Retinal degenerations are one of the most important causes of retinal dysfunction including blindness. The prevention and ultimately the cure of such diseases depends on understanding the cellular and molecular mechanisms responsible for the differentiation and maintenance of retinal cells. Formation of the retina requires orchestration of numerous complex cellular and molecular events. Cell division, migration, and differentiation result in a retina with well defined layers, each containing a limited number of cellular phenotypes. Generation of the appropriate number and type of cells is a complex problem and the cellular and molecular mechanisms which regulate these events are unknown. We propose experiments to address specific questions about retinal development using an organotypic retinal slice preparation combined with cellular and molecular probes. Using the retinal slice preparation, we will examine how cell fate is regulated in the retina. Does the fate of a retinal cell depend on its location at the time of division or is there a prior commitment to particular phenotype. To do this, we will transplant neuroepithelial progenitor cells to different locations within the retina using our newly developed retinal slice preparation to discover whether and how different microenvironments change the phenotypic fate of individual cells. We will also use the retinal slice preparation to test the effects of extracellular growth factors on retinal cell proliferation and differentiation. To understand what control the differentiation of newly generated rod photoreceptors, and what fraction of the newly divided outernuclear layer cells actually differentiate into rods, we will use in vivo experiments in the experimentally useful fish retina which continues to grow throughout life. We will label newly divided cells in the outer nuclear layer of the retina with a probe for opsin mRNA using in situ hybridization to determine what fraction of the new cells continue to differentiate into rod photoreceptors. We will also examine the effect of candidate factors on differentiation. Results from these experiments should help us understand normal retinal development but also the mechanisms leading to retinal dysfunction.
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