The components of the central nervous system exhibit the greatest diversity in structure and function than any other cells in the body. The retina is a highly studied CNS tissue, partly because there is a manageable number of well characterized cell types, and the structure is consistent across all vertebrates. In this study the frog will be used due to its availability at all developmental stages, and identified developmental genes. Experiments will measure cell cycle kinetics and determine the sequence of cell production within clonally restricted groups of retinal cells. We will determine whether there is variation in the order in which cells are formed and amongst which cell types. This will indicate where there are temporal differences in the fates adopted by frog retinal precursors as they leave the cell cycle, how much this may vary between precursors at each developmental stage, and whether the fate(s) adopted changes inexorably during development. We will test two classes of molecules endogenous to the retina, and shown to be related to cell specification, for their effect on cell cycle kinetics, timing and order of genesis. Specifically, the neurogenic genes Delta and Notch, and fibroblast growth factors -1, -2 and -8 will be tested to determine how they effect cell cycle and genesis. Genes for these proteins, and mutant constructs will be misexpressed, and parameters of cell genesis determined. This will elucidate the effects of these factors on retinal differentiation. Finally, competence is an important embryonic property in determining cell fate and Delta/Notch signaling shown by us to be a substrate for this property. Combining heterochronic culture and transplantation with misexpression of Delta and Notch will confirm that this pathway functions in responding to cell fate signals, and will suggest the extend of the effect (i.e., does the same signaling operate at all times, in all fate determination events). Health relatedness: The development of retinal cell transplantation as a therapeutic tool requires an understanding of how cells are formed, when and at what rate. This helps define the optimum time for obtaining donor cells and indicates important genes and signaling molecules for keeping cells developing normally. Knowing the limits of cell genesis in the retina will help determine its potential for regeneration and repair, and may suggest ways to boost that potential. Finally, understanding of the mechanism(s) of cell genesis and differentiation has important implications for tumorigenesis.
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