br regulates the expression of the ecdysone biosynthesis gene npc1

The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. Here we used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. We have identified a conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development.     

Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm,Bombyx mori

The insect neuropeptide prothoracicotropic hormone (PTTH) triggers the biosynthesis and release of the molting hormone ecdysone in the prothoracic gland (PG), thereby controlling the timing of molting and metamorphosis. Despite the well-documented physiological role of PTTH and its signaling pathway in the PG, it is not clear whether PTTH is an essential hormone for ecdysone biosynthesis and development. To address this question, we established and characterized a PTTH knockout line in the silkworm, Bombyx mori. We found that PTTH knockouts showed a severe developmental delay in both the larval and pupal stages. Larval phenotypes of PTTH knockouts can be classified into three major classes: (i) developmental arrest during the second larval instar, (ii) precocious metamorphosis after the fourth larval instar (one instar earlier in comparison to the control strain), and (iii) metamorphosis to normal-sized pupae after completing the five larval instar stages. In PTTH knockout larvae, peak levels of ecdysone titers in the hemolymph were dramatically reduced and the timing of peaks was delayed, suggesting that protracted larval development is a result of the reduced and delayed synthesis of ecdysone in the PG. Despite these defects, low basal levels of ecdysone were maintained in PTTH knockout larvae, suggesting that the primary role of PTTH is to upregulate ecdysone biosynthesis in the PG during molting stages, and low basal levels of ecdysone can be maintained in the absence of PTTH. We also found that mRNA levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in PTTH knockouts. Our results provide genetic evidence that PTTH is not essential for development, but is required to coordinate growth and developmental timing.     

Activité,in vivo, d''inhibiteurs de la biosynthèse de l''ecdysone chezLocusta migratoria

Summary Among about 50 compounds synthesized to inhibit enzymes involved in the biosynthesis pathway of ecdysone we selected seven molecules which showed a strong effect on ecdysone production byLocusta migratoria prothoracic glands incubatedin vitro. These molecules mostly possess a specific activity on ecdysone biosynthesis which is irreversible. The compounds were administered in one or several injections of aqueous or oily solutions at different times in the course of the two last larval instars. Three inhibitors led in a 10% ratio to a prolongation (sometimes more than 3 times the standard length) of the instar, pointing out a decrease in the ecdysone biosynthesis. Two other inhibitors induced some morphogenetic modifications in the adults, as size reduction or wing alterations, and metamorphosis difficulties. Thein vivo low activity compared with the strong onein vitro could be due to difficulties for the compounds to reach the prothoracic glands without degradation. The variation of inhibitory activity which appearsin vivo between the seven compounds studied is not linked either with the chemical structures of the molecules (which are very near one another) or with theirin vitro activity.

     

bantam miRNA Promotes Systemic Growth by Connecting Insulin Signaling and Ecdysone Production

1. Download : Download high-res image (104KB)
2. Download : Download full-size image

Highlights? bantam is required in ecdysone-producing cells to promote body growth ? bantam inhibits ecdysone biosynthesis ? Insulin signaling regulates ecdysone production by repressing bantam activity ? Both the systemic and tissue-autonomous activities of bantam promote organ growth     

Phantom,a cytochrome P450 enzyme essential for ecdysone biosynthesis,plays a critical role in the control of border cell migration in Drosophila

The border cells of Drosophila are a model system for coordinated cell migration. Ecdysone signaling has been shown to act as the timing signal to initiate the migration process. Here we find that mutations in phantom (phm), encoding an enzyme in the ecdysone biosynthesis pathway, block border cell migration when the entire follicular epithelium of an egg chamber is mutant, even when the associated germline cells (nurse cells and oocyte) are wild-type. Conversely, mutant germline cells survive and do not affect border cell migration, as long as the surrounding follicle cells are wild-type. Interestingly, even small patches of wild-type follicle cells in a mosaic epithelium are sufficient to allow the production of above-threshold levels of ecdysone to promote border cell migration. The same phenotype is observed with mutations in shade (shd), encoding the last enzyme in the pathway that converts ecdysone to the active 20-hydroxyecdysone. Administration of high 20-hydroxyecdysone titers in the medium can also rescue the border cell migration phenotype in cultured egg chambers with an entirely phm mutant follicular epithelium. These results indicate that in normal oogenesis, the follicle cell epithelium of each individual egg chamber must supply sufficient ecdysone precursors, leading ultimately to high enough levels of mature 20-hydroxyecdysone to the border cells to initiate their migration. Neither the germline, nor the neighboring egg chambers, nor the surrounding hemolymph appear to provide threshold amounts of 20-hydroxyecdysone to do so. This “egg chamber autonomous” ecdysone synthesis constitutes a useful way to regulate the individual maturation of the asynchronous egg chambers present in the Drosophila ovary.     

Synthesis of a labelled putative precursor of ecdysone—II: [3H4]3β-hydroxy-5β-cholest-7en-6-one: Critical re-evaluation of its role in Locusta migratoria

We have synthesized a tritiated form of 2,14,22,25-tetradeoxyecdysone (5β-ketol) of high specific activity (115 Ci/mmol). We have examined the capacity of various tissues of Locusta migratoria to use this 5β-ketol, a putative precursor of ecdysone, in ecdysteroid biosynthesis. While larval prothoracic glands convert the radiotracer to labelled 14-deoxyecdysone they fail to hydroxylate the molecule to ecdysone itself. Other larval tissues, embryonic tissues or vitellogenic female ovaries are unable to convert the radiotracer to ecdysone, 20-hydroxyecdysone or 2-deoxyecdysone, the terminal products of biosynthesis in different developmental stages. Using subcellular preparations of prothoracic glands or follicle cells we have been unable to show a biological C-14 hydroxylation of 5β-ketol. It thus appears that the step of C-14 hydroxylation in the biosynthesis of ecdysteroids requires a substrate other than 5β-ketol.     

Involvement of ecdysone in the control of meiotic reinitiation in oocytes of Locusta migratoria (Insecta,orthoptera)

Following their biosynthesis in the follicle cells of vitellogenic ovaries, large amounts of ecdysteroids pass into the oocytes where they accumulate and persist during ovulation and egg-laying. The present paper shows that free ecdysone is unevenly distributed in the oocytes exhibiting the highest concentrations in the region of the posterior pole where the final sequences of nuclear maturation, including germinal vesicle breakdown (GVBD), occur. A correlative study indicates that the concentrations of free ecdysone in this region are particularly high (10 to 20 μM) during two periods of meiotic reinitiation observed in the oocytes: reinitiation I, leading from prophase I to metaphase I with GVBD; and reinitiation II, from metaphase I to the end of meiosis. In vitro incubations of oocytes in meiotic arrest (prophase I) in the presence of exogenous ecdysone demonstrate that complete reinitiation (including GVBD) can be triggered in a dose-dependent manner by this hormone.     

Adult ovaries of Locusta migratoria contain the sequence of biosynthetic intermediates for ecdysone

Ovaries of adult $\text{Locusta migratoria}$ have recently been shown to produce impressive amounts of ecdysone together with low polarity ecdysteroids, some of which cross-react with ecdysone in our RIA. A gas chromatographic-mass spectrometric analysis of extracts from ovaries of $\text{Locusta}$ has shown the presence of following compounds (less polar than ecdysone): 2-deoxy-ecdysone, 2,22-bis-deoxy-ecdysone, 2, 22, 2 5-tri-deoxy-ecdysone, 2, 14, 22, 2 5-tetra-deoxy-ecdysone. No other related ecdysteroids were present in our extracts. Cholesterol is used by $\text{Locusta}$ ovaries as a precursor for ecdysone biosynthesis, as our previous studies with labelled products have shown, and we propose that the compounds detected in the present work represent biosynthetic intermediates between cholesterol and ecdysone in $\text{Locusta}$ ovaries.     

Biosynthesis and inactivation of ecdysone during the pupal-adult development of the cabbage butterfly, Pieris brassicae L.

Injection of labelled ecdysone and 20-hydroxyecdysone into Pieris pupae showed that their catabolism proceeds through 26-hydroxylation followed by conversion into acidic steroids assumed to be 26-oic compounds. This biological system is characterized by the lack of conjugation reactions and by rather long-lived hormones.In vivo biosynthesis of ecdysteroids was investigated by 24 hr [³H]cholesterol labelling, followed by HPLC analysis of the resulting [³H]ecdysone and 20-hydroxyecdysone. Active conversion (up to 0.07% in 24 hours) was observed between 48 hr and 120 hr following pupal ecdysis, a result in good agreement with the variations observed in hormone contentLong-term [³H]cholesterol incorporation experiments made it possible to monitor ecdysteroid dynamics during pupal development. Three periods were observed, corresponding to the successive accumulation of ecdysone, 20-hydroxyecdysone and an acidic metabolite. Comparison of these results with those of the experiments involving labelled ecdysone injection shows that the catabolism of injected hormones is not the same as that of endogenous hormones.     

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

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

Ecdysone receptor homologs from mollusks, leeches and a polychaete worm

The genomes of the mollusk Lottia gigantea, the leech Helobdella robusta and the polychaete worm Capitella teleta each have a gene encoding an ecdysone receptor homolog. Publicly available genomic and EST sequences also contain evidence for ecdysone receptors in the seahare Aplysia californica, the bobtail squid Euprymna scolopes and the medicinal leech Hirudo medicinalis. Three-dimensional models of the ligand binding domains of these predicted ecdysone receptor homologs suggest that each of them could potentially bind an ecdysone-related steroid. Thus, ecdysone receptors are not limited to arthropods and nematodes.     

The ecdysoid RH5849 induces yolk polypeptide synthesis in male flies

Summary

RH5849 is a benzoyl hydrazine analog which has been reported to mimic several effects of the arthropod steroid hormone ecdysone to which it is chemically totally unrelated. In adult Diptera, ecdysone is the hormone that triggers vitellogenin synthesis. We report here that RH5849, upon oral ingestion, is able to induce vitellogenin synthesis in male Drosophila, Neobellieria, Phormia and Lucilia. This contrasts to data in the literature which showed that RH5849 could not mimic the pupariation-inducing effect of ecdysone in last instar fly larvae. RH5849 neither exerts a juvenile hormone mimicking effect nor behaves as an anti-juvenile hormone in both the Colorado potato beetle and Galleria.     

Ecdysone metabolism in adult crickets,Gryllus bimaculatus

The fate of injected [³H]ecdysone has been investigated in female and male adults of the Mediterranean field cricket, Gryllus binaculatus (de Geer). The metabolism is similar in both sexes and at various stages of adult life. Several classes of apolar metabolites (A1–A5) represent the major compounds. The amount of polar conjugates is low in all tissues, as are the concentrations of 20-hydroxyecdysone. Ovaries are the only organs capable of storing considerable amounts of ecdysteroids. The amount of radiolabelled ecdysteroid activity (mostly [³H]ecdysone) excreted during the first 24 hr after injection is high.The chemical identity of the apolar metabolites is not yet known. A2, which is the major apolar compound, has recently been identified as a complex of ecdysone conjugates with abundant long-chain fatty acids (Hoffman et al., 1985 Life Sci.37, 185–192). Incubations with tissue homogenates in vitro have shown that several organs are capable of converting ecdysone into apolar compounds. Apolar ecdysteroid acyl esters represent a newly identified class of ecdysone conjugates from insects. Their role in regulation of free ecdysteroid titres during the reproductive period in female crickets is discussed.     

Mechanism of hydroxylation at C-2 during the biosynthesis of ecdysone in ovaries of the locust, Schistocerca gregaria.

The stereochemistry of hydroxylation at C-2 during the biosynthesis of ecdysone in the ovaries of Schistocerca gregaria was investigated by incorporation of [1 alpha,2 alpha-3H(n)]cholesterol in admixture with [4-14C]cholesterol into oöcyte 2-deoxyecdysone and ecdysone conjugates in maturing adult female S. gregaria. Extraction of the eggs followed by enzymic hydrolysis of the ecdysteroid conjugate fraction yielded free ecdysteroids, from which 2-deoxyecdysone and ecdysone were purified. The 3H/14C ratios in the 2-deoxyecdysone and ecdysone were similar, suggesting that the 2 alpha hydrogen of cholesterol was retained during hydroxylation at C-2. This was corroborated by oxidation at C-2 of the 3,22-diacetate derivative of the ecdysone, yielding the corresponding 2-oxo compound with removal of essentially all the 3H originally present at the 2 alpha position of cholesterol. The results indicate that the 2 beta hydrogen of cholesterol has been eliminated during the hydroxylation at C-2. Thus, during ecdysone biosynthesis, hydroxylation at C-2 is direct and occurs with retention of configuration.     

HYDROGEN PEROXIDE AND ECDYSONE IN THE CRYOPROTECTIVE DEHYDRATION STRATEGY OF Megaphorura Arctica (ONYCHIURIDAE: COLLEMBOLA)

The Arctic springtail, Megaphorura arctica, survives sub‐zero temperatures in a dehydrated state via trehalose‐dependent cryoprotective dehydration. Regulation of trehalose biosynthesis is complex; based in part on studies in yeast and fungi, its connection with oxidative stress caused by exposure of cells to oxidants, such as hydrogen peroxide (H₂O₂), or dehydration, is well documented. In this respect, we measured the amount of H₂O₂ and antioxidant enzyme activities (superoxide dismutases: copper, zinc—CuZnSOD and manganese containing–MnSOD, and catalase—CAT), as the regulatory components determining H₂O₂ concentrations, in Arctic springtails incubated at 5 °C (control) versus ?2 °C (threshold temperature for trehalose biosynthesis). Because ecdysone also stimulates trehalose production in insects and regulates the expression of genes involved in redox homeostasis and antioxidant protection in Drosophila, we measured the levels of the active physiological form of ecdysone—20‐hydroxyecdysone (20‐HE). Significantly elevated H₂O₂ and 20‐HE levels were observed in M. arctica incubated at ?2 °C, supporting a link between ecdysone, H₂O₂, and trehalose levels during cryoprotective dehydration. CAT activity was found to be significantly lower in M. arctica incubated at ?2 °C versus 5 °C, suggesting reduced H₂O₂ breakdown. Furthermore, measurement of the free radical composition in Arctic springtails incubated at 5 °C (controls) versus ?2 °C by Electron Paramagnetic Resonance spectroscopy revealed melanin‐derived free radicals at ?2 °C, perhaps an additional source of H₂O₂. Our results suggest that H₂O₂ and ecdysone play important roles in the cryoprotective dehydration process in M. arctica, linked with the regulation of trehalose biosynthesis.     

Biosynthesis and distribution of ecdysone and 20-OH-ecdysone in Aedes aegypti

The distribution and biosynthesis of ecdysone and 20-hydroxyecdysone (20-OH-ecdysone) was followed in sugar- and blood-fed female Aedes aegypti. In both sugar- and early blood-fed animals most of the ecdysteroid determined by radioimmunoassay was found outside the ovary. Twenty-four to 40 h after blood feeding, however, ecdysteroid was distributed between ovary and carcass in the ratio of 1:1.5. Ecdysteroid titer reached a plateau between 18 to 40 h after the blood meal and decreased thereafter. Analysis of the ecdysteroid titer using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) revealed that both 20-OH-ecdysone and ecdysone were synthesized after the blood meal. The ratio of 20-OH-ecdysone to ecdysone remained essentially constant and fluctuated in parallel throughout egg development. Chromatography of the early ecdysteroid peak (8 h after feeding) using TLC and HPLC indicated that although it cross-reacted with ecdysteroid antibodies, it did not have the same elution times as ecdysone and 20-OH-ecdysone and is, therefore, probably a precursor of these ecdysteroids. Injections of egg development neurosecretory hormone (EDNH) preparation purified to near homogeneity, into ligated abdomens, induced ecdysteroid synthesis only if the abdomens were first treated with methoprene (12.5 pg). Methoprene at this concentration did not stimulate ecdysteroid synthesis in these abdomens. When blood-fed females were treated with [4-¹⁴C] cholesterol and analyzed using TLC and HPLC procedures, both [¹⁴C]labeled ecdysone and [¹⁴C]labeled 20-OH-ecdysone were synthesized in the ratio of 1:1.5. This report is the first to show that both ecdysone and 20-OH-ecdysone are synthesized in vivo in female A. aegypti.