As retinal axons extend through the optic nerve and establish synaptic connections in fetal and neonatal mammals, the emergence of mature synaptic and glial relationships is accompanied by a pronounced decline in the proclivity of these axons for growth and re-organization, including adaption to altered visual experience or repair of lesioned pathways. This project is motivated by the hypothesis that an important mechanism for stabilizing retinofugal projections during maturation is the regulation od specific retinal genes by signals conveyed retrograded along axons of the optic nerve. To permit a detailed molecular dissection of at least one se of such retrograde signals, this work has focused on a single gene, encoding a protein designated GAP-43, whose expression in retinal ganglion cells has been shown to be regulated developmentally and to respond to interruption of optic nerve axons. The primary emphasis of the proposed work is a dissection of the regulatory regions of the GAP-43 gene, in order to identify DNA sequences that allow the gene to recognize and respond to retrograde signalling pathways, as well as elements that define the range of cell types competent to express GAP-43. To identify individual regulatory elements, systematic combinations of DNA sequences from the 5' flanking region of the GAP-43 gene will be ligated to the coding sequence for a bacterial marker enzyme and assayed for their ability to direct expression of the marker enzyme when transfected into neural and non-neural cells in vitro. Included in the analysis are constructs containing the first intron and the 5' untranslated region of the GAP-43 mRNA, to permit an evaluation of possible regulation at the level of RNA processing or translational control. A limited set of these constructs will be evaluated more extensively for their ability to direct appropriately regulated expression of the marker enzyme in retinal cells of transgenic mice and transgenic fish. In contrast to fish retina, it is not yet clear in mammals whether retrograde regulation of GAP-43 expression in retinal ganglion cells is mediated at the level of mRNA abundance rather through translational or post-translational control. Determination of the level at which GAP-43 is regulated is complicated by high level, constitutive expression of the protein in adult mammalian retina, where the protein's distribution is consistent with either a ganglion cell or amacrine cell origin. The most likely interpretation is that GAP-43 mRNA is developmentally regulated in ganglion cells, but constitutively expressed in some amacrine cells. To analyze retrogradely regulated expression of GAP-43 mRNA in retinal ganglion cells, the ganglion cells will be marked by retrograde labeling with fluorescent dyes and isolated by fluorescence- activated cell sorting before or after optic nerve lesions known to induce GAP-43 accumulation in ganglion cell bodies. Gap-43 mRNA in the isolated ganglion cells will be measured by reverse transcription and amplification of the cDNA through a polymerase chain reaction (PCR). Together these experiments define in molecular terms the recognition end of a retrograde signalling pathway linking retinal gene expression to events in the optic nerve and synaptic terminals.

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National Eye Institute (NEI)
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Neurology C Study Section (NEUC)
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Stanford University
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