ABSTRACT: Expression analysis of multiple mouse strains across two developmental timepoints to determine both strain specific and temporal expression of genes in the mouse retina Overall design: A total of 29 retinas were collected from 15 mice of each strain at similar time points during the day. Equimolar amounts of RNA isolated from ten retinas were pooled into three separate pools from each strain and time point, then hybridized to Affymetrix Mouse 420A 2.0 GeneChip arrays.
Project description:Expression analysis of multiple mouse strains across two developmental timepoints to determine both strain specific and temporal expression of genes in the mouse retina A total of 29 retinas were collected from 15 mice of each strain at similar time points during the day. Equimolar amounts of RNA isolated from ten retinas were pooled into three separate pools from each strain and time point, then hybridized to Affymetrix Mouse 420A 2.0 GeneChip arrays.
Project description:Proteomic experiments were performed on the outer segment (OS) and the remaining retina (RR). For the OS proteomics, three biological replicates were performed for mouse at the age of P15, which included 24, 26 and 26 retinas respectively. We also performed the OS proteomic experiment on 40 retinas from mice at age P13. For the RR proteomic experiments, two biological replicates were performed independently using 30 and 30 retinas respectively.
Project description:Pigment regeneration is critical for the function of cone photoreceptors in bright and rapidly-changing light conditions. This process is facilitated by the recently-characterized retina visual cycle, in which Müller cells recycle spent all-trans-retinol visual chromophore back to 11-cis-retinol. This 11-cis-retinol is oxidized selectively in cones to the 11-cis-retinal used for pigment regeneration. However, the enzyme responsible for the oxidation of 11-cis-retinol remains unknown. Here, we sought to determine whether retinol dehydrogenase 10 (RDH10), upregulated in rod/cone hybrid retinas and expressed abundantly in Müller cells, is the enzyme that drives this reaction. We created mice lacking RDH10 either in cone photoreceptors, Müller cells, or the entire retina. In vivo electroretinography and transretinal recordings revealed normal cone photoresponses in all RDH10-deficient mouse lines. Notably, their cone-driven dark adaptation both in vivo and in isolated retina was unaffected, indicating that RDH10 is not required for the function of the retina visual cycle. We also generated transgenic mice expressing RDH10 ectopically in rod cells. However, rod dark adaptation was unaffected by the expression of RDH10 and transgenic rods were unable to use cis-retinol for pigment regeneration. We conclude that RDH10 is not the dominant retina 11-cis-RDH, leaving its primary function in the retina unknown. Overall design: Retinas from rd7 and wild-type (C57BL/6J) mice at age 21 days were harvested. Two biological replicates per strain were collected. Each replicate consisted of 8 retinas total from two female and two male mice. RNA was extracted with Trizol, polyA-selected, and processed for mRNA-seq. All four samples were sequenced on a single lane of Illumina HiSeq 2000 (1x50 bp). Note that Nr2e3 transcript levels are higher in the rd7 mutant, as previously reported (Chen et al 2006 Hum Mol Genet 15(13):2146-56).
Project description:Transcriptional profiling of mouse retina comparing naïve mouse and experimental autoimmune uveoretinitis (EAU)-developing mouse. EAU was induced by immunization of retinal autoantigens in adjuvants. Retinas were harvested 14 days after immunization. Overall design: Two-condition experiment, Naïve retinal cells vs. EAU-developing mouse retinal cells on day 14. 3 control, 3 EAU-developing, independently harvested.
Project description:We have analyzed the transcript expression in different LCM-dissected cell layers isolated from mouse retinas adapted to light or dark in order to identify transcripts potentially targetted by retinal microRNAs which are regulated in response to light treatment Overall design: Retinas were isolated from adult mice adapted to light or dark following LCM dissection of photoreceptors (ONL+OS/IS), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL) and whole retina samples. Experiments were performed in duplicates. Total RNA was extracted and hybridization on Affymatrix array was performed.
Project description:A fundamental challenge in genomics is to map DNA sequence variants onto changes in gene expression. Gene expression is regulated by cis-regulatory elements (CREs, i.e., enhancers, promoters, and silencers) and the trans factors (e.g., transcription factors) that act upon them. A powerful approach to dissecting cis and trans effects is to compare F1 hybrids with F0 homozygotes. Using this approach and taking advantage of the high frequency of polymorphisms in wild-derived inbred Cast/EiJ mice relative to the reference strain C57BL/6J, we conducted allele-specific mRNA-seq analysis in the adult mouse retina, a disease-relevant neural tissue. We found that cis effects account for the bulk of gene regulatory divergence in the retina. Many CREs contained functional (i.e., activating or silencing) cis-regulatory variants mapping onto altered expression of genes, including genes associated with retinal disease. By comparing our retinal data with previously published liver data, we found that most of the cis effects identified were tissue-specific. Lastly, by comparing reciprocal F1 hybrids, we identified evidence of imprinting in the retina for the first time. Our study provides a framework and resource for mapping cis-regulatory variants onto changes in gene expression, and underscores the importance of studying cis-regulatory variants in the context of retinal disease. Retinas from four classes of 8 week old male mice were collected: F0 C57BL/6J (B6), F0 Cast/EiJ (Cast), F1 B6xCast, and F1 CastxB6. Three replicates per class were generated. Each replicate consisted of a pool of 6-8 retinas. The mRNA-seq was conducted with paired-end 2x101 sequencing on the Illumina HiSeq 2000 platform. One lane of sequencing was run for all twelve samples. An additional lane of sequencing was run for the six F1 samples.
Project description:Purpose: Investigate the molecular determinants of retinal regeneration in adult vertebrates by analyzing the gene expression profiles of control and post-lesion retina of adult zebrafish, a system that regenerates following injury. Methods: Gene expression profiles of zebrafish retina and brain were determined with DNA microarray, RT-PCR, and real-time quantitative PCR analyses. Damaged retinas and their corresponding controls were analyzed 2-5 days post-lesion (acute injury condition) or 14 d post-lesion (cell regeneration condition). Results: Expected similarities and differences in the gene expression profile of zebrafish retina and brain were observed, confirming the applicability of the gene expression techniques. Mechanical lesion of retina triggered significant, time-dependent changes in retinal gene expression. The induced transcriptional changes were consistent with cellular phenomena known to occur, in a time-dependent manner, subsequent to retinal lesion, including cell cycle progression, axonal regeneration, and regenerative cytogenesis. Conclusions: The results indicate that retinal regeneration in adult zebrafish involves a complex set of induced, targeted changes in gene transcription, and suggest that these molecular changes underlie the ability of the adult vertebrate retina to regenerate. Keywords: time course; injury response; cellular correlation Overall design: Control brain and retina (unlesioned); Control and lesioned retina (matched animals, at least n = 8 for each condition).
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).
Project description:To characterize both short- and long-term (7 days and 28 days) gene expression profiling differences between control and MPTP-treated retina, we determined levels of gene expression using microarray analysis and real-time PCR. We found 60 genes relatively regulated in retinas treated with MPTP for 7 days (26 up-regulated and 34 down-regulated), whereas 54 genes were regulated in retinas treated with MPTP for 28 days (18 up-regulated and 36 down-regulated) when compared with the non-treated retina in each control groups. Total 26 annotated differently expressed genes were chosen for further validation by quantitative real-time PCR, and 4 genes in the 7-day treatment group (Clec2e; Dio2; Hmcn1; Rlbp1) and 3 genes in the 28-days treatment group (Pnmt; Tmem121; Ssxb3) were confirmed. Male C57BL/6 mice were randomly divided into four treatment groups (n= 6 in each group): the 7 days and 28 days saline-injection (control) groups (C-7 and C-28, respectively), and 7 days and 28 days MPTP-injection groups (M-7 and M-28, respectively). 4 retinas for 1 samples (1 gene chip), (4 experimental group × 2 samples of each experimental group = total 8 samples).
Project description:NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures. NeuroD1 mRNA is highly abundant in the adult mammalian pineal gland and exhibits a developmental expression pattern similar to the retina. This is consistent with the common evolutionary origin of pinealocytes and retinal photoreceptors. Pinealocytes and retinal photoreceptors express a shared set of phototransduction genes and submammalian pinealocytes are photosensitive. In contrast to the retina, the pineal gland is a relatively homogeneous structure, composed 95% of pinealocytes. This makes the pineal gland a particularly useful model for understanding photoreceptor cell biology. The loss of NeuroD1 in the retina results in progressive photoreceptor degeneration and the molecular mechanisms underlying this retinal degeneration phenotype remain unknown. Similarly, the role that NeuroD1 plays in the pineal gland is unknown. To determine the function of NeuroD1 in the pineal gland and retina, a Cre/loxP recombination strategy was used to selectively target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, a transcription factor selectively expressed in the pineal gland and retina. Pineal and retinal tissues from two-month-old NeuroD1 cKO and control animals were used in microarray studies to identify candidate genes responsible for the photoreceptor degeneration phenotype. The Cre/loxP recombination strategy was used to target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout mice (NeuroD1floxed::Crx-Cre+/-). NeuroD1 floxed mice (NeuroD1floxed::Crx-Cre-/-) served as the controls. Pineal glands and retinas from two-month-old control and conditional knockout mice were collected at ZT6 and ZT20. 3 pools of 6 pineal glands per genotype and respective time of day were collected for each sample. Similarly, 3 pools of 6 retinas each were also collected. RNA from each pool was extracted and hybridized on the Affymetrix Mouse 430 2.0 array.