Juvenile Hormone Signaling Pathway Research Articles

Developmental Link between Sex and Nutrition; doublesex Regulates Sex-Specific Mandible Growth via Juvenile Hormone Signaling in Stag Beetles

Sexual dimorphisms in trait expression are widespread among animals and are especially pronounced in ornaments and weapons of sexual selection, which can attain exaggerated sizes. Expression of exaggerated traits is usually male-specific and nutrition sensitive. Consequently, the developmental mechanisms generating sexually dimorphic growth and nutrition-dependent phenotypic plasticity are each likely to regulate the expression of extreme structures. Yet we know little about how either of these mechanisms work, much less how they might interact with each other. We investigated the developmental mechanisms of sex-specific mandible growth in the stag beetle Cyclommatus metallifer, focusing on doublesex gene function and its interaction with juvenile hormone (JH) signaling. doublesex genes encode transcription factors that orchestrate male and female specific trait development, and JH acts as a mediator between nutrition and mandible growth. We found that the Cmdsx gene regulates sex differentiation in the stag beetle. Knockdown of Cmdsx by RNA-interference in both males and females produced intersex phenotypes, indicating a role for Cmdsx in sex-specific trait growth. By combining knockdown of Cmdsx with JH treatment, we showed that female-specific splice variants of Cmdsx contribute to the insensitivity of female mandibles to JH: knockdown of Cmdsx reversed this pattern, so that mandibles in knockdown females were stimulated to grow by JH treatment. In contrast, mandibles in knockdown males retained some sensitivity to JH, though mandibles in these individuals did not attain the full sizes of wild type males. We suggest that moderate JH sensitivity of mandibular cells may be the default developmental state for both sexes, with sex-specific Dsx protein decreasing sensitivity in females, and increasing it in males. This study is the first to demonstrate a causal link between the sex determination and JH signaling pathways, which clearly interact to determine the developmental fates and final sizes of nutrition-dependent secondary-sexual characters.

保幼激素在昆虫中的分子作用机理

PDF HTML阅读 XML下载 导出引用 引用提醒 保幼激素在昆虫中的分子作用机理 DOI: 10.5846/stxb201306061380 作者: 作者单位: 中国计量学院生命科学学院,中国计量学院生命科学学院 作者简介: 通讯作者: 中图分类号: 基金项目: 国家重点基础研究发展计划（2010CB126200） Molecular mechanisms of the insect juvenile hormone Author: Affiliation: College of Life Sciences,China Jiliang University,Provincial Key Laboratory of Biometrology and Inspection Quarantine,College of Life Sciences,China Jiliang University,Provincial Key Laboratory of Biometrology and Inspection Quarantine Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:随着分子生物学技术的快速发展，对生态环境中各类生物的研究，包括对生物某些特定基因结构和功能的研究等逐步拓展和加深。保幼激素（Juvenile Hormone，JH）是由咽侧体（Corpus Allatum，CA）分泌的，在昆虫发育、变态和生殖过程中起重要作用的激素。目前对JH信号传导途径的作用机理还不十分清楚。现有研究表明，Kruppel homolog-1（Kr-h1）是一种含C2H2锌指结构的转录因子，处于保幼激素信号途径下游，在保幼激素信号通路中起着重要作用。已报道的Kr-h1基因的功能主要包括：调控幼虫生长发育和变态，与蜜蜂的觅食行为密切相关，参与果蝇幼体神经细胞的形成等等。对就近十年来Kr-h1基因的特性和功能研究作一个综述以了解不同昆虫中保幼激素的分子作用机制，为开发生物农药奠定理论基础，也为维护良好的生态环境作出理论贡献。 Abstract:Following the rapid development of molecular biology techniques, many studies have been carried out of specific gene structures and functions, including those in ecological environments. Juvenile hormone (JH) is an important insect hormone that regulates insect larval development, reproduction, and metamorphosis, although the molecular mechanism behind this remains unclear. The transcription factor Kruppel-homolog 1(Kr-h1) acts downstream of the JH receptor and plays a key role in the JH signaling pathway. It has been identified in over 21 insect species, and sequence analysis has shown it to contain 2-8 C2H2-type zinc-finger motifs, which are highly conserved among Kr-h1 proteins. We previously found that zinc-fingers Zn2-Zn7 are more conserved than others in all insect species we compared. Interestingly, we found that the jewel wasp Nasonia vitripennis only contains two zinc-fingers, Zn4 and Zn7, while the body louse Pediculus humanus corporis contains all zinc-fingers except Zn7. As N. vitripennis and P. humanus were the only two parasitoids among the 21 insect species examined, it is possible that zinc fingers might be lost during evolution. Studies in Drosophila melanogester, Bombyx mori, Tribolium castaneum, and Frankliniella occidentalis revealed that Kr-h1 is required for embryonic development and metamorphosis, and that its down-regulation results in development of the precocious phenotype. Knockdown of Kr-h1 by RNAi in the brown planthopper Nilaparvata lugens showed that this gene is required for normal wing and external genitalia development. Kr-h1 is also involved in neuron development, the regulation of photoreceptor maturation, larval growth, insect development and metamorphosis, and the formation of Drosophila melanogaster neurons. In addition, it controls the foraging behavior of honey bees through affecting the expression of the cGMP-mediated activation of protein kinase G. The expression of Kr-h1 is induced by JH and its mimics. The Bombyx mori juvenile hormone response element (bmJHRE), located 141 bp upstream of Kr-h1, includes an E-box with the sequence CACGTG. bmJHRE was found in the genomic sequence of all seven insect species that we previously analyzed, and the E-box was highly conserved in all species (100%). The E-boxes of Tribolium castaneum Kr-h1(TcKr-h1) and Apis mellifera Kr-h1(AmKr-h1) are close to the Kr-h1 promoter, while those of Bombyx mori Kr-h1(BmKr-h1), Acyrthosiphon pisum Kr-h1(ApKr-h1), Drosophila melanogester Kr-h1(DmKr-h1), Nasonia vitripennis Kr-h1(NvKr-h1), and Nilaparvata lugens Kr-h1(NlKr-h1) are not. JH also interacts with the hormone ecdysone (Ecd), and research from D. melanogaster, B. mori, and T. castaneum suggests that Kr-h1 is a key factor in the JH-Ecd interaction. JH mimics methoprene treatment, which increases the expression of Kr-h1 while reducing that of Broad (B), a transcription factor located downstream of Ecd and its receptor that plays an important role in the ecdysone signaling pathway. This indicates that Br expression might be inhibited by Kr-h1. Although much progress has been made in understanding insect hormone signaling pathways, several key questions still remain. For example, what is the role of Kr-h1 in linking the JH and Ecd signaling pathways? How many other genes form a close connection in the JH signaling cascade? This review highlights the characteristics and functions of Kr-h1 that have been elucidated during the past decade. It also summarizes our understanding of the molecular mechanism of JH, establishes a theoretical basis for the development of biological pesticide, and contributes theoretically to the maintenance of a good ecological environment. 参考文献 相似文献 引证文献

Juvenile hormone signaling during reproduction and development of the linden bug, Pyrrhocoris apterus

Juvenile hormone (JH), a sesquiterpenoid produced by the insect corpus allatum gland (CA), prevents metamorphosis in larvae and stimulates vitellogenesis in adult females. Whether the same JH signaling pathway regulates both processes is presently unknown. Here, we employ the robust JH response during reproduction and development of the linden bug, Pyrrhocoris apterus, to compare the function of key JH-signaling genes encoding the JH receptor, Methoprene-tolerant (Met), its binding partner Taiman (Tai), and a JH-inducible protein, Krüppel-homolog 1 (Kr-h1). RNA interference (RNAi) with Met or Tai, but not Kr-h1, blocked ovarian development and suppressed vitellogenin gene expression in the fat body of females raised under reproduction-inducing conditions. Loss of Met and Tai matched the effects of CA ablation or the natural absence of JH during reproductive diapause. Stimulation of vitellogenesis by treatment of diapausing females with a JH mimic methoprene also required both Met and Tai in the fat body, whereas Kr-h1 RNAi had no effect. Therefore, the Met-Tai complex likely functions as a JH receptor during vitellogenesis. In contrast to Met and Kr-h1 that are both required for JH to prevent precocious metamorphosis in P. apterus larvae, removal of Tai disrupted larval ecdysis without causing premature adult development. Our results show that while Met operates during metamorphosis in larvae and reproduction in adult females, its partner Tai is only required for the latter. The diverse functions of JH thus likely rely on a common receptor whose actions are modulated by distinct components.

The hormone-dependent function of Hsp90 in the crosstalk between 20-hydroxyecdysone and juvenile hormone signaling pathways in insects is determined by differential phosphorylation and protein interactions

BackgroundHeat shock protein 90 (Hsp90) interacts with steroid hormone receptors, signaling kinases, and various transcription factors. However, the mechanism by which Hsp90 interacts with different proteins in various pathways remains unclear. MethodsWestern blot was used to study Hsp90 expression profile in Helicoverpa armigera (Lepidoptera). RNA interference was performed to investigate the function of Hsp90 in 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal pathways. The binding of Hsp90 to the transcription factor ultraspiracle protein (USP1) and JH candidate receptor methoprene-tolerant (Met1) was analyzed by co-immunoprecipitation. Phospho-(Ser) PKC substrate antibody was used to detect Hsp90 phosphorylation. ResultsHsp90 participated in 20E- or JH-induced gene expression. 20E induced the interaction between Hsp90 and USP1, whereas JH III and methoprene induced the interaction between Hsp90 and Met1, respectively. 20E and JH counteracted each other for these protein interactions. Both JH III and methoprene induced protein kinase C (PKC) phosphorylation of Hsp90. This process could be inhibited by phospholipase C (PLC) and PKC inhibitors. 20E suppressed JH III- or methoprene-induced PKC phosphorylation of Hsp90. Conclusion20E maintained the non-PKC-phosphorylation status of Hsp90. Hsp90 interacted with USP1 to induce gene expression in the 20E pathway. JH regulated the PKC-phosphorylation status of Hsp90. Hsp90 also interacted with Met1 to induce gene expression in the JH pathway. General significanceOur study describes a novel mechanism of Hsp90 action by altering phosphorylation and protein interaction in various hormonal signaling pathways.

Transgenic Drosophila melanogaster Carrying RNAi Against gce Gene Proved the Involvement of gce in Juvenile Hormone Action

Juvenile hormone (JH) is important for multiple aspects of insect development and physiology. Although roles of JH have received considerable studies, JH signaling pathway at the molecular level is still not well understood. Methoprenetolerant (Met) in Drosophila melanogaster fulfills many of the requirements as a hormone receptor gene. A paralogous gene, germ-cell expressed (gce), possesses homology to Met and is a candidate as a Met partner in mediating JH action. To probe roles of this gene in JH action, we carried out in vivo gce underexpression studies using RNAi technique. Precocious expression of broad (br) gene was found to occur when gce was knocked down in the young larvae. We also demonstrated that RNA interference-driven knockdown of gce expression in transgenic flies results in appearance of resistance to pyriproxyfen (IGRs). These results show that gce is a vital gene and appears to promote JH action in larval stages.

The Juvenile Hormone Signaling Pathway in Insect Development

The molecular action of juvenile hormone (JH), a regulator of vital importance to insects, was until recently regarded as a mystery. The past few years have seen an explosion of studies of JH signaling, sparked by a finding that a JH-resistance gene, Methoprene-tolerant (Met), plays a critical role in insect metamorphosis. Here, we summarize the recently acquired knowledge on the capacity of Met to bind JH, which has been mapped to a particular ligand-binding domain, thus establishing this bHLH-PAS protein as a novel type of an intracellular hormone receptor. Next, we consider the significance of JH-dependent interactions of Met with other transcription factors and signaling pathways. We examine the regulation and biological roles of genes acting downstream of JH and Met in insect metamorphosis. Finally, we discuss the current gaps in our understanding of JH action and outline directions for future research.

How does juvenile hormone control insect metamorphosis and reproduction?

In insects juvenile hormone (JH) regulates both metamorphosis and reproduction. This lecture focuses on our current understanding of JH action at the molecular level in both of these processes based primarily on studies in the tobacco hornworm Manduca sexta, the flour beetle Tribolium castaneum, the mosquito Aedes aegypti, and the fruit fly Drosophila melanogaster. The roles of the JH receptor complex and the transcription factors that it regulates during larval molting and metamorphosis are summarized. Also highlighted are the intriguing interactions of the JH and insulin signaling pathways in both imaginal disc development and vitellogenesis. Critical actions of JH and its receptor in the timing of maturation of the adult optic lobe and of female receptivity in Drosophila are also discussed.

Common and Distinct Roles of Juvenile Hormone Signaling Genes in Metamorphosis of Holometabolous and Hemimetabolous Insects

Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis.

Juvenile Hormone Regulates Vitellogenin Gene Expression through Insulin-like Peptide Signaling Pathway in the Red Flour Beetle, Tribolium castaneum

Our recent studies identified juvenile hormone (JH) and nutrition as the two key signals that regulate vitellogenin (Vg) gene expression in the red flour beetle, Tribolium castaneum. Juvenile hormone regulation of Vg synthesis has been known for a long time in several insects, but the mechanism of JH action is not known. Experiments were conducted to determine the mechanism of action of these two signals in regulation of Vg gene expression. Injection of bovine insulin or FOXO double-stranded RNA into the previtellogenic, starved, or JH-deficient female adults increased Vg mRNA and protein levels, thereby implicating the pivotal role for insulin-like peptide signaling in the regulation of Vg gene expression and possible cross-talk between JH and insulin-like peptide signaling pathways. Reduction in JH synthesis or its action by RNAi-mediated silencing of genes coding for acid methyltransferase or methoprene-tolerant decreased expression of genes coding for insulin-like peptides (ILPs) and influenced FOXO subcellular localization, resulting in the down-regulation of Vg gene expression. Furthermore, JH application to previtellogenic female beetles induced the expression of genes coding for ILP2 and ILP3, and induced Vg gene expression. FOXO protein expressed in baculovirus system binds to FOXO response element present in the Vg gene promoter. These data suggest that JH functions through insulin-like peptide signaling pathway to regulate Vg gene expression.

Wnt Signaling Cross-Talks with JH Signaling by Suppressing Met and gce Expression

Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, we designed a novel Drosophila genetic screen to isolate mutations that derepress JH-mediated br suppression at early larval stages. We found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous JHA. A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression was suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, we conclude that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages.

The Participation of Calponin in the Cross Talk between 20-Hydroxyecdysone and Juvenile Hormone Signaling Pathways by Phosphorylation Variation

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to mediate insect development, but the mechanism of this interaction is poorly understood. Here, a calponin homologue domain (Chd) containing protein (HaCal) is reported to play a key role in the cross talk between 20E and JH signaling by varying its phosphorylation. Chd is known as an actin binding domain present in many proteins including some signaling proteins. Using an epidermal cell line (HaEpi), HaCal was found to be up-regulated by either 20E or the JH analog methoprene (JHA). 20E induced rapid phosphorylation of HaCal whereas no phosphorylation occurred with JHA. HaCal could be quickly translocated into the nuclei through 20E or JH signaling but interacted with USP1 only under the mediation of JHA. Knockdown of HaCal by RNAi blocked the 20E inducibility of USP1, PKC and HR3, and also blocked the JHA inducibility of USP1, PKC and JHi. After gene silencing of HaCal by ingestion of dsHaCal expressed by Escherichia coli, the larval development was arrested and the gene expression of USP1, PKC, HR3 and JHi were blocked. These composite data suggest that HaCal plays roles in hormonal signaling by quickly transferring into nucleus to function as a phosphorylated form in the 20E pathway and as a non-phosphorylated form interacting with USP1 in the JH pathway to facilitate 20E or JH signaling cascade, in short, by switching its phosphorylation status to regulate insect development.

The juvenile hormone analogue methoprene up-regulates the Ha-RNA-binding protein

RNA-binding proteins (RBPs) associate with RNA in cells to form ribonucleoprotein (RNP) complexes. The RBPs are involved in various aspects of RNA metabolism, but their roles in the juvenile hormone (JH) signaling pathway are not well known. An RNA-binding protein ( Ha-RBP) was obtained from Helicoverpa armigera, a lepidopteran insect, which could be up-regulated by the juvenile hormone analogue methoprene at the mRNA level. Immunohistochemistry showed that Ha-RBP was mainly distributed in cells where protein synthesis was active, and its knockdown decreased the protein levels of JH-responsive genes. Immunocytochemistry showed that Ha-RBP was located in both the nucleus and the cytoplasm of normal cells, and methoprene could promote the translocation of Ha-RBP from the nucleus to the cytoplasm. This process was mediated by the JH receptor candidate HaMet but not by the ultraspiracle protein (USP). The knockdown of HaMet by RNAi decreased the expression of Ha-RBP and blocked its translocation from the nucleus to the cytoplasm. Together these findings indicate that Ha-RBP is involved in the juvenile hormone signaling pathway and Met mediates JH signaling by regulating Ha-RBP translocation from nucleus to cytoplasm, which may allow Ha-RBP to modify protein translation.

Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression

Juvenile hormone (JH) plays crucial roles in many aspects of insect life. The Methoprene-tolerant (Met) gene product, a member of the bHLH-PAS family of transcriptional regulators, has been demonstrated to be a key component of the JH signaling pathway. However, the molecular function of Met in JH-induced signal transduction and gene regulation remains to be fully elucidated. Here we show that a transcriptional coactivator of the ecdysteroid receptor complex, FISC, acts as a functional partner of Met in mediating JH-induced gene expression. Met and FISC appear to use their PAS domains to form a dimer only in the presence of JH or JH analogs. In newly emerged adult female mosquitoes, expression of some JH responsive genes is considerably dampened when Met or FISC is depleted by RNAi. Met and FISC are found to be associated with the promoter of the early trypsin gene (AaET) when transcription of this gene is activated by JH. A juvenile hormone response element (JHRE) has been identified in the AaET upstream regulatory region and is bound in vitro by the Met-FISC complex present in the nuclear protein extracts of previtellogenic adult female mosquitoes. In addition, the Drosophila homologs of Met and FISC can also use this mosquito JHRE to activate gene transcription in response to JH in a cell transfection assay. Together, the evidence indicates that Met and FISC form a functional complex on the JHRE in the presence of JH and directly activate transcription of JH target genes.

DrosophilaCG10527 mutants are resistant to juvenile hormone and its analog methoprene

Juvenile hormone (JH) is critical for development, metamorphosis, and reproduction in insects. While the physiological importance of JH has been appreciated for decades, its biosynthetic pathway and molecular action remain poorly understood. DrosophilaCG10527 encodes a protein with high homology to crustacean farnesoic acid methyltransferase (FAMeT) that converts farnesoic acid to methyl farnesoate (MF), a precursor of JH, but its in vivo functions remain unclear. Here we report that CG10527 is expressed widely in secondary cells in the male accessory glands, in ovarian follicle cells, and in glial cells in the nervous system. Furthermore, CG10527 is expressed abundantly in the corpora allata where JH is synthesized. To understand the physiological functions of CG10527, we generated specific CG10527 deletions. Phenotypic analysis showed that CG10527 null mutants are fully viable and fertile in both sexes, indicating that CG10527 is not essential for survival and fertility. Surprisingly, CG10527 mutants showed no defects in the biosynthesis of MF and JH. However, CG10527 mutants were 3–5 times more resistant than wild-type flies to topically applied MF and JH as well as the JH analog methoprene at both sub-lethal and lethal doses. Taken together, our data indicate that DrosophilaCG10527 plays little, if any, role in JH biosynthesis but may participate in the JH signaling pathway.

Ecdysteroid regulation of ovarian growth and oocyte maturation in the red flour beetle, Tribolium castaneum

Previous studies from our laboratory showed the involvement of juvenile hormone (JH) and ecdysteroid signaling in the regulation of female reproduction in the red flour beetle, Tribolium castaneum. JH regulates vitellogenin (Vg) synthesis in the fat body but the role of ecdysteroid signaling is not known. Here, we report on ecdysteroid regulation of ovarian growth and oocyte maturation. Microarray analysis of RNA isolated from ovaries showed the up-regulation of several genes coding for proteins involved in ecdysteroid signaling on the 4th day after female adult eclosion. The functional analyses of genes coding for proteins involved in ecdysteroid and JH signaling pathways by RNA interference (RNAi) revealed that ecdysteroids but not JH regulate ovarian growth and primary oocyte maturation. Ultrastructural studies showed the temporal sequences of key events in oogenesis including the development of primary oocytes, the differentiation and development of follicle epithelial cells, and the formation of intercellular spaces to facilitate uptake of Vg protein. RNAi studies showed that ecdysone receptor (EcR) and ultraspiracle (USP) are required for the ovarian growth, primary oocyte maturation and the growth and migration of the follicle cells. These studies suggest important roles for ecdysteroids in the regulation of oocyte maturation in the beetle ovaries.

Identification of juvenile hormone target genes in the adult female mosquitoes

Many aspects of reproductive maturation in newly emerged adult female mosquitoes are controlled by juvenile hormone (JH). However, the molecular basis of this hormonal regulation remains poorly understood. In this study, we analyzed transcriptome changes in young female adults after topical application of exogenous JH. mRNA levels of 16 and 72 genes were enhanced at 3 h and 12 h after hormone administration, respectively. The observed upregulations can be also induced by the JH analogues, methoprene and pyriproxyfen, but not by farnesol, indicating that these responses are JH-specific. Among the genes activated by JH is the mosquito Krüppel homolog 1 (Kr-h1) gene. Studies in the beetle Tribolium castaneum have indicated that Kr-h1 is transcriptionally activated by JH during metamorphosis and encodes a gene expression regulator acting downstream of the methoprene-tolerant ( Met) gene in the JH signaling pathway. We found that the upregulation of AaKr-h1 after eclosion relied on the function of the mosquito Met protein, suggesting that a conserved JH signaling pathway is utilized both in the metamorphosis of T. castaneum and in the adult reproductive maturation of the Aedes aegypti mosquito. This study lays a foundation for a better understanding of the mechanisms of juvenile hormone action.

Rosy Function Is Required for Juvenile Hormone Effects in Drosophila melanogaster

Application of a high dose of juvenile hormone (JH) III or its mimics (JHM) to Drosophila at the white puparium stage causes the formation of a pupal-like abdomen with few or no short bristles. We report here that the rosy (ry) gene encoding the enzyme xanthine dehydrogenase (XDH), which catalyzes the final two-step oxidation in purine catabolism, is required for this effect of JH on the epidermis. In ry506 (null allele) homozygotes or hemizygotes, JH III or pyriproxifen (a JHM) had little effect on abdominal bristle or cuticle formation, but disrupted the development of the central nervous system as in wild-type flies. Wild-type ry rescued the JH sensitivity of the abdominal epidermis in ry506 mutants. Inhibition of XDH activity phenocopied the ry null mutant's insensitivity to JH. Larvae fed on hypoxanthine or xanthine showed a decreased JH sensitivity. ry506 clones were sensitive to JH, indicating that ry is required non-cell autonomously for the JH effects. Normally JH applied at pupariation causes the aberrant reexpression of the transcription factor broad in the abdominal epidermis during adult development, but in the ry506 mutant most of the cells in the dorsal tergite showed no broad reexpression, indicating that ry is upstream of broad in the JH signaling pathway.

The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, major plasma proteins referred to as 30K proteins are the most abundant proteins in the hemolymph of final (fifth) instar larvae. Surgical extirpation of corpora allata, the source of a juvenile hormone (JH), causes rapid accumulation of 30K proteins in the hemolymph of fourth instar larvae. The 30K protein 6G1 (30K6G1) gene was repressed in primary cultured fat body cells treated with a JH analog (JHA), methoprene. To identify the JH response element present in the promoter region of the 30K6G1 gene, we performed transfection analyses of the 5′-deletion mutants of the 30K6G1 gene using primary cultured fat body cells, gel retardation assays and in vivo footprinting analysis. The results from those analyses revealed that a JH response element exists in the sequence between positions −147 and −140. When the promoter construct mutated at positions −143, −142, and −141 was transfected to fat body primary cultured cells, the suppression effect on the reporter gene expression caused by JHA was reduced. Gel retardation assay using specific antibody revealed that a PBX protein binds to the JH response element. Northern blot analysis revealed that the gene expression of Bombyx PBX is enhanced in the fat body cells by JHA treatment. These results indicate that PBX proteins are involved in the JH signaling pathway and play an important role in suppressing 30K protein gene expression in the fat body of B. mori.

Juvenile hormone regulation of the E75 nuclear receptor is conserved in Diptera and Lepidoptera

Despite longstanding efforts, the juvenile hormone (JH) signaling pathway remains unknown. In Drosophila melanogaster (Diptera), JH activates expression of the E75A nuclear receptor. The E75 gene encodes a family of related proteins. A homologue of Drosophila E75 was previously identified and two isoforms, mE75A and mE75B, were reported in Manduca sexta (Lepidoptera). Here, we describe the identification of two additional isoforms, mE75C and mE75D, and the hormonal regulation of mE75 gene expression in Manduca CH1 cultured cells. mE75A and mE75B isoforms are specifically induced by ecdysone in CH1 cells. One isoform, mE75C, shows constitutive expression. The mE75D isoform exhibits dual hormonal regulation; it can be activated by either ecdysone or the JH analog, methoprene. E75-encoded proteins represent the first example of transcription factors directly induced by JH. E75 activation by JH, in both Diptera and Lepidoptera, suggests a conserved function in the JH signaling pathway.

Hormonal regulation and functional role of Drosophila E75A orphan nuclear receptor in the juvenile hormone signaling pathway

Ecdysone and juvenile hormone (JH) are important regulators of insect growth and development. While ecdysone initiates a transition from one developmental stage to another, JH determines the nature of the transition. How these two hormones interact at the molecular level is not known. Here we report the JH inducibility of the E75A nuclear receptor encoded by the E75 early ecdysone-inducible gene. In Drosophila S2 cells, E75A transcription is specifically activated by JH at concentrations well within the physiological range found in larvae and adults. The induction is rapid and does not require a concurrent protein synthesis, and thus represents a primary hormone response. Consistent with JH regulation, E75A mRNA levels are reduced in ovaries of apterous 4 mutant adults defective in JH secretion. Expression is rescued by topical methoprene application. We further provide evidence that ectopic E75A is sufficient to perform several functions in the JH signaling pathway. First, it can down-regulate its own transcription. Second, E75A can potentiate the JH inducibility of a secondary response gene, JhI-21. Finally, in the presence of JH, E75A can repress ecdysone activation of early genes including Broad-Complex. Based on these data, we propose a model for the role of E75A in the ecdysone-JH regulatory interplay.