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PubMed1999

E75 expression in mosquito ovary and fat body suggests reiterative use of ecdysone-regulated hierarchies in development and reproduction.

The steroid hormone ecdysone controls genetic regulatory hierarchies underlying insect molting, metamorphosis and, in some insects, reproduction. Cytogenetic and molecular analysis of ecdysone response in Drosophila larval salivary glands has revealed regulatory hierarchies including early genes which encode transcription factors controlling late ecdysone response. In order to determine whether similar hierarchies control reproductive ecdysone response, we have investigated ecdysone-regulated gene expression in vitellogenic mosquito ovaries and fat bodies. Here, we identify the homologue of the Drosophila E75 early ecdysone inducible gene in the yellow fever mosquito Aedes aegypti, and show that, as in Drosophila, the mosquito homologue, AaE75, consists of three overlapping transcription units with three mRNA isoforms, AaE75A, AaE75B, and AaE75C, originating as a result of alternative splicing. All three AaE75 isoforms are induced at the onset of vitellogenesis by a blood meal-activated hormonal cascade, and highly expressed in the mosquito ovary and fat body, suggesting their involvement in the regulation of oogenesis and vitellogenesis, respectively. Furthermore, in vitro fat body culture experiments demonstrate that AaE75 isoforms are induced by 20-hydroxyecdysone, an active ecdysteroid in the mosquito. These findings suggest that related ecdysone-triggered regulatory hierarchies may be used reiteratively during developmental and reproductive ecdysone responses.

Aedes↗
PubMed2001

Regulation of ecdysteroid signaling: cloning and characterization of ecdysone oxidase: a novel steroid oxidase from the cotton leafworm, Spodoptera littoralis.

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid followed by irreversible reduction by 3-dehydroecdysone 3alpha-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase polymerase chain reaction-based approach has been used to clone the cDNA (2.8 kilobases) encoding this 65-kDa protein. Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and data base searching revealed that the enzyme is an FAD flavoprotein that belongs to the glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P)(+)-linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5'-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a single-copy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.

3-Hydroxysteroid Dehydrogenases↗
PubMed1989

Effects of juvenile hormone on the ecdysone response of Drosophila Kc cells.

Drosophila Kc cells are ecdysone-responsive: hormone treatment leads rapidly to increased synthesis of several ecdysone-inducible polypeptides (EIPs) and to commitment to eventual proliferative arrest. Later, the treated cells undergo morphological transformation, cease to proliferate, and develop new enzymatic activities, notably, acetylcholinesterase (AChE) activity. These responses have proven useful as models for studying ecdysone action. Here we report the sensitivity of Kc cells to another important insect developmental regulator--juvenile hormone (JH). We find that JH inhibits some, but not all, aspects of the ecdysone response. When Kc cells are treated with ecdysone in the presence of either natural JHs or synthetic analogues, the morphological and proliferative responses are inhibited and AChE induction is blocked. Most striking is that JHs protect the cells from the rapid proliferative commitment induced by ecdysone alone. The JH effects exhibit reasonable dose-response curves with half-maximal responses occurring at very low JH concentrations. Nonetheless, even at high JH concentrations the inhibitory effects are incomplete. It is interesting that EIP induction appears to be refractory to JH. It seems clear that JH is not simply a generalized inhibitor of ecdysone-induced responses.

Acetylcholinesterase↗
PubMed2002

Dynamic development of Trypanosoma cruzi in Rhodnius prolixus: role of decapitation and ecdysone therapy.

Decapitation and ecdysone therapy on the population dynamics of the Trypanosoma cruzi Dm28c clone in the stomach, small intestine and rectum of fifth-instar larvae of Rhodnius prolixus were investigated. Parasites were not found in the small intestine and rectum of decapitated insects after 10 days post-infection (p.i.). Decapitated ecdysone-supplemented insects sustained the flagellate infection in both gut compartments. In the rectum, the population density of parasites increased 5-fold in ecdysone-treated decapitated larvae and 7-fold in control insects. Epimastigote forms dominated with 40-65%, intermediate stages and round forms varied over 10-35% in the stomach, small intestine and rectum in both insect groups. Low numbers of metacyclic trypomastigotes were observed in the stomach and small intestine of the control group and decapitated insects supplemented with ecdysone but, at 15 days p.i., this form of flagellate reached about 20% in the rectum of the control insects. In the entire gut, at 30 days p.i., 23% of parasites in the control group and 8% in the decapitated insects treated with ecdysone were found. These results indicate that a head factor, possibly the prothoracicotropic hormone from the brain which stimulates ecdysone production by the prothoracic glands, may act directly or indirectly to stimulate the development of epimastigotes and round forms of the parasite and that a single ecdysone treatment is not able to fully reverse metacyclogenesis in decapitated R. prolixus.

Animals↗
PubMed1978

Thin-layer chromatographic in situ analysis of insect ecdysones via fluorescence-quenching.

The possibility of quantitating insect ecdysones in situ on thin-layer chromatographic plates was examined. Two approaches were evaluated: 1) the induction of ecdysone fluorescence by sulfuric acid treatments and 2) the fluorescence-quenching of fluorescent thin-layer chromatographic plates by ecdysones. The fluorescence-quenching method was found to be most suitable and had a linear response range from 0.5 to 3 microgram for alpha-ecdysone and 20-hydroxyecdysone. Fluorescence-quenching and high pressure liquid chromatographic analyses obtained from extracts of alpha-ecdysone 20-hydroxylase incubations gave similar results. New data concerning the acid-induced fluorescence of ecdysones showed alpha-ecdysone to be twice as fluorescent as 20-hydroxyecdysone.

Animals↗
PubMed1975

The ovary as a source of alpha-ecdysone in an adult mosquito.

The ovaries of the mosquito Aedes aegypti cultured in vitro secrete material that behaves like ecdysone in a radioimmunoassay. The material was identified as alpha-ecdysone by high-resolution liquid and gas-liquid chromatography. Secretion reached a maximum 16 hr after a blood meal as shown by bioassay and direct determination. Ovariectomy reduced the concentration of ecdysone in the adult after a blood meal. Qualitative analysis of whole-body extracts indicated beta-ecdysone to be the principal species present. Thus the ovaries appear to secrete a prohormone, alpha-ecdysone, which is converted to beta-ecdysone. Beta-ecdysone plays a significant role in stimulating egg development in the adult mosquito and may have reproductive roles in other insects.

Adipose Tissue↗
PubMed2004

An arginine-histone methyltransferase, CARMER, coordinates ecdysone-mediated apoptosis in Drosophila cells.

Developmentally programmed cell death is regulated by a balance between pro- and anti-death signaling. During Drosophila metamorphosis, the removal of larval tissues is dependent on the steroid hormone ecdysone, which controls the levels of pro- and anti-death molecules. Ecdysone binds to its heterodimeric receptor ecdysone receptor/ultraspiracle to mediate transcription of primary response genes. Here we show that CARMER, an arginine-histone methyltransferase, is critical in coordinating ecdysone-induced expression of Drosophila cell death genes. Ablation of CARMER blocks ecdysone-induced cell death in Drosophila cells, but not apoptosis induced by cell stress. We demonstrate that CARMER associates with the ecdysone receptor complex and modulates the ecdysone-induced transcription of a number of apoptotic genes. Thus, the chromatin-modifying protein, CARMER, modulates cell death by controlling the hormone-dependent expression of the core cell death machinery.

Amino Acid Sequence↗
PubMed1990

Transcription at the ecdysone-inducible locus 2B5 in Drosophila.

The Broad-Complex (BR-C) of D. melanogaster, mapping at the 2B5 early ecdysone puff, mediates ecdysone-induced processes. We present here the transcriptional analysis of the locus in both wild type and representative mutants. Two well defined regions are transcribed, only one of which has a pattern consistent with the proposed 2B5 located BR-C function. The 2B5 region is actively transcribed in early third instar larvae before ecdysone levels increase. Ecdysone switches this early pattern to a complex late type which requires the presence of functional BR-C product. Therefore, BR-C self-regulates its own ecdysone-induced transcription. The effect of 2B5 mutations on transcription at several intermolt, early and late puffs is also described. Null mutations at the 2B5 locus prevents ecdysone inducible transcription. This evidence supports the fact that 2B5 codes for an ecdysone-dependent transcriptional regulator.

Animals↗
PubMed2003

rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development.

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development.

Amino Acid Sequence↗
PubMed1993

Puffs and PCR: the in vivo dynamics of early gene expression during ecdysone responses in Drosophila.

The steroid hormone ecdysone orchestrates insect development by regulating gene networks. In Drosophila the most detailed description of ecdysone action is the sequential activation of early and late puffs in the polytene chromosomes of the late larval salivary gland. A number of these early puffs (2B5, 74EF and 75B) contain complex transcription units (Broad-Complex, E74 and E75 respectively) encoding families of regulatory proteins which are expressed in most if not all tissues. In vitro, transcripts of the different isoforms of these early genes as well as the ecdysone receptor (EcR) present varying dose response characteristics (Karim and Thummel, 1992, EMBO J. 11, 4083-4093). We have developed an in vivo approach using a reverse transcription-polymerase chain reaction assay (RT-PCR) so as to visualise these transcripts in the RNA extracted from a single salivary gland. Using one salivary gland lobe for developmental puff staging and the sister lobe for RT-PCR, we have obtained precise developmental profiles for these transcripts and have extended our study to other tissues and stages where puffing studies were not possible. In the salivary gland we have characterised three distinct ecdysone responses. For the mid and late third larval instar responses our results confirm and extend the conclusions of the in vitro studies concerning the temporal expression of the early gene isoforms. The relatively brief prepupal response contains elements in common with each of the larval responses and all three can be explained by the profiles of the respective ecdysone peaks. Interestingly EcR transcripts respond differently during each response. The analysis of different tissues of the same animal reveals subtle differences in the timing of the ecdysone response and isoform expression and suggests that this may reflect tissue differences in the ecdysone profiles. As these molecules have homologues in vertebrates, our analysis may have general implications for the organisation of hormonal responses in vivo.

Animals↗
PubMed1998

Ecdysone pathway is required for furrow progression in the developing Drosophila eye.

In Drosophila, secretion of the steroid hormone ecdysone from the prothoracic ring gland coordinates and triggers events such as molting and metamorphosis. In the developing Drosophila compound eye, pattern formation and cell-type specification initiate at a moving boundary known as the morphogenetic furrow. We have investigated the role of ecdysone in eye development and report here that the ecdysone signaling pathway is required for progression of the morphogenetic furrow in the eye imaginal disc of Drosophila. Genetic disruption both of the ecdysone signal in vivo with the ecdysoneless1 (ecd1) mutant and of ecdysone response with a Broad-Complex mutant result in disruption of morphogenetic furrow progression. In addition, we show that ecdysone-dependent gene expression, both of a reporter of transcriptional activity of the Ecdysone Receptor and of the Z1 isoform of the Broad Complex, are localized in and close to the furrow. These results suggest that, in the morphogenetic furrow, temporal hormonal signals are integrated into genetic pathways specifying spatial pattern.

Animals↗
PubMed1975

[The effect of alpha- and beta-ecdysone on differentiation of imaginal disks of Drosophila melanogaster cultivated in vitro].

The effect of alpha- and beta-ecdysone was studied upon differentiation of eye and leg imaginal discs of Drosophila cultivated together with neural ganglia in the medium C-39. In the medium without hormone, eye and leg imaginal discs from larvae of the early 3rd instar are not capable of differentiation. In these conditions, eye imaginal discs from larvae of the late 3rd instar become pigmented in 5 weeks and leg imaginal discs form sometimes segmented limbs on the 4th week of cultivation. In the medium with alpha- and beta-ecdysone, eye and leg imaginal discs from larvae of the early 3rd instar reveal only initial stages of differentiation. The effect of beta-ecdysone concentration was studied upon differentiation of leg and eye imaginal discs from larvae of the late 3rd instar. On decrease of beta-ecdysone concentration from 0.05 down to 0.005 mug/ml, initial stages of differentiation of leg imaginal discs proceed at a slower rate but, then, more complete differentiation with the formation of chitin and neuromuscular tissue is observed. In eye imaginal discs alpha-ecdysone at a concentration of 0.5 mug/ml and beta-ecdysone at a concentration of 0.005-0.5 mug/ml induce the pigment accumulation already at the 1st week of cultivation and beta-ecdysone at a concentration of 5 mug/ml fully inhibits the formation and accumulation of pigment.

Drosophila melanogaster↗
PubMed1976

[Comparison of the effects of alpha and beta ecdysone on a diploid clone of cultured Drosophila melanogaster cells in vitro: induction of proteins and morphological changes].

The specific induction of a similar (class of) protein(s) by alpha or beta-ecdysone in the cells of a "sensitive" clone of Drosophila melanogaster cultured in vitro was shown by polyacrylamide gel electrophoresis. However, differences can be seen in the action of the two hormones. (1) The threshold concentration is about 10 times higher for alpha-ecdysone (10 nM) than for beta-ecdysone (1 nM). (2) Beyond this threshold (i.e. 100 nM) the rate of induction is greater for beta-ecdysone (less than or equal to 24 h) than for alpha-ecdysone (larger than or equal to 48 h). The morphological modifications and the protein induction are seen simultaneously after 1 day of 100 nM beta-ecdysone treatment. On the contrary, a dissociation of these two phenomena is noted with the same concentration of alpha-ecdysone.

Animals↗
PubMed1995

Ecdysone Regulated RNA synthesis in Drosophila larval salivary glands.

The changes in total and ribosomal RNA (rRNA) synthesis in cultured larval salivary glands of Drosophila in response to steroid hormone ecdysone have been studied. By using the incorporation of [3H]-uridine or [32P]-UTP into salivary gland cells, followed by RNA extraction and electrophoretic or sucrose gradient fractionation it was found that physiological concentrations of ecdysone stimulate the synthesis of rRNA. Administration of selective transcription inhibitors (actinomycin D, alpha-amanitin, cordycepin, DRB, and 5- fluorouridine) as well as protein synthesis inhibitors (cycloheximide, puromycin) showed that early response to ecdysone involved a direct stimulation of rDNA transcription, probably by ecdysone-ecdysone receptor complex, and is independent of protein synthesis. In later stages of the response to ecdysone, as revealed again using transcription and protein synthesis inhibitors, the synthesis of rRNA is dependent on the factor(s) produced during early stages of the response, in the manner which is very similar to sequential gene activation in chromosomal puffs. Experiments with the withdrawal of ecdysone from culture medium in early stages of its action show similar negative effects on rRNA synthesis as inhibitors of RNA polymerase II, thus proving the existence of parallel mechanism controlling induction of specific chromosomal puffs and transcription of rDNA. The data presented suggest that, in addition to the expression of tissue and developmentally-specific genes, the regulation of rDNA transcription, along with the production of ribosomes, might be required for Drosophila metamorphosis. The results are discussed in the light of vertebrate's steroid hormone action on ribosomal genes.

Journal Article↗
PubMed2002

The expression of the let-7 small regulatory RNA is controlled by ecdysone during metamorphosis in Drosophila melanogaster.

In Caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. A component of this pathway, the let-7 small regulatory RNA, is expressed at the late stages of development and promotes the transition from larval to adult (L/A) stages. The stage-specificity of let-7 expression, which is crucial for the proper timing of the worm L/A transition, is conserved in Drosophila melanogaster and other invertebrates. In Drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. To test whether let-7 expression is regulated by ecdysone in Drosophila, we used Northern blot analysis to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and S2 cultured cells. Experiments were conducted to test the role of Broad-Complex (BR-C), an essential component in the ecdysone pathway, in let-7 expression. We show that ecdysone and BR-C are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in Drosophila. These results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects.

Animals↗
PubMed1995

Multiple interacting elements delineate an ecdysone-dependent regulatory region with secondary responsive character.

Within the 2.2 kb region between hsp23 and gene 1 of the small heat shock gene locus 67B1 of Drosophila melanogaster, an approximately 1 kb perturbation of the chromatin architecture has previously been observed to occur in response to the steroid hormone ecdysone. Transient expression assays in hormonally-responsive Drosophila tissue culture cells utilizing hsp70-lacZ chimeric reporter constructs revealed the presence of ecdysone-dependent regulatory sequences in this hsp23-gene 1 intergenic region. The analysis delimited five functional segments: three core regions which were completely encompassed within the region of chromatin perturbation, and two gene-proximal regions which appear to be functionally equivalent under some circumstances. None of the delineated regions was capable of stimulating expression independently, while sub-maximal expression was obtained when combinations of two or three regions were monitored. This requirement for multiple DNA segments to drive maximal transcription suggested that cooperative interactions between the regions were essential for full hormonal responsiveness. Unexpectedly, no binding of the ecdysone receptor was detectable within any of the delineated regions, implying the involvement of multiple non-receptor factors in the observed hormonal responsiveness. The ecdysone-dependent activation of reporter constructs driven by these sequences showed a significant time lag and was coupled with a marked sensitivity to low concentrations of cycloheximide. The data obtained strongly suggest that the cis-acting elements delimited within the hsp23-gene 1 intergenic region respond to ecdysone in a secondary manner, presumably by requiring interaction with the product(s) of primary ecdysone-responsive genes.

Animals↗
PubMed1995

Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene.

The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the third instar to the end of adult life. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. To study the molecular basis for ecdysone regulated Lsp-2 activity, deletion mutants of the Lsp-2 5'-flanking region were constructed by fusion to either the Escherichia coli chloramphenicol acetyltransferase (CAT) gene or to an hsp70-lacZ hybrid gene encoding beta-galactosidase. Constructs transfected into Drosophila S2/M3 cells were shown to confer transient ecdysone inducibility on the reporter genes. A single functional ecdysone response element (EcRE) was localized at position -75 relative to the Lsp-2 transcription initiation site. In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27-bp sequence harboring the EcRE bound both the Drosophila ecdysone receptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a cooperative manner. Competition experiments indicate that the affinity of the Lsp-2 EcRE for the ecdysone receptor complex is comparable to that of the canonical EcRE of the hsp27 gene and is at least 4-fold greater than that of Fbp1, another fat body-specific Drosophila gene. Our results suggest that structural features of this EcRE determine its ability to induce ecdysone responsiveness at a lower ligand concentration and may form the basis for differential hormone responsiveness within the fat body.

Animals↗
PubMed1988

Synthesis of apolar ecdysone esters by ovaries of the cockroach Periplaneta americana.

Ecdysteroid levels detected by RIA in extracts of mature ovaries from Periplaneta americana increased approximately fourfold (53 +/- 10 to 184 +/- 38 ng/g; +/- SEM, n = 3) on treatment with Helix pomatia "sulphatase" enzymes. HPLC analysis showed that this increase in immunoreactivity resulted from the hydrolysis of six apolar compounds that cochromatographed with the ecdysteroid esters previously shown to be present in newly laid oothecae (A1, A2, A3, A4, A5, and A6; A. J. Slinger, L. N. Dinan, and R. E. Isaac (1986). Insect Biochem. 16 (i), 115-119). Intact ovaries cultured in saline were able to take up [3H]ecdysone from the medium and synthesize ecdysone esters, most of which cochromatographed with immunoreactive peaks from ovaries and oothecae. Crude homogenates and membranes prepared from mature ovaries were also able to esterify ecdysone in vitro. The enzyme activity associated with a high-speed pellet was greatly enhanced by the addition of coenzyme A fatty acyl esters, each reaction resulting in the synthesis of a single major metabolite. The three esters formed on incubating ecdysone with coenzyme A-palmitate, -lineate, and -oleate could be characterized by their retention times on HPLC which were identical to compounds A2, A5, and A6, respectively. These compounds were the three quantitatively important immunoreactive esters found in ovaries and newly laid oothecae. The data presented indicates that ovaries can esterify ecdysone with palmitic, linoleic, and oleic acids and that these apolar derivatives are transferred to the egg. The esters appear to be different from the ecdysone 22-fatty acyl esters that have been isolated from ticks and other insects.

Acyl Coenzyme A↗