Abstract
Abstract: :
Purpose: Retinoic acid (RA) plays an important role in photoreceptor development. We investigated the effects of RA on the development of rods and the different types of cones in zebrafish. The objective of this study was to determine the mechanisms of these effects. The main hypothesis was that RA causes fate changes in the cells in the retina before determination. The alternative hypothesis was that RA affects transcription of opsin genes after determination. Methods: Whole mount in situ hybridization analysis of opsin expression was performed to determine the effects of RA upon the different types of photoreceptors. To test the phenotype change hypothesis, we performed quantitative spatial pattern analysis upon 2D patterns of labeled photoreceptors, and compared these results to those of theoretical photoreceptor patterns resulting from phenotype change. In order to indirectly test if RA affects opsin transcription in determined photoreceptors, we evaluated the intensity of opsin mRNA in labeled photoreceptors. To investigate which retinal cells respond to RA, we examined the expression pattern of retinoic acid response element (RARE) driven YFP in transgenic embryos and the expression patterns of three RA receptors (RARa, RARg, RXRa). The expression pattern of an enzyme involved in RA synthesis, RALDH2, was analyzed by indirect immunofluorescence. Results: RA treatment resulted in increases in the numbers of photoreceptors expressing rod opsin and red cone opsin, and decreases in the numbers of photoreceptors expressing blue cone opsin and UV cone opsin. When theoretical patterns of photoreceptors were altered to create hypothetical fate change patterns, the pattern regularity was enormously disrupted. In contrast, the regularity of experimental patterns of photoreceptors from RA treated embryos was not very different from that of experimental control patterns. Treatment with RA differentially influenced opsin labeling intensity. The retinoid receptor RXRalpha was localized to nascent photoreceptors, while RALDH2 was localized to retinal progenitor cells. The RARE driven transgene is expressed in neuroepithelial cells and nascent photoreceptors. Conclusions: Our results are not consistent with the hypothesis that RA regulates photoreceptor phenotype, but instead support the hypothesis that RA differentially affects opsin transcription in determined photoreceptors. A cellular source of RA, and an RA receptor that may mediate these effects have been identified.
Keywords: retinal development • photoreceptors • retinoids/retinoid binding proteins