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Nicola de Prisco, Salvatore Botta, Elena Marrocco, Martina Sofia, Annamaria Carissimo, Settimio Rossi, Carlo Gesualdo, Francesca Simonelli, Enrico Maria Surace; Transcriptional blueprints of rod and cones. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1185.
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© ARVO (1962-2015); The Authors (2016-present)
Rods and Cones convert light stimuli in information signaling for visual perception. Specific gene mutations primarily affecting both photoreceptors types lead to retinal dystrophies. Photoreceptors are generated from common precursor cells and their differentiation program is strongly imposed by transcription factors (TF).The aim is to investigate the transcription regulation of rod- and cone-specific networks controlling their identities in adult retina. Retrieving transcription factor sets has biological relevance, and may be instrumental to design gene-based and/or regenerative therapeutic strategies.
We developed a method to isolate rods and cones from the same retinal samples by fluorescence-based sorting using Adeno-associated viral (AAV) vectors. We designed a "double fluo" expression cassette encoding for mCherry and eGFP transgenes under the control of rod and cone GRK1 and GNAT1 rod-specific promoter elements, respectively. Adult pigs were subretinally injected with AAV 2/8 GRK1-mCherry_GNAT1-eGFP, at the dose of 1x1012 GC per eye (n=3), and sacrificed 15 days after injection. Histological and FACS analysis were performed to isolate rods and cones evaluate the reliability of the method. Furthermore, we evaluated, on sorted populations, rods and cone specific transcriptomes by RNA sequencing (RNA-seq) using Illumina Hiseq 1500 (n=3).
Histological analysis showed expression of both mCherry and eGFP in rods, whereas cones showed exclusive expression of mCherry. Furthermore, immunofluorescence (IF) with cone arrestin showed exclusive co-localization only with mCherry signal (cones). qRT-PCR on FACS sorted photoreceptors confirmed rod/cones specific identities: RHO, NRL, GNAT1expression in rods; S-opsin, M-opsin, GNAT2 in cones and the absence of cross-contaminations. RNA-seq differential expression gene (DEGs) analysis showed a total differential expression of 905 genes in rods compared with cones, out of which 127 DEGs were up regulated and 778 genes down regulated.
We showed that the “double fluo” provided a robust method to isolate and cross-compare rod from cone photoreceptors in adult large animal model Sus scrofa, upon photoreceptor somatic gene transfer via AAV vectors. Furthermore, RNA-seq data sets analysis showed that the cone and rod specific transcriptomes differ quantitatively by 905 transcripts. We are currently focusing on TF sets whose expression determine rod and cone identity maintenance.
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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