InteractiveFly: GeneBrief

Methoprene-tolerant & germ cell-expressed bHLH-PAS Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The Methoprene-tolerant (Met) bHLH-PAS gene also termed Resistance to Juvenile Hormone (Rst(1)JH) -- is involved in juvenile hormone (JH) action in Drosophila as a likely component of a JH receptor. Met was expressed in Drosophila S2 cells, and MET partners were sought using pull-down assays. MET-MET interaction was found to occur. The germ-cell expressed (gce) gene is another Drosophila bHLH-PAS gene with high homology to Met; GCE forms heterodimers with MET. In the presence of JH or either of two JH agonists, MET-MET and MET-GCE formation is drastically reduced. Interaction between GCE and MET having N- or C-terminus truncations, bHLH or PAS-A domain deletions, or a point mutation in the PAS-B domain fail to occur. However, JH-dependent interaction occurs between GCE and MET having point mutations in bHLH or PAS-A. During development, changes in JH titer may alter partner binding by MET and result in different gene expression patterns (Godlewski, 2006).

Hormonal regulation of insect development involves the steroid 20-hydroxyecdysone (20-HE) and the sesquiterpenoid juvenile hormone (JH). The molecular biology and action of 20-HE are relatively well described. In contrast, neither the JH receptor nor the mechanism of action is well understood. JH is present during larval development of probably all insects; its role during this time period is best understood in lepidopteran and hemimetabolous insects, when it acts to maintain the developmental 'status quo' prior to metamorphosis, probably to allow proper larval molting and prevent premature metamorphosis. Its absence at the end of larval development is thought to permit 20-HE control of metamorphosis. JH can act at the molecular level to regulate gene expression (Godlewski, 2006).

One gene, Methoprene-tolerant (Met), is known to be directly involved in the manifestation of the JH effects in D. melanogaster. Met mutants show resistance to both the toxic and morphogenetic effects of JH and several JH analogs (Wilson, 1986; Riddiford, 1991; Restifo, 1998; Wilson, 1996). Ligand-binding studies have shown MET to bind JH III with specificity and nanomolar affinity (Shemshedini, 1990a; Miura, 2005), suggesting that MET is a component of a JH receptor. Met encodes a bHLH-PAS transcriptional regulator family member (Ashok, 1998), and MET can activate a reporter gene in transfected Drosophila S2 cells (Miura, 2005). Although Met is expressed throughout development (Ashok, 1998), roles for MET during embryonic and larval development are not readily apparent. The gene germ-cell expressed (gce) is another bHLH-PAS gene in D. melanogaster with high Met homology (70%-86% amino acid identity in the conserved domains) but unknown function (Godlewski, 2006).

bHLH-PAS proteins typically function as heterodimers with other bHLH-PAS proteins (Gu, 2000). Binding partners for MET were explored by co-expressing Met with either gce (heterodimer) or another Met construct (homodimer) and examining binding interaction using pull-down assays. Except in certain Met mutant constructs, MET-GCE interaction was apparent, and MET-MET interaction was also detected. However, in the presence of JH, both interactions were greatly diminished. These results suggest how JH might act during preadult development in insects (Godlewski, 2006).

The Met gene is clearly involved in the response of D. melanogaster to JH or JH agonist exposure during the sensitive period at the onset of metamorphosis. At this time of development, endogenous JH is absent, and secretion of 20-HE is initiating metamorphosis by activation of the primary-response genes. The presence of JH or JH analog (JHA) during this time disrupts metamorphosis probably by disrupting expression (Zhou, 2002) or function (Wilson, 2006) of one or more genes necessary for the completion of the adult form, resulting in the JH pathology seen in the pharate adult. Met mutants are resistant to the effects of JH (Wilson, 1986), and overexpression of Met⁺ increases sensitivity to exogenous hormone (TGW, unpublished), clearly implicating MET in the molecular action of JH/JHA. Since MET binds JH III at physiological concentration and can act as a transcriptional regulator (Miura, 2005), this protein possesses characteristics expected of a JH receptor component (Godlewski, 2006).

This work has demonstrated interaction between MET and GCE proteins co-expressed in Drosophila S2 cells. Heterodimer formation between very similar bHLH-PAS proteins is not unique: the vertebrate aryl hydrocarbon receptor functions as a heterodimer of two homologous proteins, AHR and ARNT (Gu, 2000). Since PAS proteins normally function as heterodimers, it was surprising to find MET homodimer formation. Again, MET is not unique in this property: ARNT homodimer formation has been shown (Godlewski, 2006).

JH or JHAs can block MET homodimer and heterodimer formation or stability. The effect is both time- and dose-dependent. Since MET has been shown to bind JH III, the expressed MET seems likely to be the JH binder, presumably resulting in disrupted partner protein binding. However, an effect of the added hormone on S2 cell physiology that results in disrupted binding cannot be ruled out (Godlewski, 2006).

The MET mutants that were evaluated in this study served two purposes. First, the failure of many of them to heterodimerize with GCE provided further evidence that the MET-GCE interaction is specific. Second, they provided information about the conserved domains in the MET protein. As expected, the severe N- and C-terminal deletions failed to heterodimerize. The mutations having small deletions in the bHLH and PAS-A domains also failed to heterodimerize. Other studies have demonstrated involvement of these domains in PAS protein heterodimer formation, corroborated here for MET and GCE. Met¹²⁸ (with a point mutation in PAS-B) showed poor dimer formation, but Met³ (having a point mutation in bHLH) and Met¹ (having a point mutation in PAS-A) heterodimerized. Both Met³ and Met¹ flies show strong methoprene resistance (Wilson, 1986), suggesting that the mutant phenotype results from a separate problem, such as faulty interaction with DNA, but not from faulty JH binding, since MET-GCE heterodimerization was blocked by methoprene in both of these mutants. Future studies examining methoprene-sensitive heterodimerization following site-directed mutagenesis may be valuable in identifying the JH-binding domain of MET (Godlewski, 2006).

These results have suggested how MET and GCE might function during insect development as JH receptor components. During larval development when the JH titer is high, MET either acts as a monomer or forms a dimer with an unknown protein (Godlewski, 2006).

Obviously, this dimer would be hormone insensitive, unlike MET-GCE. MET could then act as a transcriptional regulator for one or more genes involved in larval development, allowing JH to fulfill its 'status quo' role. At the end of larval development when the JH titer is depleted, MET could then homodimerize or dimerize with GCE (or both) to regulate either a set of pupal genes involved in metamorphosis or express a different pattern, having a different function, from the larval genes. If JH/JHA were artificially present during early metamorphosis, then a larval-state MET is produced, leading to either larval gene expression or failure of correct pupal gene expression, resulting in the pathology seen. Thus, in this scenario, JH presence or absence provides the signal for a change in gene expression through MET and probably other transcription factors and the resultant physiology in larvae or pupae (Godlewski, 2006).

Regulation of onset of female mating and sex pheromone production by juvenile hormone in Drosophila melanogaster

Juvenile hormone coordinates timing of female reproductive maturation in most insects. In Drosophila, JH plays roles in both mating and egg maturation. However, very little is known about the molecular pathways associated with mating. Behavioral analysis of females genetically lacking the corpora allata (CAX), the glands that produce JH, showed that they were courted less by males and mated later than control females. Application of the JH mimic, methoprene, to the allatectomized females just after eclosion rescued both the male courtship and the mating delay. Studies of the null mutants of the JH receptors, Methoprene tolerant (Met) and germ cell-expressed (gce), showed that lack of Met in Met²⁷ females delayed the onset of mating, whereas lack of Gce had little effect. The Met²⁷ females were shown to be more attractive but less behaviorally receptive to copulation attempts. The behavioral but not the attractiveness phenotype was rescued by the Met genomic transgene. Analysis of the female cuticular hydrocarbon profiles showed that corpora allata ablation caused a delay in production of the major female-specific sex pheromones (the 7,11-C27 and -C29 dienes) and a change in the cuticular hydrocarbon blend. In the Met²⁷ null mutant, by 48 h, the major C27 diene was greatly increased relative to wild type. In contrast, the gce^2.5k null mutant females were courted similarly to control females despite changes in certain cuticular hydrocarbons. These findings indicate that JH acts primarily via Met to modulate the timing of onset of female sex pheromone production and mating (Bilen, 2013).

This study has shown that JH plays a critical role in the normal timing of onset of female mating and sex pheromone production. Removal of JH through genetic ablation of the CA in the developing adult female delayed the onset of mating behaviors. This change was coupled to a decrease in male courtship, suggesting a decrease in female attractiveness. Drastic changes were found in the CHC profiles in the CAX females. Some of these changes are likely due to the temperature shift regime used for the genetic allatectomy. Treating CAX females with the JH mimic (JHM) methoprene both advanced the onset of mating and increased the attractiveness of the females, apparently by increasing the production of C27 monoenes and dienes. Therefore, JH dynamically regulates the synthesis of specific cuticular hydrocarbons (CHCs), particularly the major female sex pheromones, the 7,11-C27 and 7,11-C29 dienes. These findings are consistent with previous studies showing induction of precocious mating by CA implants and reduced mating in apterous mutants and reduced female-specific pheromones in flies overexpressing the JH esterase-binding protein DmP29, both having reduced JH levels (Bilen, 2013).

Interestingly, the CAX females slowly became attractive so that by 96 h, time to onset of copulation was similar to controls. At this time the C27 monoene and the 7,11-C29 diene were still significantly reduced, whereas the C27 dienes were significantly increased, suggesting that there was a change in the CHC blend. The C27 dienes at 96 h may act alone or together with other CHCs in the pheromone blend to increase female attractiveness and decrease time to copulation in the CAX females (Bilen, 2013).

How does JH regulate CHC synthesis? CHCs are synthesized from fatty acids via elongation, desaturation, reduction to aldehyde, and oxidative decarbonylation reactions in oenocytes in D. melanogaster. The developmental appearance of the CHCs in CAX females in this study clearly shows decreases in the long-chain n-alkanes (C23- C27), the C25 and C27 monoenes, and the C27 and C29 dienes with corresponding increases in the shorter-chain dienes. Similar changes in diene profiles coupled with an increased time to copulation was seen after reduction of Elongase-F in female oenocytes (Chertemps, 2007). Reduction of Desaturase 1 in the oenocytes caused the loss of both monoenes and dienes with a large increase in the n-alkanes, whereas reduction of Desaturase-F caused a loss of the female-specific dienes with a doubling of monoenes and some increase in n-alkanes (Chertemps, 2006; Wicker-Thomas, 2009). Both manipulations significantly increased time to mating. Thus, JH apparently influences biosynthetic enzymes important in the synthesis of the long-chain alkanes as well as Elongase-F and the desaturases. These regulatory effects of JH on pheromone synthesis are similar to its effects on aggregation pheromone synthesis in bark beetles. In these beetles, JH III regulates the transcription of many of the genes encoding the pheromone biosynthetic enzymes, especially the genes encoding geranyldiphosphate synthase/myrcene synthase (GPPS/MS), CYP9T2, and an oxidoreductase (Bilen, 2013).

Application of 0.64 pmoles of the JHM methoprene just after eclosion rescued mating of the CAX females at 24 h after eclosion to the level of about 31% as seen in the parental controls, and a 10- fold higher dose caused about 58% to mate. After treatment with the higher dose of methoprene, the 7- and 9-C27 monoenes significantly increased at 24 h, but the two female-specific 7,11 dienes were not higher until 48 h. The increased C27 monoenes which have been implicated as aphrodisiacs (Marcillac, 2004) possibly made the CAX females more attractive at 24 h. Whether there is also an effect of the exogenous JH on the maturation of the female nervous system so that she becomes receptive to male courtship earlier is not known and warrants further study. At least three hormones (JH, ecdysone, and pheromone biosynthesis- activating neuropeptide) have been shown to regulate sex pheromone biosynthesis in different insects. This study has demonstrated that JH regulates sex pheromone 7,11-diene production in D. melanogaster females. Interestingly, in another dipteran, the house fly Musca domestica, the primary female sex pheromone is Z-9-tricosene. Females ovariectomized immediately after eclosion do not synthesize this compound, but synthesis is restored by either ovarian implants or multiple injections of 20-hydroxyecdysone. These two families of flies are evolutionarily distant, but the basis for this difference in hormonal regulation is unknown (Bilen, 2013).

The duplication of the JH receptor Gce occurred in the higher Diptera, and the two have partially redundant functions in the larval fat body of D. melanogaster and at metamorphosis (Abdou, 2011). Met plays a distinct role in adult optic lobe development during metamorphosis, whereas Gce has no effect. Met is also the predominant receptor required for the effects of JH on ovarian maturation, both the normal timing and normal fecundity. This behavioral analysis of female mating and attractiveness of Met and gce mutants indicates that JH is also acting primarily via Met in its regulation of mating and pheromone production. Surprisingly, in courtship assays using CS males, Met²⁷ females lacking Met were more attractive than the wildtype CS females, whereas the CAX females were less attractive, likely due to increased C27 dienes in Met²⁷ females and decreased 7,11-C27 and -C29 dienes in the CAX females. These findings suggest that JH acts mainly via Met in mating and pheromone synthesis similarly to the roles of Met and Gce in ovarian maturation where lack of Met delays maturation and reduces fecundity, whereas lack of Gce has relatively little effect (Bilen, 2013).

This study has demonstrated that JH acting through its receptor Met plays important roles in the initiation of sex pheromone production and the maturation of female mating behavior in Drosophila. Further investigation into these two aspects of JH action -- in the peripheral tissues involved in sex pheromone production and in the neuronal circuitry underlying the mating behavior -- is necessary to elucidate the details of its critical roles in modulation of the onset of mating. An understanding at the molecular level of this coordination in this Drosophila model should shed insights into how hormones regulate pheromone production and reproductive behavior in the vertebrates (Bilen, 2013).

Genetic evidence for function of the bHLH-PAS Protein Gce/Met as a juvenile hormone receptor

Juvenile hormones (JHs) play a major role in controlling development and reproduction in insects and other arthropods. Synthetic JH-mimicking compounds such as methoprene are employed as potent insecticides against significant agricultural, household and disease vector pests. However, a receptor mediating effects of JH and its insecticidal mimics has long been the subject of controversy. The bHLH-PAS protein Methoprene-tolerant (Met), along with its Drosophila melanogaster paralog Germ cell-expressed (Gce), has emerged as a prime JH receptor candidate, but critical evidence that this protein must bind JH to fulfill its role in normal insect development has been missing. This study shows that Gce binds a native D. melanogaster JH, its precursor methyl farnesoate, and some synthetic JH mimics. Conditional on this ligand binding, Gce mediates JH-dependent gene expression and the hormone's vital role during development of the fly. Any one of three different single amino acid mutations in the ligand-binding pocket that prevent binding of JH to the protein block these functions. Only transgenic Gce capable of binding JH can restore sensitivity to JH mimics in D. melanogaster Met-null mutants and rescue viability in flies lacking both Gce and Met that would otherwise die at pupation. Similarly, the absence of Gce and Met can be compensated by expression of wild-type but not mutated transgenic D. melanogaster Met protein. This genetic evidence definitively establishes Gce/Met in a JH receptor role, thus resolving a long-standing question in arthropod biology (Jindra, 2015).

Hormonal modulation of pheromone detection enhances male courtship success

During the lifespans of most animals, reproductive maturity and mating activity are highly coordinated. In Drosophila melanogaster, for instance, male fertility increases with age, and older males are known to have a copulation advantage over young ones. The molecular and neural basis of this age-related disparity in mating behavior is unknown. This study shows that the Or47b odorant receptor is required for the copulation advantage of older males. Notably, the sensitivity of Or47b neurons to a stimulatory pheromone, palmitoleic acid, is low in young males but high in older ones, which accounts for older males' higher courtship intensity. Mechanistically, this age-related sensitization of Or47b neurons requires a reproductive hormone, juvenile hormone, as well as its binding protein Methoprene-tolerant in Or47b neurons. Together, this study identifies a direct neural substrate for juvenile hormone that permits coordination of courtship activity with reproductive maturity to maximize male reproductive fitness (Lin, 2016).

Endocrine remodelling of the adult intestine sustains reproduction in Drosophila

The production of offspring is energetically costly and relies on incompletely understood mechanisms that generate a positive energy balance. In mothers of many species, changes in key energy-associated internal organs are common yet poorly characterized functionally and mechanistically. This study shows that in adult Drosophila females, the midgut is dramatically remodelled to enhance reproductive output. In contrast to extant models, organ remodelling does not occur in response to increased nutrient intake and/or offspring demands, but rather precedes them. By performing spatially and temporally directed manipulations, juvenile hormone (JH) was identified as an anticipatory endocrine signal released after mating. Acting through intestinal bHLH-PAS domain proteins Methoprene-tolerant (Met) and Germ cell-expressed (Gce), JH signals directly to intestinal progenitors to yield a larger organ, and adjusts gene expression and sterol regulatory element-binding protein (SREBP) activity in enterocytes to support increased lipid metabolism. These findings identify a metabolically significant paradigm of adult somatic organ remodelling linking hormonal signals, epithelial plasticity, and reproductive output (Reiff, 2015).

Genetic tools to study juvenile hormone action in Drosophila

The insect juvenile hormone receptor is a basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domain protein, a novel type of hormone receptor. In higher flies like Drosophila, the ancestral receptor germ cell-expressed (gce) gene has duplicated to yield the paralog Methoprene-tolerant (Met). These paralogous receptors share redundant function during development but play unique roles in adults. Some aspects of JH function apparently require one receptor or the other. To provide a foundation for studying JH receptor function, this study has recapitulated endogenous JH receptor expression with single cell resolution. Using Bacteria Artificial Chromosome (BAC) recombineering and a transgenic knock-in, a spatiotemporal expressional atlas was generated of Met and gce throughout development. JH receptor expression was demonstrated in known JH target tissues, in which temporal expression corresponds with periods of hormone sensitivity. Larval expression largely supports the notion of functional redundancy. Furthermore, the neuroanatomical distribution of JH receptors is provided in both the larval and adult central nervous system serving as a platform for future studies regarding JH action on insect behavior (Baumann, 2017).

This study used a set of genetic tools to probe developmental expression of the duplicate Drosophila JH receptors Met and gce, by recapitulating their expression with fluorescent reporters. Larval and adult tissues were systematically surveyed, with emphasis on neuronal expression. Coexpression experiments in which the GAL4 and LexA gce reagents drove simultaneous GFP and tdTomato constructs demonstrated extensive expressional overlap between these reagents, but also illustrated some differences. The T2A-GAL4 knock-in and BAC reagents showed substantially divergent expression patterns in key CNS structures in both larvae and adults, likely a consequence of position effects and construct architecture, though there were shared features. Peripheral tissue expression was essentially the same between the T2A and BAC iterations of the gce constructs, indicating that at least in non-CNS tissues, BAC expression is a reliable proxy for endogenous gce expression. Only a single Met-GAL4::p65 BAC reagent was obtained; several efforts to produce a Met knock-in reagent were fruitless. Given the differences between BAC and knock-in data for gce, it is proposed that the Met BAC reagent faithfully recapitulates peripheral tissue expression but likely only approximates Met expression in the CNS, since it was not possible to generate the 'gold standard' genomic knock-in reagent for Met (Baumann, 2017).

Overall, the confocal data are consistent with characterized aspects of tissue-specific JH action and therefore represent a working foundation for future studies regarding JH action at the cellular level in Drosophila. An overview of key expression features in the larva is presented. JHR redundancy during larval life is supported by their near global coexpression. A key exception to this finding was that gce but not Met was conspicuously absent from the imaginal discs. Following JHA challenge, disc-derived structures develop normally in Drosophila. In contrast, exogenous JHA reprograms the abdominal histoblasts by maintaining the expression of the pupal specifier Broad, promoting a second pupal, rather than adult, epidermis. This study demonstrates that Met and gce coexpress in histoblasts during the window of JH sensitivity, following which their expression declines, with gce levels declining followed by Met, beginning at 9 h APF. Under the rationale that JH sensitivity requires both receptors, lines were created in which Met, gce, or both were ectopically expressed specifically in the wing disc. However, following dietary JH treatment, wing development in both experimental and balancer flies was normal, indicating that ectopic JHR expression was not sufficient to confer JH sensitivity to this tissue. To address the possibility that another component of the JH receptor machinery was absent in wing discs, the discs with antibodies against the JH-dependent Met binding partner Taiman (Tai). Tai was observed in discs in late wandering larvae, before the onset of pupariation. Therefore, the canonical JH signaling pathway is present. Perhaps unidentified inhibitory factors block JH action in wing discs or downstream components are missing (Baumann, 2017).

Removing JH signaling in Drosophila larvae, through genetic ablation of the corpus allatum [allatectomy (CAX)] or by suppressing Met/gce function, cannot prevent the onset of metamorphosis, but does alter developmental timing in the third instar. During this instar, the PG establishes the timing of pupariation by assessing larval size to determine whether the critical weight checkpoint, which corresponds to the time after which starvation cannot prevent metamorphosis, has been attained. By suppressing Met expression specifically in the PG, Mirth (2014) demonstrated that this treatment is insufficient to alter time to pupariation relative to phm/+ control larvae. This study found that Met and Gce were coexpressed in the PG during larval development, suggesting the possibility that Mirth (2014) failed to override functional redundancy between JHRs in the PG. To address this, the same manipulation were performed in gce mutants, eliminating PG expression of both JHRs. There was no difference in pupariation time between the Met RNAi larvae and the Tm6b balancer sibs although both were delayed slightly in comparison to the parental stock, indicating that the phm-GAL4 construct itself can influence the time to pupariation. The finding that the gce mutant with two copies of the phm-GAL4 construct showed a large delay in pupariation time indicates that this delay is dose-dependent. Thus, the phm-GAL4 construct alone is sufficient to shift time to pupariation, a phenotype that is consistent with perturbed ecdysone synthesis or PG function. When attempts were made to create a wvMet 27;;phm/TM6b line, the stock was fragile and showed extensive preadult mortality, indicating that Met flies are sensitive to PG manipulation, and that expression of GAL4 alone in the PG has developmental consequences (Baumann, 2017).

Reciprocal biosynthetic regulation between JH and ecdysone pathways has been demonstrated in several species, though the mechanisms segregate along taxonomic lines. In the hemimetabolous Diploptera punctata, expression of the ecdysone receptor proteins EcR and RXR (USP) coincides with an elevation in JH titer in 5-day old adult females; suppressing these genes lowers ecdysteroid titer and elevates JH biosynthesis in 6-day old females. In holometabolous insects, 20E has an evolutionarily-conserved allatotropic role. JH biosynthesis is irreversibly stimulated in cultured Aedes aegypti corpus allatum (CA) following 20-hydroxyecdysone (20E) addition at a concentration of 10^-6 M. Fourth instar Bombyx mori corpus cardiacum-corpus allatum complexes likewise increased JH biosynthesis when cultured with 20E in a narrow concentration range, and this JH synthesis profile was concomitant with the elevation and subsequent decline in the transcript abundance of two JH biosynthetic enzymes. In both A. aegypti and B. mori, 20E overrides an inhibitory factor from the brain that prevents JH synthesis at a developmentally inappropriate time (Baumann, 2017).

In Drosophila such a factor(s) may be a product of the CA-LP1/2 neurons that innervate the CA, and which according to the present study, show JHR expression in WL3 larvae. Further, JH receptor expression in CA/PG cells, as well as in CA-LP and PTTH neurons suggests a potential role in coordinating hormone biosynthesis. For instance, by responding to changes in hormone titer, JHRs may participate in a feedback mechanism through which one or both receptors monitor the rate of biosynthesis or release of JH, ecdysone, or both. Experiments in other insects have shown that JH can inhibit PTTH release to indirectly suppress ecdysone production, or act directly on the PG to suppress ecdysteroid synthesis. Met and Gce both express in the PTTH cells and PG during the third instar. The coexpression of JH and ecdysone receptor machinery in the endocrine cells and associated neurons supports these tissues as critical sites for the convergence of signaling pathways, and the sensitivity of wvMet 27;;phm/TM6b flies to PG manipulation suggests that Met may be the key player in integrating these signals (Baumann, 2017).

In adult flies, JH-Met regulates reproductive physiology in both sexes. Met mutants produce and oviposit fewer vitellogenic oocytes than wild type flies, while flies lacking gce exhibit considerably milder reproductive consequences. Widespread Met and gce coexpression was observed in both male and female reproductive tracts (Baumann, 2017).

In Drosophila oogenesis, JH is necessary for entry into the vitellogenic phase. The vitellogenic checkpoint occurs in stage 9 oocytes where JH is required for both yolk protein synthesis by the follicle cells and yolk protein uptake from the hemolymph. It also suppresses 20E-induced apoptosis of the oocyte at stages 8-9 when the females are starved. This apoptosis is mediated by the ecdysone-induced E75A. Both Met and gce are expressed in the follicle cells surrounding the primary oocyte in stage 8-10 egg chambers, which corresponds with the vitellogenic checkpoint). Met and gce in stage 8 and 9 follicle cells may mediate the action of JH, 20E, or both to oppose apoptosis of egg chambers. The comparatively severe oogenesis phenotype exhibited by Met mutants argues for a scenario under which Met-JH suppresses ecdysone-driven apoptosis, possibly by downregulating the apoptosis inducer E75A, which is a JH and 20E responsive gene, and a demonstrated transcriptional target of JH-Gce in cell culture. If gce regulates E75A expression in check-point stage egg chambers, gce overexpression, rather than loss of function, might be expected to promote apoptosis in egg chambers (Baumann, 2017).

Virtually nothing is known about Met and gce function in the brain, but a handful of studies have addressed the more general issue of JH action on nervous system development. Widespread and dramatic neurological damage results from a lethal dose of the JH analog methoprene in OR-C wildtype flies, including fusion of the subesophageal ganglion with the thoracicoabdominal ganglion, an incomplete fusion of the optic lobe neuropils with the central brain, and disorganization and degeneration of the optic lobe, particularly in the chiasma. Aross malformation of the lobula has been described in Met null mutants, evident as finger-like projections that encroach laterally into the inner chiasm, attributed partially to premature EcR B1 expression. gce mutants do not display this phenotype. Accordingly, gce expression in the developing optic lobe of WL3 larvae is sparse, and the T5 cells that arborize along the margin of the lobula likewise show Met but not gce expression, suggesting a potential maintenance function for Met-JH in the morphological integrity of the developing lobula (Baumann, 2017).

REGULATION

Characterization of the Drosophila Methoprene-tolerant gene product

The larval fat body of newly eclosed adults of Drosophila was found to contain a single major binding protein specific for juvenile hormone (JH). Binding to this protein was saturable, of high affinity, and specific for JH III. The protein has a subunit molecular weight (Mr) of 85,000, as determined by photoaffinity labeling. The same or similar JH-binding protein was found in larval fat body and cuticle of third instar larvae and in male accessory glands and heads of newly eclosed adults. It was not found in several other tissues in adults. Male accessory gland cytosol from wild-type flies was found to contain a single binder with a dissociation constant (KD) of 6.7 nM for JH III; a binder in similar preparations from the methoprene-tolerant (Met) mutant had a KD value 6-fold higher. JH III stimulated protein synthesis in glands cultured in vitro, but this effect was reduced in Met flies as compared to wild-type flies, establishing a correlation between JH binding and biological activity of the hormone. In addition, glandular protein accumulation during the first 2 days of adult development was less in Met flies than in wild-type flies. These results strongly suggest that the binding protein identified here mediates this JH effect in male accessory glands and thus is acting as a JH receptor (Shemshedini, 1990a).

Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a great diversity of processes in development and reproduction. As yet the molecular modes of action of JH are poorly understood. The Methoprene-tolerant (Met) gene of Drosophila melanogaster has been found to be responsible for resistance to a JH analogue (JHA) insecticide, methoprene. Previous studies on Met have implicated its involvement in JH signaling, although direct evidence is lacking. This study examined the product of Met (MET) in terms of its binding to JH and ligand-dependent gene regulation. In vitro synthesized MET directly binds to JH III with high affinity, consistent with the physiological JH concentration. In transient transfection assays using Drosophila S2 cells the yeast GAL4-DNA binding domain fused to MET exerted JH- or JHA-dependent activation of a reporter gene. Activation of the reporter gene was highly JH- or JHA-specific with the order of effectiveness: JH III JH II > JH I > methoprene; compounds which are only structurally related to JH or JHA did not induce any activation. Localization of MET in the S2 cells is nuclear irrespective of the presence or absence of JH. These results suggest that MET may function as a JH-dependent transcription factor (Miura, 2005).

The MET protein was obtained by using coupled in vitro transcription/translation (TNT) reaction. The production of the full-length polypeptide was confirmed by analysing the product of reaction in the presence of ³⁵S-methionine by SDS/PAGE and autoradiography. The principal product had the expected full-length molecular mass of 79 kDa. The programmed lysate was used as a protein source for binding assay by the dextran-coated charcoal (DCC) method. Specific binding of ³H-labeled JH III to the TNT protein showed saturable profile. By these experiments, it was demonstrated that MET binds to JH III directly with a nanomolar K_d value although the possibility that factors in the rabbit reticulocyte lysate may influence binding cannot be ruled out. The specific binding was competed away by 100-fold molar excess of cold JH III (Miura, 2005).

The Met gene product was examined for its transactivation ability. The binding sequence motif of MET is presently uncertain. In addition, it is unknown whether MET functions as a homo- or heterodimer. So, a heterologous approach was used with the yeast GAL4-DBD (DNA binding domain) fusion/UAS (upstream activating sequence) system. The GAL4-DBD possesses a zinc finger that directs homodimerization and binding to UAS elements, and a potent nuclear localization sequence. By using this system, it was possible to assess the transactivation potential of MET independently of its dimerization properties or nuclear localization signals. The MET protein was expressed in S2 cells as a fusion with GAL4-DBD, and a luciferase reporter construct possessing five tandem copies of the UAS in its regulatory region was used. In this system, the effect of JH on transcription from the reporter gene was tested (Miura, 2005).

When an empty expression vector was transfected, the addition of 5 µm JH III to the culture medium caused no elevation of reporter activity over that of controls given the vehicle ethanol. Expression of native MET lacking GAL4-DBD slightly elevated the reporter activity, but did not show any JH dependency. Next, only the GAL4-DBD was expressed. In this case, the reporter activity was elevated about twofold above the empty vector control in either the presence or absence of JH III, indicating that the GAL4-DBD translocates into the nucleus and functions as a moderate, constitutive activator of transcription in a JH-independent manner. The GAL4-DBD-MET fusion in the presence of ethanol did not bring about any enhanced reporter activity relative to the empty vector control, but when JH III was added, the reporter activity was elevated about fivefold over the case of the empty vector control or the case of the GAL4-DBD-MET with ethanol. This activation by JH III can also be described as about twofold when compared to the case of GAL4-DBD with JH III. This indicates that MET has transactivation domain(s), and its transactivation function is JH dependent. It is noteworthy that in the absence of JH III the MET moiety of the fusion protein repressed the moderate transactivation produced by GAL4-DBD. This suggests that unliganded MET may function as a transcriptional repressor (Miura, 2005).

If MET represents a JH-dependent transcription factor, it should show stringent ligand specificity. To test this, several compounds that are structurally related to JH or JHA but show no JH activity were examined in the GAL4-MET fusion/UAS system. The effects of these potential ligands on the reporter activity are shown as fold induction by dividing the activity obtained with the pAcGAL4-DBD-Met by that in negative controls using empty pAc vectors. Addition of squalene, farnesol, farnesyl acetate and geraniol at a final concentration of 5 µm did not result in any activation of the reporter. JH III, however, again brought about enhanced reporter activity. Interestingly, a JHA (methoprene) showed weaker ligand activity than JH III. Thus, the transactivation exerted by MET shows stringent ligand specificity apparently related to JH activity, ruling out nonspecific transactivation by lipid-soluble compounds (Miura, 2005).

The binding assay showed that MET has a nanomolar level K_d for JH III and several potential ligands weere used at 5 µm in the experiments described above. If MET functions as a JH-dependent transcription factor, it should respond to nanomolar levels of ligand, consistent with its high affinity for JH III. Three natural JHs, JH I, JH II, and JH III, and the JHA methoprene were tested in varying concentrations using the GAL4-MET fusion/UAS transfection assay. The effects on the reporter activity were recored as fold induction. Every compound tested showed ligand activity on transactivation, nearing saturation at 500 nm while showing only marginal increase at 5 µm. Among these, JH III, which is one of the native JHs of Drosophila, was found to be the most effective over the range of concentrations tested. Of note is that JH III was conspicuously active in the range of 5-50 nm, whereas the other JHs or JHA showed only slight effects. The other native JH of Drosophila, JH-bisepoxide was not tested. The induction activities are in the following order: JH III  JH II > JH I > methoprene. The most effective transcriptional activation produced by Drosophila MET with its native JH species further supports the putative role of MET as a JH-dependent transcription factor. These data, thus, indicate that the threshold activity concentration of JHs is reasonably low in this transient transfection system (Miura, 2005).

In these transfection assays, MET was fused to GAL4-DBD, which has a nuclear localization sequence. To test the subcellular localization of MET, a fusion to enhanced green fluorescent protein (EGFP), which does not have a nuclear localization sequence, was used. S2 cells were transfected with the expression plasmid pAcMET-EGFP together with the reporter and coreporter constructs used above. After transfection, cells were incubated for 24 h in the presence or absence of JH III, then observed by Nomarski DIC (differential-interference contrast) or fluorescence microscopy. In both cases, the fluorescence of the fusion proteins was seen in the nucleus. In these experiments the use of cultured cells allows for complete depletion of JH. These observations are consistent with the previous report in vivo (Pursley, 2000) and rule out the ligand-dependent nuclear translocation reported for the aryl hydrocarbon receptors (Ahrs) of vertebrates. Then, how is JH transported to the nucleus? A process such as vertebrate retinoid transport including cellular retinol-binding protein may be involved (Miura, 2005).

The principal new contributions of this study are: (1) demonstration of direct, reversible binding of JH III to MET; (2) demonstration of its JH-dependent transactivation potential. The former was enabled by the use of coupled in vitro transcription and translation, since difficulty was experienced in obtaining soluble preparations of full-length bHLH-PAS proteins from mosquitoes using prokaryotic expression systems. The binding of JH III by MET showed high affinity with a nanomolar K_d value, and was competed away by an excess of cold JH III (Miura, 2005).

In the present study, MET was tethered to a promoter by using the GAL4-DBD fusion/UAS reporter system. In this heterologous system, the MET fusion exhibited specific ligand-dependent activation of a reporter gene placed downstream of the UAS. The MET fusion responded only to JH or the JHA methoprene while compounds that are structurally related but hormonally inactive elicited no response. Among compounds tested, JH III was the most effective ligand, even at nanomolar concentrations, which is in accordance with its nature as one of Drosophila's native JHs. The typical range of concentration for JH In insect haemolymph is 0.3-180 nm. Further, the maximal JH titre in the Drosophila life cycle is 5-7 pmol·g^-1 wet weight, which would correspond to 25-35 nm in the haemolymph, assuming that haemolymph occupies one-fifth of the body weight. In view of these physiological JH titres, it is thus notable that in this study JH III was found to be overwhelmingly active in the physiological range of 5-50 nm over the other JHs or JHA. The ligand-dependent transactivation profile exhibited by MET clearly rules out the possibility that it is simply a JH binder, like cytosolic JH binding proteins, and suggests that it might play a role in JH signaling in vivo (Miura, 2005).

Conformational changes of recombinant Drosophila USP exposed to several different farnesoid compounds including natural JHs have been reported. JH III and JH I at 100 µm elicit the conformational changes to a similar degree whereas JH II is the least effective. Furthermore, by using Drosophila white puparial bioassay it has been have demonstrated that the biochemical differences in the three JHs mentioned above parallel the respective biological activity. For example, in prevention of adult emergence the 50% effective doses (ED₅₀s) for JH I-III are 153, 678 and 143 pmol·puparium^-1, respectively. The ED₅₀ of methoprene has been reported as 5 pmol·puparium, and Met mutant flies are more resistant to another JHA, S31183, than the parental fly stock, suggesting the involvement of the Met locus in this resistance (Riddiford, 1991). Based on these studies, the order of efficacy of these compounds in this bioassay seems to be methoprene  JH III ~ JH I > JH II. In contrast, the order was JH III  JH II > JH I > methoprene in the current transfection assay. Methoprene is the most effective in the former and the least effective in the latter. This is not surprising since methoprene is often highly active over naturally occurring JHs when applied topically. For example, the early trypsin gene of Aedes aegypti is upregulated by JH, and low doses of methoprene, but higher doses of its native JH III are required to restore its expression in the ligated abdomens. The higher efficacy of methoprene in these bioassays may be due to its higher resistance to enzymatic degradation and possible higher penetration through the cuticle than natural JHs (Miura, 2005).

Another point is that JH I has been shown to be more active than JH II in the white puparial assay whereas JH II is more active in the current transfection assay. The difference between these two studies is the concentrations of JH used. In the current assay JH I and JH II were similarly much less effective than JH III in the physiological range while these differences were somewhat obscured at higher doses, although JH II was still more effective than JH I. At present this discrepancy is not explained. Possibly, there might be more than one pathway of JH signaling underlying in the white puparial bioassay, one mediated by USP and another by MET (Miura, 2005).

In the reporter assays, it was noted that unliganded MET repressed the intrinsic activation function possessed by GAL4-DBD. Although GAL4-DBD is believed to lack transactivation domains, it showed moderate transactivation potential in Drosophila S2 cells in this study. The nuclear localization of MET was confirmed by the finding that the MET-EGFP fusion is concentrated in the nuclei of transfected S2 cells. In addition, GAL4-DBD has a nuclear localization sequence. Therefore, it is reasonable to consider that the GAL4-DBD fusion of MET sits on the UAS of the reporter construct even in the absence of ligand, and that the MET moiety is responsible for the observed repression. In the case of vertebrate Ahr, a multimeric complex including hsp90 anchors the unliganded Ahr in the cytoplasm, thereby preventing its transactivation function. Upon ligand binding, Ahr translocates to the nucleus and forms a transcription factor complex with Arnt. In fact, the C-terminal portion of Ahr fused to GAL4-DBD has been shown to act as a constitutive activator of gene regulation. Contrary to this, MET exists in the nucleus even in the absence of ligand. Upon ligand binding, it becomes a transcriptional activator. This resembles the ligand-dependent activation that has been shown in the activation function of many nuclear hormone receptors, rather than the case of the vertebrate Ahr whose activation function is regulated by its subcellular localization (Miura, 2005).

Two questions arise here as to whether MET functions as a homo- or heterodimer, and as to what DNA sequences are responsible for the binding of this transcriptional regulator complex. These questions are related since DNA-binding specificities of bHLH-PAS proteins are determined by their dimerization properties. For example, the dioxin receptor complex Ahr/Arnt heterodimer binds to TNGCGTG. Ahr recognizes the 5'-half-site TNGC, while Arnt recognizes the 3'-half-site GTG. Arnt is also capable of forming a homodimer that recognizes a consensus palindromic E-box sequence, CACGTG. Drosophila Sim protein forms a heterodimer with Tango (a Drosophila Arnt-like protein) and binds to ACGTG core sequence. Thus, DNA binding specificities of bHLH-PAS dimers are dependent upon the dimer configuration while Arnt or Tango always recognize the GTG motif. In the present study, the GAL4-DBD fusion of MET was used in transfection assays. Under these conditions MET is likely to behave as a homodimer because of its overexpression and because of dimerization interfaces provided by the GAL4-DBD moiety. Therefore, the natural dimerization partner and binding sequence of MET are unknown at present. Since the bHLH domain of MET shows relatively high similarity to vertebrate Arnts, the use of the consensus sequence CACGTG may be a good starting point to answer these questions (Miura, 2005).

Based on the framework by Wilson and coworkers, these results have further supported the notion that MET may function as a JH-dependent transcription factor. In further studies, identification of its target genes will help elucidate its in vivo function. Molecular dissection of MET and structural studies may lead to the development of new biologically active JHA and new strategies for pest management (Miura, 2005).

Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis

Metamorphosis of holometabolous insects, an elaborate change of form between larval, pupal and adult stages, offers an ideal system to study the regulation of morphogenetic processes by hormonal signals. Metamorphosis involves growth and differentiation, tissue remodeling and death, all of which are orchestrated by the morphogenesis-promoting ecdysteroids and the antagonistically acting juvenile hormone (JH), whose presence precludes the metamorphic changes. How target tissues interpret this combinatorial effect of the two hormonal cues is poorly understood, mainly because JH does not prevent larval-pupal transformation in the derived Drosophila model, and because the JH receptor is unknown. The red flour beetle Tribolium castaneum has been used to show that JH controls entry to metamorphosis via its putative receptor Methoprene-tolerant (Met). This study demonstrates that Met mediates JH effects on the expression of the ecdysteroid-response gene Broad-Complex (BR-C). Using RNAi and a classical mutant, it has been show that Tribolium BR-C is necessary for differentiation of pupal characters. Furthermore, heterochronic combinations of retarded and accelerated phenotypes caused by impaired BR-C function suggest that besides specifying the pupal fate, BR-C operates as a temporal coordinator of hormonally regulated morphogenetic events across epidermal tissues. Similar results were also obtained when using the lacewing Chrysopa perla (Neuroptera), a member of another holometabolous group with a primitive type of metamorphosis. The tissue coordination role of BR-C may therefore be a part of the Holometabola groundplan (Konopova, 2008).

In both Tribolium and Chrysopa, BR-C RNAi compromises the larval-pupal transition without affecting earlier development, regardless of the time of dsRNA injection. The TcBR-C^KS342 homozygotes die at the same stage. These data suggest that the moderate levels of BR-C mRNAs, detectable during premetamorphic stages in both species, has no essential role. This scenario would agree with the fact that zygotic BR-C function is not required in Drosophila BR-C null nonpupariating mutants until the onset of metamorphosis. However, as neither RNAi nor the likely hypomorphic TcBR-C^KS342 allele present a complete loss-of-function situation, a possibility that BR-C plays some additional role, not visualized by the phenotypes cannot be excluded. Importantly, the lethal phase correlates with a strong upregulation of BR-C expression. At least in beetles, this stage coincides with a peak of ecdysteroid titer that causes larvae to initiate prepupal development (Konopova, 2008).

In contrast to Drosophila npr1 mutants, metamorphosis was not completely blocked by BR-C deficiency in Tribolium or Chrysopa. Instead the arrested prepupae showed a blend of larval, pupal, and partially even adult features. Based on the absence of the pupal-specific gin traps in Tribolium and on the surface microsculpture, the cuticle was apparently larval in both species, thus confirming the requirement of BR-C for the pupal commitment of the epidermis. Interestingly, although the thorny cuticle in Chrysopa BR-C(RNAi) animals was distinctly larval, similar to in Tribolium, the body pigmentation resembled that of pupae. It is not certain whether this mixed character of the epidermis might be due to persisting CpBR-C function, or might be because CpBR-C is not necessary for the pupal pigmentation (Konopova, 2008).

Pupal characters in BR-C(RNAi) animals included rudimentary wings. In particular, the weak phenotypes in Tribolium (produced with either isoform-specific or diluted common-core dsRNAs) revealed that wing elongation was highly sensitive to BR-C depletion. A similar effect of BR-C RNAi was described for pupal appendages in Bombyx mori. BR-C silencing prevented the gradual wing enlargement even in larvae of the hemimetabolous milkweed bug Oncopeltus fasciatus. Imaginal discs fail to elongate in Drosophila br mutants with disrupted BR-C Z2 function. The short legs and wings are not due to insufficient proliferation of the disc cells but are due to their inability to change shape in response to the ecdysteroid. This cell shape change requires cytoskeletal components whose mutations enhance the effect of br. The rudimentary wings, present even in animals most severely affected by TcBR-C^KS342 mutation or by RNAi, suggest that cell shape changes, rather than cell proliferation may be disrupted by the loss of BR-C in Tribolium as well. Growing wings marked by EGFP in arrested beetle prepupae support this idea. The legs in Tribolium BR-C(RNAi) animals were short also but were distally specified as pupal with two tarsal claws. By contrast, the arrested Chrysopa prepupae retained pretarsi with the larval-specific elongated arolium, thus suggesting a stronger requirement for BR-C function in the Chrysopa leg (Konopova, 2008).

Except for small deviations, gross morphology of Tribolium genital segments with the pupal genital papillae was pupal in BR-C(RNAi) animals. In addition, the larval-pupal transformation of the visual system was initiated, as larval stemmata were replaced with ommatidia of the compound eyes. However, as in Drosophila, TcBR-C was important for compound eye differentiation. These observations suggest that not all aspects of pupal development are completely blocked by BR-C depletion (Konopova, 2008).

While the above described structures are retarded in their development in BR-C(RNAi) animals, others appeared accelerated in their development towards the adult state, although none could be unambiguously defined as adult. For instance, the antennae in Tribolium or the compound eyes in Chrysopa resembled their adult counterparts, but in fact were intermediates between pupal and adult organs. These heterochronic phenotypes suggest that BR-C may not only be a pupal specifier, but rather a temporal coordinator of the extensive morphogenesis in diverse tissues during metamorphosis (Konopova, 2008). Drosophila organs require a temporally regulated balance between both inductive and repressive BR-C functions, represented by the individual isoforms. Two alternative explanations are seen for the heterochronically advanced phenotypes. First, these structures may require BR-C to repress precocious adult morphogenesis in them, but the inductive BR-C function is dispensable for development beyond larval state. Consequently, loss of BR-C accelerates their development. Second, if both functions are required but the repressive one is more sensitive to reduced BR-C dose, then the inductive function will prevail under an incomplete BR-C knockdown. The first alternative alternative is favored, because progression beyond the pupal stage seems to depend on BR-C downregulation (Konopova, 2008).

Periods of JH absence are required first in larvae to initiate the pupal program, and later in pupae to exit it. BR-C in both cases promotes the pupal fate, and therefore JH must regulate BR-C differently in larvae and in pupae. In lepidopteran, as well as in Tribolium larvae, JH prevents BR-C expression until the onset of metamorphosis, and presumably that is how JH prevents pupal differentiation. Conversely, removal of the JH source (allatectomy) causes both BR-C misexpression and precocious pupal development. In pupae, ectopic JH induces BR-C, and in many insects, including Tribolium, such JH application causes reiteration of the pupal stage. In Drosophila, BR-C misexpression alone is sufficient to inhibit adult cuticle formation. BR-C is therefore a prime target of JH signaling, but how JH regulates BR-C expression is unknown (Konopova, 2008).

Precocious pupation, triggered by interference with the putative JH receptor Met, coincided with precocious TcBR-C mRNA increase in the sixth instar. Thus, disrupted JH signaling induced TcBR-C similarly to allatectomy in lepidopteran larvae. As expected, TcBR-C not only marked but also was necessary for the untimely pupation, as TcMet; TcBR-C double-RNAi resulted in a phenotype similar to TcBR-C RNAi alone, i.e. entry to a lethal prepupal stage, except one or two instars too early. Therefore, although the metamorphic program could be prematurely induced by silencing of TcMet, it could not be completed without TcBR-C. However, loss of Met has been shown to worsen the effect of BR-C mutations in Drosophila, without altering BR-C expression. This again might reflect the different response to JH in the fly (Konopova, 2008).

The evidence that TcMet is required for regulation of TcBR-C came from pupae, where the JH mimic methoprene induced TcBR-C mRNA, but not after TcMet knockdown. This result places TcBR-C downstream of TcMet in JH signaling. Importantly, the averting of ectopic TcBR-C expression by TcMet RNAi also rescued the methoprene-treated animals from repeating the pupal stage and allowed them to become adult. Together, these findings suggest that, similar to in Drosophila, downregulation of BR-C is required to exit the pupal state in Tribolium (Konopova, 2008).

The following model for BR-C function in holometabolan metamorphosis. In larvae, JH acts through Met to prevent BR-C induction until the final instar, when JH decline relieves the repression, and BR-C coordinates pupal morphogenesis. Loss of BR-C function causes both retardation and acceleration of development in diverse epidermal tissues, thus producing a mix of larval-, pupal- and adult-like features. In early pupae, low JH titer normally allows BR-C expression to drop, which is necessary for proper adult differentiation. Exogenous JH, again acting via Met, causes BR-C misexpression, which in turn promotes another round of pupal, instead of adult, development. Whether Met regulates BR-C expression directly, and what determines whether BR-C will be repressed or activated requires further work (Konopova, 2008).

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, a novel Drosophila genetic screen was designed to isolate mutations that derepress JH-mediated br suppression at early larval stages. It was 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 juvenile hormone analogs (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 are 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, it is concluded 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 (Abdou, 2011).

JH transduces its signal through Methoprene-tolerant (Met), Germ cell-expressed (Gce) and Krüppel-homolog 1 (Kr-h1) and the p160/SRC/NCoA-like molecule (Taiman in Drosophila and FISC in Aedes). The Drosophila Met and gce genes encode two functionally redundant bHLH-PAS protein family members, which have been proposed to be components of the elusive JH receptor. Both Met and gce mutants are viable and resistant to JH analogs (JHA) as well as to natural JH III. However, Met-gce double mutants are prepupal lethal and phenocopies CA-ablation flies. The Met protein binds JH III with high affinity. In Tribolium, suppression of Met activity by injecting double-stranded (ds) Met RNA causes precocious metamorphosis. Kr-h1 is considered as a JH signaling component working downstream of Met. In both Drosophila and Tribolium, Kruppel-homolog1 (Kr-h1) mRNA exhibits high levels during the embryonic stage and is continuously expressed in the larvae; then, it disappears during pupal and adult development. Kr-h1 expression can be induced in the abdominal integument by exogenous JH analog (JHA) at pupariation. Suppression of Kr-h1 by dsRNA in the early larval instars of Tribolium causes precocious br expression and premature metamorphosis after one succeeding instar. Thus, Kr-h1 is necessary for JH to maintain the larval state during a molt by suppressing br expression. Studies in Aedes, Drosophila and Tribolium have demonstrated that the p160/SRC/NCoA-like molecule is also required for JH to induce expression of Kr-h1 and other JH response genes. For example, Aedes FISC forms a functional complex with Met on the JH response element in the presence of JH and directly activates transcription of JH target genes (Abdou, 2011 and references therein).

In an attempt to isolate other genes involving JH signaling, a novel genetic screen was conducted, and mutations in were identified in three Wnt signaling component genes, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), induced precocious br expression, which was similar to a loss of JH activity. The evolutionarily conserved Wnt signaling pathway controls numerous developmental processes. The key mediator of the Drosophila Wnt pathway is Armadillo (Arm, the homolog of vertebrate β-catenin). When the Wnt signaling ligand, Wingless (Wg), is absent, the destruction complex is active and phosphorylates Arm, earmarking it for degradation. Upon Wg stimulation, the destruction complex is inactivated; as a result, unphosphorylated Arm accumulates in the cytosol and is targeted to the nucleus to stimulate transcription of Wnt target genes. Many players in the Wnt signaling pathway negatively regulate its activity. For example, Axin (Axn) is one of the main components of the destruction complex. Supernumerary limbs (Slmb) recognizes phosphorylated Arm and targets it for polyubiqitination and proteasomal destruction. Naked cuticle (Nkd) antagonizes Wnt signaling by inhibiting nuclear import of Arm. The current investigations reveal that the high activity of Wnt signaling in the Axn, slmb, and nkd mutants suppresses the transcription of Met and gce, genes encoding for putative JH receptors, thus linking Wnt signaling to JH signaling and insect metamorphosis for the first time (Abdou, 2011).

The 'status quo' action of JH in controlling insect metamorphosis is conserved in hemimetabous and most holometabous insects. However, the larval-pupal transition in higher Diptera, such as Drosophila, has largely lost its dependence on JH. For instance, in most insects, the addition of JH in larvae at the last instar causes the formation of supernumerary larvae. However, exogenous JH does not prevent pupariation and pupation in Drosophila, and instead disrupts the development of only the adult abdominal cuticle and some internal tissues. The molecular mechanisms underlying these differential responses to JH are not clear (Abdou, 2011).

Broad is a JH-dependent regulator that specifies pupal development and mediates the 'status quo' action of JH. In the relatively basal holometabolous insects, such as beetles and moths, JH is both necessary and sufficient to repress br expression during all of the larval stages. These studies revealed that JH is also required during the early larval stages in the more derived groups of the holometabolous insects, such as Drosophila, but it is not sufficient to repress br expression at the late 3^rd instar. During the early larval stages, overexpression of the JH-degradative enzyme JHE, reduction of JH biosynthesis or disruption of the JH signaling always causes precocious br expression in the fat body. However, exogenous JHA treatment can not repress br expression in the fat body of late 3^rd instar larvae. The molecular mechanism underlying the developmental stage-specific responses of the br gene to JH signaling remains to be clarified (Abdou, 2011).

As knowledge of signal transduction increases, the next step is to understand how individual signaling pathways integrate into the broader signaling networks that regulate fundamental biological processes. In vertebrates, Wnt signaling has been found to interact with different hormone signaling pathways to mediate various developmental events. For example, the Wnt/beta-catenin signaling pathway interacts with thyroid hormones in the terminal differentiation of growth plate chondrocytes and interacts with estrogen to regulate early gene expression in response to mechanical strain in osteoblastic cells. In insects, both Wnt and JH signaling are important regulatory pathways, each controlling a wide range of biological processes. This study reports that the Wnt signaling pathway interacts with JH in regulating insect development. During the Drosophila early larval stages, elevated Wnt signaling activity in the Axn, slmb, nkd mutants and arm-GAL4/UAS-arm^S10 flies represses Met and gce expression, which down-regulates Kr-h1 and causes precocious br expression in the fat body. Ectopic expression of UAS-gce in the arm-GAL4/UAS-arm^S10 larvae is sufficient for restoring Kr-h1 expression and then repressing br expression (Abdou, 2011).

Arm is a co-activator that interacts with Drosophila TCF homolog Pangolin (Pan), a Wnt-response element-binding protein, to stimulate expression of Wnt signaling target genes. In the absence of nuclear Arm, Pan interacts with Groucho, a co-repressor, to repress transcription of Wingless-responsive genes. Upon the presence of nuclear Arm, it binds to Pan, converting it into a transcriptional activator to promote the transcription of Wingless-responsive genes. It is proposed that Wnt signaling indirectly suppresses Met and gce expression by activating an unknown transcriptional repressor (Abdou, 2011).

JH signaling is well known to be a systemic factor that decides juvenile versus adult commitment. Wg is a morphogen that tissue-autonomously promotes proliferation and patterning during organogenesis. The current studies show that ectopically activating Wg signaling, either by mutations of negative regulators or by the ectopic expression of Arm, results in br derepression via loss of Met and Gce. How and why does the localized Wg signaling regulate the global JH signaling during insect development? It is hypothesized that though JH signaling activity is globally controlled by JH titer in the hemolymph, distinct tissues may response to JH with different sensitivity, which could be regulated by Wnt signaling-mediated Met and gce expression. Actually, it was found that precocious br expression is detectible in the fat body but not midgut of the Axn mutant 2^nd instar larvae. This is one line of evidence to support that Wnt signaling regulates Met and gce expression in a tissue-specific manner (Abdou, 2011).

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) results in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), are significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, this study demonstrates that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

The status quo action of JH has been well documented in several insect orders, particularly in Coleoptera, Orthoptera and Lepidoptera, in which JH treatment causes supernumerary larval molting and JH deficiency triggers precocious metamorphosis. However, as JH does not cause supernumerary larval molting in flies, evidence for the status quo action of JH in Drosophila has remained elusive. From past studies and from the experimental data presented in this study, it is concluded that the status quo hypothesis does indeed apply to JH action in Drosophila. First, although JH application during the final larval instar or during the prepupal stage has little effect on the differentiation of adult head and thoracic epidermis in Drosophila, it does prevent normal adult differentiation of the abdominal epidermis. After JH treatment, a second pupal, rather than an adult, abdominal cuticle is formed in Diptera. Second, JH or a JH agonist applied to Drosophila at the onset of metamorphosis results in lethality during pupal-adult metamorphosis. Similarly, global overexpression of jhamt (Juvenile hormone acid methyl transferase) results in severe defects during the pupal-adult transition and eventually death (Niwa, 2008). Third, CA ablation leading to JH deficiency causes precocious and enhanced fat body PCD. Fourth, JH deficiency results in pupal lethality and delayed larval development, although JH deficiency is not sufficient to cause precocious metamorphosis. The composite data demonstrate that JH in Drosophila does have status quo actions on the abdominal epidermis during pupal-adult metamorphosis and on the fat body during larval-pupal metamorphosis. It is concluded that the status quo action of JH in Drosophila is functionally important, but more subtle than that in Coleoptera, Orthoptera and Lepidoptera. However, it is not clear whether JH is essential for embryonic and earlier larval development because the CA cells are not completely ablated in the JH-deficient animals until the early-wandering (EW) stage. To address this question, it would be necessary to generate a mutant (i.e., of jhamt) that interrupts JH but not the farnesyl pyrophosphate biosynthesis pathway (Liu, 2009).

The insect fat body is analogous to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. In response to 20E pulses, Drosophila larval organs undergo a developmental remodeling process during metamorphosis. Blocking the 20E signal specifically in the fat body during the larval-pupal transition (Lsp2>; UAS-EcR^DN) prevented the fat body from undergoing PCD and cell dissociation (Liu, 2009).

The experimental data in this paper demonstrates that JH prevents caspase-dependent PCD in the fat body during the larval-pupal transition in Drosophila. First, JH deficiency in Aug21>, UAS-grim resulted in the fat body undergoing precocious and enhanced PCD and cell dissociation. Aug21> is a GAL4 driver that specifically targets gene expression to the CA. Precocious and enhanced apoptosis appeared as early as L3D1 in the JH-deficient animals. Methoprene application on L3D1 was able to rescue ~40% of the pupae to adults, but it failed to rescue post-EW. Second, 2D-DIGE/MS and qPCR analyses indicated that the fat body in the JH-deficient animals has multiple developmental defects. The upregulation of the caspase genes Dronc and Drice should account for the PCD in the fat body, as overexpression of Dronc in the fat body causes PCD, cell dissociation, and thus lethality. Overexpression of Dronc or Drice in cells and tissues is sufficient to cause caspase-dependent PCD. Third, the 20E-triggered transcriptional cascade in the fat body was downregulated in the JH-deficient animals, indicating that JH does not suppress the 20E-triggered transcriptional cascade in preventing caspase-dependent PCD in the fat body (Liu, 2009).

The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of EcR. One might expect that perfect timing, titer and receptor response of JH and 20E are required to ensure accurate PCD in a tissue- and stage-specific manner during Drosophila metamorphosis. In the JH-deficient animals, the upregulation of Dronc and Drice resulted in precocious and enhanced PCD, such that the JH-deficient animals are committed to die during the larval-pupal transition. This hypothesis was strengthened by overexpression of Dronc specifically in the fat body, which caused larval lethality. Taken together, it is concluded that JH antagonizes 20E-induced caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

Based on the phenotypes and gene expression profiles in the four fly lines used, it is concluded that JH counteracts MET and GCE to prevent caspase-dependent PCD. First, the Met-overexpressing animals died during larval life, with precocious and enhanced PCD and cell dissociation in the fat body. Dramatic upregulation of Dronc and Drice was observed when Met was specifically overexpressed in the fat body and this upregulation was significantly decreased by methoprene application demonstrating that JH is epistatic to MET and GCE. Moreover, the Dronc-overexpressing animals exhibited similar phenotypes to the Met-overexpressing animals. Second, in the fat body of the JH-deficient animals, PCD and the expression of Dronc and Drice were upregulated but not as significantly as in the Met-overexpressing animals. This might explain why the JH-deficient animals did not die until early pupal life. Third, both the global JH-overexpressing animals and the Met/gce-deficient animals died during the pupal-adult transition. In these animals, Dronc and Drice were downregulated and caspase-dependent PCD was decreased in the fat body, implying that these animals died from a lack of caspase-dependent PCD. Weak mutants of Dronc and Drice mutants die during pupal life, showing that caspase-dependent PCD is essential for Drosophila metamorphosis. In addition, it was also observed that methoprene application at the onset of metamorphosis results in delayed fat body remodeling (Liu, 2009).

In the future, it will be crucial to elucidate the detailed molecular mechanism of how JH counteracts MET and GCE to prevent caspase-dependent PCD. In Drosophila S2 cells, the transcriptional activity of MET is dependent on the JH concentration and both MET-MET and MET-GCE interactions can be greatly diminished by JH. The bHLH-PAS transcription factors typically function as hetero- or homodimers. If MET/GCE is the Juvenile Hormone Receptor (JHR), the transcriptional activities of the dimerized MET/GCE and the JH-MET/GCE complex should differ. In other words, the dimerized MET/GCE should induce transcription of Dronc and Drice and, in turn, JH binding to form the JH-MET/GCE complex should reduce this induction. Although there are no examples in the literature in which a receptor, without ligand, acts as a transcriptional activator and the transcriptional activity of the receptor is diminished when the ligand is bound, it could be speculated that the JHR is a unique hormone receptor and perhaps that is the reason why it has yet to be isolated and characterized. Unfortunately, the experiments described here were conducted in Drosophila S2 cells, where the possibility of an endogenous JHR could not be eliminated. Although MET/GCE is definitely a key component in the JH signal transduction pathway, whether MET/GCE is the bona fide JHR remains conjecture (Liu, 2009).

It is very likely that MET cross-talks with EcR-USP via a large molecular complex. One can hypothesize that MET promotes 20E action in the absence of JH and suppresses 20E action in the presence of JH, a model which is favored. Drosophila FKBP39 (FK506-BP1) could be a key component in this complex because it physically interacts with MET, EcR and USP, and binds the D. melanogaster JH response element 1. Moreover, Drosophila FKBP39 inhibits 20E-induced autophagy (Juhász., 2007). Further analysis of the complex will be crucial to precisely define the molecular mechanism of cross-talk between the action of JH and 20E (Liu, 2009).

In summary, it is concluded that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila. The Drosophila fat body has provided an excellent model for studying the long-standing question of JH signal transduction. To finally settle the question of the bona fide JHR and to understand the precisely defined molecular mechanism of JH action requires further research at a variety of levels in several species of insects that can be genetically manipulated, such as Drosophila, Bombyx and Tribolium (Liu, 2009).

Heat shock protein 83 (Hsp83) facilitates Methoprene-tolerant (Met) nuclear import to modulate juvenile hormone signaling

Juvenile hormone (JH) receptors, Met and Gce, transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified the chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with the PAS-B and bHLH domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of the larval fat body cells when the JH titer is low and JH induces Met nuclear import. Hsp83 was also accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuates JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to the reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR (He, 2014).

Juvenile hormone activates the transcription of Cell-division-cycle 6 (Cdc6) for polyploidy-dependent insect vitellogenesis and oogenesis

Although juvenile hormone (JH) is known to prevent insect larval metamorphosis and stimulate adult reproduction, the molecular mechanisms of JH action in insect reproduction remain largely unknown. The JH-receptor complex, composed of methoprene-tolerant and steroid receptor co-activator, acts on mini-chromosome maintenance (Mcm) genes Mcm4 and Mcm7 to promote DNA replication and polyploidy for the massive vitellogenin (Vg) synthesis required for egg production in the migratory locust. This study has investigated the involvement of cell-division-cycle 6 (Cdc6) in JH-dependent vitellogenesis and oogenesis, as Cdc6 is essential for the formation of prereplication complex. Cdc6 was shown to be expressed in response to JH and methoprene-tolerant, and Cdc6 transcription is directly regulated by the JH-receptor complex. Knockdown of Cdc6 inhibits polyploidization of fat body and follicle cells, resulting in the substantial reduction of Vg expression in the fat body as well as severely impaired oocyte maturation and ovarian growth. These data indicate the involvement of Cdc6 in JH pathway and a pivotal role of Cdc6 in JH-mediated polyploidization, vitellogenesis, and oogenesis (Wu, 2016).

Protein Interactions

Interaction of bHLH-PAS proteins involved in juvenile hormone reception in Drosophila

Drosophila S2 cells were screened for the presence of endogenous MET, but neither Western blotting with MET polyclonal antibody nor Met mRNA amplification by RT-PCR performed with S2 cell-derived RNA detected MET protein or mRNA. Therefore, the proteins of interest were introduced into S2 cells by expression of the respective cDNAs following transfection. The interaction between MET and GCE proteins co-expressed in S2 cells was examined as GST-MET and GCE-V5 fusion proteins. Using a GSH pull-down technique, the GCE-V5 fusion protein was readily detected with V5 antibody as an immunoprecipitation product, demonstrating interaction between the two proteins (Godlewski, 2006).

Since MET is capable of binding JH in nanomolar concentration (Miura, 2005), the influence of JH treatment on MET-GCE interaction was examined. In the initial experiment, the transfected cell cultures were treated with JH III in final concentrations ranging from 10 μM to 1 nM for 1 h prior to homogenization and pull-down. JH III had no influence on the appearance of the MET-GCE protein complex. Conversely, analogous experiments with the JH agonists methoprene and pyriproxyfen revealed a disruptive effect of each of these juvenile hormone antagonists (JHAs) on the MET-GCE interaction. The strongest effect was observed at the highest concentrations examined. At the lowest concentration of 1 nM, pyriproxyfen showed no effect on the MET-GCE interaction, but methoprene showed a slight effect at 10 nM (Godlewski, 2006).

Both of the JH agonists are thought to be more resistant to catabolism by endogenous enzymes than is JH III. Therefore, the JH III treatment experiment was repeated using a series of time periods shorter than the previous 1-h incubation. At shorter incubation times, especially 15 min, JH III also disrupted MET-GCE interaction as did methoprene. However, the effect of JH III is reversible after 1 h, whereas methoprene requires 4 h for complete reversibility. At this shorter time period of 15 min for JH III, a dose–response effect is seen. The lowest detectable effect on MET-GCE interaction was 10 nM, an amount that is within the physiological concentration range for hormone action (Godlewski, 2006).

To determine if the hormone effect is specific to JH analogs, analogous experiments were carried out using farnesol and geraniol. Both of these compounds share overall chemical similarity with JH, but neither has JH activity in cell culture (Miura, 2005). Even when present in high concentration (10 μM) for the most effective time of JH III treatment (15 min), neither had an effect on MET-GCE interaction. Therefore, the effect on the interaction appears to be specific for JH III and JH agonists (Godlewski, 2006).

To determine if MET can form homodimers, S2 cells were co-transfected with a plasmid expressing GST-MET and one expressing MET-V5. Following incubation and V5 antibody immunoprecipitation, a strong band of MET was detected. To ensure that the MET-MET interaction was specific, a plasmid expressing MET was substituted for GST-MET and subjected to MET antibody detection following incubation and GSH pull-down. Since the MET antibody also recognizes the GST-MET fusion protein, two bands were seen on the Western blot, and the larger GST-MET band was far more intense, as expected. However, a band of the appropriate size for MET (79 kDa) was also evident. Therefore, MET-MET homodimers can form from different fusion proteins for MET and detected with different immunoprecipitation techniques (Godlewski, 2006).

When JH III was included for a 15-min preincubation, far less MET-MET was detected, similar to its effect on MET-GCE heterodimer formation. Therefore, MET homodimers can form in S2 cells, and they are also sensitive to the presence of JH III (Godlewski, 2006).

It was of interest to examine the effect of mutations of Met on the MET-GCE interaction. A variety of types of mutants were constructed, including severe truncations of Met, 10-amino acid deletions in conserved domains, and finally point mutations in each of the conserved domains. S2 cells were transfected with each mutant together with gce. Following incubation, GSH pull-down and V5 antibody detection of GCE were carried out to detect interaction (Godlewski, 2006).

Neither the truncated halves of MET nor the mutants with deletions in the bHLH and PAS-A domains interacted with GCE. The presence of JH III had no effect on the failure of interaction, as expected. However, Met mutants having a point mutation in either the bHLH (Met³) or PAS-A (Met¹) domain expressed MET proteins capable of heterodimerization with GCE, and the presence of JH III greatly diminished the interaction. The Met¹²⁸ mutant having an R433G point mutation in the PAS-B region showed a slight, almost undetectable interaction. So, it appears that severe mutations in Met as well as the R433G mutation in the PAS-B domain can block heterodimer formation or stability, but certain point mutations in the bHLH or PAS-A domains do not affect it (Godlewski, 2006).

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. This study, analyzing mosquito Met, shows that a transcriptional coactivator of the ecdysteroid receptor complex, FISC (Drosophila homolog: Taiman), acts as a functional partner of Met in mediating JH-induced gene expression. Mosquito 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 (Taiman) 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 (Li, 2011).

Genetic studies have shown that Met is required for proper expression of JH target genes in fruit flies, red flour beetles, and mosquitoes. Although the protein structure of Met suggests that it may act as a JH-activated transcriptional regulator, the binding of Met to JH-responsive promoters has not been definitively demonstrated so far. In this study, a chromatin immunoprecipitation experiment indicated that Met was indeed associated with the early trypsin promoter when this gene was activated by endogenous juvenile hormone in the newly emerged adult female mosquitoes. This is a unique demonstration of Met directly regulating a JH target gene (Li, 2011).

To elucidate the molecular roles of Met in JH signaling, a number of proteins have been tested in vitro or in the cultured insect cells for their abilities to bind Met. The protein interactions with Met were largely independent of the presence of JH, or even repressed by JH. Using a library screening approach, a mosquito bHLH-PAS protein (FISC) was identified that binds to Met in a JH-dependent manner. EMSA and ChIP experiments have demonstrated that the Met-FISC complex forms in vivo and binds to a JH-regulated promoter in previtellogenic mosquitoes only in the presence of high titers of juvenile hormone. This observation is consistent with the RNAi results showing that both Met and FISC are required in adult mosquitoes for activation of JH target genes, such as AaET and AaKr-h1. The GBD-Met fusion (without the GAD-FISC fusion) activated the UASx4-188-cc-Luc reporter gene after the JH treatment. This activation also relied on the endogenous Taiman protein in the L57 cells; the JH induction was severely dampened when Taiman was depleted by RNAi. Formation of the Met-FISC complex thus constitutes a key step in signal transduction of juvenile hormone. It is also worth noting that not all of the JH target genes are affected by RNAi knockdown of Met or FISC, implying that JH might act through several distinct pathways even in a single tissue at a particular developmental stage (Li, 2011).

Transient transfection and gel shift assays indicated that Met-FISC activated the AaET promoter by binding to the JHRE. It is currently under investigation whether the two proteins are directly binding to the JHRE or are recruited to the JHRE via protein interaction with other transcription factors. Because of the relative large sizes of the two proteins, it is difficult to obtain full-length and functional recombinant Met and FISC proteins. EMSA experiments using in vitro-synthesized proteins turned out to be problematic, because both rabbit reticulocyte lysate and wheat germ extract displayed high background binding to the labeled JHRE. A preliminary study showed that the JH-induced transcriptional activation by Met-FISC was completely abolished in cell transfection assays if the DNA binding domain (bHLH region) of either Met or FISC was truncated. However, the possibility cannot be ruled out that the bHLH regions are also required for interactions with other proteins (Li, 2011).

A distal regulatory region of AaET was also shown to be indispensable for JH-dependent activation of the AaET promoter. Intriguingly, when four copies of JHRE were placed upstream of the minimal promoter (TATA box) of AaET, the JHRE seemed to be sufficient for the Met-FISC mediated JH activation. This discrepancy implies that regulation of JH target genes is more sophisticated than the binding of Met-FISC to JHRE. More studies are needed to elucidate the underlying molecular mechanisms (Li, 2011).

In vitro experiments have shown that Met can bind to both EcR and USP, two components of the ecdysteroid receptor. This study found that FISC, a coactivator of the EcR/USP, also binds to Met and plays an important role in juvenile hormone signaling. Whether these protein interactions are involved in the crosstalk of ecdysone and JH signaling is awaiting further experimental evidence. Because the binding of FISC to EcR/USP and Met relies on the presence of 20-hydroxyecdysone and juvenile hormone respectively, the shuffling of FISC between the two signaling pathways may account for the antagonistic actions of these two hormones (Li, 2011).

A sequence similar to the AaET JHRE is also found in the promoter region of AaJHA15, another JH-regulated gene in adult female mosquitoes. The common motif 2 discovered in a group of JH-activated Drosophila promoters also shares high sequence similarity with the AaET JHRE, suggesting an evolutionarily conserved mechanism underneath the JH-induced transcriptional activation. Indeed, the Drosophila Met and Taiman activated the 4×JHRE-luc reporter gene in a JH-dependent manner. Although DmMet-AaFISC appeared comparable to DmMet-DmTAI in mediating JH-induced gene expression, AaMet-DmTAI was completely unable to activate expression of the reporter gene after JH treatment. This observation suggests that the intricate protein interactions between Met and FISC/TAI determine the affinity of the dimers to the JHRE and/or their ability to activate transcription of the JH target genes (Li, 2011).

Unlike mosquitoes, two Met-like genes (Met and gce) exist in fruit flies. Combination of gce and Taiman also led to considerable activation of the reporter gene in response to JH. This observation is in line with a recent report showing that gce can partially substitute for Met in vivo (Baumann, 2010). It would be interesting to test next whether Met-TAI and gce-TAI preferentially bind to distinct JH responsive promoters in vivo (Li, 2011).

Mapping of the sequences directing localization of the Drosophila germ cell-expressed protein (GCE)

Drosophila melanogaster germ cell-expressed protein (GCE) belongs to the family of bHLH-PAS transcription factors that are the regulators of gene expression networks that determine many physiological and developmental processes. GCE is a homolog of D. melanogaster methoprene tolerant protein (MET), a key mediator of anti-metamorphic signaling in insects and the putative juvenile hormone receptor. Recently, it has been shown that the functions of MET and GCE are only partially redundant and tissue specific. The ability of bHLH-PAS proteins to fulfill their function depends on proper intracellular trafficking, determined by specific sequences, i.e., the nuclear localization signal (NLS) and the nuclear export signal (NES). Nevertheless, until now no data has been published on the GCE intracellular shuttling and localization signals. Confocal microscopy analysis was performed of the subcellular distribution of GCE fused with yellow fluorescent protein (YFP) and YFP-GCE derivatives which allowed characterization of the details of the subcellular traffic of this protein. GCE was shown to possess specific pattern of localization signals, only partially consistent with presented previously for MET. The presence of a strong NLS in the C-terminal part of GCE, seems to be unique and important feature of this protein. The intracellular localization of GCE appears to be determined by the NLSs localized in PAS-B domain and C-terminal fragment of GCE, and NESs localized in PAS-A, PAS-B domains and C-terminal fragment of GCE. NLSs activity can be modified by juvenile hormone (JH) and other partners, likely 14-3-3 proteins Greb-Markiewicz, 2015).

EFFECTS OF MUTATION

The Methoprene-tolerant (Met) gene product in Drosophila melanogaster facilitates the action of juvenile hormone (JH) and JH analog insecticides. The gene as a member of the bHLH-PAS family of transcriptional regulators. A Met(+) cDNA was expressed in Escherichia coli, and polyclonal antibody was prepared against the purified protein. A single band on a Western blot at the expected size of 79kD was detected in extracts from Met(+) larvae but not from Met(27) null mutant larvae, demonstrating the antibody specificity. Antibody detected MET in all stages of Drosophila development and showed tissue specificity of its expression. MET is present in all cells of early embryos but dissipates during gastrulation. In larvae it is present in larval fat body, certain imaginal cells, and immature salivary glands. In pupae it persists in fat body cells and imaginal cells, including abdominal histoblast cells. In adult females MET is present in ovarian follicle cells and spermathecae; in adult males it is present in male accessory gland and ejaculatory duct cells. In all of these tissues MET is found exclusively in the nucleus. Some of these tissues are known JH target tissues but others are not, suggesting either the presence of novel JH target tissues or another function for MET (Pursley, 2000).

EVOLUTIONARY HOMOLOGS

The Met mutant of Drosophila melanogaster is highly resistant to juvenile hormone III (JH III) or its chemical analog, methoprene, an insect growth regulator. Five major mechanisms of insecticide resistance were examined in Met mutant and susceptible Met+ flies. These two strains showed only minor differences when penetration, excretion, tissue sequestration, or metabolism of [3H]JH III was measured. In contrast, when JH III binding by a cytosolic binding protein from a JH target tissue was examined, Met mutant strains had a 10-fold lower binding affinity than did Met+ strains. Studies using deficiency-bearing chromosomes provide strong evidence that the Met locus controls the binding protein characteristics and may encode the protein. These studies indicate that resistance in Met flies results from reduced binding affinity of a cytosolic binding protein for JH III (Shemshedini, 1990b).

To determine if prolonged larval exposure to juvenile hormone (JH) could influence the decision to metamorphose, Drosophila larvae were reared from hatching on medium containing either of the JH mimics, methoprene or 2-[1-methyl-2-(4-phenoxyphenoxy)-ethoxy]-pyridine (S31183). The latter was 23 times more active as a JH mimic in the white puparial assay (ED50 = 0.22 pmole). Larval development and pupariation were unaffected except at 1000 ppm methoprene and 10 ppm or higher S31183 where larval life was prolonged with increased mortality in the second instar. Adult eclosion was prevented by concentrations greater than 1 ppm methoprene and 0.1 ppm S31183. At low concentrations only adult abdominal development was affected, but at the higher concentrations an increasing percentage was blocked at the pupal stage. This latter effect was considerably diminished when the treatment was begun in the mid second instar. The methoprene-resistant mutations, Met1 and Met2, were 10 and 6 times more resistant to S31183 in the white puparial assay and about 20 times more resistant in the larval feeding experiments than the wild-type, indicating that the effects seen are typical of JH. These studies suggest that excess JH may affect adult development of imaginal structures if present at the onset of postembryonic cell proliferation of the imaginal discs or histoblasts. Thus, commitment for adult differentiation must occur early during this proliferative phase (Riddiford, 1991).

The Methoprene-tolerant (Met) mutation of Drosophila results in a high (100-fold) level of resistance to the insecticide methoprene, a chemical analog of juvenile hormone. Pest species that are under control with methoprene may therefore have the potential to evolve resistance via a mutation homologous to Met. To evaluate the potential of such mutants to persist in wild populations, the fitness of flies carrying Met must be understood. In the absence of methoprene, Met flies were outcompeted by a wild-type strain both in a multigeneration population cage and in single-generation competition experiments. To determine which fitness component(s) is responsible for the competitive disadvantage, the survival, time of development, and fecundity of flies homozygous for each of five Met alleles were compared with wild type. Small but significant differences were found between the pooled Met alleles and wild type for pupal development time, pupal mortality, and early adult fecundity. These differences result in a large competitive disadvantage. Although Met flies were found to have reduced fitness by these measures, the phenotype is not as severe as might be expected from a knowledge of the disruption of juvenile hormone regulation seen in Met flies. It is concluded that (1) although Met flies have a large advantage under methoprene selection, they will quickly become outcompeted upon relaxation of methoprene usage, (2) even a seemingly severe disruption of juvenile hormone regulation has no drastic effect on the vital functions of the insect and (3) small differences in fitness components can translate into a large competitive disadvantage (Minkoff, 1992).

Juvenile hormone (JH) is an important regulator of insect development that, by unknown mechanisms, modifies molecular, cellular, and organismal responses to the molting hormone, 20-hydroxyecdysone (20E). In dipteran insects such as Drosophila, JH or JH agonists, administered at times near the onset of metamorphosis, cause lethality. This study tested the hypothesis that the JH agonist methoprene acts by interfering with function of the Broad Complex (BRC), a 20E-regulated locus encoding BTB/POZ-zinc finger transcription factors essential for metamorphosis of many tissues. Methoprene, administered by feeding or by topical application, was found to disrupt the metamorphic reorganization of the central nervous system, salivary glands, and musculature in a dose-dependent manner. Methoprene phenocopies a subset of previously described BRC defects; it also phenocopies Deformed and produces abnormalities not associated with known mutations. Interestingly, methoprene specifically disrupts those metamorphic events dependent on the combined action of all BRC isoforms, while sparing those that require specific isoform subsets. Thus, these data provide independent pharmacological evidence for the model, originally based on genetic studies, that BRC proteins function in two developmental pathways. Mutations of Methoprene-tolerant (Met), a gene involved in the action of JH, protect against all features of the 'methoprene syndrome'. These findings have led to the proposal of novel alternative models linking BRC, juvenile hormone, and MET (Restifo, 1998).

Genetic changes in insects that lead to insecticide resistance include point mutations and up-regulation/amplification of detoxification genes. This study reports a third mechanism, resistance caused by an absence of gene product. Mutations of the Methoprene-tolerant (Met) gene of Drosophila result in resistance to both methoprene, a juvenile hormone (JH) agonist insecticide, and JH. Previous results have demonstrated a mechanism of resistance involving an intracellular JH binding protein that has reduced ligand affinity in Met flies. A gamma-ray induced allele, Met27, completely lacks Met transcript during the insecticide-sensitive period in development. Although Met27 homozygotes have reduced oogenesis, they are viable, demonstrating that Met is not a vital gene. Most target-site resistance genes encode vital proteins and thus have few mutational changes that permit both resistance and viability. In contrast, resistance genes such as Met that encode nonvital insecticide target proteins can have a variety of mutational changes that result in an absence of functional gene product and thus should show higher rates of resistance evolution (Wilson, 1998).

Pleiotropic effects of methoprene-tolerant (Met), a gene involved in juvenile hormone metabolism, on life history traits in Drosophila melanogaster

Life history theory assumes that there are alleles with pleiotropic effects on fitness components. Although quantitative genetic data are often consistent with pleiotropy, there are few explicit examples of pleiotropic loci. The Drosophila melanogaster gene Methoprene-tolerant(Met) may be such a locus. The Met gene product, a putative juvenile hormone receptor, facilitates the action of juvenile hormone (JH) and JH analogs; JH affects many life history traits in arthropods. Quantitative complementation was used to investigate effects of Met mutant and wildtype alleles on female developmental time, onset of reproduction, and fecundity. Whereas the alleles did not differ in their effects on developmental time, allelic variation was detected for the onset of reproduction and for age-specific fecundity. Alleles influenced phenotypic covariances among traits (developmental time and onset of reproduction; onset of reproduction and both early and late fecundity; early and late fecundity), suggesting that alleles of Met vary in their pleiotropic effects upon life history. Furthermore, the genetic covariance between developmental time and early fecundity attributed to alleles of Met was negative, indicating consistent pleiotropic effects among alleles on these traits. The allelic effects of Met support genetic models where pleiotropy at genes associated with hormone regulation can contribute to the evolution of life history traits (Flatt, 2004).

The main finding of this study is that allelic variation at Met affects multiple life history traits. First, it was found that Met alleles differed significantly in their effects on the onset of reproduction. The difference between the tester and control cross (t-c), a measure of the heterozygous allelic effect, depends on the allele and is either positive (t>c), negative (t<c), or zero (t=c). This suggests that some alleles delay the onset of reproduction, others shorten it, and still others do not affect this trait (Flatt, 2004).

Although JH titers or JH binding affinities of the MET proteins for the Met strains used in these experiment were not measured, evidence suggests that alleles of Met specifically differ in their binding affinities for JH, but not in other aspects of JH metabolism (Shemshedini, 1990a; Shemshedini, 1990b). Consequently, it is believed that the phenotypic variation and covariation among Met alleles observed in this experiment may, at least partially, be explained by variation in binding affinity of MET for juvenile hormone. Variation in binding affinities may have similar phenotypic consequences as variation in JH signaling, for example as differences in juvenile hormone titers. Clearly, this remains to be tested in future work. Yet, the interpretation of the results is consistent with physiological models of JH action (Flatt, 2004).

For instance, Manning (1967) found that adult D. melanogaster show an earlier onset of reproduction when JH-producing corpora allata are injected at the pupal stage, suggesting that JH regulates the onset of reproduction. Similarly, it has been suggested that the early sexual maturation of the aa (abnormal abdomen, ur^aa) genotype of Hawaiian Drosophila mercatorum is due to reduced JHE activity, leading to an unusually high JH titer (Templeton, 1978; DeSalle, 1986). Recent evidence also indicates that JH regulates the onset and termination of reproductive diapause in D. melanogaster (Tatar, 2001a; Tatar, 2001b). Most interestingly, however, Wilson (1998) found that females homozygous for the Met null allele (wv Met²⁷) genotype lag behind wildtype females for the onset of oviposition. This defect can be rescued by a transgene carrying an ectopic wildtype allele, suggesting that the effect on the onset of oviposition is specific to the Met locus. Thus, this study confirms that allelic variation at Met can affect the onset of reproduction, presumably through genetically caused variation in JH metabolism. In particular, it has been shown that wildtype alleles show detectable allelic variation for this trait (Flatt, 2004).

Quantitative complementation revealed that Met alleles differ in their effects on early, late, and total fecundity. Again, some alleles seemed to decrease components of age-specific fecundity while others seemed to increase them. Given that JH (or methoprene) regulates oogenesis by stimulating vitellogenesis (e.g., Wilson, 1983; Salmon, 2001), allelic variation for JH-binding is expected to lead to variation in fecundity. In particular, the current results are consistent with previous work by Minkoff (1992) and Wilson (1998), showing that Met specifically affects early fecundity. For instance, Wilson (1998) demonstrated that the strong ovipositional defect observed in wv Met²⁷ null mutants can be specifically rescued using a wild-type transgene. This ovipositional defect has been shown to be caused by a reduced number of vitellogenic oocytes (Wilson, 1998), consistent with the role of JH in vitellogenic oocyte development. This study has shown that, for a broad range of alleles, variation at Met not only affects early fecundity, but also late fecundity, suggesting that the effects of the Met locus are not restricted to early life. Thus, Met is clearly implied in having specific effects on several age-specific components of fecundity, probably because of its role in JH metabolism (Flatt, 2004).

Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between Methoprene-tolerant and Broad-Complex

Juvenile hormone (JH) regulates insect development by a poorly understood mechanism. Application of JH agonist insecticides to Drosophila melanogaster during the ecdysone-driven onset of metamorphosis results in lethality and specific morphogenetic defects, some of which resemble those in mutants of the ecdysone-regulated Broad-Complex (BR-C). The Methoprene-tolerant (Met) bHLH-PAS gene mediates JH action, and Met mutations protect against the lethality and defects. To explore relationships among these two genes and JH, double mutants were constructed between Met alleles and alleles of each of the BR-C complementation groups: broad (br), reduced bristles on palpus (rbp), and 2Bc. Defects in viability and oogenesis were consistently more severe in rbp Met or br Met double mutants than would be expected if these genes act independently. Additionally, complementation between BR-C mutant alleles often fail when MET is absent. Patterns of BRC protein accumulation during metamorphosis reveals essentially no difference between wild-type and Met-null individuals. JH agonist treatment does not block accumulation of BRC proteins. It is proposed that MET and BRC interact to control transcription of one or more downstream effector genes, which can be disrupted either by mutations in Met or BR-C or by application of JH/JH agonist, which alters MET interaction with BRC (Wilson, 2005).

Met is essential for the manifestation of the toxic and morphogenetic effects of JH/JHA in Drosophila (Wilson, 1986; Riddiford, 1991; Wilson, 1996; Restifo, 1998). Met mutants are resistant to these effects of methoprene (Wilson, 1986). MET can bind JH III with specificity and nanomolar affinity (Shemshedini, 1990a; Miura, 2005), suggesting that it is a component of a JH receptor. Met encodes a bHLH-PAS transcriptional regulator family member (Ashok, 1998) and MET can activate a reporter gene in transfected Drosophila S-2 cells (Miura, 2005; Wilson, 2005 and references therein).

Evidence was found for interaction between Met and BR-C as reflected by synergistically reduced viability and oogenesis seen in double mutants. Consistent results were seen with different combinations of Met and br or Met and rbp alleles, indicating that the interactions are not allele-specific in either direction. Met interacts with both the weak viable alleles br¹ and rpb² as well as the severe alleles br⁵ and rbp¹ during pupal development. Each of the weak alleles possesses sufficiently functional gene product to permit completion of pupal development; but this amount is insufficient when MET is absent or defective. The more severe rbp¹ homogygotes are pupal-lethal, but only at late metamorphosis, in the pharate adult stage. Lethality was shifted in rbp¹ Met²⁷ pupae to prepupal/early pupal development, suggesting that MET absence causes the rbp¹ product to be inadequate during these earlier stages in pupal development. Homozygotes of br⁵ and 2Bc die in the early and late prepupal stage, respectively, and the double mutants with Met²⁷ show a similar phenotype, demonstrating that the interaction cannot shift lethality to an earlier stage, late third-instar larvae. These observations are consistent with the interaction between BR-C and Met beginning in prepupal or early pupal development. While the Met-BR-C interaction is interpreted as enhancing the lethality of br and rbp mutations, it is also possible that Met becomes an essential gene when BR-C function is reduced, or that the interaction is mutual, such that both mutations become more severe in phenotype when they are present together (Wilson, 2005).

Genetic interaction became strikingly evident when complementation failures between mutant alleles from different BR-C complementation groups occurred in the presence of Met²⁷. Without MET, developing animals may be less able to make use of the partial functional redundancy among BRC isoforms. The interaction between mutant alleles of BR-C and Met is also evident in the adult stage when oogenesis is examined. Both the rate of oviposition and the paucity of vitellogenic oocytes in ovaries of br¹ Met²⁷ and rpb² Met²⁷ females reflects almost complete failure of oogenesis, with only a few eggs oviposited during the lifetime of the female (Wilson, 2005).

Previous studies have also detected BR-C interaction with other genes. Double mutants ofBR-C with another primary response gene, E74, show interaction for some but not all of the phenotypic characters. In addition to interactions among transcription regulators of the ecdysone cascade, br alleles interact with genes involved in imaginal disc morphogenesis, including those encoding an atypical serine protease, Stubble-stubboid, non-muscle myosin II heavy chain (Zipper), the Drosophila serum response factor transcription factor (Blistered), the small GTPase Rho1, cytoplasmic tropomyosin and 22 others. Although BR-C expression and function overlap the JH/JHA-sensitive period, methoprene treatment does not block BRC expression in either wild-type or Met null mutants. Furthermore, the methoprene phenocopy, which excludes complementation group-specific defects (e.g., larval salivary gland persistence, which is rbp-restricted), is not consistent with methoprene simply reducing BRC expression. It is proposed that JH application results in abnormal function of BRC proteins, thus phenocopying certain characteristics common to all BR-C mutants. Therefore, the link between BR-C mutant phenotypes and JH-induced defects could be abnormal regulation of target genes, resulting in the phenotypic characteristics observed. Several possibilities have been suggested to explain methoprene pathology and BR-C phenocopy, including BRC interaction with an unidentified partner, perhaps MET (Restifo 1998). It is believed that the Met-BR-C genetic interaction can be explained best by this hypothesized protein-protein interaction between MET and BRC to regulate one or more target genes. Supporting this hypothesis are the following findings: first, both proteins are located in the nucleus, so there is no compartmental barrier to interaction. Second, both proteins appear to be transcription factors: BRC isoforms bind specific DNA sequences and regulate transcription. BR-C mutants have misexpressed secondary-response and other target genes. MET is a member of the bHLH-PAS family of transcription factors (Ashok, 1998) and was recently shown to act as one (Miura, 2005). Third, both are found at common times during development, such as prepupae and during vitellogenic oocyte development. Finally, PAS domains in bHLH-PAS proteins are thought to promote protein-protein interaction, either with other PAS proteins or as coactivators with nuclear receptor proteins, and the BTB/POZ domain of BRC has been implicated in proteinprotein interaction (Wilson, 2005 and reference therein).

In Met²⁷ mutants, BRC protein accumulation profiles are normal. Since metamorphosis is not derailed in Met²⁷ pupae, then BRC⁺ function in these pupae does not seem to be adversely affected. The fly may be protected from absence of MET by functional redundancy (Wilson, 1998). A candidate for the redundant substitute is the PAS gene germ cell expressed (gce), a gene with high (~70% amino acid identity) homology to Met that could substitute for MET to rescue larval and/or pupal development. However, this substitute does not appear to be satisfactory if BR-C is mutant. When a gce mutant becomes available, its phenotype could help evaluate this hypothesis (Wilson, 2005).

How does the application of exogenous JH act to phenocopy BR-C? It is clear that the action of these compounds occurs through MET, probably acting as a JH receptor component. JH is present during larval development when it presumably acts to prevent premature metamorphosis resulting from each wave of 20E secretion that triggers a molt. This fail-safe mechanism may occur by JH binding by and conformational change of MET, resulting in regulation of genes necessary for molting or perhaps simply blocking expression of metamorphic genes. Studies with Drosophila S-2 cells have implicated the transcription factor E75A in promoting JH regulation of larval development. At metamorphosis, when little or no JH is present, BR-C is expressed, and it is proposed that BRC dimerizes with the non-liganded MET protein to regulate a different set of target genes, promoting the initiation of metamorphosis. If exogenous JH is present during this time, it binds to MET and results in a more larval conformation, resulting in inappropriate binding to BRC and leading to a change in target gene expression patterns consequently seen as defects characteristic of BR-C mutants (Wilson, 2005).

Other work has implicated BR-C in the action of the JH agonist pyriproxyfen during metamorphic disruption. Application of this compound to white prepupae results in re-expression of BRC-Z1 in the abdomen during late pupal development, which in turn causes abnormal development of abdominal epidermis, including bristle disturbances. Those findings differ from those with methoprene in two significant ways. First, a lethal dose of methoprene causes a mild enhancement and prolongation of BRC protein accumulation in young pupae, but no re-expression at later times. Second, the modest effect of methoprene on BRC protein profiles cannot mediate the developmental effects of this JHA because the same mild persistence of BRC is seen in Met²⁷ mutants, that are protected against methoprene-induced defects. It is not clear what underlies the difference in response of BR-C to methoprene and pyriproxyfen. It is noted that pyriproxyfen is a more powerful JH agonist than methoprene (Riddiford, 1991), but qualitative differences in the actions of the two compounds may exist as well (Wilson, 2005).

In summary, the results provide genetic evidence that supports other studies implicating BR-C as a focal point for interaction of JH and 20E signaling pathways, and they suggest that BRC and MET interact to regulate expression of one or more effector genes involved in metamorphic development (Wilson, 2005).

A role for juvenile hormone in the prepupal development of Drosophila melanogaster

To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met²⁷ larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells 'misinterpreting' the ecdysteroid peaks that drive metamorphosis (Riddiford, 2010).

Insect molting and metamorphosis are governed primarily by ecdysone (used in the generic sense) and juvenile hormone (JH), with ecdysone causing molting and JH preventing metamorphosis. Juvenile hormone has a classic 'status quo' action in preventing the program-switching action of ecdysone during larval molts and in maintaining the developmental arrest of imaginal primordia during the intermolt periods. Its effects at the outset of metamorphosis, though, are more complex. Studies mainly on Lepidoptera show that for selected tissues JH needs to be present to allow them to undergo pupal differentiation, rather than undertaking a precocious adult differentiation (Riddiford, 2010).

The mechanism through which JH maintains the status quo and directs early development at metamorphosis is still poorly understood. Whether JH has one or multiple receptors, and the nature of these receptors, is still controversial. The best candidate for a receptor is the product of the Methoprene-tolerant (Met) gene, a PAS domain protein that was originally isolated in Drosophila melanogaster. In vitro transcribed and translated Met protein has been shown to bind JH with high affinity, and RNAi knock-down experiments in Tribolium castaneum show that Met is essential for mediating the status quo action of JH in this beetle (Riddiford, 2010).

In D. melanogaster, JH is thought to have no role in the onset of metamorphosis, since exogenous JH only delays but does not prevent pupariation. Although it has no apparent effect on the development of the imaginal discs, JH prevents normal adult development of the abdominal integument when given at pupariation. Internally, JH at this time affects normal reorganization of the central nervous system and development of the thoracic musculature. These effects of JH on metamorphosis do not occur in Met mutants, unless at least 100 times the dose is given. The Met²⁷-null mutants proceed through larval development and metamorphosis apparently normally. However, if in addition, RNAi is used to suppress expression of Germ-Cell Expressed (Gce), a related bHLH protein with a high similarity to Met that heterodimerizes with it, Met-null mutants die as pharate adults. In the Met-deficient mutant, the adult eye shows a few (<12) defective ommatidia in the posterior region. Also, the females mature fewer eggs at a slower rate than do wild-type females, indicating that Met is also important for JH effects in egg maturation (Riddiford, 2010).

This study genetically allatectomized Drosophila larvae by targeting expression of a cell death gene to the corpora allata (CA), the gland that produces JH. These larvae form smaller puparia and showed precocious maturation of the visual system, but die around head eversion (Riddiford, 2010).

Although a number of studies have reported the effects of applying exogenous JH or JH mimics to Drosophila, there are only two very recent studies of the effects of manipulating endogenous JH on larval growth and metamorphosis, both of which appeared while this paper was under review. JH is normally present in the early larval instars, declines substantially during the last (third) larval stage and then returns transiently around the time of pupariation. The allatectomized (CAX) larvae undergo the expected two larval molts, but because sometimes the remains of degenerating CA cells are seen at the start of the last larval stage, nothing can be concluded about the requirements of JH for these larval molts. Recently, Jones (2010) using 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) RNAi to depress the level of JH and its farnesoid precursors in early larvae, showed that the larvae mainly die during the molt to the third instar, indicating that JH may be required for that molt (Riddiford, 2010).

The destruction of the CA by the third instar allowed examination of the role of JH during the last instar and early metamorphosis. The finding that these larvae were smaller than their CyO, UAS-grim siblings at pupariation could be explained by either the loss of JH or by the loss of the salivary glands, since these glands are also destroyed. Because dietary JH in the final instar rescued these larvae to normal size, the lack of the CA, rather than the lack of the salivary glands, is the cause of their reduced growth. Preliminary studies show that CAX larvae grow more slowly in the third instar, but the underlying basis for this retardation is not yet understood. Similarly, allatectomized third instar larvae display premature apoptosis of the fat body and downregulation of several enzymes involved in energy metabolism at the onset of wandering. These fat body effects could underlie the reduced larval growth seen in CAX larvae (Riddiford, 2010).

A major effect of the removal of JH was on the timing of events during the prepupal period. Studies on the wild silkmoth Hyalophora cecropia first showed that removal of the CA in the last larval stage resulted in the formation of a pupa with adult characteristics. Other moths, like Manduca sexta, showed more subtle responses to allatectomy, with premature adult differentiation most evident in the patterned region of the compound eye, posterior to the morphogenetic furrow. Subsequent studies on a variety of tissues in Manduca showed that the eye, the optic lobe and the ventral diaphragm each had a prolonged period of proliferation that extended from the prepupal period through early adult differentiation. This proliferation is maintained by α-ecdysone or low levels of 20-hydroxyecdysone (20E), but is terminated by high levels of 20E, which induces differentiation. These tissues are exposed to differentiation-inducing titers of 20E that occur during the larval-pupal transition early in their growth, but studies on the ventral diaphragm showed that JH 'protects' them from these high 20E levels, allowing them to continue proliferating. Removal of JH results in these tissues undergoing premature termination of tissue growth and precocious adult differentiation (Riddiford, 2010).

The response of Drosophila larvae to the loss of JH is in line with the effects seen in Manduca, and is also most evident in the developing visual system. In normal individuals, the appearance of EcR-B1 in the optic lobe and the termination of proliferation in the outer proliferation zone coincide with the ecdysteroid peak at head eversion and become more pronounced at 18 hours APF with the rise of ecdysteroid for adult differentiation. The separation of the R7 and R8 growth cones also begins about this latter time. The only one of these tissues that has been directly tested in vitro for 20E sensitivity is the optic lobe and in this case high levels of 20E do indeed suppress proliferation. It is assumed that the other processes also respond to the changing ecdysteroid levels. The lack of JH results in a heterochronic advance of these events by 10 to 12 hours, consistent with the tissues now responding to the earlier ecdysteroid peak that causes pupariation. Although the removal of JH advances these processes, it was found that the application of JH mimics delays them. Consequently, in selective tissues in Drosophila, JH acts to direct the nature of tissue responses to ecdysone (Riddiford, 2010).

The removal of JH or of one its receptors, Met, has a mixed effect on the developing visual system. No effect on proliferation or inductive events was seen in the eye disc itself, in that in CAX animals the morphogenetic furrow continues to move and similar rows of ommatidia have sent R8 axons into the medulla by 6 hours APF, as compared with controls. Likewise, in Met²⁷ individuals there is only a slight advance (about 2 hours) in the schedule of lamina interneuron ingrowth into the medulla. However, for some of the cellular and molecular events, like the appearance of EcR-B1 and the separation of R7 from R8, there is a 10- to 18-hour advance in their occurrence. Hence, the lack of JH or of its receptor Met causes a heterochronic shift within the developing visual system with some differentiation responses being advanced relative to the normal schedule of neuronal birth and axon ingrowth. At least in the case of the photoreceptors, the effect of Met removal is largely cell autonomous, with the reduction of Met function in just those cells being sufficient to cause the precocious appearance of EcR-B1 and the early separation of R7 from R8. By contrast, the reduction of Met in lamina interneurons allowed these cells to precociously express EcR-B1 but did not affect the behavior of the R7 and R8 growth cones. This suggests that the separation of R7 and R8 is an active response of the photoreceptors, which is likely to be caused by the rising ecdysteroid titer driving adult differentiation. Although the lack of JH or Met function at the outset of metamorphosis results in the cell-autonomous expression of EcR-B1 in the photoreceptors, misexpression experiments show that the appearance of this receptor alone is not sufficient to bring about the early separation of R7 and R8. Therefore, although the upregulation of EcR-B1 is a prominent response to rising ecdysteroid titers, it is not the key change responsible for the repositioning of the receptor terminals (Riddiford, 2010).

As it is viable, the Met mutant allowed the final results of the mistiming of development in the optic lobes to be seen. No permanent effect was seen of the early separation of the R7 and R8 growth cones on the final anatomy of these projections in the medulla, or on the structure of the later neuropil. However, the lobula was grossly distorted and the normal layering of dendritic arbors disrupted. This aberrant morphogenesis also starts early, being already evident by 12 hours APF. The cellular basis for the lobula distortion, however, is not yet known (Riddiford, 2010).

Heterochronic shifts in the timing of development that extend beyond the visual system are likely to be the cause of the lethality seen in the CAX puparia. Puparia appear normal through the first 6 to 7 hours after pupariation but then abruptly undergo tissue collapse. In normal flies, the early part of metamorphosis is accomplished by a complicated replacement of histolyzing larval tissues by the growing adult tissues. Diverse tissues show individualized times of histolysis that are tied to the ecdysteroid titer. For instance, the larval midgut cells degenerate in response to the pupariation peak of ecdysone, whereas the larval salivary gland degeneration is triggered by the small rise of ecdysteroid at the end of the prepupal period. It is suspected that without JH, some of the histolysis events are mistimed, leading to the rapid death of the prepupa. It has been shown in CAX larvae that the fat body undergoes precocious programmed cell death beginning in the third larval instar. Interestingly, this lethal effect was not seen in animals in which Aug21-GAL4 drove RNAi for JH acid O-methyltransferase, the enzyme that converts JH acid to JH, in the CA (Niwa, 2008). Whether this indicates that JH acid plays a role in prepupal development or merely reflects the incomplete loss of JH in these animals is unknown (Riddiford, 2010).

All these effects of allatectomy can be rescued by JH either fed during the third instar or applied at the time of early wandering, but not at pupariation. A decline of JH III occurs in the third instar; this is followed by a peak of JH during late wandering. When JH begins to rise is unknown, as measurements were made every 24 hours. Presumably it is the lack of this JH during wandering when the ecdysteroid titer is rising and peaking that leads to the optic lobe anomalies and the premature histolysis (Riddiford, 2010).

The finding that the Met²⁷ null mutant has the same defects in optic lobe development as are found in CAX prepupae strongly suggests that JH is acting via the Met pathway in controlling the timing of some events in the optic lobe. Accordingly, JHM treatment cannot suppress most of the premature development seen in prepupae lacking Met. However, a major difference between the CAX animals and the Met²⁷ mutants is that the CAX prepupae died before head eversion, whereas the Met²⁷ animals are viable. This difference is also seen in the precocious cell death of the fat body caused by allatectomy, which does not occur in the Met null mutant even in the presence of gce RNAi. Instead precocious cell death of the fat body was seen when Met was overexpressed in that tissue and the death could be suppressed by exogenous methoprene (a JH mimic). This latter finding suggests that JH would act in this case to suppress Met-mediated cell death. This idea was tested by seeing whether the removal of Met would protect the prepupa from the death caused by early allatectomy. When Met²⁷; Aug21-GAL4>UAS-GFP/CyO females were crossed with UAS-grim males, 44% eclosed, all showing the CyO phenotype. The remainder died at head eversion, and should have been half CAX, Met-heterozygous females and half CAX, Met-null males. Another group was separated by sex prior to pupariation. Forty-nine percent of the females and 48% of the males died at head eversion. All of the adults that emerged were CyO, showing that all the CAX prepupae died regardless of whether or not they were lacking Met function (Riddiford, 2010).

These results together with the findings that JHM treatment of the Met²⁷ mutant gave a partial rescue of the premature separation of R7 and R8, and of the decreased proliferation in the inner proliferation zone, indicate that there may be more than one receptor for JH. Thus, JH might act through multiple pathways. A major pathway involves Met, but Gce or some other mediator may serve as an alternate pathway in some tissues. A similar protective role of JH at pupation mediated by Met is found in Tribolium; injection of Met RNAi into either fourth instar larvae or final instar larvae caused the precocious appearance of adult eyes, adult antennae and other features in the resulting pupae (Riddiford, 2010).

These studies show that JH has an endogenous function in regulating Drosophila metamorphosis, a specific example being in orchestrating the timing of differentiation events in the developing visual system. These effects of JH are primarily mediated through the Met pathway. JH also is necessary for normal larval growth and has another, as yet undefined, crucial role in prepupal development that prevents death at head eversion. The latter effect is not mediated through Met, indicating that JH might act through multiple pathways (Riddiford, 2010).

Paralogous genes involved in juvenile hormone action in Drosophila melanogaster

Juvenile hormone (JH) is critical for multiple aspects of insect development and physiology. Although roles for the hormone have received considerable study, an understanding of the molecules necessary for JH action in insects has been frustratingly slow to evolve. Methoprene-tolerant (Met) in Drosophila melanogaster fulfills many of the requirements for a hormone receptor gene. A paralogous gene, germ-cell expressed (gce), possesses homology and is a candidate as a Met partner in JH action. Expression of gce was found to occur at multiple times and in multiple tissues during development, similar to that previously found for Met. To probe roles of this gene in JH action, in vivo gce over- and underexpression studies were carried out. Overexpression studies showed that gce can substitute in vivo for Met, alleviating preadult but not adult phenotypic characters. RNA interference-driven knockdown of gce expression in transgenic flies results in preadult lethality in the absence of MET. These results show that (1) unlike Met, gce is a vital gene and shows functional flexibility and (2) both gene products appear to promote JH action in preadult but not adult development (Baumann, 2011).

Is the rescue due to an abundance of GCE or to expression in tissue not normally expressing endogenous gce? To address the issue of the tissue specificity of Met/gce expression, recent results were used that demonstrated that larval fat body catabolism, required for completion of metamorphosis, is initiated by ecdysone, MET, and GCE and can be blocked by JH application. Perhaps high pupal survival of Met mutants following methoprene application results from an absence of methoprene-induced blockage of catabolism and the lowered pupal survival in Met²⁷; gce transgenic flies results from substitution of GCE for the absent MET. Overexpression of GCE specifically in larvae fat body was carried out using a larval fat body GAL4 driver, and resistance to methoprene-induced mortality and male genitalia malrotation was examined. Methoprene-treated Met²⁷; UAS-gce/lfb-GAL4 was found to be completely (50/50 examined) resistant to the male genitalia defect, indicating no blockage of the Met²⁷ mutation. However, the progeny were only partially resistant to pupal death, showing that GCE can partially substitute for MET in this tissue to block the Met²⁷ mutation and suggesting that tissue-specific, not widespread gce overexpression, may underlie the basis of the GCE substitution effect. Since the larval fat body shows little or no gce, then supplying GCE to this tissue can explain the tissue-specific effect seen in the pupal death phenotype. The eye phenotype was not rescued by larval fat body promoter-driven GCE, but it was completely rescued by compound-eye promoter-driven GCE. Therefore, GCE expressed in the larval fat body can partially substitute for MET in the tissue(s) responsible for pharate adult death, but not for eye or male genitalia, demonstrating tissue specificity of expression or utilization of GCE/MET (Baumann, 2011).

Little effect of gce overexpression was found on adult reproductive phenotypes of Met²⁷. JH plays roles in both male and female reproduction in D. melanogaster as well as in many other insects. Clearly, overexpressed GCE can substitute for MET in Met preadults, but since the adult transgenic fly reproductive phenotypes were similar to those in adults not overexpressing the gce transgene, this substitution appears to be unproductive in adults. This result suggests that MET may be the major player in adults. The presence of only a single Met/gce homolog in three mosquito species and in the beetle T. castaneum with higher similarity to gce than to Met suggests that this reproductive role for MET evolved following the gene duplication seen in higher Diptera. Both Met and gce are present in the 12 species of D. melanogaster whose genomes have been sequenced; therefore, this duplication occurred earlier than the evolutionary divergence of these species that occurred as much as 60 million years ago (Baumann, 2011).

Clearly, gce underexpression can be lethal to either larvae or pupae, especially pupae, and especially in the absence of Met⁺. The presence of background Met⁺ allowed greater pupal development, shifting pupal death in RNAi individuals from the early pupal stage to the pharate adult/adult stage, depending on the promoter used and thus presumably on the level of RNAi produced. Underexpression of gce in the absence of Met⁺, in a Met²⁷background, is more severe and results in a total loss of preadult viability. This suggests that both GCE and MET can interact to promote some vital aspect of larval/pupal development. Previous work using D. melanogaster S-2 cells has shown that MET and GCE can heterodimerize, suggesting a mechanistic basis for MET-GCE interaction. However, underexpression driven by the stronger tubulin promoter results in pupal death in a Met⁺ background, so the absence of Met⁺ is not a prerequisite for this phenotype. If both MET and GCE are necessary for JH action, then underexpression of both genes could result in death due to a failure of some JH-controlled developmental event, for example. The greater phenotypic severity of gce underexpression in a mutant Met background argues for the JH action scenario (Baumann, 2011).

Tissue-specific expression levels of Met and gce are given for 24 larval and adult tissues in the FlyAtlas database, determined by microarray analysis. The data show robust expression for gce in 12 tissues and for Met in 10 tissues. Seven tissues showed robust expression of both genes; interestingly, none are demonstrated JH target tissues. This could mean that (1) the presence of both GCE and MET are not required for JH action, (2) only one is required, or (3) few JH target tissues have been identified (perhaps the more likely explanation). However, there are some surprises in the FlyAtlas data set; for example, neither gene showed good expression in ovary or larval fat body, and only gce showed strong expression in male accessory glands, all demonstrated JH target tissues. Possibly, additional regulatory roles, independent of JH, for either of these transcription factors exist and are reflected in the FlyAtlas data set (Baumann, 2011).

Does this work show MET and GCE involvement in a JH receptor complex? Although the phenotypic characteristics of Met suggest involvement in JH reception, there is no direct evidence for involvement in a bona fide receptor. The disparate levels of transcript found for Met and gce in certain tissues might suggest separate roles for either or both of the gene products in certain tissues, and not formation of a mandatory heterodimer that might be predicted for a JH receptor. Indeed, one of the roles might involve eye development, which can be disrupted when either Met is mutated. Likewise, the lack of substantial resistance to methoprene in the Met⁺; UAS-gce dsRNA/actin-GAL4 flies might seem perplexing, considering the high resistance seen in Met mutants, but this conundrum might simply reflect a lack of strong JH binding by GCE, a possible requirement for resistance to the hormone and insecticide. Ligand binding might be the sole property of MET in a MET-GCE heterodimer, and loss of GCE does affect JH action, but not due to failure of JH binding. Future studies focusing on the role of gce may lead to more rapid progress in defining a JH receptor (Baumann, 2011).

Acp70A regulates Drosophila pheromones through juvenile hormone induction

Mated Drosophila melanogaster females show a decrease in mating receptivity, enhanced ovogenesis, egg-laying and activation of juvenile hormone (JH) production. Components in the male seminal fluid, especially the sex peptide ACP70A stimulate these responses in females. This study demonstrates that ACP70A is involved in the down-regulation of female sex pheromones and hydrocarbon (CHC) production. Drosophila G10 females which express Acp70A under the control of the vitellogenin gene yp1, produced fewer pheromones and CHCs. There was a dose-dependent relationship between the number of yp1-Acp70A alleles and the reduction of these compounds. Similarly, a decrease in CHCs and diene pheromones was observed in da > Acp70A flies that ubiquitously overexpress Acp70A. Quantitative-PCR experiments showed that the expression of Acp70A in G10 females was the same as in control males and 5 times lower than in da > Acp70A females. Three to four days after injection with 4.8 pmol ACP70A, females from two different strains, exhibited a significant decrease in CHC and pheromone levels. Similar phenotypes were observed in ACP70A injected flies whose ACP70A receptor expression was knocked-down by RNAi and in flies which overexpress ACP70A N-terminal domain. These results suggest that the action of ACP70A on CHCs could be a consequence of JH activation. Female flies exposed to a JH analog had reduced amounts of pheromones, whereas genetic ablation of the corpora allata or knock-down of the JH receptor Met, resulted in higher amounts of both CHCs and pheromonal dienes. Mating had negligible effects on CHC levels, however pheromone amounts were slightly reduced 3 and 4 days post copulation. The physiological significance of ACP70A on female pheromone synthesis is discussed (Bontonou, 2014).

REFERENCES

Search PubMed for articles about Drosophila Methoprene-tolerant & Search PubMed for articles about Drosophila gce

Abdou, M. A., He, Q., Wen, D., Zyaan, O., Wang, J., Xu, J., Baumann, A. A., Joseph, J., Wilson, T. G., Li, S. and Wang, J. (2011). Drosophila Met and Gce are partially redundant in transducing juvenile hormone action. Insect Biochem Mol Biol 41: 938-945. PubMed ID: 21968404

Ashok, M., Turner, C. and Wilson, T. G. (1998). Insect juvenile hormone resistance gene homology with the bHLH-PAS family of transcriptional regulators. Proc. Natl. Acad. Sci. U.S.A. 95: 2761-2766. 9501163

Baumann, A., et al. (2010). Paralogous genes involved in juvenile hormone action in Drosophila melanogaster. Genetics 185(4): 1327-36. PubMed Citation: 20498297

Baumann, A. A., Texada, M. J., Chen, H. M., Etheredge, J. N., Miller, D. L., Picard, S., Warner, R., Truman, J. W. and Riddiford, L. M. (2017). Genetic tools to study juvenile hormone action in Drosophila. Sci Rep 7(1): 2132. PubMed ID: 28522854

Bilen, J., Atallah, J., Azanchi, R., Levine, J. D. and Riddiford, L. M. (2013). Regulation of onset of female mating and sex pheromone production by juvenile hormone in Drosophila melanogaster. Proc Natl Acad Sci U S A 110: 18321-18326. PubMed ID: 24145432

Bontonou, G., Shaik, H. A., Denis, B. and Wicker-Thomas, C. (2014). Acp70A regulates Drosophila pheromones through juvenile hormone induction. Insect Biochem Mol Biol 56:36-49. PubMed ID: 25484200

Chertemps, T., Duportets, L., Labeur, C., Ueyama, M. and Wicker-Thomas, C. (2006). A female-specific desaturase gene responsible for diene hydrocarbon biosynthesis and courtship behaviour in Drosophila melanogaster. Insect Mol Biol 15: 465-473. PubMed ID: 16907833

Chertemps, T., Duportets, L., Labeur, C., Ueda, R., Takahashi, K., Saigo, K. and Wicker-Thomas, C. (2007). A female-biased expressed elongase involved in long-chain hydrocarbon biosynthesis and courtship behavior in Drosophila melanogaster. Proc Natl Acad Sci U S A 104: 4273-4278. PubMed ID: 17360514

DeSalle, R. and Templeton, A. R (1986). The molecular through ecological genetics of abnormal abdomen. III. Tissue specific differential replication of ribosomal genes modulates the abnormal abdomen phenotype in Drosophila mercatorum. Genetics 112: 877-886. 1311698

Flatt, T. and Kawecki, T. J. (2004). Pleiotropic effects of methoprene-tolerant (Met), a gene involved in juvenile hormone metabolism, on life history traits in Drosophila melanogaster. Genetica. 122(2): 141-60. 15609573

Godlewski, J., Wang, S. and Wilson, T. G. (2006). Interaction of bHLH-PAS proteins involved in juvenile hormone reception in Drosophila. Biochem. Biophys. Res. Commun. 342(4): 1305-11. 16516852

Greb-Markiewicz, B., Sadowska, D., Surgut, N., Godlewski, J., Zarebski, M. and Ozyhar, A. (2015). Mapping of the sequences directing localization of the Drosophila germ cell-expressed protein (GCE). PLoS One 10: e0133307. PubMed ID: 26186223

Gu, Y. Z., Hogenesch, J. B. and Bradfield, C. A. (2000). The PAS superfamily: sensors of environmental and developmental signals. Annu. Rev. Pharmacol. Toxicol. 40: 519-61. 10836146

He, Q., Wen, D., Jia, Q., Cui, C., Wang, J., Palli, S. R. and Li, S. (2014). Heat shock protein 83 (Hsp83) facilitates Methoprene-tolerant (Met) nuclear import to modulate juvenile hormone signaling. J Biol Chem 289(40):27874-85. PubMed ID: 25122763

Jindra, M., Uhlirova, M., Charles, J. P., Smykal, V. and Hill, R. J. (2015). Genetic evidence for function of the bHLH-PAS Protein Gce/Met as a juvenile hormone receptor. PLoS Genet 11: e1005394. PubMed ID: 26161662

Jones D., et al. (2010). Suppressed production of methyl farnesoid hormones yields developmental defects and lethality in Drosophila larvae. Gen. Comp. Endocrin. 165: 244-254. PubMed Citation: 19595690

Juhász, G., Puskás, L. G., Komonyi, O, Érdi, B., Maróy, P., Neufeld, T. P. and Sass, M. (2007). Gene expression profiling identifies FKBP39 as an inhibitor of autophagy in larval Drosophila fat body. Cell Death Differ. 14: 1181-1190. PubMed Citation: 17363962

Konopova, B. and Jindra, M. (2008). Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis. Development 135: 559-568. PubMed Citation: 18171683

Li, M., Mead, E. A. and Zhu, J. (2011). Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression. Proc. Natl. Acad. Sci. 108(2): 638-43. PubMed Citation: 21187375

Lin, H. H., Cao, D. S., Sethi, S., Zeng, Z., Chin, J. S., Chakraborty, T. S., Shepherd, A. K., Nguyen, C. A., Yew, J. Y., Su, C. Y. and Wang, J. W. (2016). Hormonal modulation of pheromone detection enhances male courtship success. Neuron 90: 1272-1285. PubMed ID: 27263969

Liu, Y., et al. (2009). Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila. Development 136(12): 2015-25. PubMed Citation: 19465595

Manning, A. (1967). The control of sexual receptivity in female Drosophila. Anim. Behav. 15: 239-250. 6030948

Marcillac, F. and Ferveur, J. F. (2004). A set of female pheromones affects reproduction before, during and after mating in Drosophila. J Exp Biol 207: 3927-3933. PubMed ID: 15472023

Minakuchi, C., Namiki, T. and Shinoda, T. (2009). Krüppel homolog 1, an early juvenile hormone-response gene downstream of Methoprene-tolerant, mediates its anti-metamorphic action in the red flour beetle Tribolium castaneum. Dev. Biol. 325(2): 341-50. PubMed Citation: 19013451

Minkoff, C. and Wilson, T. G. (1992). The competitive ability and fitness components of the methoprene-tolerant (Met) Drosophila mutant resistant to juvenile hormone analog insecticides. Genetics 131: 91-97. 1592245

Mirth, C. K., Tang, H. Y., Makohon-Moore, S. C., Salhadar, S., Gokhale, R. H., Warner, R. D., Koyama, T., Riddiford, L. M. and Shingleton, A. W. (2014). Juvenile hormone regulates body size and perturbs insulin signaling in Drosophila. Proc Natl Acad Sci U S A 111(19): 7018-7023. PubMed ID: 24778227

Miura, K., Oda, M., Makita, S and Chinzei, Y. (2005). Characterization of the Drosophila Methoprene-tolerant gene product. FEBS J. 272: 1169-1178. 15720391

Niwa, R., Niimi, T., Honda, N., Yoshiyama, M., Itoyama, K., Kataoka, H. and Shinoda, T. (2008). Juvenile hormone acid O-methyltransferase in Drosophila melanogaster. Insect Biochem. Mol. Biol. 38: 714-720. PubMed Citation: 18549957

Parthasarathy, R., Tan, A. and Palli, S. R. (2008). bHLH-PAS family transcription factor methoprene-tolerant plays a key role in JH action in preventing the premature development of adult structures during larval-pupal metamorphosis. Mech. Dev. 125(7): 601-16. PubMed Citation: 18450431

Pursley, S., Ashok, M. and Wilson, T. G. (2000). Intracellular localization and tissue specificity of the Methoprene-tolerant (Met) gene product in Drosophila melanogaster. Insect Biochem. Mol. Biol. 30: 839-845. 10876128

Reiff, T., Jacobson, J., Cognigni, P., Antonello, Z., Ballesta, E., Tan, K.J., Yew, J.Y., Dominguez, M. and Miguel-Aliaga, I. (2015). Endocrine remodelling of the adult intestine sustains reproduction in Drosophila. Elife 4. PubMed ID: 26216039

Restifo, L. L. and Wilson, T. G. (1998). A juvenile hormone agonist reveals distinct developmental pathways mediated by ecdysone-inducible broad complex transcription factors. Dev. Genet. 22(2): 141-59. 9581286

Riddiford, L. M. and Ashburner, M. (1991). Effects of juvenile hormone mimics on larval development and metamorphosis of Drosophila melanogaster. General Comp. Endocrinol. 82: 172-183. 1906823

Riddiford, L. M., Truman, J. W., Mirth, C. K. and Shen, Y. C. (2010). A role for juvenile hormone in the prepupal development of Drosophila melanogaster. Development 137(7): 1117-26. PubMed Citation: 20181742

Salmon, A. B., Marx, D. B. and Harshman, L. G. (2001). A cost of reproduction in Drosophila melanogaster: stress susceptibility. Evolution 55: 1600-1608. 11580019

Shemshedini, L., Lanoue, M. and Wilson, T. G. (1990a). Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster. J. Biol. Chem. 265(4): 1913-8. 2105312

Shemshedini, L. and Wilson, T. G. (1990b). Resistance to juvenile hormone and an insect growth regulator in Drosophila is associated with an altered cytosolic juvenile hormone binding protein. Proc. Natl. Acad. Sci. 87: 2072-2076. 2107540

Smykal, V., Daimon, T., Kayukawa, T., Takaki, K., Shinoda, T. and Jindra, M. (2014). Importance of juvenile hormone signaling arises with competence of insect larvae to metamorphose. Dev Biol 390: 221-230. PubMed ID: 24662045

Tatar, M., Promislow, D. E. L., Khazaeli, A. A. and Curtsinger, J. W. (1996). Age-specific patterns of genetic variance in Drosophila melanogaster. Genetics 143: 849-858. 8725233

Tatar, M. and Yin, C.-M. (2001). Slow aging during insect reproductive diapause: why butterflies, grasshoppers and flies are like worms. Exp. Geront. 36: 723-738. 11295511

Templeton, A.R. and Rankin, M. A. (1978). Genetic revolutions and the control of insect populations, pp. 83-112 in The Screw-worm Problem, edited by R.H. Richardson. University of Texas Press, Austin.

Wicker-Thomas, C., Guenachi, I. and Keita, Y. F. (2009). Contribution of oenocytes and pheromones to courtship behaviour in Drosophila. BMC Biochem 10: 21. PubMed ID: 19671131

Wilson, T. G., Landers, M. H. and Happ, G. M. (1983). Precocene I and II inhibition of vitellogenic oocyte development in Drosophila melanogaster. J. Insect Physiol. 29: 249-254.

Wilson, T. G., and Fabian, J. (1986). A Drosophila melanogaster mutant resistant to a chemical analog of juvenile hormone. Dev. Biol. 118: 190-201. 3095161

Wilson, T. G. (1996). Genetic evidence that mutants of the Methoprene-tolerant gene of Drosophila melanogaster are null mutants. Arch. Insect Biochem. Physiol. 32: 641-649. 8756311

Wilson, T. G. and Ashok, M. (1998). Insecticide resistance resulting from an absence of target-site gene product. Proc. Natl. Acad. Sci. 95: 14040-14044. 9826649

Wilson, T. G., Yerushalmi, Y., Donell, D. M. and Restifo, L. L. (2005). Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between Methoprene-tolerant and Broad-Complex. Genetics 172(1): 253-64. 16204218

Wu, Z., Guo, W., Xie, Y. and Zhou, S. (2016). Juvenile hormone activates the transcription of Cell-division-cycle 6 (Cdc6) for polyploidy-dependent insect vitellogenesis and oogenesis. J Biol Chem 291: 5418-5427. PubMed ID: 26728459

Zhou, X. and Riddiford, L. M. (2002). Broad specifies pupal development and mediates the 'status quo' action of juvenile hormone on the pupal-adult transformation in Drosophila and Manduca, Development 129: 2259-2269. 11959833

date revised: 2 January 2023

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.