EcR Isoforms Research Articles

Functional expression patterns of four ecdysteroid receptor isoforms indicate their different functions during vitellogenesis of Chinese mitten crab, Eriocheir sinensis

In arthropods, alternative splicing of ecdysteroid receptor gene (EcR) leads to multiple functions of different EcR isoforms during metamorphosis, growth and ovarian development via ecdysteroid signaling pathway. This study was conducted to investigate the expression patterns of four EcRs of Eriocheir sinensis (EsEcRs) and the changes of haemolymph ecdysteroid titer during the ovarian development. The results showed that four EsEcR isoforms had the tissue-specific expression among 12 examined tissues, and the highest transcript levels of the four EsEcR isoforms were detected in Y-organ or sinus gland. During the ovarian development, EsEcR1 showed the highest transcript abundance of the four EsEcR isoforms. The expression profiles of all the EsEcR isoforms were similar in the hepatopancreas during the ovarian maturation cycle of E. sinensis with a trend of “high-low-high-low”. In ovary, the highest expression levels of EsEcR1 and EsEcR4 were both found at stage V ovary, while the peaks of EsEcR2 and EsEcR3 were found on stage III ovary and stage IV ovary, respectively. Meanwhile, the ecdysteroid titer in haemolymph decreased gradually during ovarian maturation cycle. Further regression analysis revealed significant negative correlations were found between the ovarian EsEcR3/ EsEcR4 expression levels and haemolymph ecdysteroid titer during part or whole ovarian development cycle. These results together indicated that four EsEcR isoforms may have different functions during ovary maturation of E. sinensis. All EcR isoforms and ecdysteroid seemed to have important roles in the hepatopancreas during early ovarian development stages, while EsEcR3 and EsEcR4 were closely related to the mid-late vitellogenesis stages.

Ultraspiracle-independent anti-apoptotic function of ecdysone receptors is required for the survival of larval peptidergic neurons via suppression of grim expression in Drosophila melanogaster.

In Drosophila melanogaster a significant number of heterogenous larval neurons in the central nervous system undergo metamorphosis-associated programmed cell death, termed metamorphoptosis. Interestingly distinct groups of doomed larval neurons are eliminated at different metamorphic phases. Although ecdysone hormonal signaling via nuclear ecdysone receptors (EcRs) is known to orchestrate the neuronal metamorphoptosis, little is known about how this signaling controls such diverse neuronal responses. Crustacean cardioactive peptide (CCAP)-producing neurons in the ventral nerve cord are developmentally programmed to die shortly after adult emergence. In this study, we show that disruption of endogenous EcR function by ectopic expression of dominant negative forms of EcRs (EcRDN) causes premature death of larval CCAP neurons in a caspase-dependent manner. This event is rescued by co-expression of individual EcR isoforms. Furthermore, larval CCAP neurons are largely normal in ecr mutants lacking either EcR-A or EcR-B isoforms, suggesting that EcR isoforms redundantly function to protect larval CCAP neurons. Of surprise, a role of Ultraspiracle (Usp), a canonical partner of EcR, is dispensable in the protection of CCAP neurons, whereas both EcR and Usp are required for inducing metamorphoptosis of vCrz neurons shortly after prepupal formation. As a downstream, grim is an essential cell death gene for the EcRDN-mediated CCAP neuronal death, while either hid or rpr function is dispensable. Together, our results suggest that Usp-independent EcR actions protect CCAP neurons from their premature death by repressing grim expression until their normally scheduled apoptosis at post-emergence. Our studies highlight two opposite roles played by EcR function for metamorphoptosis of two different peptidergic neuronal groups, proapoptotic (vCrz) versus antiapoptotic (CCAP), and propose that distinct death timings of doomed larval neurons are determined by differential signaling mechanisms involving EcR.

Ligand-independent requirements of steroid receptors EcR and USP for cell survival.

The active form of the Drosophila steroid hormone ecdysone, 20-hydroxyecdysone (20E), binds the heterodimer EcR/USP nuclear receptor to regulate target genes that elicit proliferation, cell death and differentiation during insect development. Although the 20E effects are relatively well known, the physiological relevance of its receptors remains poorly understood. We show here that the prothoracic gland (PG), the major steroid-producing organ of insect larvae, requires EcR and USP to survive in a critical period previous to metamorphosis, and that this requirement is 20E-independent. The cell death induced by the downregulation of these receptors involves the activation of the JNK-encoding basket gene and it can be rescued by upregulating EcR isoforms which are unable to respond to 20E. Also, while PG cell death prevents ecdysone production, blocking hormone synthesis or secretion in normal PG does not lead to cell death, demonstrating further the ecdysone-independent nature of the receptor-deprivation cell death. In contrast to PG cells, wing disc or salivary glands cells do not require these receptors for survival, revealing their cell and developmental time specificity. Exploring the potential use of this feature of steroid receptors in cancer, we assayed tumor overgrowth induced by altered yorkie signaling. This overgrowth is suppressed by EcR downregulation in PG, but not in wing disc, cells. The mechanism of all these cell death features is based on the transcriptional regulation of reaper. These novel and context-dependent functional properties for EcR and USP receptors may help to understand the heterogeneous responses to steroid-based therapies in human pathologies.

Alternative splicing in the fiddler crab cognate ecdysteroid receptor: Variation in receptor isoform expression and DNA binding properties in response to hormone

RXR cDNA cloning from three Uca species led to the identification of 4 conserved isoforms, indicative of alternative splicing in the hinge and ligand binding domains (LBD). Sequencing of overlapping clones from a Ucapugilator genomic library identified EcR isoforms matching previously identified cDNA variants; in addition, a cryptic exon in the LBD was detected and evidence for expression of this new isoform was obtained from next-generation sequencing. RNA-seq analysis also identified a new amino terminal EcR variant. EcR and RXR transcript abundance increases throughout ovarian maturation in U. pugilator, while cognate receptor transcript abundance remains constant in a related Indo-Pacific species with a different reproductive strategy. To examine if crab RXR LBD isoforms have different physical properties in vitro, electromobility shift assays were performed with different EcR isoforms. The cognate crab and fruit fly receptors differ in their responses to hormone. Ecdysteroids did not increase DNA binding for the crab heterodimers, while ecdysteroids stimulate binding for Drosophilamelanogaster EcR/USP heterodimers. In swapping experiments, UpEcR/USP heterodimers did not show ligand-responsive differences in DNA binding; both crab RXR LBD isoforms, however, conferred ligand-responsive increases in DNA binding with DmEcRs. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding. Unresponsiveness of the cognate receptors to ecdysteroid, however, suggest additional factors may be required to mediate endogenous, perhaps isoform-specific, differences in EcR conformation, consistent with previously reported effects of UpRXR isoforms on UpEcR ligand-binding affinities.

Multidomain sumoylation of the ecdysone receptor (EcR) from Drosophila melanogaster

The 20-hydroxyecdysone receptor (EcR) is a transcription factor belonging to the nuclear receptor superfamily. Together with the ultraspiracle nuclear receptor (Usp) it coordinates critical biological processes in insects such as development and reproduction. EcR and its ligands are used in commercially available ecdysone-inducible expression systems and are considered to be artificial gene switches with potential therapeutic applications. However, the regulation of EcR action is still unclear, especially in mammals and as far as posttranslational modifications are concerned. Up until now, there has been no study on EcR sumoylation. Using bioinformatic predictors, a Ubc9 fusion-directed sumoylation system and mutagenesis experiments, we present EcR as a new target of SUMO1 and SUMO3 modification. Our research revealed that EcR undergoes isoform-specific multisumoylation. The pattern of modification remains unchanged in the presence of the ligand and the dimerization partner. The SUMO acceptor sites are located in the DNA-binding domain and the ligand-binding domain that both exhibit structural plasticity. We also demonstrated the existence of a sumoylation site in the F region and EcRA-A/B region, both revealing characteristics of intrinsically disordered regions. The consequences of modification and the resulting impact on conformation and function may be especially crucial for the disordered sequences in these two areas. The isoform-specificity of sumoylation may explain the differences in the transcriptional activity of EcR isoforms.

Ecdysone-dependent and ecdysone-independent programmed cell death in the developing optic lobe of Drosophila

The adult optic lobe of Drosophila develops from the primordium during metamorphosis from mid-3rd larval stage to adult. Many cells die during development of the optic lobe with a peak of the number of dying cells at 24h after puparium formation (h APF). Dying cells were observed in spatio-temporal specific clusters. Here, we analyzed the function of a component of the insect steroid hormone receptor, EcR, in this cell death. We examined expression patterns of two EcR isoforms, EcR-A and EcR-B1, in the optic lobe. Expression of each isoform altered during development in isoform-specific manner. EcR-B1 was not expressed in optic lobe neurons from 0 to 6h APF, but was expressed between 9 and 48h APF and then disappeared by 60h APF. In each cortex, its expression was stronger in older glia-ensheathed neurons than in younger ones. EcR-B1 was also expressed in some types of glia. EcR-A was expressed in optic lobe neurons and many types of glia from 0 to 60h APF in a different pattern from EcR-B1. Then, we genetically analyzed EcR function in the optic lobe cell death. At 0h APF, the optic lobe cell death was independent of any EcR isoforms. In contrast, EcR-B1 was required for most optic lobe cell death after 24h APF. It was suggested that cell death cell-autonomously required EcR-B1 expressed after puparium formation. βFTZ-F1 was also involved in cell death in many dying-cell clusters, but not in some of them at 24h APF. Altogether, the optic lobe cell death occurred in ecdysone-independent manner at prepupal stage and ecdysone-dependent manner after 24h APF. The acquisition of ecdysone-dependence was not directly correlated with the initiation or increase of EcR-B1 expression.

Ecdysone Receptor Acts in fruitless- Expressing Neurons to Mediate Drosophila Courtship Behaviors

In Drosophila melanogaster, fruitless (fru) encodes male-specific transcription factors (FRU(M); encoded by fru P1) required for courtship behaviors (reviewed in). However, downstream effectors of FRU(M) throughout development are largely unknown. During metamorphosis the nervous system is remodeled for adult function, the timing of which is coordinated by the steroid hormone 20-hydroxyecdysone (ecdysone) through the ecdysone receptor, a heterodimer of the nuclear receptors EcR (isoforms are EcR-A, EcR-B1, or EcR-B2) and Ultraspiracle (USP) (reviewed in). Here, we show that genes identified as regulated downstream of FRU(M) during metamorphosis are significantly overrepresented with genes known to be regulated in response to ecdysone or EcR. FRU(M) and EcR isoforms are coexpressed in neurons in the CNS during metamorphosis in an isoform-specific manner. Reduction of EcR-A levels in fru P1-expressing neurons of males caused a significant increase in male-male courtship activity and significant reduction in size of two antennal lobe glomeruli. Additional genes were identified that are regulated downstream of EcR-A in fru P1-expressing neurons. Thus, EcR-A is required in fru P1-expressing neurons for wild-type male courtship behaviors and the establishment of male-specific neuronal architecture.

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.

Phenotypic analysis of EcR-A mutants suggests that EcR isoforms have unique functions during Drosophila development

The steroid hormone ecdysone triggers transitions between developmental stages in Drosophila by acting through a heterodimer consisting of the EcR and USP nuclear receptors. The EcR gene encodes three protein isoforms (EcR-A, EcR-B1, and EcR-B2) that have unique amino termini but that contain a common carboxy-terminal region including DNA-binding and ligand-binding domains. EcR-A and EcR-B1 are expressed in a spatially complementary pattern at the onset of metamorphosis, suggesting that specific responses to ecdysone involve distinct EcR isoforms. Here, we describe phenotypes of EcR-A specific deletion mutants isolated using transposon mutagenesis. Western blot analysis shows that each of these mutants completely lacks EcR-A protein, while the EcR-B1 protein is still present. The EcR 112 strain has a deletion of EcR-A specific non-coding and regulatory sequences but retains the coding exons, while the EcR 139 strain has a deletion of EcR-A specific protein coding exons but retains the regulatory region. In these mutants, the developmental progression of most internal tissues that normally express EcR-B1 is unaffected by the lack of EcR-A. Surprisingly, however, we found that one larval tissue, the salivary gland, fails to degenerate even though EcR-B1 is the predominant isoform. This result may indicate that the low levels of EcR-A in this tissue are in fact required. We identified yet another type of mutation, the EcR 94 deletion, that removes the EcR-A specific protein coding exons as well as the introns between the EcR-A and EcR-B transcription start sites. This deletion places the EcR-A regulatory region adjacent to the EcR-B transcription start site. While EcR 112 and EcR 139 mutant animals die during mid and late pupal development, respectively, EcR 94 mutants arrest prior to pupariation. EcR-A mutant phenotypes and lethal phases differ from those of EcR-B mutants, suggesting that the EcR isoforms have distinct developmental functions.

Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor

The steroid hormone ecdysone initiates molting and metamorphosis in Drosophila via a heterodimeric receptor consisting of EcR that binds hormone, and USP, a homolog of the vertebrate RXR receptor. EcR exists in three isoforms EcRA, EcRB1 and EcRB2 that are thought to direct specific physiological responses to ecdysone. These three isoforms differ only in their N-terminal A/B domain that implies that sequences responsible for the differential physiological effects lie within the A/B domains of the EcR isoforms. In the present study, we set out to determine the capability of the three isoforms and their A/B domains to control gene transcription. When full-length EcR plasmids were cotransfected into mammalian cells with a USP expressing and a cognate reporter plasmid, the three EcR isoforms showed striking differences in their ability to control gene transcription, both in the presence and in the absence of hormone. Furthermore, the A/B domains of EcRB1 and of EcRB2 when fused to the GAL4 DNA binding domain are sufficient to activate transcription of a reporter gene, in yeast as well as in mammalian cells. In contrast, a fusion construct containing the A/B domain of EcRA represses basal transcription of the reporter gene. All these findings emphasize the importance of the A/B domains of the three EcR isoforms for differentially controlling gene transcription. Furthermore, they provide evidence for the existence of an autonomous ligand-independent activation function (AF1) in the A/B domains of EcRB1 and EcRB2 and of an inhibitory function (IF) in the A/B domain of EcRA.

The Ecdysone Regulatory Pathway Controls Wing Morphogenesis and Integrin Expression during Drosophila Metamorphosis

Drosophila imaginal discs are specified and patterned during embryonic and larval development, resulting in each cell acquiring a specific fate in the adult fly. Morphogenesis and differentiation of imaginal tissues, however, does not occur until metamorphosis, when pulses of the steroid hormone ecdysone direct these complex morphogenetic responses. In this paper, we focus on the role of ecdysone in regulating adult wing development during metamorphosis. We show that mutations in the EcR ecdysone receptor gene and crooked legs (crol), an ecdysone-inducible gene that encodes a family of zinc finger proteins, cause similar defects in wing morphogenesis and cell adhesion, indicating a role for ecdysone in these morphogenetic responses. We also show that crol and EcR mutations interact with mutations in genes encoding integrin subunits—a family of αβ heterodimeric cell surface receptors that mediate cell adhesion in many organisms. α-Integrin transcription is regulated by ecdysone in cultured larval organs and some changes in the temporal patterns of integrin expression correlate with the ecdysone titer profile during metamorphosis. Transcription of α- and β-integrin subunits is also altered in crol and EcR mutants, indicating that integrin expression is dependent upon crol and EcR function. Finally, we describe a new hypomorphic mutation in EcR which indicates that different EcR isoforms can direct the development of adult appendages. This study provides evidence that ecdysone controls wing morphogenesis and cell adhesion by regulating integrin expression during metamorphosis. We also propose that ecdysone modulation of integrin expression might be widely used to control multiple aspects of adult development.

Expression of ecdysone receptor isoforms and trehalase in the anterior silk gland ofBombyx mori during an extra larval molt and precocious pupation induced by 20-hydroxyecdysone administration

When newly molted 5th (last) instar larvae of Bombyx mori were fed an artificial diet containing 20-hydroxyecdysone (20E), an extra larval molt was induced and the cuticle layer of the anterior silk gland was renewed. In contrast, feeding 20E to 5th instar larvae allatectomized in the mid-4th instar induced pupational responses and anterior silk gland degeneration. Topical application of a juvenile hormone analogue, fenoxycarb, to allatectomized larvae before 20E treatment nullified the effects of allatectomy and induced an extra molt. In the two cases where an extra larval molt was induced, mRNA expression of the ecdysone receptor A isoform (EcR-A) preceded that of the B1 isoform (EcR-B1) in the anterior silk gland, while EcR-A and EcR-B1 were expressed synchronously during precocious pupation. Precedence of EcR-A over EcR-B1 and the synchronization of both are expression patterns observed during the normal 4th and 5th instars. These results suggest that temporal expression profiles of EcR isoforms are important for regulating stage-specific responses of this tissue to ecdysteroids. Trehalase was also identified as a metamorphosis-specific gene in the anterior silk gland by mRNA differential display between the extra larval molt and precocious pupation. During normal 4th and 5th instar, trehalase mRNA was expressed at the end of the 5th instar only. Thus, extra larval molt and precocious pupation induced by dietary application of 20E mimic normal larval molt and pupation well and can be a useful tool in isolating stage-specific ecdysteroid-regulated genes. Arch. Insect Biochem. Physiol. 41:79–88, 1999. © 1999 Wiley-Liss, Inc.

Tissue-specific and stage-specific expression of two silkworm ecdysone receptor isoforms -- ecdysteroid-dependent transcription in cultured anterior silk glands.

Previously, we isolated a cDNA clone for the ecdysone receptor B1 isoform of the silkworm, Bombyx mori (BmEcR-B1). Here we report the cloning of a cDNA that encodes the Bombyx ecdysone receptor A isoform (BmEcR-A) and mRNA expression of the two BmEcR isoforms during molting and metamorphosis. At larval-pupal transformation, mRNA expression of BmEcR-B1 was predominant in most tissues examined, including three larval tissues (midgut, epidermis, and fat body) and the wing imaginal disc. The anterior silk gland was the only tissue where BmEcR-A was predominant. These expression patterns were different from observations demonstrated in Drosophila. In the anterior silk gland, both EcR isoforms were expressed synchronously during the fifth larval instar, while expression of the A isoform preceded that of the B1 isoform by two days in the fourth instar. Precedence of BmEcR-A during the fourth instar and synchronization of both isoforms during the fifth instar were also observed in the middle and posterior silk glands, suggesting that transcription of BmEcR in the silk gland is regulated differently in these two instars. In the cultured anterior silk glands of day 0 of the fifth instar, transcription of BmEcR-A and BmEcR-B1 was induced dose dependently by more than 5 ng/ml 20-hydroxyecdysone. BmEcR-A and BmEcR-B1 mRNAs were induced within 2 h and 1 h, respectively, of the addition of 20-hydroxyecdysone. These results suggest that the increase of BmEcR mRNAs during the fifth instar is induced in vivo by a small increase in ecdysteroids.

Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms

In D. melanogaster a pulse of the steroid hormone ecdysone triggers the larval-to-adult metamorphosis, a complex process in which this hormone induces imaginal tissues to generate adult structures and larval tissues to degenerate. We show that the EcR gene encodes three ecdysone receptor isoforms (EcR-A, EcR-B1, and EcR-B2) that have common DNA- and hormone-binding domains but different N-terminal regions. We have used isoform-specific monoclonal antibodies to show that at the onset of metamorphosis different ecdysone target tissues express different isoform combinations in a manner consistent with the proposition that the different metamorphic responses of these tissues require different combinations of the EcR isoforms. We have also determined temporal developmental profiles of the EcR isoforms and their mRNAs in whole animals, showing that different isoforms predominate at different developmental stages that are marked by a pulse of ecdysone.