Unknown,Transcriptomics,Genomics,Proteomics

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Comparative analysis of gene expression in retinal layers

ABSTRACT: Purpose: The aim of this study was to give a comprehensive overview on spatial distribution of gene expression in the adult mouse retina and integrate this information into existing retinal gene expression databases. Methods: Total RNA was collected by laser capture microdissection from the ganglion cell layer, inner nuclear layer, photoreceptors and the retinal pigmented epithelium of adult mice and was analyzed by oligonucleotide microarrays. The results were validated by quantitative real time PCR and in situ hybridization. Results: The applied method resulted in good separation of cells of different retinal layers. The spatial distribution of gene expression was determined on a global scale in the retina and the RPE. Our results show good correlation with previously reported retinal gene expression and describe genes not yet characterized in the context of the retina. Conclusions: The complexity of the vertebrate retina makes it necessary to determine not only temporal but spatial distributions of gene expression .Our work expands the already significant but still incomplete knowledge of retinal gene expression and hopefully facilitates functional characterization of key factors of retinal development and maintenance. Laser capture microdissected regions of the adult mouse neuronal retina and retinal pigmented epithelium were subjected to microarray analysis. 5 conditions were investigated: ganglion cell layer (GCL, 3 biological replicates), innner nuclear layer (INL, 3 biological replicates), photoreceptor layer from Blk6 (PR-WT, 3 biological replicates) and NrlKO animals (PR-Nrl, 2 biological replicates) and retinal pigmented epithelium (RPE, 1 biological replicate). All samples are co-hybridized with a reference sample (retina). 2 or 3 technical replicates were used for each biological sample. technical replicate - labeled-extract: GC1-1, GC1-2, GC1-3 technical replicate - labeled-extract: GC2-1, GC2-2, GC2-3 technical replicate - labeled-extract: GCpool-1, GCpool-2, GCpool-3 technical replicate - labeled-extract: INL1-1, INL1-2, INL1-3 technical replicate - labeled-extract: INL2-1, INL2-2, INL2-3 technical replicate - labeled-extract: INLpool-1, INLpool-2, INLpool-3 technical replicate - labeled-extract: PR-Nrl1-1, PR-Nrl1-2 technical replicate - labeled-extract: PR-Nrl2-1, PR-Nrl2-2, PR-Nrl2-3 technical replicate - labeled-extract: PR-WT1-1, PR-WT1-2, PR-WT1-3 technical replicate - labeled-extract: PR-WT2-1, PR-WT2-2, PR-WT2-3 technical replicate - labeled-extract: PR-WTpool-1, PR-WTpool-2, PR-WTpool-3 technical replicate - labeled-extract: RPE-1, RPE-2, RPE-3

ORGANISM(S): Mus musculus

SUBMITTER: Laszlo Hackler

PROVIDER: E-GEOD-19304 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The purpose of this study was to develop a framework for analyzing RPE expression profiles from zebrafish eye mutants. Methods: The fish model we used was smarca4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), a retinal dystrophic mutant that the retinal phenotype and expression profiles were previously characterized. Histological and Affymetrix GeneChip analyses were conducted to define the RPE defects and underlying differential expression respectively. Results: Histological analysis indicates that smarca4 RPE was formed but its differentiation was abnormal. In particular, ultra-structural analysis of smarca4 RPE by transmission electron microscopy showed a number of defects in melanogenesis, suggesting that the cytoskeletal dynamics was impaired. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, their retinas and RPE-attached retinas were microdissected and the gene expression values of these tissues measured by Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples using an approach previously established by us. A factorial analysis was conducted using the expression values of RPE, retinal as well as the whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted model to select for three groups of genes: 1) Smarca4-regulated RPE genes, 2) Smarca4-regulated retinal genes, and 3) Smarca4-regulated RPE genes that are not differentially expressed in the retina. The latter group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, paracrine and intracellular signal transduction. Conclusions: Our analytical framework can potentially identify genes in zebrafish mutants that both retina and RPE are affected by the underlying mutation. The gene expression levels of three independent replicates of WT whole embryos (WA52), WT retinas (WR52), WT RPE-attached retinas (WRR52), smarca4/yng retinas (YR52), smarca4 RPE-attached retinas (YRR52) and smarca4 whole embryos (YA52) were measured by Affymetrix Zebrafish Whole Genome Arrays. All samples were collected at 52 hpf. The probe-level data of these sample groups were background adjusted, normalized and summarized by a robust multiarray average (RMA) algorithm. RPE expression values were estimated from the comparison between the retinal and RPE-attached retinal samples of the same genotype based on a method we previously developed (Leung et al., Invest Ophthalmol Vis Sci. 2007; 48:881). Then, a 3x2 factorial design was used to analyze the expression values of three kinds of tissue (T): whole embryo, retina and RPE in two kinds of mutation (M) background: WT and smarca4. Using the coefficients from the fitted models, contrasts were built to identify candidate genes that fulfilled specific criteria. The false discovery rate (FDR) q-value cutoff for a contrast was 0.001. Several contrasts were ultimately combined to allow for selection of genes that were regulated by Smarca4 in the retina and RPE. These include 1) Smarca4-regulated RPE genes, 2) Smarca4-regulated retinal genes, and 3) Smarca4-regulated RPE genes that are not differentially expressed in the retina.

Project description:Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes. Reprinted from <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=25446321">Whitmore, S.S., Wagner, A.H., DeLuca, A.P., Drack, A.V., Stone, E.M., Tucker, B.A., Zeng, S., Braun, T.A., Mullins, R.F., Scheetz, T.E., 2014. Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq. Experimental Eye Research</a>. Used under <a href="http://creativecommons.org/licenses/by-nc-sa/3.0/">Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported </a>license. Additional RNA sequencing was performed on temporal, macular, nasal, inferior, and superior retina from a fifth subject and is included in this dataset. See original publication for details.

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Comparative analysis of gene expression in retinal layers

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