A hallmark of visual development is the dramatic loss of plasticity in the primary visual pathways that follows initial axon outgrowth and the dynamic sorting out synaptic connections. A reduced capacity of mature neurons to support axon growth is reflected not only in the limited remodeling of synaptic connections in the adult visual system, but also in the inability of most retinal ganglion cells to regenerate axons after optic nerve injury in adults. Previous work has shown that the decline in growth competence is tightly linked to a sharp decrease in expression of genes coding for characteristic proteins of axonal growth cones. The long-term goal of this project is to work out the signaling pathways responsible for activation of growth-associated genes during early axon outgrowth, and for the subsequent repression of these genes as neurons mature. As a point of entry into these pathways, the proposed studies seek to identify specific DNA response elements through which growth-related signals control expression of specific genes. One gene expected to contain such elements is the gene for GAP-43, a prominent growth cone component whose expression is tightly linked to neuronal competence for axon growth, both in early development and in mature neurons. Recent results show that a 1 kb fragment of DNA from the GAP-43 gene contains elements that direct preferential expression of a marker gene in developing neurons, and chronically suppress transcription in most adult neurons. The current proposal, therefore, is to dissect this 1 kb fragment further to identify specific sequence elements that control the activation of growth-related genes during neuronal differentiation, and chronic repression of those genes in mature retinal ganglion cells. The experimental approach relies principally on functional assays in which potential regulatory sequences from the GAP-43 gene are used to direct expression of a marker gene in transiently transfected neurons in culture, and in transgenic mice at different stages of development. Systematic deletions and mutations of sequences within the 1 kb DNA fragment will be used to determine which specific sequences are required for appropriate activation or repression of the GAP-43 gene at specific stages of differentiation, maturation and regeneration by retinal neurons. The expression studies will be guided by biochemical studies to identify sites for the initiation of RNA synthesis, and recognition sites for DNA binding proteins. Functional characterization of the response elements identified in these studies will define key steps in the regulatory pathways controlling the early acquisition and later repression of growth competence by developing and adult neurons.
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