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.     

Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster

The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third larval instar. The region contains a complex genetic locus, the Broad-Complex (BR-C) composed of four groups of fully complementing (br, rbp, l(1)2Bc, and l(1)2Bd) alleles, and classes of noncomplementing (npr 1) and partially noncomplementing l(1)2Bab alleles. BR-C mutants prevent metamorphosis, including the morphogenesis of imaginal discs. Results are presented that indicate that the BR-C contains two major functional domains. One, the br domain is primarily, if not exclusively, involved in the elongation and eversion of appendages by imaginal discs. The second, the l(1)2Bc domain, is primarily involved in the fusion of discs to form a continuous adult epidermis. Nonetheless, the two domains may encode products with related functions because in some situations mutants in both domains appear to affect similar developmental processes.     

Mutations in a steroid hormone-regulated gene disrupt the metamorphosis of the central nervous system in Drosophila

The actions of steroid hormones on vertebrate and invertebrate nervous systems include alterations in neuronal architecture, regulation of neuronal differentiation, and programmed cell death. In particular, central nervous system (CNS) metamorphosis in insects requires a precise pattern of exposure to the steroid molting hormone 20-hydroxyecdysone (ecdysterone). To test whether the effects of steroid hormones on the insect nervous system are due to changes in patterns of gene expression, we examined Drosophila mutants of the ecdysterone-regulated locus, the Broad Complex (BR-C). This report documents aspects of CNS reorganization which are dependent on BR-C function. During wild-type metamorphosis, CNS components undergo dramatic morphogenetic movements relative to each other and to the body wall. These movements, in particular, the separation of the subesophageal ganglion from the thoracic ganglion, the positioning of the developing visual system, and the fusion of right and left brain hemispheres, are deranged in BR-C mutants. In addition, a subset of mutants shows disorganization of optic lobe neuropil, both within and among optic lobe ganglia. Optic lobe disorganization is found in mutants of the br and l(1)2Bc complementation groups, but not in those of the rbp complementation group. This suggests that the three complementation groups of this complex locus represent distinct but overlapping functions necessary for normal CNS reorganization. This study demonstrates that ecdysterone-regulated gene expression is essential for CNS metamorphosis, illustrating the utility of Drosophila as a model system for investigating the genetic basis of steroid hormone action on the nervous system.     

Interaction of the Stubble-Stubbloid Locus and the Broad-Complex of Drosophila Melanogaster

The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third instar. The region is coextensive with a complex genetic locus, the Broad-Complex (BR-C). The BR-C is a regulatory gene that contains two major functional domains, the br domain and the l(1)2Bc domain. BR-C mutants prevent metamorphosis, including morphogenesis of imaginal discs; br mutants prevent elongation and eversion of appendages and l(1)2Bc mutants prevent fusion of the discs. The Stubble-stubbloid (Sb-sbd) locus at 89B9-10 is best known for the effects of its mutants on bristle structure. Mutants of the BR-C and the Sb-sbd locus interact to produce severe malformation of appendages. Viable heteroallelic and homoallelic combinations of Sb-sbd mutants, including loss-of-function mutants, affect the elongation of imaginal disc appendages. Thus, the Sb-sbd(+) product is essential for normal appendage elongation. Sb-sbd mutants, however, do not affect eversion or fusion of discs. Correspondingly, only BR-C mutants deficient in br function interact with Sb-sbd mutants. The interaction occurs in deficiency heterozygotes using single, wild-type doses of the BR-C, of the Sb-sbd locus, or of both loci. These last results are formally consistent with the possibility that the BR-C acts as a positive regulator of the Sb-sbd locus. The data do not exclude other possible nonregulatory interactions between the two loci, e.g., interactions between the products of both genes.     

GFP in living animals reveals dynamic developmental responses to ecdysone during Drosophila metamorphosis

Studies of Drosophila metamorphosis have been hampered by our inability to visualize many of the remarkable changes that occur within the puparium. To circumvent this problem, we have expressed GFP in specific tissues of living prepupae and pupae and compiled images of these animals into time-lapse movies. These studies reveal, for the first time, the dynamics and coordination of morphogenetic movements that could only be inferred from earlier studies of dissected staged animals. We also identify responses that have not been described previously. These include an unexpected variation in some wild-type animals, where one of the first pairs of legs elongates in the wrong position relative to the second pair of legs and then relocates to its appropriate location. At later stages, the antennal imaginal discs migrate from a lateral position in the head to their final location at the anterior end, as leg and mouth structures are refined and the wings begin to fold. The larval salivary glands translocate toward the dorsal aspect of the animal and undergo massive cell death following head eversion, in synchrony with death of the abdominal muscles. These death responses fail to occur in rbp(5) mutants of the Broad-Complex (BR-C), and imaginal disc elongation and eversion is abolished in br(5) mutants of the BR-C. Leg malformations associated with the crol(3) mutation can be seen to arise from defects in imaginal disc morphogenesis during prepupal stages. This approach provides a new tool for characterizing the dynamic morphological changes that occur during metamorphosis in both wild-type and mutant animals.     

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.     

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

The Methoprene-tolerant (Met) bHLH-PAS gene is involved in juvenile hormone (JH) action in Drosophila melanogaster as a likely component of a JH receptor. We expressed Met in Drosophila S2 cells and explored for MET partners using pull-down assays. MET-MET interaction was found to occur. The germ-cell expressed (gce) gene is another D. melanogaster bHLH-PAS gene with high homology to Met, and GCE formed heterodimers with MET. In the presence of JH or either of two JH agonists, MET-MET and MET-GCE formation was 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 failed to occur. However, JH-dependent interaction occurred 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.     

An F1 genetic screen for maternal-effect mutations affecting embryonic pattern formation in Drosophila melanogaster

Large-scale screens for female-sterile mutations have revealed genes required maternally for establishment of the body axes in the Drosophila embryo. Although it is likely that the majority of components involved in axis formation have been identified by this approach, certain genes have escaped detection. This may be due to (1) incomplete saturation of the screens for female-sterile mutations and (2) genes with essential functions in zygotic development that mutate to lethality, precluding their identification as female-sterile mutations. To overcome these limitations, we performed a genetic mosaic screen aimed at identifying new maternal genes required for early embryonic patterning, including zygotically required ones. Using the Flp-FRT technique and a visible germline clone marker, we developed a system that allows efficient screening for maternal-effect phenotypes after only one generation of breeding, rather than after the three generations required for classic female-sterile screens. We identified 232 mutants showing various defects in embryonic pattern or morphogenesis. The mutants were ordered into 10 different phenotypic classes. A total of 174 mutants were assigned to 86 complementation groups with two alleles on average. Mutations in 45 complementation groups represent most previously known maternal genes, while 41 complementation groups represent new loci, including several involved in dorsoventral, anterior-posterior, and terminal patterning.     

Isolation of synthetic lethal mutations in the rsm1-null mutant of fission yeast

To identify mutations in genes that are genetically linked to rsm1, we performed a synthetic lethal genetic screen in the fission yeast, Schizosaccharomyces pombe. Four mutations that showed synthetic lethality in combination with the rsm1null allele were isolated from approximately 320,000 colonies and defined in three complementation groups. One mutant (SLrsm1) exhibited a significant accumulation of poly(A)⁺ RNA in the nucleus under synthetic lethal conditions, while the rest had no mRNA export defects. In addition, some genes (spmex67, rae1, or mlo3) required for mRNA export complemented the growth defects of the identified mutants. These results suggest that the isolated mutants contain mutations in genes that are involved in mRNA export and/or pre-mRNA retention.