Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression changes during retinal development and rod specification.

ABSTRACT: Photoreceptor disorders are collectively known as retinal degeneration (RD), and include retinitis pigmentosa (RP), cone-rod dystrophy and age related macular degeneration (AMD). These disorders are largely genetic in origin; individual mutations in any one of >200 genes cause RD, making mutation specific therapies prohibitively expensive. A better treatment plan, particularly for late stage disease, may involve stem cell transplants into the photoreceptor or ganglion cell layers of the retina. Stem cells from young mouse retinas can be transplanted, and can form photoreceptors in adult retinas. These cells can be grown in tissue culture, but can no longer form photoreceptors. We have used microarrays to investigate differences in gene expression between cultured retinal progenitor cells (RPCs) that have lost photoreceptor potential, postnatal day 1 (pn1) retinas and the postnatal day 5 (pn5) retinas that contain transplantable photoreceptors. We have also compared FACS sorted Rho-eGFP expressing rod photoreceptors from pn5 retinas with Rho-eGFP negative cells from the same retinas. We have identified over 300 genes upregulated in rod photoreceptor development in multiple comparisons, 37 of which have been previously identified as causative of retinal disease when mutated. It is anticipated that this research should bring us closer to growing photoreceptors in culture and therefore better treatments for RD. This dataset is also a resource for those seeking to identify novel retinopathy genes in RD patients. We extracted whole retinas from postnatal day 1 (Pn1) and postnatal day 5 (Pn5) mice, and compared them with cultured RPCs derived from pn5 retinas, using Affymetrix mouse 430A_2 arrays. We also extracted cells from Rho-eGFP Pn5 retinas and FACS sorted them. GFP+ve cells represent immature rod photoreceptors, as they express the Rho-eGFP fusion protein, which is only expressed in rods. GFP-ve cells represent all other retinal neurons. These samples were amplified and compared using Affymetrix mouse 430A_2arrays, by Source Biosciences GMBH, Berlin, Germany. Results from immature rods were then compared with those from other retinal neurons, while results from whole Pn5 retinas were compared with Pn1 retinas (which don't yet express rod specific genes), and RPCs, which are glial precursors. RPCs were also compared with Pn1 retinas. Genes which showed changed expression profiles in at least 3/4 of comparisons were prioritised for further investigation.

ORGANISM(S): Mus musculus

SUBMITTER: Fiona Mansergh

PROVIDER: E-GEOD-59201 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose. XOPS-mCFP transgenic zebrafish experience a continual cycle of rod photoreceptor development and degeneration throughout life, making them a useful model to investigate the molecular determinants of rod photoreceptor regeneration. The purpose of this study was to compare the gene expression profiles of wild type and XOPS-mCFP retinas in order to identify genes that may contribute to the regeneration of the rods. Methods. Wild type and XOPS-mCFP retinal mRNA was subjected to microarray analysis using the Agilent platform. The Ingenuity Pathway Analysis program was used to identify biologically relevant processes that were significantly represented in the dataset. Expression changes were verified by RT-PCR. Selected genes were further examined during retinal development and in adult retinas by in situ hybridization, immunohistochemistry, and using a transgenic fluorescent reporter line. Results. Over 600 genes displayed significant expression changes in XOPS-mCFP retinas compared to wild type controls. Many of the downregulated genes were associated with phototransduction, whereas upregulated genes were associated with several biological functions, including cell cycle, DNA replication and repair, cell development and cell death. RT-PCR analysis of a subset of these genes confirmed the microarray results. Three transcription factors (sox11b, insm1a, and c-myb) displaying increased expression in XOPS-mCFP retinas were also expressed throughout retinal development and in the persistently neurogenic ciliary marginal zone. Conclusions. This study identified numerous gene expression changes in response to rod degeneration in zebrafish, and further suggests a role for the transcriptional regulators sox11b, insm1a, and c-myb in both retinal development and rod photoreceptor regeneration. Two-condition experiment: wild-type vs. XOPS-mCFP retinas. Four biological replicates for each condition. RNA was prepared from one retina for each sample. Each hybridization was accompanied by a dye-swap control, for a total of eight array hybridizations.

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Gene expression changes during retinal development and rod specification.

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