Marek Jindra

The Juvenile Hormone Signaling Pathway in Insect Development

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

Juvenile hormone resistance gene Methoprene-tolerant controls entry into metamorphosis in the beetle Tribolium castaneum

Besides being a spectacular developmental process, metamorphosis is key to insect success. Entry into metamorphosis is controlled by juvenile hormone (JH). In larvae, JH prevents pupal and adult morphogenesis, thus keeping the insect in its immature state. How JH signals to preclude metamorphosis is poorly understood, and a JH receptor remains unknown. One candidate for the JH receptor role is the Methoprene-tolerant (Met) Per-Arnt-Sim (PAS) domain protein [also called Resistance to JH, Rst (1)JH], whose loss confers tolerance to JH and its mimic methoprene in the fruit fly Drosophila melanogaster. However, Met deficiency does not affect the larval–pupal transition, possibly because this process does not require JH absence in Drosophila. By contrast, the red flour beetle Tribolium castaneum is sensitive to developmental regulation by JH, thus making an ideal system to examine the role of Met in the antimetamorphic JH action. Here we show that impaired function of the Met ortholog TcMet renders Tribolium resistant to the effects of ectopic JH and, in a striking contrast to Drosophila, causes early-stage beetle larvae to undergo precocious metamorphosis. This is evident as TcMet-deficient larvae pupate prematurely or develop specific heterochronic phenotypes such as pupal-like cuticular structures, appendages, and compound eyes. Our results demonstrate that TcMet functions in JH response and provide the critical evidence that the putative JH receptor Met mediates the antimetamorphic effect of JH.

Ligand-binding properties of a juvenile hormone receptor, Methoprene-tolerant

Juvenile hormone (JH) is a sesquiterpenoid of vital importance for insect development, yet the molecular basis of JH signaling remains obscure, mainly because a bona fide JH receptor has not been identified. Mounting evidence points to the basic helix–loop–helix (bHLH)/Per-Arnt-Sim (PAS) domain protein Methoprene-tolerant (Met) as the best JH receptor candidate. However, details of how Met transduces the hormonal signal are missing. Here, we demonstrate that Met specifically binds JH III and its biologically active mimics, methoprene and pyriproxyfen, through its C-terminal PAS domain. Substitution of individual amino acids, predicted to form a ligand-binding pocket, with residues possessing bulkier side chains reduces JH III binding likely because of steric hindrance. Although a mutation that abolishes JH III binding does not affect a Met–Met complex that forms in the absence of methoprene, it prevents both the ligand-dependent dissociation of the Met–Met dimer and the ligand-dependent interaction of Met with its partner bHLH-PAS protein Taiman. These results show that Met can sense the JH signal through direct, specific binding, thus establishing a unique class of intracellular hormone receptors.

Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis

The transcription factor Broad-Complex (BR-C) is required for differentiation of adult structures as well as for the programmed death of obsolete larval organs during metamorphosis of the fruit fly Drosophila melanogaster. Whether BR-C has a similar role in other holometabolous insects could not be proven without a loss-of-function genetic test, performed in a non-drosophilid species. Here we use a recombinant Sindbis virus as a tool to silence BR-C expression in the silkmoth Bombyx mori. The virus expressing a BR-C antisense RNA fragment reduced endogenous BR-C mRNA levels in infected tissues (adult wing and leg primordia) via RNA interference (RNAi). The RNAi knock-down of BR-C resulted in the failure of animals to complete the larval–pupal transition or in later morphogenetic defects, including differentiation of adult compound eyes, legs, and wings from their larval progenitors. BR-C RNAi also perturbed the programmed cell death of larval silk glands. These developmental defects correspond to loss-of-function phenotypes of BR-C Drosophila mutants in both the morphogenetic and degenerative aspects, suggesting that the critical role of BR-C in metamorphosis is evolutionarily conserved. We also demonstrate that the Sindbis virus is a useful vehicle for silencing of developmental genes in new insect models.

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. We have recently used the red flour beetle Tribolium castaneum to show that JH controls entry to metamorphosis via its putative receptor Methoprene-tolerant (Met). Here, we demonstrate that Met mediates JH effects on the expression of the ecdysteroid-response gene Broad-Complex (BR-C). Using RNAi and a classical mutant, we 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.

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

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

The conserved nuclear receptor Ftz-F1 is required for embryogenesis, moulting and reproduction in Caenorhabditis elegans

Nuclear receptors are essential players in the development of all metazoans. The nematode Caenorhabditis elegans possesses more than 200 putative nuclear receptor genes, several times more than the number known in any other organism. Very few of these transcription factors are conserved with components of the steroid response pathways in vertebrates and arthropods. Ftz‐F1, one of the evolutionarily oldest nuclear receptor types, is required for steroidogenesis and sexual differentiation in mice and for segmentation and metamorphosis in Drosophila.

Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems.

During gene activation, the effect of binding of transcription factors to cis-acting DNA sequences is transmitted to RNA polymerase by means of co-activators. Although co-activators contribute to the efficiency of transcription, their developmental roles are poorly understood. We used Drosophila to conduct molecular and genetic dissection of an evolutionarily conserved but unique co-activator, Multiprotein Bridging Factor 1 (MBF1), in a multicellular organism. Through immunoprecipitation, MBF1 was found to form a ternary complex including MBF1, TATA-binding protein (TBP) and the bZIP protein Tracheae Defective (TDF)/Apontic. We have isolated a Drosophila mutant that lacks the mbf1 gene in which no stable association between TBP and TDF is detectable, and transcription of a TDF-dependent reporter gene is reduced by 80%. Although the null mutants of mbf1 are viable, tdf becomes haploinsufficient in mbf1-deficient background, causing severe lesions in tracheae and the central nervous system, similar to those resulting from a complete loss of tdf function. These data demonstrate a crucial role of MBF1 in the development of tracheae and central nervous system.

Cell-autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis

Steroid signaling underlies developmental processes in animals. Mutations that impair steroidogenesis in the fruit fly Drosophila melanogaster provide tools to dissect steroid hormone action genetically. The widely used temperature-sensitive mutation ecdysoneless1 (ecd1) disrupts production of the steroid hormone ecdysone, and causes developmental and reproductive defects. These defects cannot be satisfactorily interpreted without analysis of the ecd gene. Here, we show that ecd encodes an as yet functionally undescribed protein that is conserved throughout eukaryotes. The ecd1 conditional allele contains an amino acid substitution, whereas three non-conditional larval lethal mutations result in truncated Ecd proteins. Consistent with its role in steroid synthesis, Ecd is expressed in the ecdysone-producing larval ring gland. However, development of ecd-null early larval lethal mutants cannot be advanced by Ecd expression targeted to the ring gland or by hormone feeding. Cell-autonomous ecd function, suggested by these experiments, is evidenced by the inability of ecd– clones to survive within developing imaginal discs. Ecd is also expressed in the ovary, and is required in both the follicle cells and the germline for oocyte development. These defects, induced by the loss of ecd, provide the first direct evidence for a cell-autonomous function of this evolutionarily conserved protein.

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. Here, we show 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 hormones 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.