Abstract
Abstract: :
Purpose: To improve our understanding of the molecular mechanisms underlying retinal regeneration in adult vertebrates. Methods: For control and injured retinas of adult zebrafish (Danio rerio) quantitative real-time PCR was used to determine the expression levels of genes previously implicated in various aspects of retinal development. Gene expression was examined 1 and 14 days following surgical extraction of a patch of dorsotemporal retina from an eye; the contralateral unoperated retina served as a control. Total RNA was isolated from pooled injured and control eyes (n = 4-5 for each time point), single-stranded cDNA was synthesized, and real-time quantitative PCR was performed using a ABI Prism 7700 Sequence Detection System. Primers were designed to screen for the expression of the following: ß-actin, delta C, neuroD, ndr2, pax6, rx3, and vsx-1 (an analogue of Chx10). For all genes and conditions expression level was referenced to the expression level of rhodopsin; previous microspectrophotometric analyses provided no evidence for changes in rhodopsin content in the photoreceptors of injured retinas. Results: In control retinas message was detected for all of the screened genes. One day following retinal extraction there was a significant elevation in all messages, ranging from a 3-fold elevation of rx3 to a 22-fold elevation of delta C, except for ndr2 which showed no change relative to control. Relative to day 1, at day 14 the message levels in injured retinas varied considerably: delta C, neuroD, pax6, and ß-actin expression was decreased; vsx-1 displayed a sustained elevation in expression; ndr2 expression was elevated (suggesting a delayed up-regulation); and rx3 expression was below control (suggesting a delayed down-regulation). These different temporal profiles ruled out the hypothesis that injury-induced changes in rhodopsin expression could account for all the observed changes in gene expression (e.g., a decrease in rhodopsin message due to the extraction of retinal tissue). Conclusion: Retinal injury in adult zebrafish induces rapid and significant changes in the expression of genes previously implicated in retinal development. The different temporal profiles in the injury-induced changes in gene expression suggest that the expression of these genes may be differentially regulated.
Keywords: 417 gene/expression • 564 retinal development • 553 regeneration