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PubMed2000

Adenovirus-mediated inducible gene expression in vivo by a hybrid ecdysone receptor.

Precise control of transgene expression would markedly facilitate certain applications of gene therapy. To regulate expression of a transferred gene in response to an exogenous compound in vivo, we modified the ecdysone-responsive system. We combined the advantages of the Drosophila (DmEcR) and the Bombyx ecdysone receptor (BmEcR) by creating a chimeric Drosophila/Bombyx ecdysone receptor (DB-EcR) that preserved the ability to bind to the modified ecdysone promoter without exogenous retinoid X receptor (RXR). In cultured cells, DB-EcR effectively mediates ligand-dependent transactivation of a reporter gene at lower concentrations of the chemical ecdysone agonist GS-E than VgRXR (DmEcR + RXR). Transgene delivery in vivo was achieved by intramyocardial injection of recombinant adenovirus vectors in adult rats. Upon stimulation with GS-E, DB-EcR potently (>40-fold induction) activated gene expression in vivo while VgRXR was not induced. This hybrid ecdysone receptor represents an important new tool for in vivo transgene regulation with potentially diverse applications in somatic and germline transfer.

Adenoviridae↗
PubMed1993

20-OH-ecdysone swells nuclear volume by alkalinization in salivary glands of Drosophila melanogaster.

Ecdysteroids play an important role in the larval moulting process of insects. Ecdysone-induced stimulation causes specific "puffs" in polytene chromosomes of salivary gland cells resulting in nuclear swelling. During this process, changes of intracellular ion composition are thought to act as an early regulatory mechanism of gene activation. By use of video-imaging analysis and electrophysiological techniques, we examined ecdysone-induced nuclear swelling in Drosophila salivary glands in situ and its dependence on pH and calcium. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. Addition of 5 x 10(-6) mol/1 20-OH-ecdysone led, after a lag period of 50 min, to a sustained Ca(2+)-dependent increase of nuclear volume by 23.0 +/- 2.3%. Amiloride, a blocker of plasma membrane Na+/H+ exchange, prevented 20-OH-ecdysone-induced nuclear swelling. Decreasing pH in the superfusate from 7.15 to 6.8 led to nuclear shrinkage by 16.9 +/- 3.9%. Measurements of pH in salivary gland cells with ion-sensitive microelectrodes disclosed an alkalinization of 0.23 +/- 0.05 pH units after stimulation with 20-OH-ecdysone. We postulate that 20-OH-ecdysone activates the amiloride-sensitive plasma membrane Na+/H+ exchanger. This leads to intracellular alkalinization and concomitant decondensation of the nuclear chromatin visible as nuclear swelling. Thus, cell alkalinization could be a potentially important stimulatory mechanism in mediating ecdysteroid-induced activation of the cell nucleus.

Alkalies↗
PubMed2002

Ecdysone-regulated puff genes 2000.

The Ashburner model for the hormonal control of polytene chromosome puffing has provided a strong foundation for understanding the basic mechanisms of steroid-regulated gene expression (Cold Spring Harbor Symp. Quant. Biol. 38 (1974) 655). According to this model, the steroid hormone 20-hydroxyecdysone (referred here as ecdysone) directly induces the expression of a small set of early regulatory genes. These genes, in turn, induce a much larger set of late target genes that play a more direct role in controlling the biological responses to the hormone. The recent characterization of two early puff genes, E63-1 and E23, and three late puff genes, D-spinophilin, L63, and L82, provide further confirmation of the Ashburner model. In addition, these studies provide exciting new directions for our understanding of ecdysone signaling. Overexpression studies of E63-1 implicate this gene in directing calcium-dependent salivary gland glue secretion. In contrast, overexpression of E23 indicates that this ABC transporter family member may negatively regulate ecdysone signaling by actively transporting the hormone out of target cells. Finally, genetic studies of the L63 and L82 late genes reveal unexpected possible functions for ecdysone in controlling developmental timing and growth. This review surveys the recent characterization of these ecdysone-inducible genes and provides an overview of how they expand our understanding of ecdysone functions during development.

ATP-Binding Cassette Transporters↗
PubMed1989

A member of the steroid hormone receptor gene family is expressed in the 20-OH-ecdysone inducible puff 75B in Drosophila melanogaster.

Drosophila melanogaster DNA has been cloned which encompasses a major part of the 20-OH-ecdysone inducible puff 75B. One 20-OH-ecdysone responsive transcription unit was detected which is expressed into two transcripts which accumulate upon the incubation of salivary glands of 3rd instar larvae with 20-OH-ecdysone. This accumulation is correlated with the 20-OH-ecdysone induced activity of puff 75B. 75B cDNA analysis indicates that the activity of puff 75B leads to the synthesis of a protein which belongs to the steroid and thyroid hormone receptor superfamily. The highest similarity of the derived 75B protein sequence was found to the DNA and ligand binding domains of human retinoic acid receptor. A study of the tissue distribution in larvae revealed that 75B mRNA is present in most, if not all 20-OH-ecdysone target tissues. It is proposed that 75B protein is a DNA-binding protein playing a key role in mediating the regulation of the larval molt by 20-OH-ecdysone.

Amino Acid Sequence↗
PubMed1998

The RXR homolog ultraspiracle is an essential component of the Drosophila ecdysone receptor.

Pulses of the steroid hormone ecdysone function as key temporal signals during insect development, coordinating the major postembryonic developmental transitions, including molting and metamorphosis. In vitro studies have demonstrated that the EcR ecdysone receptor requires an RXR heterodimer partner for its activity, encoded by the ultraspiracle (usp) locus. We show here that usp exerts no apparent function in mid-third instar larvae, when a regulatory hierarchy prepares the animal for the onset of metamorphosis. Rather, usp is required in late third instar larvae for appropriate developmental and transcriptional responses to the ecdysone pulse that triggers puparium formation. The imaginal discs in usp mutants begin to evert but do not elongate or differentiate, the larval midgut and salivary glands fail to undergo programmed cell death and the adult midgut fails to form. Consistent with these developmental phenotypes, usp mutants show pleiotropic defects in ecdysone-regulated gene expression at the larval-prepupal transition. usp mutants also recapitulate aspects of a larval molt at puparium formation, forming a supernumerary cuticle. These observations indicate that usp is required for ecdysone receptor activity in vivo, demonstrate that the EcR/USP heterodimer functions in a stage-specific manner during the onset of metamorphosis and implicate a role for usp in the decision to molt or pupariate in response to ecdysone pulses during larval development.

Animals↗
PubMed1997

Endogenous ecdysteroid levels and rates of ecdysone acylation by intact ovaries in vitro in relation to ovarian development in adult female crickets, Acheta domesticus.

Ecdysteroid titres have been determined in adult female house crickets (Acheta domesticus) in relation to reproductive maturation. Ecdysteroid levels in newly emerged adult females are low except in the gut and carcass, which probably reflects the remnants of the preecdysial ecdysteroid peak. Ecdysteroid levels in all compartments increase markedly once ovarian weight surpasses 10 mg. Apolar ecdysteroid conjugates (ecdysone 22-fatty acyl esters) predominate in ovarian tissue throughout ovarian maturation, but low levels of free ecdysteroid and polar conjugated ecdysteroids are also present. During this period, two peaks of ecdysteroids (mainly free and apolar conjugated ecdysteroids) are observed in the haemolymph, gut, and carcass compartments. The peaks in the haemolymph occur when the ovarian mass reaches 30 and 100 mg. The gut and carcass may be acting as sinks or sites of metabolism for the hormone released from the ovaries. The rate of ecdysone acylation by ovaries was found to be developmentally regulated, increasing from low levels in the immature ovaries of newly emerged females as the ovaries increase in size. A semiquantitative assay has been developed to identify compounds which inhibit the conversion of [3H]ecdysone into 22-fatty acyl [3H]ecdysone by ovaries in vitro. A number of ecdysteroids possessing a free hydroxyl group as C-22 as well as the side-chain stereochemistry of ecdysone effectively inhibit this conversion, probably by acting as competitive substrates. In the cases of 20-hydroxyecdysone and ponasterone A, it was clearly demonstrated that these compounds are converted to a mixture of C-22 fatty acyl esters. Several other compounds which have been suggested to affect ecdysteroid metabolism/mode of action in other systems were also tested for their effects on the acyltransferase activity of ovaries in vitro.

Acylation↗
PubMed1990

Puffs and gene regulation--molecular insights into the Drosophila ecdysone regulatory hierarchy.

Sixteen years ago, Michael Ashburner and his colleagues proposed a hierarchical model for the genetic control of polytene chromosome puffing by the steroid hormone ecdysone. The recent molecular isolation and characterization of three early ecdysone-inducible genes has confirmed many aspects of this model--these genes are directly induced by ecdysone, repressed by ecdysone-induced proteins, and appear to encode DNA binding regulatory proteins. The three early genes are also remarkably similar in structure. They are all unusually long and complex, with multiple transcripts that direct the synthesis of several related proteins from each locus. Proteins encoded by two of the early genes bind to both early and late ecdysone-induced puffs, implying that they are key regulators in the hierarchy.

Animals↗
PubMed2002

Ecdysone triggers the expression of Golgi genes in Drosophila imaginal discs via broad-complex.

One of the most significant morphogenic events in the development of Drosophila melanogaster is the elongation of imaginal discs during puparium formation. We have shown that this macroscopic event is accompanied by the formation of Golgi stacks from small Golgi larval clusters of vesicles and tubules that are present prior to the onset of disc elongation. We have shown that the fly steroid hormone 20-hydroxyecdysone triggers both the elongation itself and the formation of Golgi stacks (V. Kondylis, S. E. Goulding, J. C. Dunne, and C. Rabouille, 2001, Mol. Biol. Cell, 12, 2308). Using mRNA in situ hybridisation, we show here that ecdysone triggers the upregulation of a subset of genes encoding Golgi-related proteins (such as dnsf1, dsec23, dsed5, and drab1) and downregulates the expression of others (such as dergic53, dbeta'COP, and drab6). We show that the transcription factor Broad-complex, itself an "early" ecdysone target, mediates this regulation. And we show that the ecdysone-independent upregulation of dnsf1 and dsnap prior to the ecdysone peak leads to a precocious formation of large Golgi stacks. The ecdysone-triggered biogenesis of Golgi stacks at the onset of imaginal disc elongation offers the exciting possibility of advancing our understanding of the relationship between gene expression and organelle biogenesis.

Animals↗
PubMed1976

Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster. Assay by in situ hybridization.

Cytoplasmic RNA from imaginal discs of Drosophila melanogaster, labeled by uridine incorporation in organ culture, has been assayed by hybridization to cytological preparations of polytene chromosomes. RNA labeled during the early stages (first four hours) of ecdysone stimulation was compared to RNA labeled in the absence of the hormone. For the poly(A)-containing fraction (oligo-dT bound), several loci hybridize only RNA labeled in the presence of ecdysone; one locus hybridizes only control RNA. The majority of hybridizing loci are unaffected by the hormone. Of the loci hybridizing RNA not bound to oligo-dT, several appear specific for the ecdysone-treated sample, though most are labeled more heavily with this RNA than with the control. None of the ecdysone-sensitive loci visualized by in situ hybridization are the sites of salivary gland puffs induced by ecdysone on the same time scale.

Animals↗
PubMed1990

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3 beta-reductase mediated conversion to ecdysone in representative insects.

The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3 beta-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.

3-Hydroxysteroid Dehydrogenases↗
PubMed1983

Ecdysone and 20 hydroxyecdysone: new hormones for the human parasite schistosoma mansoni.

The insect moulting hormones, ecdysone and 20 hydroxyecdysone, were detected by the combined use of radioimmunoassay and high performance liquid chromatography in the human parasite Schistosoma mansoni. On day 11 after infection only the ecdysone form is present, but, on day 40 after infection the ratio between ecdysone and 20 hydroxyecdysone changes with anatomic localization of the adult worms in mammalian host. In the eggs, the ratio of these two hormones is identical to the ratio found in sexually mature worms located in mesenteric veins. These data demonstrate for the first time that S. mansoni synthesizes the steroid hormones ecdysone and 20 OH ecdysone which are potent molecules in stimulating growth and vitello-genesis of this gonochoric trematode.

Aging↗
PubMed1993

The Drosophila 78C early late puff contains E78, an ecdysone-inducible gene that encodes a novel member of the nuclear hormone receptor superfamily.

We report the molecular definition of an early late puff locus, at position 78C, that is inducible by ecdysone at the onset of Drosophila metamorphosis. This puff contains a single ecdysone-inducible gene consisting of two nested transcription units, E78A and E78B. E78A mRNA is expressed during a brief interval in mid-pupal development and encodes a novel member of the nuclear hormone receptor superfamily. E78B encodes a truncated receptor isoform that lacks the DNA-binding domain and is predominantly expressed at puparium formation and immediately following E78A in pupae. E78B is directly inducible by ecdysone in late third instar larvae and depends on ecdysone-induced protein synthesis for its maximal level of expression. These observations indicate that E78 represents a distinct subset of early ecdysone-inducible regulatory genes.

Amino Acid Sequence↗
PubMed1994

A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone.

Two successive pulses of ecdysone signal the ends of larval and prepupal development in Drosophila, inducing early and late puffs in the salivary gland polytene chromosomes. Early puff induction in prepupae is dependent on a preceding period of protein synthesis and low ecdysone concentration. We demonstrate here that the competence acquired during this interval can be provided by beta FTZ-F1, a nuclear hormone receptor superfamily member derived from the 75CD mid-prepupal puff. We show that beta FTZ-F1 represses its own transcription and is repressed by ecdysone, explaining its brief expression in mid-prepupae. We further show that ectopic beta FTZ-F1 expression leads to enhanced levels of ecdysone-induced BR-C, E74, and E75 early gene transcription and premature induction of the stage-specific 93F early puff and E93 transcription. These findings indicate that beta FTZ-F1 plays a central role in the prepupal genetic response to ecdysone and provide a molecular mechanism for stage-specific responses to steroid hormones.

Animals↗
PubMed1985

Calcium-cyclic AMP interactions in prothoracicotropic hormone stimulation of ecdysone synthesis.

The calcium-dependence of ecdysone synthesis by the insect prothoracic glands was examined in vitro using glands from day 0 pupae of the tobacco hornworm, Manduca sexta. Stimulation of ecdysone synthesis by the cerebral neuropeptide, prothoracicotropic hormone (PTTH), requires extracellular calcium; peptide-stimulated steroidogenesis is blocked by omission of calcium or by addition of the calcium antagonist lanthanum. By contrast, basal synthesis of ecdysone is not calcium-dependent. A stimulatory, as opposed to simply a permissive, role for calcium is indicated by the ability of the calcium ionophore A23187 to mimic the steroidogenic effects of PTTH. Agents that act by increasing the intracellular levels of cAMP (dibutyryl cAMP, 1-methyl-3-isobutylxanthine, forskolin) enhance ecdysone synthesis equally well in the presence or absence of extracellular calcium, indicating that the site of action of the cation in the steroidogenic pathway occurs prior to that of cAMP. Both PTTH and A23187 enhance the formation of cAMP, as measured by the conversion of [3H]ATP to [3H]cAMP, in a manner absolutely dependent upon the presence of extracellular calcium. The results suggest sequential roles for calcium and cAMP in PTTH-stimulated steroidogenesis by the insect prothoracic glands. A model is presented in which the peptide stimulates cAMP formation in a calcium-dependent manner, with the cyclic nucleotide in turn enhancing ecdysone synthesis.

Animals↗
PubMed1985

Conversion of a radiolabelled ecdysone precursor, 2,22,25-trideoxyecdysone, by embryonic and larval tissues of Locusta migratoria.

A high specific activity tritiated ecdysone precursor, 2,22,25-trideoxyecdysone, was used to probe the capacity of various embryonic and larval tissues to perform the last 3 hydroxylation steps in ecdysone biosynthesis. Embryos at early stages of development, prior to the differentiation of their endocrine glands and embryonic heads, thoraces and abdomens of later stages, were found to have the capacity to hydroxylate the precursor to ecdysone. Larval epidermis and fat body are also able to transform 2,22,25-trideoxyecdysone into ecdysone; Malpighian tubules and midgut hydroxylate the precursor at C-2 but are apparently unable to hydroxylate both at C-22 and C-25. Larval prothoracic glands convert the precursor to ecdysone at a very efficient rate, which is 1-2 magnitudes higher than that of the other tissues investigated; several data argue for the existence of a privileged sequence of hydroxylations, C-25, C-22, C-2, in the larval prothoracic glands.

Abdomen↗
PubMed1996

Ecdysone and insect immunity: the maturation of the inducibility of the diptericin gene in Drosophila larvae.

The developmental analysis of the inducibility of the Drosophila diptericin gene promoter as a response to septic injury shows an important increase in the response during the third larval instar leading to a maximum in late larvae and early prepupae. This increase, or maturation, is temporally correlated with known ecdysone induced events of the salivary gland and we now present evidence, using wild type and mutant larvae, that it does indeed depend upon ecdysone. The response remains minimal in larvae carrying either the temperature sensitive ecdysone deficient late larval lethal allele ecd1, or l(1)t187, a deep orange allele known to be deficient in the ecdysone response. However, experiments with the late larval lethal Broad-Complex mutant l(1)t435 show that the regulation of this response is distinct from the developmental ecdysone regulated hierarchies.

Animals↗
PubMed2004

Rho-LIM kinase signaling regulates ecdysone-induced gene expression and morphogenesis during Drosophila metamorphosis.

The steroid hormone 20-hydroxyecdysone (ecdysone) is the key regulator of postembryonic developmental transitions in insects and controls metamorphosis by triggering the morphogenesis of adult tissues from larvae. The Rho GTPase, which mediates cell shape change and migration, is also an essential regulator of tissue morphogenesis during development. Rho activity can modulate gene expression, in part, by activating LIM kinase (LIMK) and consequently affecting actin-induced SRF transcriptional activity. We have established a link between Rho-LIMK-SRF signaling and the ecdysone-induced transcriptional response during Drosophila development. Specifically, we determined that the Rho GTPase, via LIMK, regulates the expression of several ecdysone-responsive genes, including those encoding the ecdysone receptor itself, a downstream transcription factor (Br-C), and Stubble, a transmembrane protease required for proper leg formation. Stubble and Br-C mutants exhibit strong genetic interactions with several Rho pathway components in the formation of adult structures, but not with Rac or Cdc42. In cultured SL2 cells, inhibition of Rho, F-actin assembly, or SRF blocks the transcriptional response to ecdysone. Together, these findings indicate a link between Rho-LIMK signaling and steroid hormone-induced gene expression in the context of metamorphosis and thereby establish a novel role for the Rho GTPase in development.

Animals↗
PubMed2006

Cellular immune response in Rhodnius prolixus: role of ecdysone in hemocyte phagocytosis.

In this paper we investigate in vivo and in vitro effects of orally administered azadirachtin and ecdysone on the phagocytic responses of Rhodnius prolixus 5th-instar larval hemocytes to the yeast Saccharomyces cerevisiae. Groups of insects fed non-treated blood (control) and insects that received azadirachtin plus ecdysone in the blood meal were inoculated with yeast cells in the hemocele. The injected yeast cells disappeared rapidly from the hemolymph, being removed completely by 90min after inoculation. In the insects treated only with azadirachtin the clearance of free yeast circulating particles was significantly delayed compared to the two previously mentioned groups. It was demonstrated that the binding of yeast cells to hemocytes was reduced in the insects treated only with azadirachtin in comparison to both non-treated control and azadirachtin plus ecdysone-treated groups. Phagocytosis occurred when yeast cells were added to hemocyte monolayers prepared with hemolymph from blood fed insects, treated or not with azadirachtin plus ecdysone, so that yeast cells were rapidly bound to hemocytes and internalized in high numbers. By contrast, insects treated with azadirachtin exhibited a drastic reduction in the quantity of yeast cell-hemocyte binding and subsequent internalization. In all groups, the hemocytes attached to the glass slides were predominantly plasmatocytes. The magnitude and speed of the cellular response suggests that hemocyte phagocytosis is one of the main driving forces for the clearance of free circulating yeast cells from the hemolymph. We propose that ecdysone modulates phagocytosis in R. prolixus larvae, and that this effect is antagonized by azadirachtin.

Animals↗