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P.F. Hitchcock, T. Vihtelic, L. Kakuk–Atkins, D. Hyde; Transcriptional Profiling of Light–Lesioned, Regenerating Retinas of Teleost Fish . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5328.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The purpose of this study was to identify genes that are differentially expressed in the teleost retina during regeneration of photoreceptors. The broad goal of this work is to identify injury– and regeneration–associated changes in gene expression and raise hypotheses of gene function that can be tested by reverse genetics. Methods:Animals were exposed to constant, bright light for 72hrs, which results in the death of photoreceptors in dorsal retina and the accumulation of injury–induced cone progenitors overlying the depleted ONL. Expression profiling was performed using Affymetrix GeneChip® Zebrafish Genome Arrays. Total RNA was isolated and processed in separate pools, and each pool was used to generate targets that were hybridized to a single chip (4 control, 4 experimental). Following hybridization, chips were scanned using an Affymetrix GeneChip Scanner 3000, and differentially–expressed genes were identified using a two–step multicriteria approach that generates a rank–ordered list of genes that show a statistically–significant difference in average fluorescence intensity on the array. Results:Ninety nine genes with 4–fold or higher change in fluorescence intensity and an additional 579 genes with a two–fold or higher difference were identified. Among the differentially–expressed genes were 32 known transcriptional regulators and growth factors. Among this subset of genes were four transcriptional inhibitors, twenty–one DNA–binding transcription factors and five growth factors/growth regulators. Predicted changes in gene expression were also observed. The expression levels of photoreceptor–specific genes were reduced, reflecting the death of photoreceptors, and the expression levels of cell–cycle control genes were increased, which we infer reflects the accretion of mitotically–active photoreceptor progenitors. Quantitative, real–time RT–PCR was used to validate array results for transcriptional repressors, transcription factors and growth factors. Conclusions: The differential expression of genes in the light–lesioned retina reflects both changes in transcriptional activity among static populations of cells as well as physical changes in the number of cells, i.e., death of photoreceptors. This study has identified numerous molecules that can be hypothesized to play critical roles in the regeneration of photoreceptors.
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