Molecular characterization of Pegarn: a <Emphasis Type="Italic">Drosophila</Emphasis> homolog of UNC-51 kinase

We have isolated and characterized the gene encoding a Drosophila melanogaster homolog of Caenorhabditis elegans UNC-51 (uncoordinated movement-51): Pegarn. Developmental Northern blot shows the Pegarn gene is expressed at all stages of development. The protein is detected throughout the Drosophila third instar larval central nervous system (CNS) in axons projecting out from the ventral ganglion and in the optic anlagen of the optic lobe. Heterozygous Pegarn mutant embryos show defects in larval axonal neuronal patterning, but survive to adulthood. Homozygous mutants have an even more deformed pattern of neuronal development and do not survive through the larval stages. The data from this research suggest the critical roles of Pegarn in CNS and PNS axonal formation in Drosophila melanogaster and indicates its similar role in other multicellular species.     

Interaction of the Stubble-Stubbloid Locus and the Broad-Complex of Drosophila Melanogaster

The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third instar. The region is coextensive with a complex genetic locus, the Broad-Complex (BR-C). The BR-C is a regulatory gene that contains two major functional domains, the br domain and the l(1)2Bc domain. BR-C mutants prevent metamorphosis, including morphogenesis of imaginal discs; br mutants prevent elongation and eversion of appendages and l(1)2Bc mutants prevent fusion of the discs. The Stubble-stubbloid (Sb-sbd) locus at 89B9-10 is best known for the effects of its mutants on bristle structure. Mutants of the BR-C and the Sb-sbd locus interact to produce severe malformation of appendages. Viable heteroallelic and homoallelic combinations of Sb-sbd mutants, including loss-of-function mutants, affect the elongation of imaginal disc appendages. Thus, the Sb-sbd(+) product is essential for normal appendage elongation. Sb-sbd mutants, however, do not affect eversion or fusion of discs. Correspondingly, only BR-C mutants deficient in br function interact with Sb-sbd mutants. The interaction occurs in deficiency heterozygotes using single, wild-type doses of the BR-C, of the Sb-sbd locus, or of both loci. These last results are formally consistent with the possibility that the BR-C acts as a positive regulator of the Sb-sbd locus. The data do not exclude other possible nonregulatory interactions between the two loci, e.g., interactions between the products of both genes.     

Hormonal control of invertebrate behavior

Invertebrates show a wide variety of behaviors that are influenced by hormones. In insects the involvement of hormones at a particular life stage is directly correlated with the complexity of the behavioral repertoire at that stage. In larval stages, the steroid hormone, ecdysone, when present with juvenile hormone, apparently causes the behaviors observed during the periodic molts. At the end of larval life, ecdysone in the absence of juvenile hormone triggers the onset of premetamorphic behaviors such as wandering behavior and cocoon-spinning behavior. In insects having complete metamorphosis, the emergence (eclosion) of the adult from the pupal case is accomplished by a stereotyped program of movements that are triggered by a peptide hormone. In moths, injection of this “eclosion hormone” into competent recipients will cause the release of the eclosion program. Also this program can be elicited by the hormone from the isolated abdominal central nervous system (CNS). The onset of reproductive behavior in females of various species requires juvenile hormone. In addition, certain peptides are then involved in the transition from virgin to mated behaviors. Also, pupatitive peptide factors trigger specific stereotyped behaviors such as those involved in mate attraction and in oviposition. In males, the control is simpler. Juvenile hormone is required for the maturation of sexual behavior in only a few species. But in at least one insect group, the cockroaches, a neurosecretory hormone serves to release directly copulatory behavior. Social behavior and migratory behavior in certain insects are also under hormonal influence. Hormones play a prominent role in regulating the behavior of gastropod mollusks. The best studied examples involve the hormonal stimulation of egg-laying behavior by CNS peptides. Also, peptide hormones cause stereotyped changes in specific identified neurons in the CNS of various gastropods. In at least some cases, these latter changes are related to arousal from aestivation.With their simple nervous systems, invertebrates are especially suited for studies on the mode of action of hormones on the nervous system. In most cases the behavioral effects of these hormones appear to be due to their direct action on the CNS. Indeed, the isolated moth CNS will respond to the eclosion hormone by generating the motor program that gives rise to the emergence behavior, and various isolated molluscan preparations will respond to hormones with stereotyped neural responses. By the direct application of hormone to the surface of identified nerve cells in mollusks it has been possible to localize target cells for specific hormones. Little is known of the mode of action of ecdysone or juvenile hormone in altering behavior. Peptide hormones appear to have effects which long outlast the actual presence of the hormone. In at least two cases, cyclic AMP has been implicated as a mediator of the hormonal response.     

Ascidians as excellent chordate models for studying the development of the nervous system during embryogenesis and metamorphosis

The swimming larvae of the chordate ascidians possess a dorsal hollowed central nervous system (CNS), which is homologous to that of vertebrates. Despite the homology, the ascidian CNS consists of a countable number of cells. The simple nervous system of ascidians provides an excellent experimental system to study the developmental mechanisms of the chordate nervous system. The neural fate of the cells consisting of the ascidian CNS is determined in both autonomous and non-autonomous fashion during the cleavage stage. The ascidian neural plate performs the morphogenetic movement of neural tube closure that resembles that in vertebrate neural tube formation. Following neurulation, the CNS is separated into five distinct regions, whose homology with the regions of vertebrate CNS has been discussed. Following their larval stage, ascidians undergo a metamorphosis and become sessile adults. The metamorphosis is completed quickly, and therefore the metamorphosis of ascidians is a good experimental system to observe the reorganization of the CNS during metamorphosis. A recent study has shown that the major parts of the larval CNS remain after the metamorphosis to form the adult CNS. In contrast to such a conserved manner of CNS reorganization, most larval neurons disappear during metamorphosis. The larval glial cells in the CNS are the major source for the formation of the adult CNS, and some of the glial cells produce adult neurons.     

Progesterone as a neurosteroid: synthesis and actions in rat glial cellsProceedings of Xth International Congress on Hormonal Steroids, Quebec, Canada, 17–21 June 1998.

The central nervous system (CNS) and the peripheral nervous system (PNS) are targets for steroid hormones where they regulate important neuronal functions. Some steroid hormones are synthesized within the nervous system, either de novo from cholesterol, or by the metabolism of precursors originating from the circulation, and they were termed ‘neurosteroids'. The sex steroid progesterone can also be considered as a neurosteroid since its synthesis was demonstrated in rat glial cell cultures of the CNS (oligodendrocytes and astrocytes) and of the PNS (Schwann cells). Both types of glial cells express steroid hormone receptors, ER, GR and PR. As in target tissue, e.g. the uterus, PR is estrogen-inducible in brain glial cell cultures. In the PNS, similar PR-induction could not be seen in pure Schwann cells derived from sciatic nerves. However, a significant PR-induction by estradiol was demonstrated in Schwann cells cocultured with dorsal root ganglia (DRG), and we will present evidence that neuronal signal(s) are required for this estrogen-mediated PR-induction. Progesterone has multiple effects on glial cells, it influences growth, differentiation and increases the expression of myelin-specific proteins in oligodendrocytes, and potentiates the formation of new myelin sheaths by Schwann cells in vivo. Progesterone and progesterone analogues also promotes myelination of DRG-Neurites in tissue culture, strongly suggesting a role for this neurosteroid in myelinating processes in the CNS and in the PNS.     

20-hydroxyecdysone stimulates proliferation of glial cells in the developing brain of the moth Manduca sexta

The steroid hormone 20-hydroxyecdysone (20-HE) controls diverse aspects of neuronal differentiation during metamorphosis in the hawkmoth Manduca sexta. In the present study we have examined the effect of 20-HE on glial cells of the brain during the metamorphic period. The antennal (olfactory) lobe of Manduca provides an ideal system in which to study effects of hormones on glial cells, since three known classes of glial cells participate in its development, and at least one type is critically important for establishment of normal neuronal morphology. These glial cells, associated with the neuropil, form boundaries for developing olfactory glomeruli as a result of proliferation and migration. We determined whether glial cells proliferate in response to 20-HE by injecting a pulse of 20-HE into the hemolymph at different stages of development and monitoring proliferation of all three types of glial cells. Hormone injections at the beginning and end of metamorphic development, when hormone titers are normally low, did not stimulate proliferation of neuropil-associated glial cells. Injections during the period when hormone titers are normally rising produced significant increases in their proliferation. Injections when hormone titers are normally high were ineffective at enhancing their proliferation. One other class of glial cells, the perineurial cells, also proliferate in response to 20-HE. Thus, glial proliferation in the brain is under the control of steroid hormones during metamorphic development. © 1995 John Wiley & Sons, Inc.     

The dare gene: steroid hormone production, olfactory behavior, and neural degeneration in Drosophila.

Steroid hormones mediate a wide variety of developmental and physiological events in insects, yet little is known about the genetics of insect steroid hormone biosynthesis. Here we describe the Drosophila dare gene, which encodes adrenodoxin reductase (AR). In mammals, AR plays a key role in the synthesis of all steroid hormones. Null mutants of dare undergo developmental arrest during the second larval instar or at the second larval molt, and dare mutants of intermediate severity are delayed in pupariation. These defects are rescued to a high degree by feeding mutant larvae the insect steroid hormone 20-hydroxyecdysone. These data, together with the abundant expression of dare in the two principal steroid biosynthetic tissues, the ring gland and the ovary, argue strongly for a role of dare in steroid hormone production. dare is the first Drosophila gene shown to encode a defined component of the steroid hormone biosynthetic cascade and therefore provides a new tool for the analysis of steroid hormone function. We have explored its role in the adult nervous system and found two striking phenotypes not previously described in mutants affected in steroid hormone signaling. First, we show that mild reductions of dare expression cause abnormal behavioral responses to olfactory stimuli, indicating a requirement for dare in sensory behavior. Then we show that dare mutations of intermediate strength result in rapid, widespread degeneration of the adult nervous system.     

The gastropod nervous system in metamorphosis

Many gastropods, including the sea hare Aplysia californica, undergo metamorphosis in passing from the larval to the juvenile phases of their life cycle. During metamorphosis, the gastropod nervous system is affected by both progressive and regressive neuronal events. In addition to this metamorphic reorganization, the nervous system appears to be centrally involved in initiating metamorphosis. We propose that gastropods not only possess temporally distinct neuronal adaptations for the specific needs of the larval and juvenile phases, but also another transient neuronal adaptation specialized to subserve the metamorphic episode.     

Control of Dopa decarboxylase gene expression by the Broad-Complex during metamorphosis in Drosophila

The induction of the Dopa decarboxylase gene (Ddc) in the epidermis of Drosophila at pupariation is a receptor-mediated response to the steroid molting hormone, ecdysone. Activity is also dependent on the Broad-Complex (BR-C), an early ecdysone response gene that functions during metamorphosis. BR-C encodes a family of zinc-finger protein isoforms, BR-C(Z1-Z4). Genetic experiments have shown that the Z2 isoform is required for epidermal Ddc to reach maximum expression at pupariation. In this paper, we report that BR-C regulates Ddc expression at two different developmental stages through two different cis-acting regions. At pupariation, BR-C acts synergistically with the ecdysone receptor to up-regulate Ddc. DNase I foot printing has identified four binding sites of the predominant Z2 isoform within a distal regulatory element that is required for maximal Ddc activity. The sites share a conserved core sequence with a set of BR-C sites that had been mapped previously to within the first Ddc intron. Using variously deleted Ddc genomic regions to drive reporter gene expression in transgenic organisms, we show that the intronic binding sites are required for Ddc expression at eclosion. At both pupariation and eclosion, BR-C releases Ddc from an active silencing mechanism, operating through two distinct cis-acting regions of the Ddc genomic domain at these stages. Transgenes, bearing a Ddc fragment from which one of the cis-acting silencers has been deleted, exhibit beta-galactosidase reporter activity in the epidermal cells prior to the appearance of endogenous DDC. Our finding that BR-C is required for Ddc activation at eclosion is the first evidence to suggest that this important regulator of the early metamorphic events, also regulates target gene expression at the end of metamorphosis.     

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.