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.     

Juvenile hormone modulates 20-hydroxyecdysone-inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta

 Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. During the molt and metamorphosis of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ecdysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and that the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high concentration (10^–5 M) of 20E equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurred at lower concentrations (effective concentrations, EC₅₀=6.3×10^–7 M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until pupation. When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 μg/ml JH I which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increased steadily with the same EC₅₀ as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, each in its own time-dependent manner, similar to that seen in vivo. These initial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected. Received: 16 July 1998 / Accepted: 5 August 1998     

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.     

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.