Retinal regeneration in adult vertebrates is a process exclusive to newts and salamanders. One source of the new retina is the retinal pigment epithelium (RPE) which is induced to divide and provide progenitor cells. The long-term objective is to identify the signals involved in the induction phase and in the subsequent proliferative and differentiation phases of retinal formation from the RPE. The two thrusts of the proposal are to generate molecular markers to monitor the process, in conjunction with histology,k and to develop culture systems in which continued differentiation and eventually in which the induction event can occur. Monoclonal antibodies, generated against normal newt retina and RPE, will be used to follow the differentiation of cells regenerating in vivo. Antibodies will also be generated against regenerating retina to identify regeneration-specific molecules. Preliminary experiments have shown that eyes put directly into culture do not show retinal degeneration or regeneration. However, culturing of eyes that had been reimplanted into the animal for 4-7 days does lead to retinal degeneration and incomplete retinal regeneration. The culture experiments will start with defining the growth conditions that will substantially improve the extent of regeneration in culture. Growth factors and conditioned medium from regenerating eyes in vivo will be tested for their ability to enhance regeneration. The molecular markers previously obtained will be used to follow differentiation in vitro and to compare regeneration in vitro with that in vivo. The last phase of the project is to induce the RPE cells in freshly isolated eyes to divide and begin forming a retina in culture. An eye cup preparation will be used and the optimum conditions for retinal regeneration in vitro will be applied. Since there is currently no short term assay for the induction event, the formation of a retina in the eye cup will signal that induction occurred. To confirm that the culture conditions were affecting the RPE cells directly, attempts will be made to induce freshly isolated RPE cells in low density cell culture. A planned extension of this proposal is to apply the techniques of molecular biology to identify and characterize the cytoplasmic and nuclear events involved in the early induction event, and in the subsequent proliferation and differentiation phases of retinal regeneration. The health related aspect of this project lies in the prospect of inducing adult mammalian RPE cells to regenerate a functional retina. During early development, some, and possibly all, vertebrates possess the ability to regenerate a retinal from the RPE but only newts and salamanders retain this capability as adults. Knowledge of the molecular events in newts can be used to direct a search for similar events in mammals and possibly to find the change that occurred in mammals during development that made the RPE incapable of regeneration in the adult. This raises the possibility of therapeutic intervention with the goal of retinal regeneration in cases of retinal damage through trauma or disease in the human population.
