The insect homologue of the amyloid precursor protein interacts with the heterotrimeric G protein Go alpha in an identified population of migratory neurons.

The amyloid precursor protein (APP) is the source of Abeta fragments implicated in the formation of senile plaques in Alzheimer's disease (AD). APP-related proteins are also expressed at high levels in the embryonic nervous system and may serve a variety of developmental functions, including the regulation of neuronal migration. To investigate this issue, we have cloned an orthologue of APP (msAPPL) from the moth, Manduca sexta, a preparation that permits in vivo manipulations of an identified set of migratory neurons (EP cells) within the developing enteric nervous system. Previously, we found that EP cell migration is regulated by the heterotrimeric G protein Goalpha: when activated by unknown receptors, Goalpha induces the onset of Ca2+ spiking in these neurons, which in turn down-regulates neuronal motility. We have now shown that msAPPL is first expressed by the EP cells shortly before the onset of migration and that this protein undergoes a sequence of trafficking, processing, and glycosylation events that correspond to discrete phases of neuronal migration and differentiation. We also show that msAPPL interacts with Goalpha in the EP cells, suggesting that msAPPL may serve as a novel G-protein-coupled receptor capable of modulating specific aspects of migration via Goalpha-dependent signal transduction.

Different isoforms of fasciclin II are expressed by a subset of developing olfactory receptor neurons and by olfactory-nerve glial cells during formation of glomeruli in the moth Manduca sexta.

During development of the primary olfactory projection, olfactory receptor axons must sort by odor specificity and seek particular sites in the brain in which to create odor-specific glomeruli. In the moth Manduca sexta, we showed previously that fasciclin II, a cell adhesion molecule in the immunoglobulin superfamily, is expressed by the axons of a subset of olfactory receptor neurons during development and that, in a specialized glia-rich "sorting zone," these axons segregate from nonfasciclin II-expressing axons before entering the neuropil of the glomerular layer. The segregation into fasciclin II-positive fascicles is dependent on the presence of the glial cells in the sorting zone. Here, we explore the expression patterns for different isoforms of Manduca fasciclin II in the developing olfactory system. We find that olfactory receptor axons express transmembrane fasciclin II during the period of axonal ingrowth and glomerulus development. Fascicles of TM-fasciclin II+ axons target certain glomeruli and avoid others, such as the sexually dimorphic glomeruli. These results suggest that TM-fasciclin II may play a role in the sorting and guidance of the axons. GPI-linked forms of fasciclin II are expressed weakly by glial cells associated with the receptor axons before they reach the sorting zone, but not by sorting-zone glia. GPI-fasciclin II may, therefore, be involved in axon-glia interactions related to stabilization of axons in the nerve, but probably not related to sorting.

Cell type-specific expression of fasciclin II isoforms reveals neuronal-glial interactions during peripheral nerve growth.

During the formation of the insect peripheral nervous system (PNS), the cell adhesion receptor fasciclin II has been shown to play a prominent role in axonal fasciculation and synapse formation during motor neuron outgrowth. In the moth Manduca, fasciclin II (MFas II) is expressed both as a transmembrane isoform (TM-MFas II) and a glycosyl phosphatidylinositol-linked isoform (GPI-MFas II). By using RNA and antibody probes, we have shown that these two isoforms are expressed in nonoverlapping patterns: TM-MFas II is expressed exclusively by neurons and becomes localized to their most motile regions, while GPI-MFas II is expressed primarily by the glial cells that ensheath the peripheral nerves. This cell-type specificity of expression allowed us to monitor the nature of neuronal-glial interactions during PNS development. The outgrowth of TM-MFas II-positive axons in many regions preceded the arrival of GPI-MFas II-expressing glial processes that enwrapped them. In a few key locations, however, GPI-MFas II-positive glial cells differentiated before the arrival of the first axons and prefigured their subsequent trajectories. Prior inhibition of GPI-MFas II expression disrupted the subsequent outgrowth of axons at these locations but not elsewhere in the PNS. Our results suggest that the two isoforms of MFas II play distinct roles with respect to cellular motility and nerve formation.

Remodeling of motor terminals during metamorphosis of the moth Manduca sexta: expression patterns of two distinct isoforms of Manduca fasciclin II.

During metamorphosis of the moth Manduca sexta, the neuromuscular system of the thoracic legs is reorganized dramatically. Larval leg muscles degenerate at the end of larval life, and new adult leg muscles develop during the ensuing pupal stage. Larval leg motoneurons persist, but undergo substantial remodeling of central and peripheral processes. As part of our on-going investigation of mechanisms underlying the remodeling of motor terminals, we have used antisera generated against Manduca-specific isoforms of the homophilic adhesion molecule fasciclin II (MFas II) to label motor terminals during metamorphosis. Antisera generated against the glycosyl-phosphatidylinositol (GPI) -linked isoform of MFas II (GPI-MFas II) labeled the motor nerves at all stages and seemed to be associated with glial cells ensheathing the peripheral nerves. In addition, the anti-GPI-MFas II antisera labeled regions associated with synaptic boutons at both larval and adult stages. In contrast, antisera generated against a transmembrane isoform of MFas II (TM-MFas II) only labeled specific neuronal processes at discrete intervals during remodeling. Identified leg motoneurons (such as the femoral depressor motoneuron) expressed detectable levels of TM-MFas II in their peripheral processes only during phases of motor-terminal retraction and initial stages of motor-terminal re-growth. Putative modulatory neurons (such as the unpaired median neurons), however, expressed TM-MFas II in their processes during larval stages as well as during remodeling. Use of the isoform-specific anti-MFas II antisera provided a novel method for visualizing remodeling of motor terminals during metamorphosis and helped distinguish different components of the motor nerves and neuromuscular junction.

Different isoforms of fasciclin II play distinct roles in the guidance of neuronal migration during insect embryogenesis.

During the formation of the enteric nervous system (ENS) of the moth Manduca sexta, identified populations of neurons and glial cells participate in precisely timed waves of migration. The cell adhesion receptor fasciclin II is expressed in the developing ENS and is required for normal migration. Previously, we identified two isoforms of Manduca fasciclin II (MFas II), a glycosyl phosphatidylinositol-linked isoform (GPI-MFas II) and a transmembrane isoform (TM-MFas II). Using RNA and antibody probes, we found that these two isoforms were expressed in cell type-specific patterns: GPI-MFas II was expressed by glial cells and newly generated neurons, while TM-MFas II was confined to differentiating neurons. The expression of each isoform also corresponded to the motile state of the different cell types: GPI-MFas II was detected on tightly adherent or slowly spreading cells, while TM-MFas II was expressed by actively migrating neurons and was localized to their most motile regions. Manipulations of each isoform in embryo culture showed that they played distinct roles: whereas GPI-MFas II acted strictly as an adhesion molecule, TM-MFas II promoted the motility of the EP cells as well as maintaining fasciculation with their pathways. These results indicate that precisely regulated patterns of isoform expression govern the functions of fasciclin II within the developing nervous system.

A role for fasciclin II in the guidance of neuronal migration.

The insect cell adhesion receptor fasciclin II is expressed by specific subsets of neural and non-neural cells during embryogenesis and has been shown to control growth cone motility and axonal fasciculation. Here we demonstrate a role for fasciclin II in the guidance of migratory neurons. In the developing enteric nervous system of the moth Manduca sexta, an identified set of neurons (the EP cells) undergoes a stereotyped sequence of migration along the visceral muscle bands of the midgut prior to their differentiation. Probes specific for Manduca fasciclin II show that while the EP cells express fasciclin II throughout embryogenesis, their muscle band pathways express fasciclin II only during the migratory period. Manipulations of fasciclin II in embryonic culture using blocking antibodies, recombinant fasciclin II fragments, and enzymatic removal of glycosyl phosphatidylinositol-linked fasciclin II produced concentration-dependent reductions in the extent of EP cell migration. These results support a novel role for fasciclin II, indicating that this homophilic adhesion molecule is required for the promotion or guidance of neuronal migration.

A delayed role for nitric oxide-sensitive guanylate cyclases in a migratory population of embryonic neurons.

Neuronal differentiation requires a coordinated intracellular response to diverse extracellular stimuli, but the role of specific signaling mechanisms in regulating this process is still poorly understood. Soluble guanylate cyclases (sGCs), which can be stimulated by diffusible free radical gasses such as nitric oxide (NO) and carbon monoxide (CO) to produce the intracellular messenger cGMP, have recently been found to be expressed within a variety of embryonic neurons and implicated in the control of both neuronal motility and differentiation. Using the enteric nervous system (ENS) of the moth, Manduca sexta, we examined the role of NO and NO-sensitive sGCs during the migration and differentiation of an identified set of migratory neurons (the EP cells). Shortly after the onset of their migration, a subset of EP cells began to express NO-sensitive sGC activity (visualized with an anti-cGMP antiserum). Unlike many neurons in the central nervous system, the expression of sGC activity in the EP cells was not transient but persisted throughout subsequent periods of axon elongation and terminal branch formation on the gut musculature. In contrast, nitric oxide synthase activity (visualized using NADPH-diaphorase histochemistry) was undetectable in the vicinity of the EP cells until the period of synapse formation. Manipulations designed to alter sGC and NOS activity in an in vivo embryonic culture preparation had no discernible effect on either the migration or axonal outgrowth of the EP cells. In contrast, inhibition of both of these enzymes resulted in a significant reduction in terminal synaptic branch formation within the postmigratory neurons. These results indicate that while NO-sensitive sGC activity is expressed precociously within the EP cells during their initial migratory dispersal, a role for this signaling pathway can only be demonstrated well after migration is complete, coincident with the formation of mature synaptic connections.

G protein-mediated inhibition of neuronal migration requires calcium influx.

Neuronal migration is an essential feature of the developing nervous system, but the intracellular signaling mechanisms that regulate this process are poorly understood. During the formation of the enteric nervous system (ENS) in the moth Manduca sexta, the migration of an identified set of neurons (the EP cells) is regulated in part by the heterotrimeric guanyl-nucleotide binding protein (G protein) Goalpha. Using an in vivo culture preparation for developing embryos that allows direct access to the ENS, we have shown that EP cell migration is similarly regulated by intracellular Ca2+; treatments that increased intracellular Ca2+ inhibited the migratory process, whereas buffering intracellular Ca2+ induced aberrant migration onto inappropriate pathways. Imaging the spontaneous changes in intracellular Ca2+ within individual EP cells showed that actively migrating neurons exhibited only small fluctuations in intracellular Ca2+. In contrast, neurons that had reached the end of migration displayed large, transient Ca2+ spikes. Similar Ca2+ spikes were induced in the EP cells by G protein stimulation, an effect that was reversed by removal of external Ca2+. Stimulation of Go in individual EP cells (by injection of either activated Goalpha subunits or mastoparan) also inhibited migration in a Ca2+-dependent manner. These results suggest that the regulation of neuronal migration by G proteins involves a Ca2+-dependent process requiring Ca2+ influx.

An identified set of visceral muscle bands is essential for the guidance of migratory neurons in the enteric nervous system of Manduca sexta.

During the formation of the enteric nervous system (ENS) in Manduca, a population of approximately 300 enteric neurons (the EP cells) become distributed along the foregut and midgut by migrating onto specific sets of visceral muscle bands. Only after their migration is complete do the neurons express a variety of position-specific phenotypes, including a peptidergic phenotype that is usually restricted to a subset of EP cells on the midgut. To investigate whether direct interactions between the EP cells and these pathways are necessary for either neuronal migration or differentiation, we have investigated the developmental origins and functional role of the muscle bands in embryonic culture. Using scanning electron microscopy, immunohistochemistry, and mitotic labeling with bromodeoxyuridine, we found that the eight major muscle bands of the midgut form by the coalescence of longitudinal muscle fibers on the midgut surface, apparently in response to regional cues associated with the underlying epithelium. These bands then serve as migratory pathways for the EP cells, which travel rapidly along the bands (but not onto adjacent interband musculature) and then complete their differentiation. Dye labeling of individual EP cells revealed that prior to migration onset, each neuron extended widely distributed filopodia onto both the band and interband regions of the midgut surface. As the muscle bands coalesced, however, the leading process of each EP cell became increasingly confined to a specific band, onto which it subsequently migrated. In a series of surgical manipulations of both the muscle bands and the migratory neurons, we demonstrated that these pathways are both necessary and sufficient to support the migratory behavior of the EP cells. Surgical interventions that prevented the neurons from contacting the muscle bands inhibited migration, while contact between isolated EP cells and a muscle band supported both their migration and differentiation. However, the acquisition of mature phenotypes by the EP cells was not strictly dependent on the migration of these neurons to their expected positions. In particular, the onset of neuropeptide expression could be detected in at least some of the neurons whose migration onto the midgut had been blocked. Thus, in the embryonic ENS, the migration and delayed differentiation of the EP cells represent precisely coordinated aspects of development that are nevertheless regulated in an independent manner.

A developmental role for the heterotrimeric G protein Go alpha in a migratory population of embryonic neurons.

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.

Developmental expression of heterotrimeric G proteins in the nervous system of Manduca sexta.

The heterotrimeric G proteins are a conserved family of guanyl nucleotide-binding proteins that appear in all eukaryotic cells but whose developmental functions are largely unknown. We have examined the developmental expression of representative G proteins in the developing nervous system of the moth Manduca sexta. Using affinity-purified antisera against different G alpha subunits, we found that each of the G proteins exhibited distinctive patterns of expression within the developing central nervous system (CNS), and that these patterns underwent progressive phases of spatial and temporal regulation that corresponded to specific aspects of neuronal differentiation. Several of the G proteins examined (including Gs alpha and G(o) alpha) were expressed in an apparently ubiquitous manner in all neurons, but other proteins (including Gi alpha) were ultimately confined to a more restricted subset of cells in the mature CNS. Although most of the G proteins examined could be detected within the central ganglia, only G(o) alpha-related proteins were seen in the developing peripheral nerves; manipulations of G protein activity in cultured embryos suggested that this class of G protein may contribute to the regulation of neuronal motility during axonal outgrowth. G(o) alpha-related proteins were also localized to the developing axons and terminals of the developing adult limb during metamorphosis. These intracellular signaling molecules may, therefore, play similar developmental roles in both the embryonic and postembryonic nervous system.

Neurogenesis in the insect enteric nervous system: generation of premigratory neurons from an epithelial placode.

The enteric plexus (EP) is a major division of the enteric nervous system (ENS) in the moth Manduca sexta and contains a dispersed population of about 360 bipolar neurons, the EP cells. Previously we showed that embryonic EP cells achieve their mature distributions by extensive migration along the gut surface and then display position-specific phenotypes. We now demonstrate that the entire EP cell population is generated from an ectodermal placode that invaginates from the embryonic foregut. Individual EP cells become postmitotic just as they leave the epithelium, but their terminal differentiation is subsequently delayed until after their migratory dispersal. Clonal analysis by injection of lineage-tracing dyes has shown that the EP cell population is derived from a large number of placodal cells, each of which contributes a limited number of neurons to the ENS. Placodally derived clones produce neurons exclusively, while clones arising from cells adjacent to the placode are incorporated into the gut epithelium. These results indicate that neurogenesis in the insect ENS involves a developmental strategy that is distinct from that seen in the insect CNS and which resembles the generation of certain cell classes in the vertebrate nervous system.