Development of the primary visual pathways and regeneration of injured optic nerves both depend on a complex interplay between axons and their environment, first in the optic nerve and later at synaptic sites. One of the most important ways in which the optic nerve and synaptic targets may exert their effects on retinal cells is by controlling the expression of specific genes, which would require that signals be conveyed retrogradely along optic axons to the neuronal nuclei. The work proposed here examines the nature of these signals and the DNA sequences that make neuronal genes responsive to them. Most of this work focuses on the gene encoding a protein called GAP-43, whose synthesis is greatly elevated during visual development in mammals and during optic nerve regeneration in lower vertebrates, but not after optic nerve injury in mammals. The close correlation between GAP-43 synthesis and axon elongation indicate that its gene is responsive to some of the same signals that control axon growth. The proposed study begins by using pharmacological manipulations in vivo and co-culture of retinal cells with optic nerves and nerve derivatives in vitro to determine whether the retrogradely transported signals controlling the GAP-43 gene include inhibitors, inducers, or both. Specific antibodies and cDNA probes will be used to assay GAP-43 expression in response to these manipulations. One hypothesis to be considered is that interaction of axons with the optic nerve environment in adult mammals directly inhibits expression of GAP-43 in retinal ganglion cells. The second phase of this work looks at the final targets of retrograde signalling pathways -- the DNA sequences controlling GAP-43 gene transcription. Potential regulatory domains in the GAP-43 gene will be identified by direct sequence analysis of mammalian (rat) and fish GAP-43 genes. Functional dissection of the putative regulatory sequences will be carried out by transfecting cultured neuron-like cells with recombinant plasmids in which DNA fragments from the GAP-43 gene control the synthesis of a marker enzyme. Functionally defined regulatory sequences from the GAP-43 gene will be used to probe for nuclear proteins that bind specifically to those sequences and hence might serve as the final carriers of retrogradely transported signals. This work provides an essential base for understanding how neuronal genes respond to glial or synaptic signals related to axon growth.
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