Microarray Analysis of XOPS-mCFP Zebrafish Retina Identifies Genes Associated with Rod Photoreceptor Degeneration and Regeneration

Photoreceptors are specialized sensory neurons in the retina that transduce the energy from photons of light into electrical signals. Rod photoreceptors mediate visual responses in dim light, whereas cone photoreceptors mediate daytime and color vision. Mutations in several genes cause retinopathic diseases such as retinitis pigmentosa (RP), a genetically inherited blinding disorder characterized by progressive degeneration of the rod photoreceptors and secondary degeneration of the cones (RetNet: http://www.sph.uth.tmc.edu/Retnet/). To develop effective treatments for diseases such as RP and to better understand its pathogenesis, we need a thorough understanding of the molecular mechanisms that regulate rod photoreceptor genesis. Although some key molecules have been identified, the transcriptional regulatory network regulating rod photoreceptor development remains to be fully defined. The zebrafish is an excellent genetic model organism in which to study the mechanisms of rod photoreceptor determination.1 The zebrafish retina possesses the same laminar organization and cell types found in higher vertebrates.2 Zebrafish embryos develop externally, and both embryos and newly hatched larvae are optically transparent, greatly facilitating the study of developmental processes and gene expression during retinogenesis. Furthermore, development is extremely rapid; by 3 days after fertilization (dpf) the zebrafish retina is fully laminated, and by 5 dpf zebrafish larvae are actively swimming and demonstrate visually evoked behavior.3,4 Finally, zebrafish (like other teleost fish) demonstrate persistent retinal neurogenesis throughout life and are able to regenerate all retinal cell types in response to acute injury.5 Studies of retinal regeneration in zebrafish are particularly relevant, because cell-based treatments for retinal degenerative diseases must enable photoreceptor replacement in the context of a fully mature tissue, and this is likely to involve overlapping yet distinct regulatory pathways from those used during embryonic photoreceptor development. To better understand the molecular mechanisms that mediate photoreceptor regeneration in zebrafish, our laboratory has focused on characterizing genetic models of selective photoreceptor loss and replacement. One such model is the XOPS-mCFP transgenic line, in which the toxic effect of a rod-targeted fluorescent reporter gene causes selective degeneration of rods.6 We have shown previously that the rod photoreceptor degeneration caused by the XOPS-mCFP transgene does not result in secondary degeneration of cones. Furthermore, in contrast to what has been observed after acute retinal injury or large-scale ablation of the rods in zebrafish, chronic, progressive rod photoreceptor loss in XOPS-mCFP retinas does not induce proliferation of retinal Muller glia.6 Rather, a population of rod progenitor cells located in the outer nuclear layer (ONL) increases proliferation in an attempt to regenerate the lost rods. In the healthy adult retina, rod progenitor cells produce new rods at a slow but steady rate.2,7 However, in XOPS-mCFP zebrafish the rod progenitor population is increased several fold, suggesting that this pool of committed progenitor cells mediates regeneration in cases of chronic rod loss.6 The specific amplification of the rod progenitor population in XOPS-mCFP retinas provides a unique opportunity to study the molecular determinants of rod photoreceptor development in adult zebrafish in greater detail. We therefore performed a microarray analysis of retinal RNA from wild-type and XOPS-mCFP adults. Several genes displaying increased expression in response to rod photoreceptor degeneration were identified, including many transcription factors with previously uncharacterized roles in the retina. RT-PCR and in situ hybridization verified the microarray results and also suggested a role for some of these genes during retinal development and regeneration.