The directed migration of neurons or their precursors is an essential feature of the developing nervous system. Besides distributing cells to their appropriate locations, the process of migration exposes immature neurons to a spectrum of differentiative cues and facilitates the establishment of appropriate synaptic connections. The critical nature of this process is underscored by the numerous pathologies that are associated with disruptions of neuronal migration, resulting in severe neuroanatomical, behavioral, and cognitive disorders. While much is known about the phenomenology of migration, however, the mechanisms that govern the migratory process at the level of the individual neuron are poorly understood. This issue can be addressed in a relatively simple preparation, the enteric nervous system (ENS) of the moth Manduca sexta. During the formation of the ENS, an identified set of 300 neurons (the EP cells) delaminate from the gut epithelium, migrate along a defined set of muscle bands, and subsequently express differentiated phenotypes in a position-specific manner. Moreover, the neurons, their pathways, and their eventual targets are accessible throughout embryonic development. These features permit a mechanistic analysis of neuronal migration to be conducted in a normal developmental context. Recently, the migration of the EP cells has been shown to coincide with the onset of expression of Go-alpha, a member of the heterotrimeric family of guanyl nucleotide binding proteins (G proteins). Among the G proteins, the Go class is particularly abundant in the nervous systems of all organisms and is expressed in a variety of motile cells, but the developmental functions of these intracellular messengers are unknown. This proposal addresses the role of G proteins in regulating the migratory process. To assess the function of Go during migration, the developmental expression of Go will be characterized in the EP cells, using affinity purified antisera and cDNA probes specific for the alpha-subunit (Go-alpha). Experimental manipulations of G protein activity in the neurons will then be implemented at key phases of migration: by transient permeabilization of semi-intact embryos and intracellular injections of identified subsets of EP cells, G protein-specific reagents and toxins will be introduced to test their effects on neuronal motility. Antibodies and antisense oligonucleotide probes against Go-alpha (or other G proteins) will also be used to confirm the specific role of Go-alpha in modulating the migratory process. The possibility that Go exerts its effects via a developmentally regulated calcium current in the EP cells will subsequently be investigated by a combination of pharmacological, electrophysiological, and fluorescent imaging techniques. Finally, the effects of these manipulations on key elements of the neuronal cytoskeleton will also be examined. These experiments should clarify the mechanisms by which Go regulates the normal sequence of neuronal migration in a simple embryo and should lend insight into how similar developmental processes are controlled in more complex systems, as well.
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