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Molecular dissection of cone photoreceptor-enriched genes encoding transmembrane and secretory proteins.

ABSTRACT: Cone photoreceptors mediate color perception and daylight vision through intricate synaptic circuitry. In most mammalian retina, cones are greatly outnumbered by rods and exhibit inter-dependence for functional maintenance and survival. Currently, we have limited understanding of cone-specific molecular components that mediate response to extrinsic signaling factors or are involved in communication with rods and other retinal cells. To fulfill this gap, we compared the recently-published transcriptomes of developing S-cone-like photoreceptors from the Nrl-/- mouse retina with those of rods and identified candidate genes responsible for cone cell functions and communication. We generated an in silico expression profile of 823 genes that encode candidate transmembrane and secretory proteins and are up-regulated in Nrl-/- cone photoreceptors compared to wild type cones. In situ hybridization analysis validated high expression of seven of the selected candidate genes in the Nrl-/- retina. To examine their relevance to cone function, we performed in vivo knockdown of Epha10 in the Nrl-/- retina and demonstrated aberrant morphology and mislocalization of the photoreceptor cell bodies. Thus, the receptor tyrosine kinase Ephrin type-A receptor 10 appears to influence cone morphogenesis. Our studies reveal novel cone-enriched genes involved in interaction of cones with other retinal cell types and provide a framework for examining molecular pathways associated with intercellular communication.

SUBMITTER: Papal S

PROVIDER: S-EPMC6239890 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Molecular dissection of cone photoreceptor-enriched genes encoding transmembrane and secretory proteins.

Papal Samantha S Monti Christopher E CE Tennison Mackenzie E ME Swaroop Anand A

Journal of neuroscience research 20180927 1

Cone photoreceptors mediate color perception and daylight vision through intricate synaptic circuitry. In most mammalian retina, cones are greatly outnumbered by rods and exhibit inter-dependence for functional maintenance and survival. Currently, we have limited understanding of cone-specific molecular components that mediate response to extrinsic signaling factors or are involved in communication with rods and other retinal cells. To fulfill this gap, we compared the recently-published transcr ...[more]

PMID: 30260491

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Project description:Cone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2TαCP:EGFP) zebrafish. Electropherograms confirmed downstream isolation of high-quality RNA with RNA integrity number (RIN) >7.6 and RNA concentration >5.7 ng/µl obtained from both populations. Reverse Transcriptase-PCR (RT-PCR) confirmed that the EGFP-positive cell populations express known genetic markers of cone photoreceptors that were not expressed in the EGFP-negative cell population. This work is an important step towards the identification of cone photoreceptor-enriched genes, protein and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness. In order to analyse and sort samples by flow cytometry, values for FSC and SSC were displayed in a logarithmic scale, as this is normally the default starting display. This allowed for the identification of different sub-populations of cells present in the retina, which were mixed with unwanted cell debris and cell fragments. Since there were multiple cell populations, different levels of auto-fluorescence were thus successfully detected. It was therefore important to change the strategy and display side scatter and fluorescence characteristics of control and EGFP samples, which ultimately allowed the identification of the extremely well-defined population of EGFP-cone photoreceptors. This improved sorting process minimised RNA degradation. The purified EGFP+ cone photoreceptors represent ~5% of the original dissociated population, which is consistent with humans, wherein the total number of cones (6 million) in the retina is approximately 20 times the one of rods (120 million) (Williamson and Cummins 1983). This work allows high-quality RNA to be obtained from sorted-adult cone photoreceptors. RNA integrity is assessed via 28S and 18S rRNA (Imbeaud et al 2005), and our electropherogram results demonstrate production of high-quality RNA with two clearly visible ribosomal peaks (28S and 18S) from EGFP-sorted cones. In addition, the RNA Integrity Number (RIN), an algorithm for assigning integrity values to RNA based on 28S to 18S rRNA ratios (Sambrook et al 1989; Imbeaud et al 2005; Schroeder et al 2006), had a value of 7.6, higher than the minimum-required 7.0. RNA yields of 5.7 ng/µl were relatively high and sufficient for downstream profiling. RT-PCR confirmed expression of the cone specific gene gnat2, and promoter fragment TαC, but not the retinal pigment epithelium specific gene rpe65 in flow cytometry-sorted GFP-positive photoreceptors (GFP+ cells). rpe65 was neither present in flow cytometry-sorted GFP-negative cones (GFP- cells) as this gene is only expressed in the retinal pigment epithelium (RPE). This study therefore permits the identification of cone photoreceptor-enriched genes, protein and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness.

Dataset Information

Molecular dissection of cone photoreceptor-enriched genes encoding transmembrane and secretory proteins.

Publications

Molecular dissection of cone photoreceptor-enriched genes encoding transmembrane and secretory proteins.

Similar Datasets

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