Directed neuronal migration is an essential process in the developing nervous system that, when misregulated, can lead to a variety of severe anatomical, behavioral, and cognitive disorders. Numerous guidance cues that affect neuronal motility have been identified, but the intracellular signaling mechanisms by which a neuron integrates its response to multiple cues in vivo remain poorly understood. This issue can be addressed using the enteric nervous system (ENS) of the moth, Manduca sexta, in which an identified set of migratory neurons (the EP cells) remains uniquely accessible to manipulation throughout development. Despite obvious morphological differences, the regulation of neuronal development in this system employs many of the same mechanisms as have been found in mammalian preparations, permitting an analysis of specific signal transduction pathways to be performed within the living embryo. In particular, the EP cells express Gomicronalpha, a member of the heterotrimeric family of guanyl nucleotide binding proteins (G proteins) that is abundantly expressed in all developing neurons but whose function is unknown. Gomicronalpha activation in the EP cells down- regulates their motility, an effect that requires calcium (Ca) influx and induces Ca oscillations within the migratory neurons; Ca elevations also inhibit their migration. To determine the precise role of G protein-mediated events during migration, an embryonic culture preparation will be used to investigate the pattern of spontaneous Ca fluctuations during EP cell development. Time lapse imaging of individual EP cells injected with Ca Green Dextran or Fura 2 will be used to monitor changes in intracellular Ca with respect to normal migration. Controlled manipulations of external and internal Ca (using ion channel blockers, chelators, and caged Ca) will then be used to demonstrate the functional relationship between Ca fluctuations and migratory behavior. The precise mechanism by which Gomicronalpha induces Ca oscillations will be investigated by injection of activated Gomicronalpha subunits into individual neurons while selectively manipulating (A) the activity of Ca channels in the plasma membrane, and (B) the activity of Ca- release channels associated with intracellular Ca stores. Lastly, three members of the Rho-related family of monomeric G proteins (Rac1, Rho, and Cdc42) have now been identified in the migratory EP cells. Modified forms of these proteins will be injected intracellularly to investigate their role in controlling EP cell migration, and to determine whether any of these monomeric G proteins act as downstream effectors for the heterotrimeric G protein Gomicronalpha. These experiments should lend insight into the fundamental mechanisms by which G proteins regulate the normal sequence of neuronal migration during embryonic development.
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