Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene profiling studies in postnatal Mfrprd6 mutant eyes reveal differential expression of Prss56, a trypsin-like serine protease, and genes involved in visual and phototransduction pathways.

ABSTRACT: Mutations in the membrane frizzled-related gene (Mfrp) are linked to posterior microphthalmia, retinitis pigmentosa and nanophthalmia in humans. In homozygous Mfrprd6 mice, a splice site mutation causes a slow photoreceptor degeneration characterized by shortening and disorganization of outer segments with eventual photoreceptor loss. To better understand the function of MFRP in the retina, microarray analysis was carried out in mutant and control mice at postnatal day14 (P14), prior to the loss of photoreceptors. Analysis of the data revealed differentially expressed RPE and neuroretina transcripts. Although the analysis of the microarray data from Mfrprd6 mutant mice compared to age-matched wild-type controls identified some transcripts with relative fold change > 5.0, most of the differentially expressed genes showed a relative fold change between1.5 - 2.0. Global gene expression analysis using Ingenuity Pathway Analysis software identified decreased levels of transcripts in phototransduction and visual pathways in Mfrprd6 mutants. Select candidate transcripts were validated by quantitative real-time PCR analyses. The expression of RPE-specific visual transduction protein, RPE65, was significantly decreased in Mfrprd6 mutants. As an indirect consequence of the primary RPE cell defect due to the Mfrprd6 mutation, retinal specific transcripts Rgr, Pde6a, GuCa1b, and Rgs9 were also significantly decreased. We also confirmed the significantly elevated levels of Prss56, a gene previously associated with myopia and open angle glaucoma. In the Mfrprd6 mutant, a progressive increase in Prss56 mRNA levels from 14- to 70-fold was observed from P7 to P21, respectively. In situ hybridization and glutamine synthetase staining of mutant eyes indirectly identified MM-CM-

ORGANISM(S): Mus musculus

SUBMITTER: Timothy Stearns

PROVIDER: E-GEOD-53411 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description:Background: To identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa. Methodology: RPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software. Principal Findings: Our previous 22k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways. Conclusions: In this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE. Six RPE samples, three phot samples, three choroid samples

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Gene profiling studies in postnatal Mfrprd6 mutant eyes reveal differential expression of Prss56, a trypsin-like serine protease, and genes involved in visual and phototransduction pathways.

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