Balancing sex chromosome expression and satisfying the sexes

One of the key aspects of functional nervous systems is the restriction of particular neural subtypes to specific regions, which permits the establishment of differential segment-specific neuromuscular networks. Although Hox genes play a major role in shaping the anterior-posterior body axis during animal development, our understanding of how they act in individual cells to determine particular traits at precise developmental stages is rudimentary. We have used the abdominal leucokinergic neurons (ABLKs) to address this issue. These neurons are generated during both embryonic and postembryonic neurogenesis by the same progenitor neuroblast, and are designated embryonic and postembryonic ABLKs, respectively. We report that the genes of the Bithorax-Complex, Ultrabithorax (Ubx) and abdominal-A (abd-A) are redundantly required to specify the embryonic ABLKs. Moreover, the segment-specific pattern of the postembryonic ABLKs, which are restricted to the most anterior abdominal segments, is controlled by the absence of Abdominal-B (Abd-B), which we found was able to repress the expression of the neuropeptide leucokinin. We discuss this and other examples of how Hox genes generate diversity within the central nervous system of Drosophila.     

Patterns of cell division and expression of asymmetric cell fate determinants in postembryonic neuroblast lineages of Drosophila

We have studied the division of postembryonic neuroblasts (Nbs) in the outer proliferation center (OPC) and central brain anlagen of Drosophila. We focused our attention on three aspects of these processes: the pattern of cellular division, the topological orientation of those divisions, and the expression of asymmetric cell fate determinants. Although larval Nbs are of embryonic origin, our results indicate that their properties appear to be modified during development. Several conclusions can be summarized: (i) In early larvae, Nbs divide symmetrically to give rise to two Nbs while in the late larval brain most Nbs divide asymmetrically to bud off an intermediate ganglion mother cell (GMC) that very rapidly divides into two ganglion cells (GC). (ii) Symmetric and asymmetric divisions of OPC Nbs show tangential and radial orientations, respectively. (iii) This change in the pattern of division correlates with the expression of inscuteable, which is apically localized only in asymmetric divisions. (iv) The spindle of asymmetrically dividing Nb is always oriented on an apical-basal axis. (v) Prospero does not colocalize with Miranda in the cortical crescent of mitotic Nbs. (vi) Prospero is transiently expressed in one of the two sibling GCs generated by the division of GMCs. The implications of these results on cell fate specification and differentiation of adult brain neurons are discussed.     

The origin of postembryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster

Embryonic and postembryonic neuroblasts in the thoracic ventral nerve cord of Drosophila melanogaster have the same origin. We have traced the development of threefold-labelled single precursor cells from the early gastrula stage to late larval stages. The technique allows in the same individual monitoring of progeny cells at embryonic stages (in vivo) and differentially staining embryonic and postembryonic progeny within the resulting neural clone at late postembryonic stages. The analysis reveals that postembryonic cells always appear together with embryonic cells in one clone. Furthermore, BrdU labelling suggests that the embryonic neuroblast itself rather than one of its progeny resumes proliferation as a postembryonic neuroblast. A second type of clone consists of embryonic progeny only.     

Aspects of the embryology and neural development of the American lobster.

It is feasible to study the anatomical, physiological, and biochemical properties of identifiable neurons in lobster embryos. To exploit fully the advantages of this preparation and to lay the foundation for single-cell studies, our recent goals have been to 1) establish a quantitative staging system for embryos, 2) document in detail the lobster's embryonic development, 3) determine when uniquely identifiable neurons first acquire their transmitter phenotypes, and 4) identify particular neurons that may serve developmental functions. Behavioral, anatomical, morphometric, and immunocytochemical studies have led to a detailed characterization of the growth and maturation of lobster embryos and to the adoption of a percent-staging system based upon the eye index of Perkins (Fish. Bull., 70:95-99, 1972). It is clear from these studies that the lobster nauplius molts at approximately 12% embryonic development (E12%) into a metanauplius, which subsequently undergoes a complete molt cycle within the egg. This molt cycle climaxes with the emergence of the first-stage larva shortly after hatching. Serotonin and proctolin, neurohormones widely distributed in the lobster nervous system, appear at different times in development. Serotonin immunoreactive neurons begin to appear at approximately E10%, with the adult complement being established by E50%. In contrast, proctolin immunoreactive neurons appear later and attain their full complement over a protracted period including larval and juvenile stages. The development of serotonergic deutocerebral neurons and their targets, the olfactory and accessory lobes in the brain, are also examined. The olfactory lobes are forming by E10% and have acquired their glomerular organization by E50%, whereas the formation of the accessory lobes is delayed; the early rudiments of the accessory lobes are seen by E50%, and glomeruli do not form until the second larval stage.     

Postembryonic development of the antennal lobes in Periplaneta americana L.

Summary The postembryonic development of the antennal lobes of Periplaneta americana L. was examined with light- and electron-microscopical methods. There is no difference in the number of glomeruli and neurons in the antennal lobes of larval and adult animals. At hatching, the first larva already possesses the adult number of approximately 125 glomeruli and 500 to 560 deutocerebral neurons in the dorsolateral cell group of each antennal lobe. During postembryonic development the volume of the deutocerebral neurons increases three- to fourfold. The glomeruli of the first larva have about 7 % of the volume of the corresponding adult glomeruli. Since number, pattern, and size ratio of glomeruli (with the exception of the macroglomerulus) are constant in all larval stages and adult animals, it is possible to identify individual glomeruli. During the whole postembryonic development the ordinary glomeruli show a continuous volume increase, which parallels the increase in antennal sensory input. The macroglomerulus develops by way of special growth of two to four neuropil units, but not before the last three to four larval stages and only in males. Its growth precedes the formation of antennal pheromone receptors during the final molt; these receptors are known to project into the macroglomerulus. The development of the macroglomerulus in the last larval stages of the male may be caused by a genetically fixed growth program of specific deutocerebral neurons.Supported by the Deutsche Forschungsgemeinschaft (Scha 291/1)     

Roles of Hox genes in the patterning of the central nervous system of Drosophila

One of the key aspects of functional nervous systems is the restriction of particular neural subtypes to specific regions, which permits the establishment of differential segment-specific neuromuscular networks. Although Hox genes play a major role in shaping the anterior-posterior body axis during animal development, our understanding of how they act in individual cells to determine particular traits at precise developmental stages is rudimentary. We have used the abdominal leucokinergic neurons (ABLKs) to address this issue. These neurons are generated during both embryonic and postembryonic neurogenesis by the same progenitor neuroblast, and are designated embryonic and postembryonic ABLKs, respectively. We report that the genes of the Bithorax-Complex, Ultrabithorax (Ubx) and abdominal-A (abd-A) are redundantly required to specify the embryonic ABLKs. Moreover, the segment-specific pattern of the postembryonic ABLKs, which are restricted to the most anterior abdominal segments, is controlled by the absence of Abdominal-B (Abd-B), which we found was able to repress the expression of the neuropeptide leucokinin. We discuss this and other examples of how Hox genes generate diversity within the central nervous system of Drosophila.Keyword: development, Hox genes, central nervous system, Drosophila, cell fate specification     

Expression of Drosophila MAGE gene encoding a necdin homologous protein in postembryonic neurogenesis

The MAGE (melanoma antigen) family is characterized by a large conserved domain termed MAGE homology domain. Originally identified MAGE genes encoding tumor rejection antigens are expressed only in cancers and male germ cells. Necdin, which contains the MAGE homology domain, is highly expressed in postmitotic cells such as neurons and skeletal muscle cells. The human necdin gene NDN is transcribed only from the paternal allele through genomic imprinting, and its deficiency is implicated in the pathogenesis of the neurodevelopmental disorder Prader-Willi syndrome. Although over 30 MAGE genes have been identified in humans, fruit fly (Drosophila melanogaster) has only a single MAGE gene that encodes a protein similar to necdin homologous MAGE proteins. In this study, we analyzed the spatiotemporal expression patterns of MAGE mRNA and the encoded protein during fly development. Whole-mount embryo in situ hybridization analysis revealed that MAGE mRNA was highly expressed at the syncytial blastoderm stage and in the ventral and procephalic neurogenic regions of the ectoderm during gastrulation. In contrast, MAGE expression was nearly undetectable in postmitotic neurons of the central nervous system at late embryonic stages. During postembryonic neurogenesis, MAGE was highly expressed in neural stem cells (neuroblasts) and their progeny (ganglion mother cells and postmitotic neurons) at larval and pupal stages. MAGE was also expressed in postmitotic neurons including mushroom body neurons and retinal photoreceptors in adulthood. These results indicate that MAGE expression lasts throughout the postembryonic neurogenesis in Drosophila.     

Segmentally distributed metamorphic changes in neural circuits controlling abdominal bending in the hawkmoth Manduca sexta

During the metamorphosis of Manduca sexta the larval nervous system is reorganized to allow the generation of behaviors that are specific to the pupal and adult stages. In some instances, metamorphic changes in neurons that persist from the larval stage are segment-specific and lead to expression of segment-specific behavior in later stages. At the larval-pupal transition, the larval abdominal bending behavior, which is distributed throughout the abdomen, changes to the pupal gin trap behavior which is restricted to three abdominal segments. This study suggests that the neural circuit that underlies larval bending undergoes segment specific modifications to produce the segmentally restricted gin trap behavior. We show, however, that non-gin trap segments go through a developmental change similar to that seen in gin trap segments. Pupal-specific motor patterns are produced by stimulation of sensory neurons in abdominal segments that do not have gin traps and cannot produce the gin trap behavior. In particular, sensory stimulation in non-gin trap pupal segments evokes a motor response that is faster than the larval response and that displays the triphasic contralateral-ipsilateral-contralateral activity pattern that is typical of the pupal gin trap behavior. Despite the alteration of reflex activity in all segments, developmental changes in sensory neuron morphology are restricted to those segments that form gin traps. In non-gin trap segments, persistent sensory neurons do not expand their terminal arbors, as do sensory neurons in gin trap segments, yet are capable of eliciting gin trap-like motor responses. Accepted: 10 January 1997     

A primary cell culture of Drosophila postembryonic larval neuroblasts to study cell cycle and asymmetric division

Drosophila melanogaster is a key model system that has greatly contributed to the advance of developmental biology through its extensive and sophisticated genetics. Nevertheless, only a few in vitro approaches are available in Drosophila to complement genetic studies in order to better elucidate developmental mechanisms at the cellular and molecular level. Here we present a dissociated cell culture system generated from the optic lobes of Drosophila larval brain. This culture system makes it feasible to study the proliferative properties of Drosophila postembryonic Nbs by allowing BrdU pulse and chase assays, as well as detailed immunocytochemical analysis with molecular markers. These immunofluorescence experiments allowed us to conclude that localization of asymmetric cell division markers such as Inscuteable, Miranda, Prospero and Numb is cell autonomous. By time-lapse video recording we have observed interesting cellular features of postembryonic neurogenesis such us the polarized genesis of the neuroblast progeny, the extremely short ganglion mother cell (GMC) cell cycle, and the last division of a neuroblast lineage. The combination of this cell culture system and genetic tools of Drosophila will provide a powerful experimental model for the analysis of cell cycle and asymmetric cell division of neural progenitor cells.     

Development of the enteric nervous system in the moth. I. Diversity of cell types and the embryonic expression of FMRFamide-related neuropeptides

The enteric nervous system (ENS) of the larval moth Manduca sexta consists of two small ganglia and several nerve networks that lie superficially along the alimentary tract. Within this system are approximately 600 neurons that exhibit a spectrum of biochemical and morphological characteristics and that express these features in a definable sequence during development. The accessibility of both the neural and nonneural components of the moth ENS throughout embryogenesis makes it a potentially useful model in which to examine the developmental regulation of transmitter phenotype. In this paper, we have focused on the differentiation of the enteric plexus (EP) cells, a heterogeneous population of enteric neurons that are distributed across the foregut-midgut boundary. Unlike many neurons of the CNS in insects, the cells of the enteric plexus are not uniquely identifiable. While the total number of EP cells is constant, their locations vary significantly from animal to animal. However, several distinct classes of neurons can be identified within this population on the basis of morphology and transmitter phenotype, including one class that contains substances related to the molluscan peptide Phe-Met-Arg-Phe-amide (FMRFamide). Expression of this FMRFamide-like material within the enteric plexus is position-specific, occurring only in neurons on the midgut and not in those on the foregut. FMRFamide-like immunoreactivity first appears in approximately one-third of these cells at 65% of development; this pattern is retained without apparent modification throughout subsequent embryonic and postembryonic development. In the following paper, we describe the sequence of stereotyped cell migration that precedes the expression of this peptidergic phenotype and that underlies the formation of the enteric plexus during embryogenesis.     

Pre-ovipositional maternal care alleviates food stress of offspring in the flower beetle <Emphasis Type="Italic">Dicronocephalus wallichii</Emphasis>

Unlike most other flower beetles, females of Dicronocephalus wallichii exhibit nesting behaviour. The female constructs a burrow in the soil, cuts dead plant leaves into small pieces to provision the nest, and then lays one egg inside the nest. Hatched larvae have been thought to feed on the nest materials prepared by their mothers, but the effects of pre-ovipositional care on larval performance have not been tested. The hatched larvae were found to stay in the nest for 15–30 days until they consumed the nest materials. We examined whether the presence of provisioned nests enhanced larval performance under both benign and food-stress conditions. With high-nutrient soil, larval survival rate and growth speed were not affected by the presence of provisioned nests. By contrast, with low-nutrient soil, mortality of the larvae was much higher in the absence than in the presence of provisioned nests. The growth speed of larvae with nests located in low-nutrient soil was as high as those reared in high-nutrient soil. These results indicate that females alleviate the food stress of larvae during their initial developmental stage by constructing provisioned nests.     

Social Behaviour of the Carpenter Bee Xylocopa pubescens (Spinola)

The development of about 20 relatively small nests of Xylocopa pubescens was studied. After the first offspring had become adult, these nests reached a social stage in which there was only one egg-layer per nest. Freshly emerged (teneral) adults eat a lot of food, collected by a forager, before they fly out of the nest. This food appears to be of major importance to these bees in that it makes them fully agile. It seems therefore, that part of the food needed for the development of a young bee is not given at the larval stage, via the beebread, but at the teneral adult stage. As a result of this, a necessary overlap of generations is accomplished. Besides, less pollen has to be collected for the provision of a brood cell, so more cells can be constructed within a short period of time. There is a high degree of nest competition. Many nests were taken over by conspecific individuals (or by females of X. sulcatipes) that were searching for nesting sites within the study area. However, although more than 50% of the solitary nests were taken over sooner or later, strangers hardly ever intruded into social nests, with more than one adult. This illustrates how important it is for a reproducing female to tolerate the presence of nestmates which guard the nest in her absence. Although in most cases the foundress of a nest proceeded to produce new brood in the presence of her offspring, it happened that a daughter took over reproduction from her mother. In two of these cases, it could be observed that a mother started a new nest elsewhere after having been thrown out by her own daughter. At least in these cases, nest competition between mother and daughter started long before the mother had reached the end of her reproductive capacities. Since nesting possibilities are scarce, it seems a logical strategy to stay in the maternal nest and wait for a chance to become the egg-layer if the mother dies or if she loses her dominance. The occurrence of several social interactions among nestmates is discussed: — trophallaxis was observed often, not only under ‘forced’ conditions, but also as a form of ‘voluntary’ feeding; — nestmates were observed to groom each other.     

Both cooperation and nest position improve larval survival in the sweat bee, <Emphasis Type="Italic">Lasioglossum</Emphasis> (<Emphasis Type="Italic">Evylaeus</Emphasis>) <Emphasis Type="Italic">baleicum</Emphasis>

Nesting by multiple females in sweat bees raises the question of the benefits associated with grouping. Adult numbers of the sweat bee, Lasioglossum (Evylaeus) baleicum, exhibited variation within a nest population. The effects of adult number and nest position on larval survival were analyzed in a nest aggregation. Both variables independently affected larval survival, with the presence of multiple adults in a nest markedly improving survival rate. Nests located near the periphery of the aggregation of nests suffered high larval mortalities. Increased frequency of cell-inspection in multiple-female nests seems to be associated with a concomitant increase in larval cell protection from external predation. Such predation pressure was assumed, given the existence of an underground ant colony that employs a recruitment system while foraging in the study area.     

Evolution of nest-weaving behaviour in arboreal nesting ants of the genus Polyrhachis Fr. Smith (Hymenoptera: Formicidae)

Abstract  Polyrhachis ants represent one of the most taxonomically and ecologically diverse ant genera, with over 500 described species organised into 12 subgenera. Nesting habits range from subterranean localities to arboreal nests incorporating silk produced by the ants' own larvae (nest weaving). In this article, we combine scanning electron microscopy and gross observations of the nests of 35 species representing nine subgenera thought to contain individuals that nest above the ground (Cyrtomyrma, Hagiomyrma, Hedomyrma, Hemioptica, Myrma, Myrmatopa, Myrmhopla, Myrmothrinax, Polyrhachis) to revaluate the relationship between nest locale, the type of nest material used and the use of larval silk for nest construction. Nesting habits are highly diverse, ranging from truly arboreal nests on or between leaves and branches, to lignicolous nests inside hollow stems or bamboo internodes and lithocolous nests on the sides of rock walls. Flat sheets of larval silk are used only by arboreal nesting species within the subgenera Cyrtomyrma , Hemioptica , Myrma , Myrmatopa , Myrmothrinax and Polyrhachis . Lignicolous nesting habits were demonstrated predominantly by Hedomyrma spp., but these habits also occur in Myrma and Myrmhopla . Lithicolous nesting habits occur within Hagiomyrma and Hedomyrma , though the actual nesting material used can be either carton or dense masses of spider silk. Based on existing phylogenetic hypotheses, the use of larval silk for nest construction has evolved independently within the genus, and has evolved independently of the construction of silk nests per se . Further examination of the exact type of silk found in the colonies of 'nest-weaving' Polyrhachis is warranted.     

Expression of two different isoforms of fasciclin II during postembryonic central nervous system remodeling in <Emphasis Type="Italic">Manduca sexta</Emphasis>

Insect metamorphosis serves as a useful model to investigate postembryonic development in the central nervous system, because the transformation between larval and adult life is accompanied by a remodeling of neural circuitry. Most changes are controlled by ecdysteroids, but activity-dependent mechanisms and cell surface signals also play a role. This immunocytochemical study investigates the expression patterns of two isoforms of the neural cell adhesion molecule, fasciclin II (FasII), during postembryonic ventral nerve cord remodeling in the moth, Manduca sexta. Both the expression of the glycosyl-phosphatidylinositol (GPI)-linked isoform and the transmembrane isoform of Manduca FasII (TM-MFasII) are regulated in a stereotyped spatio-temporal pattern. TM-MFasII is expressed in a stage-specific manner in a subset of neurons. Subsets of central axons express high levels during outgrowth supporting a functional role for TM-FasII during pathfinding. Dendritic localization is not found at any stage of metamorphosis, suggesting no homophilic interactions of TM-MFasII during central synapse development. GPI-MFasII is expressed in a stage-specific manner, most likely only in glial cells. The larval and adult stages show almost no GPI-MFasII expression, whereas during pupal life, positive GPI-MFasII labeling is present around synaptotagmin-negative tracts or commissures, so that either homophilic stabilization of glial boundaries or heterophilic neuron-glial interactions possibly stabilize the axons within their tracts. GPI-MFasII expression is not co-localized with synaptotagmin-positive central terminals, rendering a role for synapse development unlikely. Neither isoform is expressed in all neurons of a specific class at any developmental stage, indicating that MFasII functions are restricted to specific subsets of neurons or to individual neurons. The support of the German Science Foundation (Du 331/4–1) and of Arizona State University to C.D. is greatly appreciated.     

Adult-specific neurons in the nervous system of the moth, Manduca sexta: selective chemical ablation using hydroxyurea

The segmental ganglia of adults of the moth, Manduca sexta, are constructed both from remodeled larval neurons and from adult-specific cells. The latter are produced by identified stem cells (neuroblasts) during larval life and then differentiate to form functional neurons during metamorphosis. The mitotic activity of the larval neuroblasts could be irreversibly blocked by the DNA-synthesis inhibitor hydroxyurea (HU). Treatment on day 1 of the third larval stage resulted in 80-90% of the neuroblasts being blocked before they produced any progeny while leaving the functional larval neurons unaffected. Treated larvae finished growth, underwent metamorphosis, and produced an adult CNS that contained the normal set of remodeled larval neurons but lacked most of the new adult-specific cells. When HU treatment was delayed until the start of the fourth or fifth larval stage, the neuroblasts produced the early portions of their respective lineages before they were blocked. The immature neurons that were generated prior to treatment survived to contribute adult-specific neurons to the moth CNS, but the remainder of each lineage was missing. This technique therefore enables one to produce adult nervous systems containing the basic set of remodeled larval cells plus defined sets of adult-specific neurons.     

Fate of neuroblast progeny during postembryonic development of mushroom bodies in the house cricket,Acheta domesticus

Mushroom bodies represent the main sensory integrative center of the insect brain and probably play a major role in the adaptation of behavioral responses to the environment. Taking into account the continuous neurogenesis of cricket mushroom bodies, we investigated ontogenesis of this brain structure. Using BrdU labeling, we examined the fate of neuroblast progeny during the postembryonic development. Preimaginal Kenyon cells survived throughout larval and imaginal moults and persisted during adulthood. Our results indicate that the location of labelled Kenyon cells in the cortex of the adult cricket mainly depends upon the period when they were produced during development. The present data demonstrate that cricket mushroom bodies grow from the inside out and that, at any developmental stage, the center of the cortex contains the youngest Kenyon cells. This study also allowed us to observe the occurrence of quiescent neuroblasts. Kenyon cell death during postembryonic and adult life seems to be reduced. Although preimaginal Kenyon cells largely contribute to adult mushroom body structure, a permanent remodeling of the mushroom body occurs throughout the whole insect life due to the persistence of neurogenesis in the house cricket. Further studies are needed to understand the functional significance of these findings.     

Development of the embryo nest ofVespa affinis (Hymenoptera: Vespidae) in Southern Japan

Summary The aerial nests ofVespa affinis on Ishigaki Island, Japan, were found mainly within 1 m of the ground, attached to a twig roughly 4 mm in diameter. These are probably adaptations to survive adverse weather conditions. The favoured nesting habitat was in open farmland areas rather than in the natural forest. The entire envelope and more than half of the comb of the embryo nest is constructed during the first 10–12 days. The first workers emerge around 34 days after nest initiation. The duration of larval development was found to be linked to the number of larvae present in the nest, with the earlier progeny developing faster. The changes in the ovarian index of the mother queen during the early stages of nesting are described.     

Azadirachtin potentiates the action of ecdysteroid agonist RH-2485 in Spodoptera littoralis

Edysteroid agonist RH-2485 induces an immediate and fatal molt in Spodoptera littoralis when added to the diet of the 2nd and 4th instar larvae at 1 ppm, and to that of the 6th instar larvae at 0.001 ppm concentration. Ten times lower doses fed to the larvae continuously allow an apparently normal larval development that is terminated by a supernumerary larval molt. The other effects of RH-2485 include death during metamorphosis and impaired fertility of emerged adults. The number of progeny is reduced even with low RH-2485 doses that do not interfere with moltings; e.g., insects fed 0.0001 ppm since the 2nd, 4th, and 6th instar produce 72%, 62%, and 22%, respectively, less progeny than the controls. Feeding larvae with 10-1000 ppm Suneem oil (containing about 0.1-10 ppm azadirachtin) causes, in a stage- and dose-dependent manner, a cessation or reduction of feeding, delay of molts, death of larvae and pupae, and sterility of emerged adults; with 10 ppm Suneem oil, the number of progeny is reduced by 20-32%. Presence of Suneem oil in the diet does not influence the potential of RH-2485 to induce a prompt molt, but it increases ten times the potency to elicit a supernumerary larval molt. Certain combinations of RH-2485 with Suneem oil provoke up to 3 extra larval molts. Lethal developmental derangements and sterility are more frequent, and the response of larvae of different age is more uniform, when Suneem oil and RH-2485 are combined than when each of these agents is administered alone.