was specifically expressed in adult zebrafish cone photoreceptors. Knockdown of the gene clul1 resulted in a significant impairment of visual behaviour without substantial morphological differences in the retina.
Twentyseven novel, conserved cone photoreceptor-enriched factors were identified and spatiotemporal expression elucidated. Knockdown of clul1 indicates it is not required for normal cone photoreceptor morphogenesis but is required for cone photoreceptor mediated visual function. Cone photoreceptor specific expression is aiding the development of CRISPR knockout models to elucidate the role these factors play in cone morphogenesis and function.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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