Analysis of opsin mRNA and protein expression in adult and regenerating newt retina by immunology and hybridization

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Abstract
Rod photoreceptor cells were analysed in adult and regeneratingNotophthalmus viridescens retinae using a combination of immunochemical and molecular biological approaches. Monoclonal anti-rhodopsin antibody (Rho-4D2) labelled rod but not cone photoreceptors in adult newt retinae. The antibody bound to the entire cell body, from the synaptic ending through to the outer segment, as examined by light and electron immunocytochemistry. The antibody labelled two bands of 35 and 56 kDa in Western blots of urodele retinal preparations separated by gel electrophoresis.In situ hybridization with radiolabelled anti-sense riboprobes specific for bovine opsin revealed intense patches of silver grains overlying approximately 50% of photoreceptor inner segment myoid regions; no signal above background was detected elsewhere in the retina, or with radiolabelled sense riboprobe controls. Northern blot analysis using the probe on poly A(+) mRNA of adult newt retinae indicated a single band of 1.5 kb, corresponding to the opsin transcript. Following surgical removal of the original retina in test animals, retinal regeneration was studied by sampling tissue from 0–50 days post-surgery. The reappearance of opsin immunoreactivity was examined by light microscopical techniques. No opsin expression was detected in regenerating tissue prior to 16 days. Subsequent to this time, rho-4D2 bound to cells in central areas in which substantial lamination of the new retina had already occurred, and was limited to the scierai border. At no time was any immunoreactivity observed in more peripheral undifferentiated tissue. Thus the reformation of a functional retina seems to follow the same control mechanisms as during development, photoreceptor redifferentiation being at least partly governed by positional or environmental cues.