DEVELOPMENTAL ROLE OF G PROTEINS IN NEURONAL MIGRATION

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.