A Developmental Role for the Heterotrimeric G Protein Goαin a Migratory Population of Embryonic Neurons

Abstract

The heterotrimeric G proteins are an extended family of guanyl nucleotide-binding proteins that serve essential functions in the mature nervous system but whose contributions to neuronal development remain poorly understood. We have investigated the potential role of one specific G protein, Go(alpha), in the control of neuronal migration. During embryogenesis of the moth, Manduca sexta, an identified population of undifferentiated neurons (the EP cells) migrate along sets of visceral muscle bands to form part of the enteric nervous system. Previously, immunohistochemical studies indicated the presence of Go(alpha)-related proteins in the EP cells during migration. We have now verified this result, using probes derived from the Go(alpha) gene in Manduca. A clone containing the full-length coding domain for Go(alpha) was sequenced from a Manduca cDNA library; digoxigenin-labeled probes were then made from this clone and used to examine the developmental expression of the Go(alpha) gene during embryogenesis. Go(alpha)-specific transcripts could first be detected in the EP cells several hours before the onset of their migration. The level of Go(alpha) expression in all of the EP cells continued to increase during migration, but subsequently was down-regulated in a subset of the postmigratory neurons at the time of their terminal differentiation. This pattern of regulated expression is consistent with the distribution of Go(alpha)-related protein in the EP cells. We also used a semi-intact culture preparation of staged embryos to investigate the effects of G protein-specific toxins on the migratory process. Intracellular injections of the wasp toxin mastoparan, a specific activator of Go(alpha)-and Gi(alpha)-related proteins, inhibited the migration of individual EP cells. Injections of pertussis toxin (an inhibitor of Go(alpha) and Gi(alpha)) or cholera toxin (a selective activator of Gs(alpha)) had no effect on migration, although pertussis toxin treatments did cause a measurable increase in the subsequent outgrowth of axonal processes. However, co-injection of mastoparan with pertussis toxin blocked the inhibitory effects of mastoparan alone. These results suggest that Go(alpha)-coupled signaling events within the EP cells may down-regulate their migratory behavior, possibly in response to inhibitory cues that normally guide migration in the developing embryo.

Mechanisms of Dendritic Elaboration of Sensory Neurons inDrosophila: Insights fromIn VivoTime Lapse

“…The migration of neurons has been widely described in vertebrates (Hatten, 1999) but is less common in insects [e.g., migration of aCC and pCC in the grasshopper CNS (Bastiani et al, 1986) and of the enteric neurons (Horgan et al, 1994;Haase and Bicker, 2003)]. We report for the first time neuronal migration in the peripheral nervous system of Drosophila.…”

Reverse Signaling via a Glycosyl-Phosphatidylinositol-Linked Ephrin Prevents Midline Crossing by Migratory Neurons during Embryonic Development inManduca

“…At the end of each experiment, the preparations were re-dissected to expose the ENS and immunostained with anti-MsFas II, using the ABC HRP kit from Vector Laboratories (Burlingame, CA). This procedure allowed the EP cells and their processes to be visualized unambiguously (Horgan et al, 1995;Wright and Copenhaver, 2000;Coate et al, 2007). The extent of EP cell migration, axon outgrowth, and number of midline crossover events was then analyzed using photomicrographic and camera lucida techniques (Wright et al, 1999;Wright and Copenhaver, 2000).…”

Eph receptor expression defines midline boundaries for ephrin‐positive migratory neurons in the enteric nervous system of Manduca sexta

“…6, 7). Previous studies showed that these midline cells can be distinguished by a variety of antigenic markers (Horgan et al, 1995;and unpublished data), but their identity and function remained unclear. The histological and electron microscopic analysis presented in this paper (Fig.…”

“…Dissected embryos were fixed for 1 hr in PBS (pH 8.0) plus 4% paraformaldehyde (electron microscopy grade; Electron Microscopy Sciences), rinsed, and incubated with the riboprobes (1:100 -1:250 in hybridization buffer) overnight at 60ºC. After extensive rinsing, bound probes were detected using an alkaline phosphatase (AP)-conjugated anti-digoxigenin antibody (1:2000, Roche) and NBT/BCIP substrates (Bio-Rad; Hercules, CA; Horgan et al, 1995).…”