We have examined the morphology and neuronal elements of the cerebral neuroendocrine system in the larval, pupal, and adult stages of the moth Manduca sexta with a variety of neuroanatomical techniques. The larval brain contains several discrete groups of neurosecretory and non-neurosecretory cells that project to the associated neurohemal organs (the corpora cardiaca-allata complex, or CC-CA) and to a variety of more peripheral structures. A previously undescribed set of cells in the subesophageal ganglion have also been found that project out the neurosecretory nerves. During metamorphosis, the cerebral neuroendocrine system undergoes a dramatic structural reorganization, including the reduction or loss of many larval nerves and a repositioning of the cell groups and their dendritic fields. Despite these changes, most of its central elements are retained. In addition, by the completion of adult development a new cluster of cells can be found on either side of the dorsal midline of the brain. We have also determined the relative contributions of the different cell groups to the moth neuroendocrine system by intracellular iontophoresis of dye into individual cells. Within the dorsal protocerebrum, five separate morphological types of cells can be recognized, each with a distinctive pattern of dendritic arborization in the brain and terminal neurohemal processes that project to the CC, the CA, the aorta, or to a combination of these regions. The large intrinsic cells of the CC have also been filled, revealing an unusual set of morphological features in these peripheral neurosecretory cells.
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.
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.
Amyloid precursor protein (APP) belongs to a family of evolutionarily conserved transmembrane glycoproteins that has been proposed to regulate multiple aspects of cell motility in the nervous system. Although APP is best known as the source of -amyloid fragments (A) that accumulate in Alzheimer's disease, perturbations affecting normal APP signaling events may also contribute to disease progression. Previous in vitro studies showed that interactions between APP and the heterotrimeric G protein Go␣-regulated Go␣ activity and Go-dependent apoptotic responses, independent of A. However, evidence for authentic APP-Go interactions within the healthy nervous system has been lacking. To address this issue, we have used a combination of in vitro and in vivo strategies to show that endogenously expressed APP family proteins colocalize with Go␣ in both insect and mammalian nervous systems, including human brain. Using biochemical, pharmacological, and Bimolecular Fluorescence Complementation assays, we have shown that insect APP (APPL) directly interacts with Go␣ in cell culture and at synaptic terminals within the insect brain, and that this interaction is regulated by Go␣ activity. We have also adapted a well characterized assay of neuronal migration in the hawkmoth Manduca to show that perturbations affecting APPL and Go␣ signaling induce the same unique pattern of ectopic, inappropriate growth and migration, analogous to defective migration patterns seen in mice lacking all APP family proteins. These results support the model that APP and its orthologs regulate conserved aspects of neuronal migration and outgrowth in the nervous system by functioning as unconventional Go␣-coupled receptors.
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