Remodeling of an identified motoneuron during metamorphosis: hormonal influences on the growth of dendrites and axon terminals

During metamorphosis of the tobacco hawkmoth Manduca sexta, the femoral depressor motoneuron (FeDe MN) undergoes remodeling of its dendrites and motor terminals. Previous studies have established that remodeling of MNs during metamorphosis is mediated by the same hormones that control metamorphosis: the ecdysteroids and juvenile hormone (JH). During the pupal stage, the ecdysteroids promote adult-specific growth of MNs in the absence of JH, but JH or its synthetic mimics can interfere with ecdysteroid-mediated growth if applied during early sensitive periods. Hence, the application of a JH mimic (JHM) either systemically or locally to a target muscle has been used to distinguish those aspects of motor-terminal remodeling that are controlled by ecdysteroid action on the CNS from those that are influenced by ecdysteroid action on the peripheral targets. Here, we have extended the analysis of central and peripheral hormonal influences on MN remodeling by injecting JHM locally into the CNS thus altering the hormonal environment of the FeDe MN soma without altering the hormonal environment of its target muscle. Our results demonstrate that adult dendritic growth and motor-terminal growth can be experimentally uncoupled, suggesting that each is regulated independently. JHM application to the CNS perturbed dendritic growth, but had no measurable impact on motor-terminal growth. Peripheral actions of ecdysteroids, therefore, appear sufficient to promote the development of adult-specific motor terminals but not the development of an adult-specific dendritic arbor.     

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.     

家蚕蜕皮与变态的内分泌调控

家蚕的蜕皮与变态是由前胸腺分泌的脱皮素（ｍｏｌｔｉｎｇ ｈｏｒｍｏｎｅ或 ｅｃｄｙｓｔｅｒｏｉｄ简称 ＭＨ）及由咽侧体分泌的保幼激素（ｊｕｖｅｎｉｌｅ ｈｏｒｍｏｎｅ）控制的,而促有前胸腺激素（ｐｒｏｔｈｏｒａｃｉｃｏｔｒｏｐｉｃ ｈｏｒｍｏｎｅ,以下简称ＰＴＴＨ）的功能为刺激前胸腺分泌蜕皮素。笔者近１０年来从家蚕内分泌体系的一系列研究中发现,蜕皮素浓度的变化可以通过控制咽侧体的保幼激素的生物合成来影响幼虫发育,而ＰＴＴＨ的信息传递可通过调控前胸腺的功能,进而影响血淋巴中蜕皮素浓度。     

Autoradiographic identification of ecdysteroid-binding cells in the nervous system of the moth Manduca sexta

The steroid hormone 20-hydroxyecdysone regulates many aspects of nervous system development in the moth Manduca sexta, including stage-specific neuronal morphology and stage-specific neuronal death. We have used steroid hormone autoradiography to study the distribution of cells that concentrate ecdysteroids in the ventral nervous system of this insect. The ligand was [3H]-ponasterone A, a bioactive phytoecdysone. Tissue was examined from three stages of development: the end of larval life (first day of wandering), the end of metamorphosis (pharate adult), and 4-day-old adults. In the abdominal ganglia of wandering larvae and pharate adults, a subset of neurons including both motoneurons and interneurons exhibited a nuclear concentration of radiolabeled hormone. The pattern of binding was reproducible but stage-specific, with a greater proportion of neurons showing binding in the larvae than in pharate adults. No labeled neurons were found in abdominal ganglia from mature (4-day-old) adults. In the case of the pharate adult ganglia, the ecdysteroid receptor content of specific, identified motoneurons was determined. These results are discussed in light of the responses of these neurons to physiological changes in levels of circulating ecdysteroids.     

Target muscles and sensory afferents do not influence steroid-regulated,segment-specific death of identified motoneurons in Manduca sexta

Programmed cell death plays a critical role in sculpting the nervous system during embryonic development. In holometabolous insects, cell death also plays an important role in the reorganization of the nervous system during metamorphosis. In Manduca sexta, cell death and the factors that regulate it can be studied at the level of individually identified neurons. The accessory planta retractor (APR) motoneurons undergo segment-specific death during the larval-pupal transformation. APRs in abdominal segments 1, 5, and 6 die at pupation; those in abdominal segments 2, 3, and 4 survive until adulthood. Juvenile hormone and ecdysteroids regulate the metamorphic restructuring of the nervous system, but the factors that determine which APRs will live and which will die are not known. The present study assessed the possible importance of cell-cell interactions in determining APR survival at pupation by removing APR's target muscle or mechanosensory input early in the final larval instar, prior to the hormonal cues that trigger the larval-pupal transformation. The motoneurons showed their normal, segment-specific pattern of death in nearly all cases. These results suggest that target muscles and sensory input play little or no role in determining the segment-specific pattern of APR survival at pupation. © 1996 John Wiley & Sons, Inc.     

Early events in adult eye development of the moth, Manduca sexta

The eye imaginal disc of Manduca sexta is created early in the final larval instar from the adult eye primordium, which is composed of fully differentiated cells of the larval head capsule epidermis. Concomitant with the down-regulation of the larval epidermal program, expression of broad, a marker of pupal commitment, is activated in the primordium. The cells then detach from the cuticle, fold inward, and begin to proliferate at high levels to produce the inverted, eye imaginal disc. These and other events that begin on the first day of the final larval instar appear to mark the initiation of metamorphosis. Little is known about the endocrine control of the initiation of metamorphosis in any insect. The hemolymph titer of juvenile hormone (JH) declines to low levels during this period and the presence of JH is sufficient to repress development in cultured eye primordia. However, maintenance of JH at high levels in vivo by treatment with long-lasting JH mimics has no apparent effect on early steps in eye imaginal disc development. We discuss our findings in the context of the endocrine control of metamorphosis. The initiation of metamorphosis in Manduca, and perhaps a wide range of insect species, appears to involve the overcoming of JH repression by an unidentified, nutrient-dependent, hormonal factor.     

Hormonally mediated changes in simple reflex circuits during metamorphosis in Manduca

During insect metamorphosis, the nervous system must be reorganized to allow the production of unique behaviors during each life stage. In the hawkmoth, Manduca sexta, it has been possible to follow this postembryonic phase of neuronal development at the level of identified neurons. Of particular interest in the present context are sensory neurons, motoneurons, and interneurons which persist through metamorphosis, but participate in different types of behavior at different stages of life. Many of these neurons undergo striking changes in their dendritic arborizations and axonal projection patterns, which can be correlated with changes in their synaptic interactions with other neurons. Manipulations of the ecdysteroid and juvenile hormone titers, both in vivo and in vitro, implicate these hormones in the regulation of metamorphic changes within the nervous system. Taking advantage of this endocrine control, it has been possible to create heterochronic mosaic animals that allow the relationship between specific cellular changes and behavioral alterations to be tested directly.     

Steroid control of muscle remodeling during metamorphosis in Manduca sexta

During metamorphosis in the tobacco hornworm, Manduca sexta, the abdominal body-wall muscle DEO1 is remodeled to form the adult muscle DE5. The degeneration of muscle DEO1 involves the dismantling of its contractile apparatus followed by the degeneration of muscle nuclei. As some nuclei are degenerating, others begin to incorporate 5-bromodeoxyuridine (BrdU), indicating the onset of nuclear proliferation. This proliferation is initially most evident at the site where the motoneuron contacts the muscle remnant. The developmental events involved in muscle remodeling are under the control of the steroid hormones, the ecdysteroids. The loss of the contractile elements of the larval muscle requires the rise and fall of the prepupal peak of ecdysteroids, whereas the subsequent loss of muscle nuclei is influenced by the slight rise in ecdysteroids seen after pupal ecdysis. Incorporation of BrdU by muscle nuclei depends on both the adult peak of the ecdysteroids and contact with the motoneuron. Unilateral axotomy blocks proliferation within the rudiment, but it does not block its subsequent differentiation into a very thin muscle in the adult. © 1996 John Wiley & Sons, Inc.     

Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.     

Hormonal regulation and segmental specificity of motoneuron phenotype during metamorphosis of the tobacco hornworm, Manduca sexta.

The abdominal prolegs of Manduca sexta larvae are eliminated at the onset of metamorphosis. Previous work showed that the prepupal peak of ecdysteroids in the hemolymph causes the dendritic arbors of proleg motoneurons to regress and a stereotyped subset of the motoneurons to die. In the present study we investigated the parameters of ecdysteroid exposure that are important for eliciting these responses by directly infusing 20-hydroxyecdysone (20-HE) into the hemolymph of insects deprived of their own endocrine glands. Doses of 20-HE that were near threshold for evoking regression or death were consistently more effective when infused over a longer duration. Theoretical calculations of hemolymph hormone profiles produced by the infusions support a model of ecdysteroid action in which the hormone concentration must remain above a threshold level for a critical duration of time to be physiologically effective. We further found that segmental location can influence both the metamorphic fate and the hormonal sensitivity of Manduca motoneurons.     

Interendocrine regulation of the corpora allata and prothoracic glands of Manduca sexta

Regulation of the haemolymph titres of ecdysteroids and the juvenile hormones (JH) during larval-pupal development of the tobacco hornworm, Manduca sexta, involves the interendocrine control of the synthesis of each hormone by the other. Temporal relationships between the ecdysteroid titre peaks in the fourth and early fifth larval instar and the increases in corpora allata (CA) activity at these times suggests that ecdysteroids are evoking the increases. Incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes and isolated CA from these stages with 20-hydroxyecdysone (20-HE) revealed that 20-HE stimulates CA activity and that it does this indirectly via the Br-CC. The resulting increase in the JH titre after the commitment (first) peak in the fifth instar stimulates the fat body to secrete a factor which appears to be the same as a haemolymph stimulatory factor for the prothoracic glands. This moiety acts as a secondary effector that modulates the activity of the prothoracic glands and thus the ecdysteroid titre. These findings together have begun to elucidate the mechanisms by which the principal developmental hormones in the insect interact to regulate postembryonic development.     

Effects of ecdysteroid agonist RH-2485 reveal interactions between ecdysteroids and juvenile hormones in the development of Sesamia nonagrioides

Larvae of Sesamia nonagrioides developing under long day (LD) conditions pupate in the 5th or 6th instar, whereas under the short day (SD) conditions, they undergo several supernumerary larval molts and are regarded as diapausing. The development in early larval instars occurs in the LD larvae at a moderate and in the SD larvae at a high juvenile hormone (JH) titer; ecdysteroid titer cycles similarly under both conditions. The transformation to pupa is initiated by a burst of ecdysteroids at undetectable JH levels, whereas extra larval molts in the diapausing larvae are associated with moderate JH titer and irregular rises of ecdysteroids. Application of 0.2 ppm RH-2485 to the diet of the 6th instar larvae promotes hormonal changes supporting metamorphosis in the LD larvae and slightly accelerates larval molts in the diapausing SD larvae. The 0.5- and 1-ppm doses revert these patterns of endocrine regulations to a mode typical for early larval instars. Particularly dramatic is a JH titer increase provoked within 24 h in the LD larvae. After the treatment, both the LD and SD larvae undergo a series of larval molts, suggesting that hormonal programming of the larval development has been stabilized. A few insects receiving 1 ppm RH-2485, and a high proportion of those fed with 5 ppm RH-2485, deposit two cuticles within a single apolysis and die.     

The morphostatic actions of juvenile hormone

The maintenance of "status quo" in larvae by juvenile hormone (JH) involves both the programming of ecdysteroid-dependent synthesis during the molt and the suppression of morphogenetic growth during the intermolt. The latter morphostatic action does not require ecdysteroids, and has been studied in the formation of imaginal discs in Manduca sexta. Preultimate larval instars have both invaginated discs and imaginal primordia, both of which grow isomorphically with the larva. In the last instar, the young discs/primordia initiate the morphogenesis and patterning that results in a mature disc. JH suppresses both the initiation and progression of the signaling that transforms immature discs or primordia into a fully patterned imaginal disc. This transformation normally occurs in the context of the rapid growth of the last larval stage, and nutrient-dependent factors appear to be able to override the JH suppression. The morphostatic action of JH may have been important for the evolution of the larval stage. Studies on embryos of basal, hemimetabolous insects show that their premature exposure to JH can truncate patterning programs and cause precocious tissue maturation, factors essential for organizing a novel larval form. This suppression of embryonic patterning then results in embryonic fields that remain dormant as long as JH is present. These are the primordia that can transform into imaginal discs once JH disappears in preparation for metamorphosis.     

The POU Factor Ventral Veins Lacking/Drifter Directs the Timing of Metamorphosis through Ecdysteroid and Juvenile Hormone Signaling

Although endocrine changes are known to modulate the timing of major developmental transitions, the genetic mechanisms underlying these changes remain poorly understood. In insects, two developmental hormones, juvenile hormone (JH) and ecdysteroids, are coordinated with each other to induce developmental changes associated with metamorphosis. However, the regulation underlying the coordination of JH and ecdysteroid synthesis remains elusive. Here, we examined the function of a homolog of the vertebrate POU domain protein, Ventral veins lacking (Vvl)/Drifter, in regulating both of these hormonal pathways in the red flour beetle, Tribolium castaneum (Tenebrionidae). RNA interference-mediated silencing of vvl expression led to both precocious metamorphosis and inhibition of molting in the larva. Ectopic application of a JH analog on vvl knockdown larvae delayed the onset of metamorphosis and led to a prolonged larval stage, indicating that Vvl acts upstream of JH signaling. Accordingly, vvl knockdown also reduced the expression of a JH biosynthesis gene, JH acid methyltransferase 3 (jhamt3). In addition, ecdysone titer and the expression of the ecdysone response gene, hormone receptor 3 (HR3), were reduced in vvl knockdown larvae. The expression of the ecdysone biosynthesis gene phantom (phm) and spook (spo) were reduced in vvl knockdown larvae in the anterior and posterior halves, respectively, indicating that Vvl might influence ecdysone biosynthesis in both the prothoracic gland and additional endocrine sources. Injection of 20-hydroxyecdysone (20E) into vvl knockdown larvae could restore the expression of HR3 although molting was never restored. These findings suggest that Vvl coordinates both JH and ecdysteroid biosynthesis as well as molting behavior to influence molting and the timing of metamorphosis. Thus, in both vertebrates and insects, POU factors modulate the production of major neuroendocrine regulators during sexual maturation.     

Hormonal regulation of gametogenesis in insect

The review discusses the role of juvenile hormone (JH), ecdysone and brain in the regulation of oogenesis and spermatogenesis in insects. The early period of gametogenesis (gonial mitoses, the meiotic prophase) in both sexes is controlled mainly by ecdysone and neurosecretory cells of the brain. In periods of cytoplasmic growth of oocytes and vitellogenesis the main role in the regulation belongs to JH. The modern views on hormonal regulation of vitellogenin synthesis and follicular epithelium differentiation are under consideration with a special reference of the role of ecdysteroids in Diptera and Lepidoptera oogenesis.