Over-expression of Hsp83 in grossly depleted hsrω lncRNA background causes synthetic lethality and l(2)gl phenocopy in Drosophila.

We examined interactions between the 83 kDa heat-shock protein (Hsp83) and hsrω long noncoding RNAs (lncRNAs) in hsrω66 Hsp90GFP homozygotes, which almost completely lack hsrω lncRNAs but over-express Hsp83. All +/+; hsrω66 Hsp90GFP progeny died before the third instar. Rare Sp/CyO; hsrω66 Hsp90GFP reached the third instar stage but phenocopied l(2)gl mutants, becoming progressively bulbous and transparent with enlarged brain and died after prolonged larval life. Additionally, ventral ganglia too were elongated. However, hsrω66 Hsp90GFP/TM6B heterozygotes, carrying +/+ or Sp/CyO second chromosomes, developed normally. Total RNA sequencing (+/+, +/+; hsrω66/hsrω66, Sp/CyO; hsrω66/ hsrω66, +/+; Hsp90GFP/Hsp90GFP and Sp/CyO; hsrω66 Hsp90GFP/hsrω66 Hsp90GFP late third instar larvae) revealed similar effects on many genes in hsrω66 and Hsp90GFP homozygotes. Besides additive effect on many of them, numerous additional genes were affected in Sp/CyO; hsrω66 Hsp90GFP larvae, with l(2)gl and several genes regulating the central nervous system being highly down-regulated in surviving Sp/CyO; hsrω66 Hsp90GFP larvae, but not in hsrω66 or Hsp90GFP single mutants. Hsp83 and several omega speckle-associated hnRNPs were bioinformatically found to potentially bind with these gene promoters and transcripts. Since Hsp83 and hnRNPs are also known to interact, elevated Hsp83 in an altered background of hnRNP distribution and dynamics, due to near absence of hsrω lncRNAs and omega speckles, can severely perturb regulatory circuits with unexpected consequences, including down-regulation of tumoursuppressor genes such as l(2)gl.

Menin: a tumor suppressor that mediates postsynaptic receptor expression and synaptogenesis between central neurons of Lymnaea stagnalis.

Neurotrophic factors (NTFs) support neuronal survival, differentiation, and even synaptic plasticity both during development and throughout the life of an organism. However, their precise roles in central synapse formation remain unknown. Previously, we demonstrated that excitatory synapse formation in Lymnaea stagnalis requires a source of extrinsic NTFs and receptor tyrosine kinase (RTK) activation. Here we show that NTFs such as Lymnaea epidermal growth factor (L-EGF) act through RTKs to trigger a specific subset of intracellular signalling events in the postsynaptic neuron, which lead to the activation of the tumor suppressor menin, encoded by Lymnaea MEN1 (L-MEN1) and the expression of excitatory nicotinic acetylcholine receptors (nAChRs). We provide direct evidence that the activation of the MAPK/ERK cascade is required for the expression of nAChRs, and subsequent synapse formation between pairs of neurons in vitro. Furthermore, we show that L-menin activation is sufficient for the expression of postsynaptic excitatory nAChRs and subsequent synapse formation in media devoid of NTFs. By extending our findings in situ, we reveal the necessity of EGFRs in mediating synapse formation between a single transplanted neuron and its intact presynaptic partner. Moreover, deficits in excitatory synapse formation following EGFR knock-down can be rescued by injecting synthetic L-MEN1 mRNA in the intact central nervous system. Taken together, this study provides the first direct evidence that NTFs functioning via RTKs activate the MEN1 gene, which appears sufficient to regulate synapse formation between central neurons. Our study also offers a novel developmental role for menin beyond tumour suppression in adult humans.

Postembryonic development of centrally generated flight motor patterns in the hawkmoth, Manduca sexta.

This study analyses the maturation of centrally generated flight motor patterns during metamorphosis of Manduca sexta. Bath application of the octopamine agonist chlordimeform to the isolated central nervous system of adult moths reliably induces fictive flight patterns in wing depressor and elevator motoneurons. Pattern maturation is investigated by chlordimeform application at different developmental stages. Chlordimeform also induces motor patterns in larval ganglia, which differ from fictive flight, indicating that in larvae and adults, octopamine affects different networks. First changes in motoneuron activity occur at the pupal stage P10. Rhythmic motor output is induced in depressor, but not in elevator motoneurons at P12. Adult-like fictive flight activity in motoneurons is observed at P16 and increases in speed and precision until emergence 2 days later. Pharmacological block of chloride channels with picrotoxin also induces fictive flight in adults, suggesting that the pattern-generating network can be activated by the removal of inhibition, and that proper network function does not rely on GABA(A) receptors. Our results suggest that the flight pattern-generating network becomes gradually established between P12 and P16, and is further refined until adulthood. These findings are discussed in the context of known physiological and structural CNS development during Manduca metamorphosis.

Developmental changes in dendritic shape and synapse location tune single-neuron computations to changing behavioral functions.

During nervous system development, different classes of neurons obtain different dendritic architectures, each of which receives a large number of input synapses. However, it is not clear whether synaptic inputs are targeted to specific regions within a dendritic tree and whether dendritic tree geometry and subdendritic synapse distributions might be optimized to support proper neuronal input-output computations. This study uses an insect model where structure and function of an individually identifiable neuron, motoneuron 5 (MN5), are changed while it develops from a slow larval crawling into a fast adult flight motoneuron during metamorphosis. This allows for relating postembryonic dendritic remodeling of an individual motoneuron to developmental changes in behavioral function. Dendritic architecture of MN5 is analyzed by three-dimensional geometric reconstructions and quantitative co-localization analysis to address the distribution of synaptic terminals. Postembryonic development of MN5 comprises distinct changes in dendritic shape and in the subdendritic distribution of GABAergic input synapses onto MN5. Subdendritic synapse targeting is not a consequence of neuropil structure but must rely on specific subdendritic recognition mechanisms. Passive multicompartment simulations indicate that postembryonic changes in dendritic architecture and in subdendritic input synapse distributions may tune the passive computational properties of MN5 toward stage-specific behavioral requirements.

A conserved nuclear receptor, Tailless, is required for efficient proliferation and prolonged maintenance of mushroom body progenitors in the Drosophila brain.

The intrinsic neurons of mushroom bodies (MBs), centers of olfactory learning in the Drosophila brain, are generated by a specific set of neuroblasts (Nbs) that are born in the embryonic stage and exhibit uninterrupted proliferation till the end of the pupal stage. Whereas MB provides a unique model to study proliferation of neural progenitors, the underlying mechanism that controls persistent activity of MB-Nbs is poorly understood. Here we show that Tailless (TLL), a conserved orphan nuclear receptor, is required for optimum proliferation activity and prolonged maintenance of MB-Nbs and ganglion mother cells (GMCs). Mutations of tll progressively impair cell cycle in MB-Nbs and cause premature loss of MB-Nbs in the early pupal stage. TLL is also expressed in MB-GMCs to prevent apoptosis and promote cell cycling. In addition, we show that ectopic expression of tll leads to brain tumors, in which Prospero, a key regulator of progenitor proliferation and differentiation, is suppressed whereas localization of molecular components involved in asymmetric Nb division is unaffected. These results as a whole uncover a distinct regulatory mechanism of self-renewal and differentiation of the MB progenitors that is different from the mechanisms found in other progenitors.

Hormone-dependent expression of fasciclin II during ganglionic migration and fusion in the ventral nerve cord of the moth Manduca sexta.

The ventral nerve cord of holometabolous insects is reorganized during metamorphosis. A prominent feature of this reorganization is the migration of subsets of thoracic and abdominal larval ganglia to form fused compound ganglia. Studies in the hawkmoth Manduca sexta revealed that pulses of the steroid hormone 20-hydroxyecdysone (20E) regulate ganglionic fusion, but little is known about the cellular mechanisms that make migration and fusion possible. To test the hypothesis that modulation of cell adhesion molecules is an essential component of ventral nerve cord reorganization, we used antibodies selective for either the transmembrane isoform of the cell adhesion receptor fasciclin II (TM-MFas II) or the glycosyl phosphatidylinositol-linked isoform (GPI-MFas II) to study cell adhesion during ganglionic migration and fusion. Our observations show that expression of TM-MFas II is regulated temporally and spatially. GPI-MFas II was expressed on the surface of the segmental ganglia and the transverse nerve, but no evidence was obtained for regulation of GPI-MFas II expression during metamorphosis of the ventral nerve cord. Manipulation of 20E titers revealed that TM-MFas II expression on neurons in migrating ganglia is regulated by hormonal events previously shown to choreograph ganglionic migration and fusion. Injections of actinomycin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor) blocked ganglionic movement and the concomitant increase in TM-MFas II, suggesting that 20E regulates transcription of TM-MFas II. The few neurons that showed TM-MFas II immunoreactivity independent of endocrine milieu were immunoreactive to an antiserum specific for eclosion hormone (EH), a neuropeptide regulator of molting.

The Drosophila Perlecan gene trol regulates multiple signaling pathways in different developmental contexts.

BACKGROUND: Heparan sulfate proteoglycans modulate signaling by a variety of growth factors. The mammalian proteoglycan Perlecan binds and regulates signaling by Sonic Hedgehog, Fibroblast Growth Factors (FGFs), Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF), among others, in contexts ranging from angiogenesis and cardiovascular development to cancer progression. The Drosophila Perlecan homolog trol has been shown to regulate the activity of Hedgehog and Branchless (an FGF homolog) to control the onset of stem cell proliferation in the developing brain during first instar. Here we extend analysis of trol mutant phenotypes to show that trol is required for a variety of developmental events and modulates signaling by multiple growth factors in different situations. RESULTS: Different mutations in trol allow developmental progression to varying extents, suggesting that trol is involved in multiple cell-fate and patterning decisions. Analysis of the initiation of neuroblast proliferation at second instar demonstrated that trol regulates this event by modulating signaling by Hedgehog and Branchless, as it does during first instar. Trol protein is distributed over the surface of the larval brain, near the regulated neuroblasts that reside on the cortical surface. Mutations in trol also decrease the number of circulating plasmatocytes. This is likely to be due to decreased expression of pointed, the response gene for VEGF/PDGF signaling that is required for plasmatocyte proliferation. Trol is found on plasmatocytes, where it could regulate VEGF/PDGF signaling. Finally, we show that in second instar brains but not third instar brain lobes and eye discs, mutations in trol affect signaling by Decapentaplegic (a Transforming Growth Factor family member), Wingless (a Wnt growth factor) and Hedgehog. CONCLUSION: These studies extend the known functions of the Drosophila Perlecan homolog trol in both developmental and signaling contexts. These studies also highlight the fact that Trol function is not dedicated to a single molecular mechanism, but is capable of regulating different growth factor pathways depending on the cell-type and event underway.

Neurogenesis of cephalic sensory organs of Aplysia californica.

The opisthobranch gastropod Aplysia californica serves as a model organism in experimental neurobiology because of its simple and well-known nervous system. However, its nervous periphery has been less intensely studied. We have reconstructed the ontogeny of the cephalic sensory organs (labial tentacles, rhinophores, and lip) of planktonic, metamorphic, and juvenile developmental stages. FMRFamide and serotonergic expression patterns have been examined by immunocytochemistry in conjunction with epifluorescence and confocal laser scanning microscopy. We have also applied scanning electron microscopy to analyze the ciliary distribution of these sensory epithelia. Labial tentacles and the lip develop during metamorphosis, whereas rhinophores appear significantly later, in stage 10 juveniles. Our study has revealed immunoreactivity against FMRFamides and serotonin in all major nerves. The common labial nerve develops first, followed by the labial tentacle base nerve, oral nerve, and rhinophoral nerve. We have also identified previously undescribed neuronal pathways and other FMRFamide-like-immunoreactive neuronal elements, such as peripheral ganglia and glomerulus-like structures, and two groups of conspicuous transient FMRFamide-like cell somata. We have further found two distinct populations of FMRFamide-positive cell somata located both subepidermally and in the inner regions of the cephalic sensory organs in juveniles. The latter population partly consists of sensory cells, suggesting an involvement of FMRFamide-like peptides in the modulation of peripheral sensory processes. This study is the first concerning the neurogenesis of cephalic sensory organs in A. californica and may serve as a basis for future studies of neuronal elements in gastropod molluscs.

Remodeling of an identified motoneuron during metamorphosis: central and peripheral actions of ecdysteroids during regression of dendrites and motor terminals.

During metamorphosis of the moth Manduca sexta, an identified leg motoneuron, the femoral depressor motoneuron (FeDe MN), undergoes reorganization of its central and peripheral processes. This remodeling is under the control of two insect hormones: the ecdysteroids and juvenile hormone (JH). Here, we asked whether peripheral or central actions of the ecdysteroids influenced specific regressive aspects of MN remodeling. We used stable hormonal mimics to manipulate the hormonal environment of either the FeDe muscle or the FeDe MN soma. Our results demonstrate that motor-terminal retraction and dendritic regression can be experimentally uncoupled, indicating that central actions of ecdysteroids trigger dendritic regression whereas peripheral actions trigger terminal retraction. Our results further demonstrate that discrete aspects of motor-terminal retraction can also be experimentally uncoupled, suggesting that they also are regulated differently.

Developmental changes in the electrophysiological properties and response characteristics of Manduca antennal-lobe neurons.

Using whole cell patch-clamp recordings, we have examined changes in the electrophysiological properties and response characteristics of antennal lobe (AL) neurons associated with the metamorphic adult development of the sphinx moth, Manduca sexta. Whole cell current profiles and electrical excitability were examined in dispersed AL neurons in vitro, and in medial-group AL neurons in situ in semi-intact brain preparations. Around stages 2-4 of the 18 stages of metamorphic adult development, whole cell current profiles were dominated by large outward (K+) currents. Calcium-dependent action potentials could be elicited at this stage, but only a small percentage of cells exhibited sodium spikes. From stages 3 to 10, there was a rapid increase in the proportion of AL neurons exhibiting rapidly activating, transient sodium currents, and many cells in vitro exhibited spontaneous bursts of spike activity at this time. As development progressed, action-potential waveforms became shorter in duration and larger in amplitude. Cell-type-specific differences in the prevalence of spontaneous activity, and in the electrophysiological properties and response characteristics of AL neurons, were most apparent late in metamorphosis. While removal of antennal sensory input to the ALs early (stage 1-2) in metamorphosis had no detectable effect on the development of cell excitability, a significantly higher percentage of neurons in vitro from stage 4 pupae exhibited sodium-based action potentials following the addition of serotonin to the culture medium. Characteristic forms of electrical excitability in developing Manduca AL neurons, and their modulation by serotonin, seem likely to play a central role in the functional development of the ALs.

Developmental changes in the density of ionic currents in antennal-lobe neurons of the sphinx moth, Manduca sexta.

Early in metamorphic adult development, action potentials elicited from Manduca sexta antennal lobe neurons are small in amplitude, long in duration, and calcium dependent. As development proceeds, the action potential waveform becomes larger in amplitude, shorter in duration, and increasingly sodium dependent. Whole cell voltage-clamp analysis of Manduca antennal-lobe neurons in vitro has been used to identify voltage-activated currents that contribute to developmental changes in the electrical excitability of these cells. Proximal Branching neurons [putative projection (output) neurons] and Rick Rack neurons (putative local antennal-lobe interneurons) are examined in detail early (pupal stage 5) and late (pupal stage 14) in adult metamorphosis. In both cell types, four voltage-gated and two calcium-dependent ionic currents have been identified. Cell-type-specific changes in the density of sodium, calcium, and potassium currents correlate temporally with changes in cell excitability and spike waveform. Developmental changes in ionic current profiles are accompanied also by the emergence of cell-type-specific response characteristics in the cells. Together with the accompanying paper, this study provides an important foundation for examining the impact of developmental changes in electrical excitability on the growth, electrical properties and connectivity of neurons in central olfactory pathways of the moth.

Molecular characterization and cell-specific expression of a Manduca sexta FLRFamide gene.

FMRFamide-related peptides (FaRPs) are a large group of neuropeptides containing a common RFamide C-terminus; they have been identified in vertebrates and invertebrates. We have isolated the cDNA that encodes three FaRPs in the tobacco hornworm, Manduca sexta, including the amidated decapeptide F10. The larger FaRPs are the partially processed precursors of F10, a neuropeptide belonging to the myosuppressin family of peptides. The presence of all three FaRPs in different tissues suggests differential utilization of typical dibasic processing sites and atypical processing sites C-terminal to leucine residues. F10 mRNA was detected in the brain, nerve cord, and midgut, and the mRNA levels in the nervous system are dynamically regulated during development. In situ hybridization analysis localized the F10 mRNA to a variety of cell types within the central nervous system (CNS), a peripheral neurosecretory cell (L1), and midgut endocrine cells, which suggests diverse functions. Distribution of the F10-containing neurons within the central nervous system is segment-specific, and the developmental profile suggests that the F10 gene products may have stage-specific functions. Molecular characterization of the F10 gene has provided insights into its regulation and cell-specific distribution that will enhance our understanding of how these FaRPs modulate different physiological systems and ultimately behavior.

Modulation of ecdysis in the moth Manduca sexta: the roles of the suboesophageal and thoracic ganglia.

The sequential behaviours shown by insects at ecdysis are due to the sequential release of various hormones, but the transition from one phase to the next can be fine-tuned by inhibitory influences. The ecdysis sequence in the moth Manduca sexta was initiated by injecting sensitive animals with the neuropeptide ecdysis-triggering hormone (ETH). Exposure to ETH stimulates the release of eclosion hormone (EH) which, in turn, activates a set of neurons containing crustacean cardioactive peptide (CCAP) by elevating their levels of intracellular cyclic GMP. We characterized a set of non-CCAP containing neurons that also appear to be EH targets because of their response to cyclic GMP at ecdysis. The neurons did not display leucokinin-, diuretic-hormone- or FMRFamide-like immunoreactivity. They are probably the bursicon-containing cells described previously. After release of EH, there is a transient inhibition of the abdominal centers responsible for ecdysis. Transection experiments suggested that this suppression is via descending inhibitory units from the suboesophageal and thoracic ganglia. The duration of this inhibition appears to depend on the levels of cyclic GMP and can be extended by pharmacologically suppressing cyclic GMP breakdown. We further found that brief exposure to CO(2) caused premature ecdysis. Since the CO(2) treatment was effective only after EH release, it probably acts by suppressing descending inhibition. Studies on adult eclosion suggest that CO(2), given at the appropriate time, can uncouple the basic larval motor program from modulatory influences provided by the adult pterothoracic ganglion. CO(2) therefore appears to be a novel and non-invasive tool for studies of ecdysis behavior in insects.

Adult-like complexity of the larval antennal lobe of D. melanogaster despite markedly low numbers of odorant receptor neurons.

We provide a detailed analysis of the larval head chemosensory system of Drosophila melanogaster, based on confocal microscopy of cell-specific reporter gene expression in P[GAL4] enhancer trap lines. In particular, we describe the neuronal composition of three external and three pharyngeal chemosensory organs, the nerve tracts chosen by their afferents, and their central target regions. With a total of 21 olfactory and 80 gustatory neurons, the sensory level is numerically much simpler than that of the adult. Moreover, its design is different than in the adult, showing an association between smell and taste sensilla. In contrast, the first-order relay of the olfactory afferents, the larval antennal lobe (LAL), exhibits adult-like features both in terms of structure and cell number. It shows a division into approximately 30 subunits, reminiscent of glomeruli in the adult antennal lobe. Taken together, the design of the larval chemosensory system is a "hybrid," with larval-specific features in the periphery and central characteristics in common with the adult. The largely reduced numbers of afferents and the similar architecture of the LAL and the adult antennal lobe, render the larval chemosensory system of Drosophila a valuable model system, both for studying smell and taste and for examining the development of its adult organization.

Neuronal development in larval chiton Ischnochiton hakodadensis (Mollusca: Polyplacophora).

Chitons are the most primitive molluscs and, thus, a matter of considerable interest for understanding both basic principles of molluscan neurogenesis and phylogeny. The development of the nervous system in trochophores of the chiton Ischnochiton hakodadensis from hatching to metamorphosis is described in detail by using confocal laser scanning microscopy and antibodies raised against serotonin, FMRFamide, and acetylated alpha tubulin. The earliest nervous elements detected were peripheral neurons located in the frontal hemisphere of posthatching trochophores and projecting into the apical organ. Among them, two pairs of unique large lateral cells appear to pioneer the pathways of developing adult nervous system. Chitons possess an apical organ that contains the largest number of neurons among all molluscan larvae investigated so far. Besides, many pretrochal neurons are situated outside the apical organ. The prototroch is not innervated by larval neurons. The first neurons of the developing adult central nervous system (CNS) appear later in the cerebral ganglion and pedal cords. None of the neurons of the larval nervous system are retained in the adult CNS. They cease to express their transmitter content and disintegrate after settlement. Although the adult CNS of chitons resembles that of polychaetes, their general scenario of neuronal development resembles that of advanced molluscs and differs from annelids. Thus, our data demonstrate the conservative pattern of molluscan neurogenesis and suggest independent origin of molluscan and annelid trochophores.

Innervation of the heart and aorta of Manduca sexta.

Innervation of the heart and aorta of Manduca sexta was studied by using anatomic, neuronal tracing and immunocytochemical techniques. The study was undertaken to provide a foundation for investigating the neural mechanisms controlling cardiac reversal in adults. Lateral cardiac nerves were not found in the larval or adult heart. The larval heart and aorta seem to lack innervation, but a neurohemal system for the release of a cardioactive peptide is associated with the larval alary muscles. At adult metamorphosis, this neurohemal system regresses, and, at the same time, processes grow onto the anterior aorta. These processes seem to be neurohemal and originate from two pairs of neurosecretory cells located in the subesophageal ganglion. This system is immunoreactive to cardioactive peptides and may function, therefore, in hormonal modulation of the activity of the adult heart. Also during metamorphosis, synaptic innervation develops on the terminal heart chamber, and this innervation is from axons extending through the seventh and eighth dorsal nerves of the terminal abdominal ganglion. These axons originate from cells that have been identified as serial homologs of motor neuron-1 of other abdominal ganglia. These neurons are immunoreactive to a cardioactive peptide, and this peptide probably modulates the synaptic innervation of the terminal heart chamber. During metamorphosis, the target of the motor neurons-1 of the seventh and eighth segments becomes respecified from larval skeletal muscles to the terminal chamber of the adult heart.

Olfactory receptor axons influence the development of glial potassium currents in the antennal lobe of the moth Manduca sexta.

In the olfactory (antennal) lobe of the moth Manduca sexta, olfactory receptor axons strongly influence the distribution and morphology of glial cells. In the present study, we asked whether the development of the electrophysiological properties of the glial cells is influenced by the receptor axons. Whole-cell currents were measured in antennal lobe glial cells in acute brain slices prepared from animals at different stages of metamorphic development (stages 3, 6, and 12). Outward currents were induced by depolarizing voltage steps from a holding potential of -70 mV. At all developmental stages investigated, the outward currents were partly blocked by bath application of the potassium channel blocker 4-aminopyridine (4AP, 10 mM) or by including tetraethylammonium (TEA, 30 mM) in the pipette solution. The relative contribution of the 4AP-sensitive current to the outward current increased from 18% at stages 3 and 6 to 42% at stage 12, while the TEA-sensitive current increased from 18% at stage 3 to 81% at stage 6, and then declined again to 40% at stage 12. In contrast, in the absence of receptor axons, these changes in the contribution of the TEA- and 4AP-sensitive currents to the total outward current did not occur; rather, the current profile remained in the most immature state (stage 3). The results suggest that olfactory receptor axons are essential for development of the mature pattern of glial potassium currents.

Fenvalerate treatment affects development of olfactory glomeruli in Manduca sexta.

Low doses of fenvalerate, a widely used type-II pyrethroid insecticide, have been shown previously to produce abnormal olfactory centers in the brain and abnormal olfactory-mediated behavior in beetles (Wegerhoff et al. [1998] Neuroreport 9:3241-3245). Here, we use the experimental advantages of the moth Manduca sexta to explore the cellular changes that lead to these abnormalities. Our results indicate that treatment with fenvalerate may affect multiple aspects of the development of the primary olfactory centers, the antennal lobes, in Manduca, including ingrowth of olfactory receptor axons, axon fasciculation, and targeting within the antennal lobe, and intercellular signaling between the receptor axons and the glial cells that ordinarily surround and stabilize the developing olfactory glomeruli.

Identification of the cellular target for eclosion hormone in the abdominal transverse nerves of the tobacco hornworm, Manduca sexta.

The isolated abdominal central nervous system of Manduca sexta undergoes an increase in cyclic GMP (cGMP) when exposed to the insect peptide eclosion hormone (EH) before pupal ecdysis. Previously, cGMP immunocytochemistry revealed that the EH-stimulated increase in cGMP was contained in numerous filamentous processes within the transverse nerve associated with each abdominal ganglion. These processes seemed to be the axons of neurosecretory cells projecting to this neurohemal organ. In the present paper, we now show that the EH-stimulated cGMP is not present in neurosecretory terminals. There is no colocalization of the EH-stimulated cGMP with immunoreactivity of two peptides, known to be present in axons in the transverse nerves. Furthermore, there is no colocalization of EH-stimulated cGMP with the synaptic vesicle protein, synaptotagmin. The neurosecretory axons are localized to a narrow band at the anterior margin of the transverse nerve, whereas the cellular elements showing an EH-stimulated cGMP increase are primarily present in the posterior region. There are two cell types in this region: a granular and a nongranular type. The cGMP immunoreactivity seems to be contained within the nongranular type. During adult development, the cells of the posterior compartment spread in a thin layer between the transverse and dorsal nerves, become positive for myosin immunoreactivity between pupal stages 5 and 8, and seem to form the adult ventral diaphragm muscles. We conclude that the EH-sensitive filaments in the transverse nerves of Manduca are most likely to be intrinsic cells that subsequently develop into the ventral diaphragm muscles of the adult.

Development of the labial pit organ glomerulus in the antennal lobe of the moth Manduca sexta: the role of afferent projections in the formation of identifiable olfactory glomeruli.

Iterated neuropil modules called glomeruli are characteristic of primary olfactory centers in both vertebrates and invertebrates. To gain insight into the developmental mechanisms underlying the formation of such structured, organized neuropil, we have examined the development of an identified glomerulus in the olfactory (antennal) lobe of the moth Manduca sexta. The labial pit organ glomerulus (LPOG) receives bilateral sensory projections from the labial pit organs in the labial palps of the mouthparts, while other glomeruli in the antennal lobe receive unilateral projections from the antenna. Here, we chronicle the development of the LPOG under normal and perturbed conditions. Our findings suggest that the sensory axons of the labial pit organ, like those of the antenna, induce and shape growth of interneuronal arborizations, but specific features of interneuronal arborizations such as the relative position of glomerular arborizations within the antennal lobe are independent of both classes of afferent innervation. Labial pit organ axons and antennal axons exhibit a high degree of specificity for their respective target regions, independent of the presence or absence of the other class of afferent axon or the route taken to the antennal lobe. Specification of glomerular position is intrinsic to the antennal lobe rather than a consequence of competition between afferent axons.