Many gene products critically involved in development elicit their effects through their unique spatial and temporal patterning in tissues. The complexity of these developmental processes is highlighted by neurogenesis, which involves the actions of dozens of temporally and spatially patterned molecular guidance cues. The co-spatial presentation of multiple molecular cues involved in the guidance of neurons makes controlled study of these molecular interactions in vivo a formidable challenge. We have recently developed a new method to control the spatial and temporal expression of genes in cultured cells (in vitro) in a light-directed manner using photo-caged agonists of nuclear hormone receptors. This method will be used to investigate the effects of spatially and temporally patterned axon guidance cues that cannot be adequately addressed by any current method. NgCAM is the avian homolog of mammalian L1, a biomedically important protein associated with the X-linked mental retardation syndrome CRASH (Corpus callosum agenesis, Retardation, Abducted thumbs, Spastic paraplegia, Hydrocephalus). NgCAM is an important cell surface protein that is involved in guiding axons during brain development. The ability of spatially discrete patterns of NgCAM to direct neurite outgrowth when presented in a biologically relevant manner on cell surfaces in cultured cell monolayers will be explored. The ability of both binary (on/off) patterns and gradients of NgCAM to direct and influence axon extension will be investigated through controlled in vitro co-culture experiments. The unique ability of this light-activated expression system to create transient expression patterns will be used to dissect the role of NgCAM in promoting axon extension versus sustaining extended axons. """"""""Moving"""""""" patterns of expressed NgCAM will be used to assess the ability of NgCAM to redirect axons once extended. Because many events in neurogenesis involve the combined actions of multiple genes expressed within the same tissue, light-activated gene expression will be further developed to permit the independent cospatial control of multiple genes products in vitro.
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