A conditional rescue system reveals essential functions for the ecdysone receptor (EcR) gene during molting and metamorphosis in Drosophila

In Drosophila, pulses of the steroid hormone ecdysone trigger larval molting and metamorphosis and coordinate aspects of embryonic development and adult reproduction. At each of these developmental stages, the ecdysone signal is thought to act through a heteromeric receptor composed of the EcR and USP nuclear receptor proteins. Mutations that inactivate all EcR protein isoforms (EcR-A, EcR-B1, and EcR-B2) are embryonic lethal, hindering analysis of EcR function during later development. Using transgenes in which a heat shock promoter drives expression of an EcR cDNA, we have employed temperature-dependent rescue of EcR null mutants to determine EcR requirements at later stages of development. Our results show that EcR is required for hatching, at each larval molt, and for the initiation of metamorphosis. In EcR mutants arrested prior to metamorphosis, expression of ecdysone-responsive genes is blocked and normal ecdysone responses of both imaginal and larval tissues are blocked at an early stage. These results show that EcR mediates ecdysone signaling at multiple developmental stages and implicate EcR in the reorganization of imaginal and larval tissues at the onset of metamorphosis.     

Isoform specific control of gene activity in vivo by the Drosophila ecdysone receptor

The steroid hormone 20-hydroxyecdysone induces metamorphosis in insects. The receptor for the hormone is the ecdysone receptor, a heterodimer of two nuclear receptors, EcR and USP. In Drosophila the EcR gene encodes 3 isoforms (EcR-A, EcR-B1 and EcR-B2) that vary in their N-terminal region but not in their DNA binding and ligand binding domains. The stage and tissue specific distribution of the isoforms during metamorphosis suggests distinct functions for the different isoforms. By over-expressing the three isoforms in animals we present results supporting this hypothesis. We tested for the ability of the different isoforms to rescue the lack of dendritic pruning that is characteristic of mutants lacking both EcR-B1 and EcR-B2. By expressing the different isoforms specifically in the affected neurons, we found that both EcR-B isoforms were able to rescue the neuronal defect cell autonomously, but that EcR-A was less effective. We also analyzed the effect of over-expressing the isoforms in a wild-type background. We determined a sensitive period when high levels of either EcR-B isoform were lethal, indicating that the low levels of EcR-B at this time are crucial to ensure normal development. Over-expressing EcR-A in contrast had no detrimental effect. However, high levels of EcR-A expressed in the posterior compartment suppressed puparial tanning, and resulted in down-regulation of some of the tested target genes in the posterior compartment of the wing disc. EcR-B1 or EcR-B2 over-expression had little or no effect.     

EcR expression in the prothoracicotropic hormone-producing neurosecretory cells of the Bombyx mori brain

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis through binding with a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle (USP). Expression of the specific isoforms EcR-A and EcR-B1 governs steroid-induced responses in the developing cells of the silkworm Bombyx mori. Here, analysis of EcR-A and EcR-B1 expression during larval-pupal development showed that both genes were up-regulated by 20E in the B. mori brain. Whole-mount in situ hybridization and immunohistochemistry revealed that EcR-A and EcR-B1 mRNAs and proteins were exclusively located in two pairs of lateral neurosecretory cells in the larval brain known as the prothoracicotropic hormone (PTTH)- producing cells (PTPCs). In the pupal brain, EcR-A and EcR-B1 expression was detected in tritocerebral cells and optic lobe cells in addition to PTPCs. As PTTH controls ecdysone secretion by the prothoracic gland, these results indicate that 20E-responsive PTPCs are the master cells of insect metamorphosis.     

The Drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis

Oogenesis in Drosophila is regulated by the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone. Response to ecdysone is mediated by a heteromeric receptor composed of the EcR and USP proteins. We have identified a temperature-sensitive EcR mutation, EcR(A483T), from a previously isolated collection of EcR mutations. EcR(A483T) is predicted to affect all EcR protein products (EcR-A, EcR-B1, and EcR-B2) since it maps to a common exon encoding the ligand-binding domain. In wild-type females, we find that both EcR-A and EcR-B1 are expressed in nurse cells and follicle cells throughout oogenesis. EcR mutant females raised at permissive temperature and then shifted to restrictive temperature exhibit severe reductions in fecundity. Oogenesis in EcR mutant females is defective, and the spectrum of oogenic defects includes the presence of abnormal egg chambers and loss of vitellogenic egg stages. Our results demonstrate a requirement for EcR during female reproduction and suggest that EcR is required for normal oogenesis.     

Molecular cloning,expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis

PCR techniques were used to clone and identify cDNAs for ecdysone receptor A and B1 (EcR-A and EcR-B1) isoforms from the rice stem borer, Chilo suppressalis. They differ only in the N-terminal A/B regions and show high sequence identities to other insects' EcRs. At the wandering stage, EcR-B1 mRNA was expressed more abundantly in the midgut than in the epidermis and fat body, whereas expression levels of EcR-A mRNA were similar in the three tissues. In the epidermis of the last instar larvae, the maximal mRNA expression of both EcR-A and EcR-B1 was observed from the wandering to prepupal stages prior to the peak of ecdysteroid titer in the hemolymph. In gel mobility shift assays, in vitro translated C. suppressalis EcR-B1 (CsEcR-B1) and Bombyx mori ultraspiracle (BmUSP) proteins bound to the Pal 1 and Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. These results indicate that the cDNAs isolated here encode functional ecdysone receptors.     

Modulation of the ecdysteroid-induced cell death by juvenile hormone during pupal wing development of Lepidoptera

Females of the tussock moth Orgyia recens have only vestigial wings, whereas the males have normal wings. We previously found that ecdysteroid induces both apoptotic events and phagocytotic activation in sex-specific and region-specific manners. To investigate whether different responses to ecdysteroid are controlled at the receptor level, we cloned ecdysteroid receptor isoforms, EcR-A and EcR-B1, in O. recens. In both male and female wings, EcR-A signal was detected in the distal region of the bordering lacuna (BL), whereas EcR-B1 signal was detected in the proximal region of the BL. The similar expression patterns of both EcR isoforms suggested that molecules other than EcR should be involved in different ecdysteroid responses between male and female of O. recens. We next tested juvenile hormone (JH) effects on pupal wing morphogenesis in O. recens. Interestingly, both JH and 20E addition induced wing degeneration not only in females but also in males. In addition, higher concentration of JH pre-treatment of the pupal wings of the silkworm, Bombyx mori, also caused wing degeneration under ecdysteroid treatment. These results indicate that JH modulates the ecdysteroid action to induce the cell death on pupal wings, generally in Lepidoptera.     

The ecdysone receptor and ultraspiracle regulate the timing and progression of ovarian morphogenesis during Drosophila metamorphosis

 Ecdysteroids regulate insect metamorphosis through the edysone receptor complex, a heterodimeric nuclear receptor consisting of the ecdysone receptor (EcR) and its partner ultraspiracle (USP). Differentiation in the Drosophila ovary at metamorphosis correlates with colocalization of USP and the EcR-A isoform in all but one of eight mesoderm-derived somatic cell types. The one exception is the larval terminal filament (TF) cells, in which only USP is detectable during cell differentiation. In cells destined to form the basal stalks and anterior oviduct, USP colocalizes with what appears to be the EcR-B2 isoform. Flies heterozygous for a deletion of the EcR gene exhibit several defects in ovarian morphogenesis, including a heterochronic delay in the onset of terminal filament differentiation. Flies heterozygous for a strong usp allele exhibit accelerated TF differentiation. Flies simultaneously heterozygous for both EcR and usp have additional phenotypes, including several heterochronic shifts, delayed initiation and completion of terminal filament morphogenesis and delayed ovarian differentiation during the first day of metamorphosis. Terminal filament morphogenesis is severely disrupted in homozygous usp clones. Our results demonstrate that proper expression of the ecdysone receptor complex is required to maintain the normal progression and timing of the events of ovarian differentiation in Drosophila. These findings are discussed in the context of a developmental and evolutionary role for the ecdysone receptor complex in regulating the timing of ovarian differentiation in dipteran insects. Received: 12 February 1998 / Accepted: 5 May 1998     

The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily

The steroid hormone ecdysone triggers coordinate changes in Drosophila tissue development that result in metamorphosis. To advance our understanding of the genetic regulatory hierarchies controlling this tissue response, we have isolated and characterized a gene, EcR, for a new steroid receptor homolog and have shown that it encodes an ecdysone receptor. First, EcR protein binds active ecdysteroids and is antigenically indistinguishable from the ecdysone-binding protein previously observed in extracts of Drosophila cell lines and tissues. Second, EcR protein binds DNA with high specificity at ecdysone response elements. Third, ecdysone-responsive cultured cells express EcR, whereas ecdysone-resistant cells derived from them are deficient in EcR. Expression of EcR in such resistant cells by transfection restores their ability to respond to the hormone. As expected, EcR is nuclear and found in all ecdysone target tissues examined. Furthermore, the EcR gene is expressed at each developmental stage marked by a pulse of ecdysone.