Transcriptional profiling of the adult mouse retina in wild-type and Nrl deficient mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE136564 | GEO | 2020/01/08
REPOSITORIES: GEO
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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 rod photoreceptor-specific neural retina leucine zipper protein Nrl is essential for rod differentiation and plays a critical role in regulating gene expression. In the mouse retina, rods account for 97% of the photoreceptors; however, in the absence of Nrl (Nrl-/-), no rods are present and a concomitant increase in cones is observed. Using mouse GeneChips (Affymetrix), we have generated expression profiles of the wild-type and Nrl-/- retina at three time-points representing distinct stages of photoreceptor differentiation. Experiment Overall Design: Both the Nrl-/- mice and wild type controls were of a matched mixed genetic background (R1 and C57BL/6 strains).
2009-10-30 | E-GEOD-8972 | biostudies-arrayexpress
Project description:The rod photoreceptor-specific neural retina leucine zipper protein Nrl is essential for rod differentiation and plays a critical role in regulating gene expression. In the mouse retina, rods account for 97% of the photoreceptors; however, in the absence of Nrl (Nrl-/-), no rods are present and a concomitant increase in cones is observed. Using mouse GeneChips (Affymetrix), we have generated expression profiles of the wild-type and Nrl-/- retina at three time-points representing distinct stages of photoreceptor differentiation. Keywords: time course
2007-09-08 | GSE8972 | GEO
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.
2011-11-15 | GSE33674 | GEO
Project description:Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene RHO (rhodopsin). Transcriptional regulatory activity of NRL is modulated by post-translational modifications (PTMs), specially phosphorylation, and mutations in phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified Protein Kinase CK2 in NRL-enriched complexes bound to RHO promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. Bioinformatic and mass spectrometry analysis indicated potential phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding CK2 with NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells, and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene expression. In concordance, overexpression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter gene expression. RNA-seq analysis of CK2-electroporated retina revealed altered expression of many transcriptional targets of NRL. Thus, our studies demonstrate an important role of CK2 in context-dependent fine-tuning of NRL function, consequently modulating the expression of rod photoreceptor genes.
2024-04-06 | GSE171665 | GEO
Project description:Rod and cone photoreceptors in mammalian retina are generated from common pool(s) of neuroepithelial progenitors. NRL, CRX and NR2E3 are key transcriptional regulators that control photoreceptor differentiation. Mutations in NR2E3, a rod-specific orphan nuclear receptor, lead to loss of rods, increased density of S-cones, and supernormal S-cone-mediated vision in humans. To better understand its in vivo function, NR2E3 was expressed ectopically in the Nrl-/- retina, where post-mitotic precursors fated to be rods develop into functional S-cones similar to the human NR2E3 disease. Expression of NR2E3 in the Nrl-/- retina completely suppressed cone differentiation and resulted in morphologically rod-like photoreceptors, which were not functional. Gene profiling of FACS-purified photoreceptors confirmed the role of NR2E3 as a strong suppressor of cone genes and an activator of a subset of rod genes (including rhodopsin) in vivo. Ectopic expression of NR2E3 in cone precursors and differentiating S-cones of wild type retina also generates rod-like cells. The dual regulatory function of NR2E3 is not dependent upon the presence of NRL and/or CRX, but on the timing and level of its expression. Our studies reveal a critical role of NR2E3 in establishing functional specificity of post-mitotic photoreceptor precursors during retinal neurogenesis. Experiment Overall Design: We mated the Crx::Nr2e3/Nrlko mice with the Nrl::GFP transgenic mice, in which the expression of GFP is driven by an Nrl promoter. Mouse retinas were dissected at 4 wk. GFP+ photoreceptors were enriched by FACS (FACSAria, BD Biosciences, Franklin Lakes, NJ). RNA was extracted from 1~5x105 flow-sorted cells using Trizol (Invitrogen). Total RNA (40-60 ng) was used for linear amplification with Ovation Biotin labeling system (Nugen), and 2.75 ug of biotin-labeled fragmented cDNA was hybridized to mouse GeneChips MOE430.2.0 (Affymetrix) having 45,101 probesets (corresponding to over 39,000 transcripts, and 34,000 annotated mouse genes). Experiment Overall Design: Four independent samples were used at 4 weeks. We normalized Experiment Overall Design: Crx::Nr2e3/Nrl-ko-Gfp data along with Nrl-ko-Gfp 4 weeks samples ( 4 replicates, refer to Series submission GSE4051). The normalized data was then subjected to two stage analysis based on False Discovery Rate Confidence Interval (FDR-CI) for screening differentially expressed genes (24, 27) with a minimum fold change of 4.
2008-06-13 | E-GEOD-5338 | biostudies-arrayexpress
Project description:We performed non-coding transcriptional profiling to identify Nrl-dependent ncRNAs by deep sequencing non-polyadenylated RNA from the same control and Nrl-/- retinal samples. non-coding RNA profiling is a robust method for the identification of cell-type specific functional enhancer elements in the mouse retina
2020-01-08 | GSE136563 | GEO
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. Methods: Retinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl-/-) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Retinal mRNA profiles of 21-day old wild type (WT) and Nrl-/- mice were generated by deep sequencing, in triplicate, using Illumina GAIIx.
2011-10-25 | E-GEOD-33141 | biostudies-arrayexpress
Project description:The initial aim of this work was to understand the pathophysiology of Enhanced S-cone Syndrome (ESCS) that leads to retinal degeneration. Although ESCS was identified in humans decades ago and since then the causative genes have been elucidated, our understanding of the accompanying retinal degeneration is still poorly understood. Knockout of the Nrl transcription factor in mice produces a retina overpopulated with S-cone like photoreceptors along with absence of rod photoreceptors and recapitulates many of the phenotypic features seen in human ESCS patients. We wanted to study this murine model through a combinatorial genetic and structural approach to improve understanding of the disease process that leads to photoreceptor degeneration and blindness, potentially guiding future therapies. By using RNA-Sequencing (RNA-Seq) to examine mature wild type and Nrl-/- ocular tissues, we were able to determine global changes in their transcriptomes. The massively parallel RNA-sequencing experiment revealed new insight into the transcriptional mis-regulation in the ESCS murine model and revealed a change in gene expression in putative proteins involved in photoreceptor phagocytosis. Key photoreceptor ligands necessary for phagocyotsis, Tub and Tulp1, were down-regulated in the Nrl-/- retina. Down regulation of key retinoid metabolic genes, coupled with down-regulation of Tub and Tulp1, suggested a potential mechanism involving defective phagocytosis underlies the photoreceptor degeneration seen in ESCS. We report RNA-Seq experiments of whole eye and retinal tissues from wild type and Nrl transcription factor knockout mice on the C57BL/6 background. Examine two different ocular tissues in two mouse models of varying photoreceptor populations
2011-06-07 | E-GEOD-29752 | biostudies-arrayexpress
Project description:The basic motif-leucine zipper (bZIP) transcription factor NRL determines rod photoreceptor cell fate during retinal development, and its loss leads to cone-only retina in mice. NRL works synergistically with homeodomain protein Cone-Rod Homeobox and other regulatory factors to control the transcription of most genes associated with rod morphogenesis and functional maturation, which span over a period of several weeks in the mammalian retina. We predicted that NRL gradually establishes rod cell identity and function by temporal and dynamic regulation of stage-specific transcriptional targets. Therefore, we mapped the genomic occupancy of NRL at four stages of mouse photoreceptor differentiation by CUT&RUN analysis. Dynamics of NRL binding revealed concordance with the corresponding changes in transcriptome of the developing rods. Notably, we identified c-Jun proto-oncogene as one of the targets of NRL, which could bind to specific cis-elements in the c-Jun promoter and modulate its activity in HEK293 cells. Coimmunoprecipitation studies showed the association of NRL with c-Jun, also a bZIP protein, in transfected cells as well as in developing mouse retina. Additionally, shRNA-mediated knockdown of c-Jun in the mouse retina in vivo resulted in altered expression of almost 1000 genes, with reduced expression of phototransduction genes and many direct targets of NRL in rod photoreceptors. We propose that c-Jun-NRL heterodimers prime the NRL-directed transcriptional program in neonatal rod photoreceptors before high NRL expression suppresses c-Jun at later stages. Our study highlights a broader cooperation among cell-type restricted and widely expressed bZIP proteins, such as c-Jun, in specific spatiotemporal contexts during cellular differentiation.
2022-09-23 | GSE197420 | GEO