Dataset Information

Identification of Genes With Enriched Expression in Early Developing Mouse Cone Photoreceptors.

ABSTRACT: Purpose:The early transcriptional events that occur in newly generated cone photoreceptors are not well described. Knowledge of these events is critical to provide benchmarks for in vitro-derived cone photoreceptors and to understand the process of cone and rod photoreceptor diversification. We sought to identify genes with differential gene expression in embryonic mouse cone photoreceptors. Methods:The specificity of expression of the LHX4 transcription factor in developing cone photoreceptors was examined using immunofluorescence visualization in both mouse and chicken retinas. A LHX4 transgenic reporter line with high specificity for developing mouse cone photoreceptors was identified and used to purify early-stage cone photoreceptors for profiling by single-cell RNA sequencing. Comparisons were made to previous datasets targeting photoreceptors. Results:The LHX4 transcription factor and a transgenic reporter were determined to be highly specific to early developing cone photoreceptors in the mouse. Single-cell transcriptional profiling identified new genes with enriched expression in cone photoreceptors relative to concurrent cell populations. Comparison to previous profiling datasets allowed for further characterization of these genes across developmental time, species, photoreceptor type, and gene regulatory network. Conclusions:The LHX4 gene is highly enriched in developing cone photoreceptors as are several new genes identified through transcriptional profiling, some of which are expressed in subclusters of cones. Many of these cone-enriched genes do not show obvious de-repression in profiling of retinas mutant for the rod-specific transcription factor NRL, highlighting differences between endogenous cones and those induced in NRL mutants.

SUBMITTER: Buenaventura DF

PROVIDER: S-EPMC6607928 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Identification of Genes With Enriched Expression in Early Developing Mouse Cone Photoreceptors.

Buenaventura Diego F DF Corseri Adrianne A Emerson Mark M MM

Investigative ophthalmology & visual science 20190701 8

<h4>Purpose</h4>The early transcriptional events that occur in newly generated cone photoreceptors are not well described. Knowledge of these events is critical to provide benchmarks for in vitro-derived cone photoreceptors and to understand the process of cone and rod photoreceptor diversification. We sought to identify genes with differential gene expression in embryonic mouse cone photoreceptors.<h4>Methods</h4>The specificity of expression of the LHX4 transcription factor in developing cone ...[more]

PMID: 31260032

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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

Identification of Genes With Enriched Expression in Early Developing Mouse Cone Photoreceptors.

Publications

Identification of Genes With Enriched Expression in Early Developing Mouse Cone Photoreceptors.

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