Vertebrate eye formation is a complex process that requires specification of the anterior neural plate, formation of the optic vesicle, and cellular differentiation of the lens and retina. Each of these developmental processes is likely to be mediated by an interaction of transcriptional regulators, growth factors and their receptors. It is the goal of this research to identify genes and processes that play a critical role in the initial stages of eye formation. Specific emphasis will be placed on the role of the homeobox gene Rx in the formation of retinal progenitor cells and in the maintenance of retinal stem cells. We have found that Rx is essential for normal eye development and its misexpression has profound effects on eye morphology. Xenopus embryos injected with Rx RNA develop ectopic retinal tissue and display hyperproliferation in the neuroretina. Mouse embryos carrying a null allele of this gene do not form optic cups and consequently do not develop eyes. Given the vital role of Rx during eye development, we propose three specific goals related to its function during retinal formation:
Specific aim 1 : Study of the Rx regulatory pathway. This will be achieved by analyzing gene expression in Xenopus embryos and animal caps injected with Rx RNA and by analyzing gene expression in Rx null mouse mutant.
Specific aim 2 : Characterization of sequences that direct Rx expression into the retinal progenitor cells and retinal stem cells. For this purpose we will ligate the 5' flanking regions of the Rx gene to Beta-galactosidase reporter system. Transgenic mice made using this construct will be monitored for eye specific expression of Beta-galactosidase.
Specific aim 3 : Immortalization of retinal stem cells. Retinal stem cells will be immortalized either by fusing Rx regulatory sequences to the SV40 T antigen or by linking Rx coding sequences to a constitutively activated promoter. This research is of importance for better understanding of normal and abnormal eye development such as anophthalmia and microphthalmia, and is likely to provide us with information necessary for replacement of retinal cells in degenerative eye diseases using retinal stem cell population.
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