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).
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).
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, uraa) 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).
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), 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).
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).
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).
Reference names in red indicate recommended papers.
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
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
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
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
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
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
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
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
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.
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
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: 5 August 2011
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