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During metamorphosis, the reorganization of the nervous system of Drosophila melanogaster proceeds in part through remodeling of larval neurons. In this study, we used in-vitro imaging techniques and immunocytochemistry to track the remodeling of the thoracic ventral neurosecretory cells. Axons of these neurons prune their larval arbors early in metamorphosis and a larger, more extensive adult arbor is established via branch outgrowth. Expression of EcR dominant negative constructs and an EcR inverted repeat construct resulted in pruning defects of larval axon arbors and a lack of filopodia during pruning, but showed variable effects on outgrowth depending on the construct expressed. Cells expressing either UAS-EcR-B1W650A or UAS-EcR-AW650A lacked filopodia during the outgrowth period and formed a poorly branched, larval-like arbor in the adult. Cells expressing UAS-EcR-B1F645A,UAS-EcR-B2W650A or UAS-IR-EcR (core) showed moderate filopodial activity and normal, albeit reduced, adult-like branching during outgrowth. These results are consistent with the role of activation versus derepression via EcR for successive phases of neuronal remodeling and suggest that functional ecdysone receptor is necessary for some, but not all, remodeling events.

Regressive events that refine exuberant or inaccurate connections are critical in neuronal development. We used multi-photon, time-lapse imaging to examine how dendrites of Drosophila dendritic arborizing (da) sensory neurons are eliminated during early metamorphosis, and how intrinsic and extrinsic cellular mechanisms control this deconstruction. Removal of the larval dendritic arbor involves two mechanisms: local degeneration and branch retraction. In local degeneration, major branch severing events entail focal disruption of the microtubule cytoskeleton, followed by thinning of the disrupted region, severing and fragmentation. Retraction was observed at distal tips of branches and in proximal stumps after severing events. The pruning program of da neuron dendrites is steroid induced; cell-autonomous dominant-negative inhibition of steroid action blocks local degeneration,although retraction events still occur. Our data suggest that steroid-induced changes in the epidermis may contribute to dendritic retraction. Finally, we find that phagocytic blood cells not only engulf neuronal debris but also attack and sever intact branches that show signs of destabilization.

The genesis of dendritic shape during development sets in place key characteristics of a neuron's physiology and connectivity. During this construction, a cell interprets intrinsic cell-specific developmental programs and cues from the environment to generate its final phenotype. In insects that undergo complete metamorphosis certain neurons function in the larval nervous system and then remodel to generate an adult-specific arbor. By studying the dendrites of neurons that undergo such a cellular metamorphosis, one can explore the mechanisms that underlie both stereotyped pruning and local remodeling. Live imaging techniques in intact Drosophila have been especially useful in examining the outgrowth of the adult-specific dendritic arbors in remodeling dendritic arborizing (da) sensory neurons. These neurons show an initial scaffold-building phase during which the cell establishes the overall shape of the arbor and then switch to an elaboration phase where the arbor is filled out with higher order branches. The cellular machinery employed during these two phases is different, with branch retraction being a prominent feature of the scaffold building phase but absent from the elaboration phase. The transition between these two modes does not appear to be "hard-wired" but is plastic and under the extrinsic control of developmental hormones. This transition in branch dynamics may also involve changes in calcium signaling in the growing arbor. The potential relationship between hormone-induced transcriptional change and the calcium dynamics in dendritic morphogenesis is discussed.

Publisher Summary This chapter presents an overview of the ecdysis control system, emphasizing the lessons from ecdysis control that may help in understanding behavioral organization in animals in general. The first indication of a hormonal control over the ecdysis sequence came from studies on the giant silk moths, Hyalophora cecropia and Antheraea pernyi . Brain extirpation and implantation experiments within and between species showed that the brain controlled the timing of the behavior and could exert this control even if transplanted to the abdomen. Among insects, the wealth of variation in the factors that control ecdysis is evident at both the species and the stage level. Manduca and Drosophila have received the most attention in this regard, and there is only scattered information available for other species. In Manduca , ETH and PETH appear in the blood coincident with the onset of pre‐ecdysis movements. Experiments with isolated CNS preparations in Manduca show the onset of pre‐ecdysis motor bursts a few minutes after addition of the peptides and the pre‐ecdysis behaviors fail to appear in mutant Drosophila that lack a functional ETH gene. Temporal ordering is also evident in the interaction of the cells that produce and release the various modulators. A crucial feature of the behavioral program is that it occurs at the appropriate time relative to the developmental processes of the molt.

SUMMARYInsect molting is triggered by ecdysteroids, which are produced in the prothoracic glands (PG). The broad (br) gene is one of the`early genes' directly regulated by ecdysteroids. Ectopic expression of the BR-Z3 isoform in early second instar Drosophila larvae (L2) before the rise of the ecdysteroid titer prevented molting to the third instar, but the larvae subsequently formed L2 prepupae after prolonged feeding. When these larvae were fed on diet containing 20-hydroxyecdysone (20E), they formed pharate third instar larvae. The critical weight for normal L3 pupariation of w1118 larvae was found to be 0.8 mg and that for L2 pupariation was 0.45 mg. We also defined a threshold weight for metamorphosis of 0.3 mg, above which L2 larvae will metamorphose when provided with 20E. BR-Z3 apparently works through the PG cells of the ring gland but not the putative neurosecretory cells that drive ecdysone secretion, because ectopic expression of BR-Z3 specifically in the ring gland caused 53% of the larvae to become permanent first instar larvae. Driving other BR isoforms in the ring gland prevented larval molting or pupariation to varying degrees. These molting defects were rescued by feeding 20E. Overexpression of each of the BR isoforms caused degeneration of the PG cells but on different time courses,indicating that BR is a signal for the degeneration of the PG cells that normally occurs during the pupal–adult transition.

In vivotime-lapse multiphoton microscopy was used to analyze the remodeling of the dendritic arborizing (da) sensory neuron known as dorsal dendritic arborizing neuron E (ddaE) during metamorphosis. After its larval processes have been removed, the cell body of ddaE repositions itself on the body wall between 25 and 40 hr after puparium formation (APF) and begins its adult outgrowth at 40 hr APF. The scaffold of the arbor is laid down between 40 and 54 hr APF, when growth is characterized by high filopodial activity at both terminal and interstitial positions and by branch retraction along with branch establishment. Later in development, filopodial activity remains high but is confined to terminal branches, and branch retraction is no longer seen. Treatment with the insect hormone juvenile hormone (JH), a key regulator of metamorphosis, alters the shape and complexity of the adult dendritic tree in a time-dependent manner. Early treatments with juvenile hormone mimic (JHm) appear to repress extension programs and maintain retraction programs. With later JHm treatments, extension programs appear normal, but retraction programs are maintained beyond their normal time. The JH treatments show the importance of retraction programs in establishing the overall arbor shape.

During embryogenesis of hemimetabolous insects, the sesquiterpenoid hormone, juvenile hormone (JH), appears late in embryogenesis coincident with formation of the first nymphal cuticle. We tested the role of embryonic JH by treating cricket embryos with JH III, or the JH-mimic (JHM) pyriproxifen, during early embryogenesis. We found two discrete windows of JH sensitivity. The first occurs during the formation of the first (E1) embryonic cuticle. Treatment with JHM prior to this molt produced small embryos that failed to complete the movements of katatrepsis. Embryos treated after the E1 molt but before the second embryonic (pronymphal) molt completed katatrepsis but then failed to complete dorsal closure and precociously formed nymphal, rather than pronymphal characters. This second sensitivity window was further assessed by treating embryos with low doses of JH III prior to the pronymphal molt. With low doses, mosaic cuticles were formed, bearing features of both the pronymphal and nymphal stages. The nymphal characters varied in their sensitivity to JH III, due at least in part to differences in the timing of their sensitivity windows. Unexpectedly, many of the JH III-treated embryos with mosaic and precocious nymphal cuticles made a second nymphal cuticle and successfully hatched. JH treatment also affected the growth of the embryos. By focusing on the developing limb, we found that the effect of JH upon growth was asymmetric, with distal segments more affected than proximal ones, but this was not reflected in misexpression of Distal-less or Bric-a-brac, which are involved in proximal-distal patterning of the limb.

In Drosophila, two intersecting molecular loops constitute an autoregulatory mechanism that oscillates with a period close to 24 hr. These loops touch when proteins from one loop, PERIOD (PER) and TIMELESS (TIM), repress the transcription of their parent genes, period (per) and timeless (tim), by blocking positive transcription factors from the other loop. The arrival of PER and TIM into the nucleus of a clock cell marks the timing of this interaction between the two loops; thus, control of PER:TIM nuclear accumulation is a central component of the molecular model of clock function. If a light pulse occurs early in the night as the heterodimer accumulates in the nucleus of clock cells, TIM is degraded, PER is destabilized, and clock time is delayed. Alternatively, if TIM is degraded during the later part of the night, after peak accumulation, clock time advances. Current models state that the effect of a light pulse depends on the state of the PER:TIM oscillation, which turns on the changing levels of TIM. However, previous studies have shown that light:dark (LD) regimes mimicking seasonal changes cause behavioral adjustments while altering clock gene expression. This should be reflected in the adjustment of PER and TIM dynamics. We manipulated LD cycles to assess the effects of altered day length on PER and TIM dynamics in clock cells within the central brain as well as light-induced resetting of locomotor rhythms.

The steroid hormone 20-hydroxyecdysone induces metamorphosis in insects. The receptor for the hormone is the ecdysone receptor, a heterodimer of two nuclear receptors, EcR and USP. In Drosophila the EcR gene encodes 3 isoforms (EcR-A, EcR-B1 and EcR-B2) that vary in their N-terminal region but not in their DNA binding and ligand binding domains. The stage and tissue specific distribution of the isoforms during metamorphosis suggests distinct functions for the different isoforms. By over-expressing the three isoforms in animals we present results supporting this hypothesis. We tested for the ability of the different isoforms to rescue the lack of dendritic pruning that is characteristic of mutants lacking both EcR-B1 and EcR-B2. By expressing the different isoforms specifically in the affected neurons, we found that both EcR-B isoforms were able to rescue the neuronal defect cell autonomously, but that EcR-A was less effective. We also analyzed the effect of over-expressing the isoforms in a wild-type background. We determined a sensitive period when high levels of either EcR-B isoform were lethal, indicating that the low levels of EcR-B at this time are crucial to ensure normal development. Over-expressing EcR-A in contrast had no detrimental effect. However, high levels of EcR-A expressed in the posterior compartment suppressed puparial tanning, and resulted in down-regulation of some of the tested target genes in the posterior compartment of the wing disc. EcR-B1 or EcR-B2 over-expression had little or no effect.

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.

Publisher Summary This chapter reviews briefly the general endocrine regulation of the postembryonic development in insects and the characteristics of insect hormones, and then discusses what is known about their cellular and molecular modes of action. It focuses primarily on their roles in the development of the nervous system. Here, the primary emphasis is on two model insects, the moth Manduca sexta and the fruit fly Drosophila melanogaster. Insect development is regulated by two families of hormones: steroid ecdysones and sesquiterpenoid juvenile hormones (JHs). The ecdysones control the molting of exoskeleton (cuticle) that is secreted by the underlying epidermis, and has both rigid and extensible regions. To accommodate the growth and changes in morphology that accompany metamorphosis, a new exoskeleton is periodically formed followed by the shedding of the old one. These molts are evident during the late stages of embryogenesis, and continue through the growth and metamorphic phases of postembryonic life. The adult insect does not molt.

A requirement for nitric oxide (NO) in visual system development has been demonstrated in many model systems, but the role of potential downstream effector molecules has not been established. Developing Drosophila photoreceptors express an NO-sensitive soluble guanylate cyclase (sGC), whereas the optic lobe targets express NO synthase. Both of these molecules are expressed after photoreceptor outgrowth to the optic lobe, when retinal growth cones are actively selecting their postsynaptic partners. We have previously shown that inhibition of the NO-cGMP pathway in vitro leads to overgrowth of retinal axons. Here we examined flies mutant for the alpha subunit gene of the Drosophila sGC (Gcalpha1). This mutation severely reduced but did not abolish GCalpha1 protein levels and NO-stimulated sGC activity in the developing photoreceptors. Although few mutant individuals possessed a disorganized retinal projection pattern, pharmacological NOS inhibition during metamorphosis increased this disorganization in mutants to a greater degree than in the wild type. Adult mutants lacked phototactic behavior, and the off-transient component of electroretinograms was frequently absent or greatly reduced in amplitude. Normal phototaxis and off-transient amplitude were restored by heat shock-mediated Gcalpha1 expression applied during metamorphosis but not in the adult. We propose that diminished sGC activity in the visual system during development causes inappropriate or inadequate formation of first-order retinal synapses, leading to defects in visual system function and visually mediated behavior.

The stomatogastric ganglion (STG) of the crab Cancer productus contains approximately 30 neurons arrayed into two different networks (gastric mill and pyloric), each of which produces a distinct motor pattern in vitro. Here we show that the functional division of the STG into these two networks requires intact NO-cGMP signaling. Multiple nitric oxide synthase (NOS)-like proteins are expressed in the stomatogastric nervous system, and NO appears to be released as an orthograde transmitter from descending inputs to the STG. The receptor of NO, a soluble guanylate cyclase (sGC), is expressed in a subset of neurons in both motor networks. When NO diffusion or sGC activation are blocked within the ganglion, the two networks combine into a single conjoint circuit. The gastric mill motor rhythm breaks down, and several gastric neurons pattern switch and begin firing in pyloric time. The functional reorganization of the STG is both rapid and reversible, and the gastric mill motor rhythm is restored when the ganglion is returned to normal saline. Finally, pharmacological manipulations of the NO-cGMP pathway are ineffective when descending modulatory inputs to the STG are blocked. This suggests that the NO-cGMP pathway may interact with other biochemical cascades to partition rhythmic motor output from the ganglion.

The steroid hormone 20-hydroxyecdysone (20E) initiates metamorphosis in insects by signaling through the ecdysone receptor complex, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP). Analysis of usp mutant clones in the wing disc of Drosophila shows that in the absence of USP, early hormone responsive genes such as EcR, DHR3 and E75B fail to up-regulate in response to 20E, but other genes that are normally expressed later, such as β-Ftz-F1 and the Z1 isoform of the Broad-Complex (BRC-Z1), are expressed precociously. Sensory neuron formation and axonal outgrowth, two early metamorphic events, also occur prematurely. In vitro experiments with cultured wing discs showed that BRC-Z1 expression and early metamorphic development are rendered steroid-independent in the usp mutant clones. These results are consistent with a model in which these latter processes are induced by a signal arising during the middle of the last larval stage but suppressed by the unliganded EcR/USP complex. Our observations suggest that silencing by the unliganded EcR/USP receptor and the subsequent release of silencing by moderate steroid levels may play an important role in coordinating early phases of steroid driven development.

Publisher Summary This chapter discusses the roles of nitric oxide (NO) in the physiology and development of the nervous system. The nervous systems of invertebrates offer the twin advantages of numerical simplicity and the presence of comparatively large and identifiable nerve cells. The identified neurons can be reliably located in different animals that make it possible to analyze the specific function of a given neuron in differing developmental and physiological contexts. This approach has been a valuable tool in the study of the cellular aspects of nervous system function and development, and it is common theme in the discussion of NO-mediated signaling in invertebrates. NO is an ancient signaling molecule, presumably evolving before the radiation of the metazoans. There is remarkable conservation of function for NO-mediated signaling, even among organisms that belong to different kingdoms. Comparative studies have found NO-producing neurons in animals from all the major phyla, suggesting that cellular signaling using NO is an ancestral character of both vertebrate and invertebrate nervous systems.

Publisher Summary This chapter discusses the ecdysone receptors and their biological actions. It also summarizes the insect endocrinology and the roles of these steroids in the molting and metamorphosis. Natural hormones that lead to molting and metamorphosis are ecdysones. Molecules whose structures resemble those of the natural hormones are called ecdysteroids. The receptor for ecdysone is a member of the nuclear receptor superfamily that acts as a ligand-dependent transcription factor. The vertebrate steroid hormone receptors act as homodimers whereas the functional ecdysone receptor is always a heterodimer of receptor for ecdysone (EcR) with another member of the nuclear receptor (NR) superfamily, Ultraspiracle, the insect homolog of the vertebrate retinoid X receptor (RXR). Two new technologies promise to transform the environment for investigations of insect hormones, ecdysone receptor, and metamorphosis. The microarrays of expressed sequence tags are constructed (ESTs) and used hybridization to catalog changes in gene expression during metamorphosis. The first technological achievement is reviewed: the complete sequence of the Drosophila genome is obtained and is about to be released. It will be the first complete insect sequence and also the first genomic sequence from an organism that has served as a model for nuclear receptor endocrinology.

The cellular response to steroid hormones is mediated by nuclear receptors which act by regulating transcription. In Drosophila melanogaster, the receptor for the insect molting hormone, 20-hydroxyecdysone, is a heterodimer composed of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The DNA binding domains of arthropod USPs and their vertebrate homologs, the retinoid X receptor (RXR) family, are highly conserved. The ligand binding domain sequences, however, divide into two distinct groups. One group consists of sequences from members of the holometabolous higher insect orders Diptera and Lepidoptera, the other of sequences from vertebrates, a crab and a tick. We here report the sequence of an RXR/USP from the hemimetabolous orthopteran, Locusta migratoria. The locust RXR/USP ligand binding domain clearly falls in the vertebrate-crab-tick rather than the dipteran-lepidopteran group. The reason for the evolutionarily abrupt divergence of the dipteran and lepidopteran sequences is unknown, but it could be a change in the type of ligand bound or the loss of ligand altogether.

Both the proliferation and differentiation of ventral diaphragm myoblasts are controlled by ecdysteroid during metamorphosis of the moth, Manduca sexta, but the responses have different hormonal requirements. Tonic exposure to moderate levels of ecdysteroid are required to stimulate myoblast proliferation. This is due to the presence of an ecdysteroid-dependent control point in the G(2) phase of the cell cycle. As a result, proliferation can be repeatedly turned on or off simply by adjusting the concentration of ecdysteroid to be above or below a critical threshold concentration. In contrast, high levels of ecdysteroid trigger irreversible proliferative arrest and differentiation of myofibers. Myoblast proliferation and differentiation also differ in their response to the juvenile hormone mimic, methoprene. Ecdysteroid-dependent proliferative arrest and differentiation are blocked by coculture with methoprene but methoprene has no effect on ecdysteroid-dependent proliferation. In the animal, premature exposure to high levels of ecdysteroid in the absence of juvenile hormone triggers precocious differentiation of the myoblasts, resulting in the formation of several thin bands of muscle rather than a complete diaphragm. Thus, ecdysteroid and juvenile hormone collaborate to determine the size and shape of the adult musculature.

Neurogenesis was examined in the central nervous system of embryos of the primitively wingless insect, the silverfish, Ctenolepisma longicaudata, using staining with toluidine blue (TB) and the incorporation of bromodeoxyuridine (BUdR). The silverfish has the same number and positioning of neuroblasts as seen in more advanced insects and the relative order in which the different neuroblasts segregate from the neuroectoderm is highly conserved between Ctenolepisma and the grasshopper, Schistocerca. Of the 31 different neuroblasts found in a thoracic segment, one (NB 6–3) has a much longer proliferative period in silverfish. Of the remainder, 14 have similar proliferative phases, while16 neuroblasts have extended their proliferative period by 10% of embryogenesis or greater in the grasshopper as compared with the silverfish. Both insects had similar periods of abdominal neurogenesis except that in the silverfish terminal ganglion a prominent set of neuroblasts continued dividing until close to hatching, possibly reflecting the importance of cercal sensory input in this insect. This comparison between silverfish and grasshopper shows that the shift from wingless to flying insects was not accompanied by the addition of any new neuronal lineages in the thorax. Instead, selected lineages underwent a proliferative expansion to supply the additional neurons presumably needed for flight. The expansion of specific thoracic lineages was accompanied by the reduction of the terminal abdominal lineages as flying insects began to de-emphasize their cercal sensory system.

During the metamorphic reorganization of the insect central nervous system, the steroid hormone 20-hydroxyecdysone induces a wide spectrum of cellular responses including neuronal proliferation, maturation, cell death and the remodeling of larval neurons into their adult forms. In Drosophila, expression of specific ecdysone receptor (EcR) isoforms has been correlated with particular responses, suggesting that different EcR isoforms may govern distinct steroid-induced responses in these cells. We have used imprecise excision of a P element to create EcR deletion mutants that remove the EcR-B promoter and therefore should lack EcR-B1 and EcR-B2 expression but retain EcR-A expression. Most of these EcR-B mutant animals show defects in larval molting, arresting at the boundaries between the three larval stages, while a smaller percentage of EcR-B mutants survive into the early stages of metamorphosis. Remodeling of larval neurons at metamorphosis begins with the pruning back of larval-specific dendrites and occurs as these cells are expressing high levels of EcR-B1 and little EcR-A. This pruning response is blocked in the EcR-B mutants despite the fact that adult-specific neurons, which normally express only EcR-A, can progress in their development. These observations support the hypothesis that different EcR isoforms control cell-type-specific responses during remodeling of the nervous system at metamorphosis.

Ecdysteroids regulate the remodeling of the dorsal external oblique 1 (DEO1) muscle during metamorphosis inManduca sexta(Hegstrom and Truman, 1996a). We show that the temporal and spatial patterning of the A and B1 isoforms of the ecdysone receptor (EcR) within muscle DEO1 corresponds with the developmental fates of the fibers. Using antibodies directed to specific isoforms of EcR, we show that the expression of various EcR isoforms in myonuclei differ among the five fibers of DEO1 and correspond with the developmental response of the muscle to the changing steroid titers and to the pattern of innervation. Muscle degeneration and apoptosis of myonuclei in all fibers are correlated with the expression of only EcR-A just before pupal ecdysis and then with the expression of low levels of both EcR-A and EcR-B1 shortly after pupation. Only the first fiber of muscle DEO1 participates in the regrowth of the adult muscle, and only this fiber shows an upregulation of EcR-B1 that is evident at 3 d after pupal ecdysis. Denervation of the muscle prevents both the upregulation of EcR-B1 and myoblast proliferation. We conclude that the developmental fate of muscle DEO1 during metamorphosis is orchestrated by interactions between rising and falling ecdysteroid titers, the pattern of expression of EcR isoforms by the muscle, and interactions with other cells in the local environment.

Cell proliferation within the optic lobe anlagen is dependent on ecdysteroids during metamorphosis of the moth Manduca sexta. We use cultured tissues to show that ecdysteroids must be maintained above a sharp threshold concentration to sustain proliferation. Proliferation can be turned on and off repeatedly simply by shifting the ecdysteroid concentration to above or below this threshold. In subthreshold hormone, cells arrest in the G2 phase of the cell cycle. Ecdysteroid control of proliferation is distinguished from differentiative and maturational responses to ecdysteroids by requiring tonic exposure to the hormone and lower levels of 20-hydroxyecdysone, and by being sensitive to either 20-hydroxyecdysone or its precursor, ecdysone. These characteristics allow optic lobe development to be divided into two ecdysteroid-dependent phases. Initially, moderate levels of ecdysteroid stimulate proliferation. Later, high levels of 20-hydroxyecdysone trigger a wave of apoptosis within the anlage that marks completion of its proliferative phase.

The photoreceptors of Drosophila express a nitric oxide–sensitive guanylate cyclase during the first half of metamorphosis, when postsynaptic elements in the optic lobe are being selected. Throughout this period, the optic lobes show NADPH-diaphorase activity and stain with an antibody to nitric oxide synthase (NOS). The NOS inhibitor L-NAME, the NO scavenger PTIO, the sGC inhibitor ODQ, and methylene blue, which inhibits NOS and guanylate cyclase, each caused the disorganization of retinal projections and extension of photoreceptor axons beyond their normal synaptic layers in vitro. The disruptive effects of L-NAME were prevented with the addition of 8-bromo-cGMP. These results suggest NO and cGMP act to stabilize retinal growth cones at the start of synaptic assembly.

At the end of each molt insects shed their old cuticle by performing the stereotyped behavior of ecdysis. In the moth, Manduca sexta, this behavior is triggered by the neuropeptide eclosion hormone (EH). Insights into the mechanism of action of EH have come from the identification of a small network of peptidergic neurons that shows increased cyclic 3',5'-guanosine monophosphate (cGMP) immunoreactivity at ecdysis in insects from many different orders. Here we present further evidence that strengthens the association between ecdysis and the occurrence of this cGMP response in Manduca. We found that the cGMP increases occurred at every ecdysis, although some of the neurons that showed a response at larval ecdysis did not participate at pupal and adult ecdysis. Both ecdysis and the cGMP increases only required an intact connection with the brain for the first 30 min after EH injection. Interestingly, ecdysis in debrained animals only occurred if the cGMP response had been initiated, suggesting that the onset of this response marks the time at which the central nervous system is first able to drive ecdysis. Finally, we found that the appearance of sensitivity to EH for triggering the cGMP response coincided with the time at which EH first triggers ecdysis.

The process by which a butterfly emerges from a chrysalis, called ecdysis, has been thought to be controlled by eclosion hormone. J. Truman discusses the discovery of a new insect hormone, reported in this issue of Science by hacek Zithacek nan et al. (p. 88), that can also trigger ecdysis—MasETH. The two hormones seem to act sequentially.

In the neural circuits that comprise the crustacean stomatogastric nervous system (STNS), synaptically delivered neurotransmitters and circulating neurohormones elicit a wide range of rhythmic motor outputs. However, functional roles for second messengers in this system are poorly understood. Here we demonstrate two different signaling pathways that control the synthesis of 3′,5′-cGMP in the crab STNS. One pathway is activated by nitric oxide (NO) and is mediated by a cytoplasmic guanylate cyclase. A second pathway is stimulated by peptide-containing extracts from a crab neurohemal organ that activate a membrane-associated guanylate cyclase. Using whole-mount immunocytochemistry to localize individual cGMP-containing cells, we find that NO elevates intracellular cGMP in a small subset of STNS neurons. Immunopositive cells are found predominantly in the stomatogastric ganglion, with a few additional cells located in the oesophageal and commissural ganglia. Crab tissues differ in their sensitivities to NO and to the peptide-containing extract. The NO- mediated pathway is apparently restricted to the nervous system, whereas the peptidemediated pathway is present in every tissue tested. The results of these experiments demonstrate that multiple signaling pathways involving cGMP are present in the STNS and suggest that this second messenger may help control the metabolic and physiological status of these motor circuits.

The motor system of the moth Manduca sexta is completely remodeled during the pupal-adult transformation (PAT). It is stable until the formation of the pupal stage (0% PAT), but larval motor end plates become disrupted by 5% PAT and are lost by 10% PAT, at the time that the muscle has begun to degenerate. Most of the axonal arbor is retracted by 15% with the first signs of adult sprouts appearing by 20% PAT, coinciding with proliferative activity in the remains of the larval muscle. Extensive growth of the axonal arbor begins after 30% PAT, with an initial phase of rapid longitudinal growth (35–50% PAT) and then the production of short transverse branches that then form sprays of end plates (50–70% PAT). Growth and maturation of the end plates occupies the remainder of metamorphosis. Neuromuscular metamorphosis was interfered with by systemic or local treatment with a mimic of the insect juvenile hormone. The results of these treatments suggest that some aspects of the removal of larval axonal branches requires cues from the target. For the sprouting response, the rapid longitudinal growth over the muscle appears to be due to ecdysteroids acting directly on the cell body of the motoneuron. By contrast, the subsequent production and maintenance of transverse sprouts and the corresponding end plates may be an indirect response to stimulation of muscle growth and differentiation by ecdysteroids.

We analyzed the development of several sets of postembryonic sex-specific motoneurons in Manduca sexta which belong to a group of homologous lineages of neurons called the imaginal midline neurons (IMNs). Adult female oviduct motoneurons and male sperm duct motoneurons are IMNs that show similar anatomical features and differentiate during metamorphosis, despite appearing in different segments: A7 for oviduct neurons, A9 for sperm duct neurons. These cells are born at the same time and, initially, similar sets are found in A7 and A9 ganglia of larvae of both sexes. The dimorphic adult pattern is generated by sex-specific production and cell death. A7 IMNs differentiate in both sexes through early pupal stages, whereupon they disappear in the male and become the oviduct motoneurons in the female. A9 IMNs are overproduced in the male, and subsequent cell death reduces male cell number and eliminates the small complement of female cells; the surviving male cells develop into the sperm duct motoneurons. Similar IMN arrays are generated in nongenital ganglia, but show non-sex-specific fates. This suggests that both the sex of these cells and their segment of residence play major roles in their subsequent differentiation.

While the majority of neurons in the adult nervous system of the moth Manduca sexta are produced postembryonically, little is known about how these cells interact with their targets during development. Few of these cells are motor neurons; most of Manduca's adult motor neurons are respecified larval motor neurons that developed embryonically. A few motor neurons do develop postembryonically, including a large class of mixed neurosecretory and motor neurons called the imaginal midline neurons (IMNs). A subset of these cells show an unusual pattern of sex-specific development and survival (Thorn and Truman, 1994, J. Neurobiol. in press), which led us to suspect that factors extrinsic to the cells were controlling their fates. We analyzed one such potential factor by altering the contacts between a subset of these developing IMNs and their adult-specific target, the male sperm duct. When we transected the nerve that innervated the sperm duct in the pupa, we observed a loss of many sperm duct IMNs. In contrast, a transection of the same nerve in larvae showed no neuron loss. Immunocytochemistry showed that the pupal nerve transections were accompanied by a loss of axon endings on the sperm duct, while the larval nerve transections showed no such loss. Using local hormone application to slow the development of the sperm duct while leaving the nerve intact still resulted in a loss of IMNs. These results suggest that these IMNs need contact with a robust developing target in the pupa to survive metamorphosis.

Eclosion hormone (EH) is a 7000 Da peptide that triggers ecdysis behavior in insects. In the moth, Manduca sexta, EH is found in two pairs of ventromedial (VM) cells in the brain which send their axons down the ventral nerve cord to a neurohemal site in the proctodeal nerve in the larva and pupa. During adult development, these cells send axon collaterals to the corpora cardiaca where they form a new release site used for adult eclosion. Studies of bioassayable peptide during the 5th larval instar and the larval-pupal transformation revealed that after depletion at ecdysis, the VM cells showed a transient increase in EH found in their cell bodies and axons. By contrast, their terminals in the proctodeal nerve showed a gradual accumulation of peptide followed by a release of over 90% of the stored material at pupal ecdysis. In situ hybridization analysis on whole mounts of the brains showed that the VM cells always contained EH mRNA with increased accumulation during the larval and pupal molting periods with a slight decline just before ecdysis. High levels of EH mRNA were found in brains of diapausing pupae. During the first two-thirds of adult development, mRNA accumulated to high levels, then slowly declined until ecdysis. EH mRNA levels then increased and remained at intermediate levels up to 3 days after adult eclosion. At no time was EH mRNA found in the lateral neurosecretory cell cluster previously reported to produce EH for adult eclosion.

n the moth Manduca sexta, the declining ecdysteroid titer on the final day of the molt from the fourth to the fifth larval instar acts on the ventromedial neurosecretory cells (VM cells) to stimulate the release of eclosion hormone (EH). EH then triggers the motor programs involved in ecdysis behavior. Intracellular recordings that were made from the VM cells throughout the intermolt and molting stages showed no spontaneous action potentials until 0.9 hr before ecdysis (during the expected time of EH release), when 50% of the VM cells fired tonically at rest. This change was associated with a marked reduction in VM cell threshold without alteration of input resistance, resting potential, or synaptic drive. The increase in VM cell excitability was dependent on the declining ecdysteroid titer, because the injection of 20- hydroxyecdysone (20-HE) 11.5 hr before ecdysis significantly delayed the expected decrease in VM cell threshold. Since the same steroid treatment given 4.6 hr before ecydysis did not delay the subsequent increase in VM cell excitability, the inhibitory actions of 20-HE appear not to be mediated by a rapid membrane mechanism. The possible involvement of genomic events in the steroid-dependent increase in VM cell excitability was examined using the RNA synthesis inhibitor actinomycin D (AcD). When injected 6.3 hr before ecdysis, AcD blocked EH release without altering the response of the nervous system to exogenous peptide. Such AcD treatments also prevented the increase in VM cell excitability. These results suggest that the declining ecdysteroid titer increases the excitability of the VM cells via a transcription-dependent process.

A decline in circulating 20-hydroxyecdysone permits the emergence of the adult Manduca sexta moth; this endocrine signal also triggers the death of approximately half of the neurons in the unfused abdominal ganglia of the moth central nervous system. This programmed death of neurons was markedly reduced by treatment with either actinomycin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor). Similar results were found after addition of these agents to ventral nerve cord cultures. The effectiveness of these treatments in delaying or blocking neuronal death depended upon their time of administration relative to the normal time of post-emergence death in the particular motoneuron under study: late-dying neurons, for example, could still be saved by these treatments even after early-dying neurons had already initiated degeneration. In both intact moths and cultured ventral nerve cords, the ability of actinomycin D to prevent neuronal death waned at the same time at which replacement of the steroid hormone could no longer block neuronal death. This suggests that the steroid commitment point represents the time at which the genes that mediate cell death are transcribed. Cycloheximide remained effective in delaying or blocking neuronal death until shortly before the onset of degeneration, suggesting that ongoing protein synthesis is essential for the initiation of the degeneration response.

Eclosion hormone (EH) is a neuropeptide that triggers the performance of ecdysis behaviors at the end of a molt. We have isolated the EH gene from Drosophila melanogaster, and localized the gene to the right arm of chromosome 3 at band position 90B1-2. The 97-amino-acid translation product contains a signal peptide followed by a 73-amino-acid prohormone. The N-terminus of the prohormone has diverged from lepidopteran EH both in its length and amino acid composition, and contains a potential endoproteolytic cleavage site. The deduced sequence of Drosophila EH is 58% identical (36 of 62 amino acids) to that of Manduca EH. The EH gene is expressed as a 0.8-kb transcript in a single pair of brain neurons which extend their processes the entire length of the central nervous system and also to the corpora cardiaca portion of the ring gland. These cells show massive depletion of immunoreactive EH at ecdysis.

During metamorphosis, the insect nervous system must change to accomodate alterations in body form and behavior. Studies primarily on moths have shown that these changes involve the death of some larval neurons, the conservation and remodeling of others, and the maturation of new, adult-specific cells. The motor and sensory sides of the adult CNS vary in this regard with the former being constructed primarily from remodeled larval components, whereas the latter arises primarily from new neurons. Neuronal remodeling has received considerable attention. Larval-specific dendritic fields are pruned back during the larval-pupal transition, followed by the sprouting of adult-specific dendrites. Simple reflexes have been used to correlate these neuronal changes with the acquisition or loss of particular behaviors. The loss of the proleg retraction reflex is associated with the regression of the dendritic arbors of the proleg motoneurons. By contrast, expansion of axon arbors of the gin-trap afferents is necessary, but not sufficient, for the assembly of the gin-trap reflex in the pupal stage. The stretch receptor reflex provides a third example in which a new dendritic field in the adult form of a neuron is associated with new adult-specific connections. Interestingly, these connections are masked by persisting larval contacts until the emergence of the adult moth. For the metamorphosis of more complex behavioral circuits, some, such as that for flight behavior, seem to be assembled de novo, whereas others, like that for adult ecdysis behavior, show conservation of some circuit elements from the larval stage but with the superposition of some adult-specific components.

Larvae of the red abalone, Haliotis rufescens, rely on external chemical cues to trigger metamorphosis; thus, the timing of metamorphosis is depedent upon the larva's chance encounter with the appropriate substrate. We examined the effect of the timing of metamorphosis on the development of the central nervous system (CNS), concentrating on the pattern of serotonin and small cardioactive peptide- (SCP) immunopositive neurons in the cerebral ganglia. By 4 days postfertilization the cerebral ganglion has five pairs of serotonin-immunoreactive (IR) neurons, one pair of which (the V cells) innervate the velum. This complement of cells remains stable for as long as the larval stage persists but metamorphosis causes the rapid loss of the V cells. In the case of SCP-IR neurons, one pair is present prior to metamorphic competency, but as larvae continue to age in the absence of inducing cues, additional pairs are gradually added. Metamorphosis causes an acceleration in SCP-IR neuron addition. This separation of developmental patterns is well adapted for the inherent uncertainty of the timing of metamorphosis in abalone larvae. © 1992 John Wiley & Sons, Inc.

Adult specific neurons in the central nervous system of holometabolous insects are generated by the postem-bryonic divisions of neuronal stem cells (neuroblasts). In the ventral nervous system of Drosophila melanogaster, sex-specific divisions by a set of abdominal neuroblasts occur during larval and early pupal stages. Animals mutant for several sex-determining genes were analyzed to determine the genetic regulation of neuroblast commitment to the male or female pattern of division and the time during development when these decisions are made. We have found that the choice of the sexual pathway taken by sex-specific neuroblasts depends on the expression of one of these genes, doublesex (dsx). In the absence of any functional dsx+ products, the sexspecific neuroblasts fail to undergo any postembryonic divisions in male or female larval nervous systems. From the analysis of intersexes generated by dominant alleles of dsx, it has been concluded that the same neuroblasts provide the sex-specific neuroblasts in both male and female central nervous systems. The time when neuroblasts become committed to generate their sex-specific divisions was identified by shifting tra-2ts flies between the male- and female-specifying temperatures at various times during larval development. Neuroblasts become determined to adopt a male or female state at the end of the first larval instar, a time when abdominal neuroblasts enter their first postembryonic S-phase.

Publisher Summary Peptides and the neurons that produce them play key roles in regulating physiology, development, and behavior of insects. This chapter describes the eclosion hormone (EH) system of insects, which coordinates the physiological and behavioral processes necessary for shedding the old exoskeleton at the end of a molt. Circulating EH has only been found just preceding the times of ecdysis, and these are the only times, when measurable EH depletions occur in the neurohemal storage sites. The nature and context of the behaviors triggered by EH are doubtlessly responsible for some of the extreme modifications of the EH system. The performance of ecdysis behavior only makes sense when an insect is covered by an old cuticle that must be shed. The appearance of EH is rapidly followed by the inactivation of the system on both the release and response sides. Both sides of the system are only then reconstituted in response to the steroid cues that also provide the physiological context for the next bout of ecdysis.

The majority of the neurons in the adult nervous system of Manduca sexta are born postembryonically, during larval life. Stereotypic arrays of identifiable neuroblasts generate their clonal families or lineages commencing at the end of the second larval instar through pupal day 2, when the neuroblasts die (Booker and Truman, 1987a). We have used immunohistochemical techniques to follow the neurochemical differentiation of GABA and a peptide similar to molluscan small cardioactive peptide B (SCPB) in identified lineages. We report here the distribution and developmental acquisition of the expression of these putative transmitters. There are 24 postembryonic lineages in the second thoracic ganglion of the larvae (Booker and Truman, 1987a). Immunoreactivity against GABA and SCPB is seen only in a subset of these 24 clonal families. GABA immunoreactivity is confined to the progeny of the E, K, M, N, T, and X neuroblasts and is expressed by most or all of the neurons in these lineages. The SCPB-like immunoreactivity is found in a subset of the neurons in only two clonal groups, the K and M groups, and is colocalized with GABA. These results show that, though heterogeneity in transmitter type exists (GABA, GABA/SCPB), members of a given lineage share at least some features (GABA) in common. The onset of transmitter expression was followed in detail for the K- and M-lineage neurons. During the larval stages, the postembryonic lineage cells are developmentally arrested in a partially differentiated state (Booker and Truman, 1987a) and do not express transmitter immunoreactivity at this time. Their maturation resumes with the onset of metamorphosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of GABA is restricted to the progeny of only six of the 24 identified postembryonic lineages in the thoracic ganglia of the tobacco hornworm, Manduca sexta (Witten and Truman, 1991). It is colocalized with a peptide similar to molluscan small cardioactive peptide B (SCPB) in some of the neurons in two of the six lineages. By combining chemical ablation of the neuroblasts at specific larval stages with birth dating of the progeny, we tested whether the expression of GABA and the SCPB-like peptide was determined strictly by cell lineage or involved cellular interactions among the members of individual clonal groups. Chemical ablation of the six specific neuroblasts that produced the GABA-positive neurons (E, K, M, N, T, and X) or of the two that produced the GABA + SCPB-like-immunoreactive neurons (K, M) prior to the generation of their lineages resulted in the loss of these immunoreactivities. These results suggest that regulation between lineages did not occur. Ablation of the K and M neuroblasts after they had produced a small portion of their lineages had no effect on the expression of GABA, but did affect the pattern of the SCPB-like immunoreactivity. Combining birth-dating techniques with transmitter immunocytochemistry revealed that it was the position in the birth order and not interactions among the clonally related neurons that influenced the peptidergic phenotype. These results suggest that cell lineage is involved in establishing the GABAergic phenotype and that both cell lineage and birth order influence the determination of the peptidergic phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Ecdysis, a behavior by which insects shed the old cuticle at the culmination of each molt, is triggered by a unique peptide hormone, eclosion hormone (EH). In pupal Manduca sexta, EH is released into the hemolymph just prior to ecdysis, and circulating hormone is sufficient to elicit this behavior. 2. Removal of the proctodeal nerves in prepupal animals eliminated the appearance of blood-borne EH, but ecdysis behavior occurred on schedule. Therefore, circulating EH is not necessary for the triggering of ecdysis. 3. In contrast, a set of dermal glands failed to show their expected bout of secretion after proctodeal nerve removal. Injection of exogenous EH rescued this secretion. Thus, circulating EH appears necessary for action on peripheral but not central targets. 4. A major reduction in EH immunostaining is seen in the proctodeal nerves just preceding ecdysis; this coincides with a >90% reduction in extractable EH from this structure and the appearance of circulating EH. A similar, concomitant reduction was seen in central EH cell processes, suggesting release of peptide within the CNS. 5. Antidromic stimulation of the proctodeal nerve stumps following proctodeal nerve removal triggered precocious ecdysis. This result further supports the conclusion that centrally released EH is sufficient to trigger the motor program.

Neuropeptides are an important class of regulatory molecules in insects which affect a number of physiological functions relating to growth, metamorphosis, and homeostasis. One of the better known neuropeptides is eclosion hormone (EH). EH is synthesized in the brain of the insect and released into the hemolymph at a defined developmental time just prior to ecdysis at the end of the molt (Reynolds and Truman, 1983). Once released into the hemolymph, its principal effect is on the nervous system of the insect to activate a program of neural activity which leads to ecdysis. Other effects of EH integrate ecdysis with the physiological events that occur at that time.

Ecdysis involves more than just the shedding of an old cuticle. The ecdysis of the adult, for example, is the time when the new imago first confronts the external world, and at this time many behavioral and physiological systems first become activated. Studies on the hormonal regulation of this event began 20 years ago with the report that a transplanted brain could control the timing of ecdysis of a debrained host (Truman and Riddiford, 1970). A factor that induces precocious ecdysis was subsequently extracted from the brain and termed the eclosion hormone (EH; Truman, 1971). The sole known function of this hormone is to coordinate the events that occur at ecdysis. It acts directly on the central nervous system to release the appropriate ecdysial behaviors (e.g., Truman, 1978) but also has ecdysis-related effects on non-neural targets such as epidermis (Reynolds, 1977) and muscle (Schwartz and Truman, 1982).

Publisher Summary This chapter deals with the use of invertebrates to study cellular and molecular aspects of neuronal death. The identified neurons in these animals that are well known for their stereotyped morphologies and patterns of connectivity may likewise have stereotyped, predictable fates. Thus, one can examine not only the changes that occur after degeneration begins, but also the events that precede death. Much of this work has been done on insects, in which neuronal death plays a prominent role both during embryogenesis and at metamorphosis. The neurons that die during the latter period include both immature cells and mature, functioning neurons that are used during larval life but then discarded. The occurrence of cell death as part of metamorphosis means that these events have come under the control of the hormones that regulate metamorphosis. Besides being able to manipulate the timing of cell death by endocrine manipulations, hormones have also provided useful tools for probing cellular interactions in the degeneration response.

At adult emergence, the ventral CNS of Drosophila shows a group of approximately 300 neurons, which are unique in that they express 10-fold higher levels of the A isoform of the ecdysone receptor (EcR-A) than do other central neurons. This expression pattern is established early in metamorphosis and persists throughout the remainder of the pupal stage. Although these cells represent a heterogeneous group of neurons, they all share the same fate of undergoing rapid degeneration after the adult emerges from the pupal case. One prerequisite for this death is the decline of ecdysteroids at the end of metamorphosis. Treatment of flies with 20hydroxyecdysone blocks the death of the cells, but only if given at least 3 hours before the normal time of degeneration. The correlation of a unique pattern of receptor isoform expression with a particular steroid-regulated fate suggests that variations in the pattern of receptor isoform expression may serve as important switches during development.

The segmentally arranged Verson's glands are epidermal derivatives comprised of three cells: the duct, saccule, and secretory cells. The development of these glands was followed through the 5th instar and larval-pupal transition of Manduca sexta. The glands are relatively small during the feeding stage, begin to grow at wandering, and undergo about a 50-fold increase in size during the prepupal period. The increase in size is due mainly to the hypertrophy of the secretory cell which synthesizes a heterogeneous set of proteinaceous secretory products. Three prominent 11 to 12 kiloDalton (kD) polypeptides are made by the pharate fifth larval gland, whereas the pupal gland produces polypeptides ranging from 14 to 75 kD with a major complex at 30 to 34 kD. The secretory product is poured out onto the surface of the new cuticle at the time of ecdysis and contains all of the major proteins detected in extracts of the whole gland. The accumulation of secretory products by the gland occurs during the prepupal peak of ecdysteroid and is blocked if this rise is prevented by abdominal isolation. Infusion of 30 micrograms 20-hydroxyecdysone (20-HE) into such isolated abdomens caused synthesis of the pupal products. Treatment with the juvenile hormone mimic, methoprene, during the fifth instar showed that the commitment of the glands to produce the pupal proteins is independent of and occurs before the overlying epidermis becomes committed to make pupal cuticle.