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.

Project description:Retinal degeneration is the leading cause of irreversible blindness. Retinitis pigmentosa (RP) is a genetically heterogenous group of diseases. In the United States, approximately one in 4000 individuals is affected. RP begins with the loss of night vision due to the loss of rod photoreceptor cells. The disease progresses slowly with the loss of peripheral vision, and eventually leads to complete debilitating and irreversible blindness. The first mutation associated with human RP was identified in the gene encoding rhodopsin, the G-protein coupled receptor of rod photoreceptor cells. Mutations within the rhodopsin gene account for significant portion of RP cases. Specifically, mutations of the proline at residue 347 in rhodopsin have been linked to human RP. We are fortunate to have access to the P347S rhodopsin mutant mice. These mice represent an excellent transgenic mouse model of retinal degeneration. The P347S rhodopsin mutation is one of the best studied mutations, yet the mechanism by which the mutation causes degeneration is still unknown. One study has demonstrated that galectin-1 plays a role in degeneration of neuronal processes (1) and another study has shown that expression level of galectin-3 is elevated in retinas of patients with age-related macular degeneration. These studies in conjunction with the availibility of the P347S mutant mice have provided impetus to examine the pathogenesis of retinal degeneration in the context of the possible role of glycans and glycan-binding proteins. The time course of photoreceptor degeneration in the P347S mouse model has been carefully studied. In these mice, degeneration is barely detectable at 1 month of age, yet biochemical evidence suggests that the rod photoreceptor cells have already begun to die. At 4 months of age, approximately half of the rod photoreceptor cells have degenerated. To distinguish involvement of glycogens at the various stages of retinal degeneration, we have collected retinas of wild type and the mutant mice at four time points (1, 2, 3, and 4 months of age). This will allow us to identify the genes that target early, mid- and late stages of the retinal degeneration process. Thus we request the analysis of total 24 samples as specified below: Age Group (months) Mice No of samples at each time point 1 Wild type 3 2 Wild type 3 3 Wild type 3 4 Wild type 3 1 P347S 3 2 P347S 3 3 P347S 3 4 P347S 3 Total 24.

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Danio rerio

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