Regulation and function of the retinal homeobox (Rx) gene in the developing and regenerating retina of pre-metamorphic Xenopus laevis

Eye development is a complex process that requires sequential induction events and the contribution of three embryonic tissues. Disruption of eye development results in the lack of eye development or congenital ocular malformations. Thus, investigating how the eye develops is essential to understanding how vision is established. Being such an intricate developmental process it is strictly regulated both at the molecular and cellular level. At the molecular level, a group of transcription factors called the eye field transcription factors (EFTFs) are essential in the establishment of the eye anlage. The retinal homeobox (Rx) gene belongs to this group of transcription factors.;Rx is essential for eye development. As one of the EFTFs, Rx function is required for the specification and maintenance of retinal progenitor cells (RPCs). Loss of Rx function leads to a lack of eye development in many different organisms. The studies presented in this dissertation aim to provide a better understanding of Rx function and regulation during development and retinal regeneration. To understand Rx function during regeneration I performed a detailed characterization of retinal regeneration after partial retinal resection in pre-metamorphic X. laevis. I show that after injury the wound is repopulated with retinal progenitor cells (RPCs) that express Rx. These RPCs are derived both from transdifferentiating RPE and the ciliary marginal zone (CMZ).;By dissection of a novel functional genomic regulatory element I demonstrate that Rx is similarly regulated in the RPCs of the developing and mature X. laevis retina. This genomic regulatory element is capable of directing Rx expression in retinal stem cells in the developing retina and in retinal progenitor cells that are induced by regeneration. Furthermore, using a novel in vivo gene silencing approach, I show that Rx function is necessary for proper retinal regeneration. I demonstrate that marked reduction in Rx expression levels impairs retinal regeneration by inhibition retinal pigment epithelium (RPE) transdifferentiation. These results strongly suggest that Rx function is not only essential during eye development, but also during retinal regeneration.;Finally, to further understand the mechanisms governing Rx transcriptional regulation during development, I describe how the X. laevis Rx2A promoter is regulated. I show that cooperative activity among POU, Sox, Otx and Fox families of transcription factors is necessary for the onset and maintenance of Rx expression. I also show that cooperation between two cis-regulatory regions is necessary to maintain specificity of Rx expression during development.;Collectively, the results from these studies support the hypothesis that a gene essential during development is also important during regeneration. Regeneration is thought to initiate early developmental events in differentiated cells. Hence, further analysis of Rx function during regeneration could also yield relevant insights about its function and regulation during eye development.