Abstract
Purpose: :
Inherited defects in retinal photoreceptor outer segment (OS) structure impair function, disrupt relationship with the retinal pigment epithelium (RPE), compromise cell viability, and result in a wide variety of progressive retinal degenerative diseases. We are investigating gene expression in the retinal degeneration slow (rds; aka prph2) mouse model; rds lacks peripherin/rds, and is unable to elaborate photoreceptor OSs. We utilized a dissociated retinal preparation, in conjunction with microarray screening, to assay how gene expression in rod photoreceptors responds to development in the absence of peripherin/rds and OSs.
Methods: :
Rods were labeled with GFP by crossing a transgenic line of Nrl-GFP mice onto a congenic line of rds mice. Fluorescence activated cell sorting (FACS) was used to enrich rod photoreceptors from dissociated retinas of rds and WT control mice, prior to and after the stage at which OSs are normally elaborated (~P10). Purified mRNA was subjected to 4-5 Affymetrix GeneChip hybridizations (mouse Genome 430 V2.0 chips). Statistical validation of the raw data was performed using Agilent’s Genespring GX software, to select transcripts with a minimum average fold change of >2 (p-value <0.05). Gene regulation networks were constructed using Ingenuity pathway analysis, and further validations were done using Taqman analysis.
Results: :
The microarray data indicates that the rds defect causes large scale gene expression changes at time points just before the onset of OS elaboration. Close to 700 transcripts show differential regulation at P9, in contrast to only 118 in case of P6. Functional annotation clustering identified the gene networks most affected as the ones related to: lipid transport and metabolism, disc morphogenesis, intraflagellar transport, and tight junction signaling.
Conclusions: :
This study tracks the broad based response of rod photoreceptor gene expression to the loss of peripherin/rds protein, and identifies major genetic networks which are significantly perturbed. These data provide a framework for further understanding the role of peripherin/rds in photoreceptor structure and viability.
Keywords: retinal degenerations: cell biology • photoreceptors • gene/expression