Zebrafish cone -rod homeobox (crx) and retinal development.

Abstract

The paired-class homeobox gene <italic>Crx</italic> is essential for vertebrate retinal photoreceptor differentiation and maintenance. Mutations in human CRX cause photoreceptor degeneration diseases, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Targeted disruption of the <italic>Crx</italic> gene results in failure of formation of outer segments in photoreceptors and degeneration of photoreceptors in young mice. Mammalian <italic>Crx</italic> is also required for the pineal gland function. In my thesis, I studied the function of the zebrafish <italic>crx</italic> gene in retinal development. I showed that <italic>crx</italic> expression emerged in a ventronasal patch in the retina around 24 hpf, before any retinal progenitors exit the cell cycle. The expression domain was then expanded in a wave into the dorsotemporal region of the retina. As differentiation of the retina progressed, <italic>crx</italic> became restricted to the outer layers of the retina. In adult zebrafish retina, <italic>crx</italic> is expressed in the outer nuclear layer by both rods and cones, and in the outer region of the inner nuclear layer, where bipolar cells reside. Antisense Morpholino oligonucleotides were then used to knock down the crx function. With Morpholinos, I demonstrated that <italic>crx</italic> is required for expression of opsin genes and that it positively regulates the expression of other photoreceptor-specific genes. My results also showed that <italic>crx</italic> has additional roles in retinal development in zebrafish, which have not been described previously in mammals. First, I found that zebrafish <italic>crx</italic> regulates transcription of homeobox genes that are specifically expressed in the INL. Second, <italic> crx</italic> promotes retinal determination during regionalization of the optic primordia. Third, <italic>crx</italic> promotes withdrawal of retinal progenitors from the cell cycle. These results suggest that <italic>crx</italic> not only is required for photoreceptor differentiation but also plays a role in early retinal development and is involved in multiple steps during retinal development. My research also showed that a closely related gene, <italic>otx5</italic>, plays a role in regulating pineal and retinal gene expression, with stronger regulation in the pineal gland. According to a phylogenetic study, zebrafish <italic> otx5</italic> and <italic>crx</italic> genes are co-orthologs. My results indicated that there is subfunctionalization <italic>otx5/crx</italic> gene family in zebrafish, with <italic>crx</italic> dominating in the retina, <italic> otx5</italic> in the pineal.