Inducible control of tissue-specific gene expression in Xenopus tropicalis

Vertebrate eye formation has been extensively studied in amphibian embryos as a model for embryonic induction. More recently, a number of putative regulatory genes involved in eye determination have been identified. However, their defined functions during eye formation remain to be examined. Combining recent technological breakthroughs, e.g. a procedure for frog transgenesis and a new genetic system, Xenopus tropicalis, I have performed a series of experiments to test the function of these genes in eye determination.; The primary goal of the dissertation is to define the function of an essential regulatory gene, Otx2, controlling eye determination. To accomplish this, first, the transgenesis technique was used to express engineered mutations of this gene in a tissue-specific manner, resulting in ocular defects whose analysis was complicated by the background abnormalities associated with this nuclear transfer technique. To overcome these difficulties, the feasibility of using a hormone-inducible GAL4/UAS system permitting establishment of stable lines and consistent spatiotemporal control of transgene expression was tested in X. tropicalis. The expression of a UAS-target reporter gene was efficiently activated in a tissue-specific manner by a ligand-inducible GAL4 in response to RU486.; This hormone-inducible system was then utilized to functionally inactivate Otx2 in the eye through the induced expression of a dominant negative construct as an example of tissue-specific gene repression approach. Embryos inheriting both the GAL4 activator and dominant negative effector displayed consistent profound eye defects in an RU486-dependent fashion. Early morphogenetic movements of the optic vesicle fail, which normally position the dorsal presumptive retinal pigmented epithelium (RPE) in juxtaposition to the distal neural retina, separating the double-layered optic cup from the neural tube. As a result, the RPE cells remain dorsally, the choroid fissure fails to close, and the eyes remain fused to the forebrain vesicle. These results indicate that Otx2 plays a critical role in ocular development, in particular, in controlling the morphogenesis of the transition from the optic vesicle to optic cup.; In addition to helping to clarify the role of Otx2 during eye development, these studies provide the groundwork for future experiments utilizing the inducible GAL4/UAS system to study other gene functions in the frog.