Project description:MicroRNA expression in the mouse eye.MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina . This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures. microRNA profiling of ocular tissues from mouse. In particular we analysed the cornea, lens, Retina Pigment Epithelium (RPE) and retina and compared them against RNA extracted from the entire eye. The purpose of this experiment was to understand which microRNAs are present nd/or show differential expression in the various structures of the eye (cornea, lens, RPE, retina). The samples numbered 1 & 2 (i.e. CORNEA1, CORNEA2 etc ) are biological replicates, prepared from tissues dissecyed from different groups of wild-type animals. RNA extracted from the entire eye (EYE) served as the unique reference sample. For each tissue to be analysed we performed the following hybridizations: - 2 slides for lens (LENS1, LENS2) vs entire eye (EYE) - 2 slides for RPE (RPE1, RPE2) vs entire eye (EYE) - 2 slides for retina (RETINA1, RETINA2) vs entire eye (EYE) - 2 slides for cornea (CORNEA1, CORNEA2) vs entire eye (EYE) - 1 slide for entire eye (EYE) vs entire eye (EYE)
Project description:MicroRNA expression in the mouse eye.MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina . This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures.
Project description:To identify to identify target tissues and molecules involved with refractive myopic shift and axial length elongation in a murine lens-induced myopia model, we performed comprehensive analysis by microRNA array. Negative 30diptor (-30D) lens was fixed on right eye (-30D) of C57BL/6J mice (3weeks old, N=3) for 3 weeks, the refraction and the axial length were measured using a refractometer and a SD-OCT system in all eyes. Eye balls were enucleated and separated to cornea, iris, lens, retina, choroid and sclera. Total RNA was extracted from individual ocular components. MicroRNA expression analysis was carried out using Agilent Mouse miRNA Microarray (8×60K) miRBase21.0 (Agilent). Expression ratio calculation and miRNA varying expression were extracted by GeneSpring GX 14.5 (Agilent). After 3weeks of lens fixing, a refractive change and an axial length elongation change were observed (Normal vs -30D: 0.95 ± 1.85D vs -18.42 ± 3.98D, 0.155 mm ± 0.015mm vs 0.273 ± 0.009 mm), respectively. MiRNA expression changes that induced only by -30D lens fixing was confirmed in each part of the eyeball. By expression ratio calculation and miRNA varying expression analysis, upregulated miRNA (56 in cornea, 13 in iris, 6 in lens, 0 in retina, 29 in choroid and 30 in sclera) and downregulated miRNA (7 in cornea, 28 in iris, 17 in lens, 9 in retina, 7 in choroid and 40 in sclera) were observed. Overlapping miRNAs were also found while each eye tissues. In this study, miRNA varying expression were observed in each ocular part of the murine lens-induced myopia model. These miRNAs dysregulation may be functionally involved with the refractive myopia shift and the axial length elongation.
Project description:Because refractive development is governed largely by the retina, we analyzed the retinal transcriptome in chicks wearing a spectacle lens, a well-established means to induce refractive errors, to identify gene expression alterations and to develop novel mechanistic hypotheses about refractive development. One-week-old white Leghorn chicks wore a unilateral spectacle lens of + or –15 diopters for 6 hours or 3 days (n=6 for each condition). With total RNA from the retina/retinal pigment epithelium (RPE), Affymetrix Chicken GeneChips were used to compare gene expression levels between lens-wearing and contralateral control eyes. Normalized microarray signal intensities were evaluated by an analysis of variance approach. Selected differentially expressed genes were validated by qPCR in biologically independent samples.
Project description:The principal aim of this work was to investigate the methylation profiles of specific ocular tissues, and compare this profile to matched peripheral blood. Matched human blood and eye tissue were obtained post-mortem (n=8) and DNA methylation profiling performed on blood, neurosensory retina, retinal pigment epithelium (RPE)/choroid and optic nerve tissue using the Illumina Infinium HumanMethylation450 platform.
Project description:During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358A->C mutation (Ile120Leu) in the Hist2h3c1 gene and a 71T->C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development.
Project description:We used DNA microarrays representing ~30,000 human genes to analyze gene expression in the cornea, lens, iris, ciliary body, retina, and optic nerve. Eight whole globes (G1-G8) were harvested from autopsy donors (age range=30-85 years-old) within 24 hours of death, and the tissues were immediately stored at 4◦ C in RNAlater (Ambion). Four of the globes were from female donors (G3, G6-8) and four were from male donors (G1, 2, 4, 5). Globes 4 and 5 were harvested as a set from a single donor, as were globes 6 and 7. No ophthalmologic clinical records were available for any of the globes at the time of harvest. Seven of the globes (G1-G7) were dissected into the following components: cornea, lens, iris, ciliary body, retina, and optic nerve, while only retinal tissue was available from G8. The maculas and the peripheral retinal tissues were further dissected from several of the retinal samples. The macula was defined as the visible xanthophyll-containing tissue temporal to the optic nerve, which encompassed an approximate area of 4 mm2. For comparison purposes, three post-mortem brain specimens were analyzed. Only those tissues that yielded adequate amounts of RNA were processed on the arrays. An organism part comparison experiment design type compares tissues, regions, organs within or between organisms. Differential gene expression in anatomical compartments of the human eye. Diehn et al. Genome Biology 2005, 6:R74 Using regression correlation
Project description:Retinitis Pigmentosa is a group of inherited eye disorders characterized by progressive degeneration of photoreceptor cells in the retina, leading to vision loss and eventual blindness. One of the known genetic mutations associated with RP is the c.6926A>C mutation in the RPE (retinal pigment epithelium) cells. The dataset involves multiple experimental approaches and cell types, providing a comprehensive understanding of the disease and potential corrective strategies.
Project description:Eye photoreceptor membrane discs in outer rod segments are highly enriched in the visual pigment rhodopsin and the omega-3 fatty acid docosahexaenote (DHA). The eye acquires DHA from blood, but transporters for DHA uptake across the blood-retinal barrier (BRB) or retinal pigment epithelium have not been identified. Mfsd2a is a newly described sodium-dependent lysophosphatidylcholine symporter expressed at the BRB. Microarrays were used to determine difference in gene expression between wild-type and Mfsd2a KO eye cups. RNA was extracted from eye cups from postnatal day 13 wild-type and Mfsd2a KO mice. Equal amounts of RNA from 6 wild-type eye cups were pooled for the wild-type sample, or 6 Mfsd2a KO eye cups were pooled for the Mfsd2a KO sample. Microarray profiling was done on pooled samples with a RIN cut-off of 7.0 using Mouse 430 2.0 arrays (Affymetrix).
Project description:Eye development and photoreceptor maintenance requires the retinal pigment epithelium (RPE), a thin layer of cells that underlies the neural retina. Despite its importance, RPE development has not been studied by a genomic approach. A microarray expression profiling methodology was established in this study for studying RPE development. The intact retina with RPE attached was dissected from developing embryos, and differentially expressed genes in RPE were inferred by comparing the dissected tissues with retinas without RPE using microarray and statistical analyses. We found 8810 probesets to be significantly expressed in RPE at 52 hours post-fertilization (hpf), of which 1443 might have biologically meaningful expression levels. Further, 78 and 988 probesets were found to be significantly over- or under-expressed in RPE respectively compared to retina. Also, 79.2% (38/48) of the known over-expressed probesets have been independently validated as RPE-related transcripts. The results strongly suggest that this methodology can obtain in vivo RPE specific gene expression from the zebrafish embryos and identify novel RPE markers. Keywords: Development, retina, retinal pigment epithelium