Identification of genes with enriched expression in early developing mouse cone photoreceptors
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ABSTRACT:
PROVIDER: PRJNA547437 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9208360.fastq.gz | Fastqsanger.gz | |||
| SRR9208361.fastq.gz | Fastqsanger.gz | |||
| SRR9208362.fastq.gz | Fastqsanger.gz | |||
| SRR9208359.fastq.gz | Fastqsanger.gz |
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Project description: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.
2019-06-06 | GSE132272 | GEO
Project description:We determined the accessible genome of postmitotic cone photoreceptors in the central region of the mouse retina on postnatal days P3, P6, and P10. At eachtimepoint we isolated GFP-labeled cells from three different Chrnb4-GFP mice (biological triplicates) using fluorescence-activated cell sorting. We then acquired reads of regions of accessible DNA of the sorted cones using ATAC sequencing. Our data set contained 9 samples.
2017-11-07 | GSE97535 | GEO
Project description:We determined the transcriptomes of postmitotic cone photoreceptors in the central region of the mouse retina every day between birth (P0) and eye opening (P12). At each postnatal day we isolated GFP-labeled cells from three different Chrnb4-GFP mice (biological triplicates) using fluorescence-activated cell sorting. We then acquired the transcriptomes of the sorted cones using next generation RNA sequencing. Our data set contained 39 transcriptomes.
2017-11-07 | GSE97534 | GEO
Project description:RNA sequencing and ATAC sequencing of developing cone photoreceptors
| PRJNA382178 | ENA
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.
2017-10-31 | GSE86155 | GEO
Project description: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.
| S-EPMC6607928 | biostudies-literature
Project description:Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor NRL. The loss of Nrl in mice (Nrl-/-) results in a retina with predominantly S-opsin containing cones that exhibit molecular and functional characteristics of WT cones. Here we report that Nrl-/- retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by four months of age, resulting in a thinned but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic ERG. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of stress response and inflammation genes, implying their involvement in cone death. The Nrl-/- retina illustrates the long-term viability of cones in the absence of rods and may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula. Targets were generated from a pair of retinas (one Nrl-/- mouse) per biological replicate. Four biological replicates were generated for each of the five aging timepoints (1, 2, 4, 6, and 10 months post natal).
2011-11-15 | E-GEOD-33674 | biostudies-arrayexpress
Project description:The outer segments of cones serve as light detectors for daylight color vision, and their dysfunction leads to human blindness conditions. We show that the cone-specific disruption of DiGeorge Syndrome Critical Region Gene 8 (DGCR8) in adult mice led to the loss of miRNAs and the loss of outer segments, resulting in photoreceptors with significantly reduced light responses. Using next-generation sequencing of RNA from isolated wild type P60 cones, we determine the most highly expressed miRNAs as candidates for controlling outer segment maintenance. The expression pattern of miRNAs was highly uneven, with a single miRNA, miR-182, representing 64% of all miRNA expressed in cones. Re-expression of miR-182 and miR-183 (third most abundant miRNA) prevented outer segment loss. These miRNAs were also necessary and sufficient for the formation of inner segments, connecting cilia and short outer segments, as well as light responses in stem-cell-derived retinal cultures. Our results show that miR-182- and miR-183-regulated pathways are necessary for cone outer segment maintenance in vivo and functional outer segment formation in vitro. microRNA profile in cone photoreceptors from P60 D4-cre/Ai9 tdTomato mice representing wild type control.
2014-06-20 | E-GEOD-58500 | biostudies-arrayexpress
Project description:To define molecular mechanisms underlying rod and cone differentiation, we generated H9 human embryonic stem cell line carrying a GFP reporter that is controlled by the promoter of cone-rod homeobox (CRX) gene, the first known marker of post-mitotic photoreceptor precursors. CRXp-GFP reporter in H9 line replicates endogenous CRX expression when induced to form self-organizing 3-D retina-like tissue. We define temporal transcriptome dynamics of developing photoreceptors during the establishment of cone and rod cell fate. Our studies provide an essential framework for delineating molecules and cellular pathways that guide human photoreceptor development and should assist in chemical screening and cell-based therapies of retinal degeneration.
2015-08-03 | GSE67645 | GEO
Project description:The macula of the retina has a high ratio of cones to rods and is critical for central vision and visual acuity. Here we report the generation, transcriptome profiling, and functional validation of single cells from cone-enriched human retinal organoids differentiated from hESCs. Single-cell RNA-seq of 8-month retinal organoids identified clusters of cone and rod photoreceptors and confirmed the cone enrichment initially revealed by immunostaining. Collectively, we have established cone-enriched retinal organoids and a reference of transcriptomes that are rich resources for retinal studies.
2019-04-25 | GSE119343 | GEO