The overall objective of the research is to elucidate mechanisms that control lens differentiation. This is important for our understanding of: (1) how normal lens forms, (2) how developmental processes are involved in the maintenance of its normal structure and function throughout life. This is basic to our understanding of developmental and growth abnormalities, and perhaps even more insidious aging changes that may lead to cataract. There are four specific aims for the next three year project period. The first two are concerned with the control of events in early stages of lens development. 1. To analyze the composition of the extra-cellular matrix (ECM) in the interspace between developing lens and retina. 2. To determine if the interspace ECM influences events in early lens differentiation. The composition of the interspace will be established by immunohistochemical and immunocytochemical localization of known constituents of ECM and basement membranes. Once the composition of the interspace is established presumptive lens ectoderm will be grown on synthetic ECM in organ culture to determine whether it influences lens morphogenesis and/or alpha-crystallin synthesis. Alpha-Crystallin is the first lens specific protein to be detected in rats and will be localized by immunofluorescence. Natural ECM from cell free optic vesicle will also be used as a substratum for presumptive lens ectoderm to determine whether it influences lens differentiation. The subsequent aims of the project are largely concerned with control of fibre differentiation by neural retina. 3. To further characterize factors from the neural retina that stimulate lens epithelial cell division, fibre differentiation and secretory activity. 4. Are lens epithelial cells required to migrate and/or divide before they differentiate into fibres under the influence of neural retina? Characterization of factors from the neural retina will be carried out using immune serum raised against the factors. The factors will be purified by column chromatography and enzyme digestion. To examine the influence of migration and division on fibre differentiation, drugs will be used to inhibit these activities. In all experiments immunofluorescence and ELISA methods will be used to localize and quantify, respectively, Beta- and Gamma-crystallins, the markers for fibre differentiation in rats.
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