Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila.

The steroid hormone ecdysone regulates larval development and metamorphosis in Drosophila melanogaster through a complex genetic hierarchy that begins with a small set of early response genes. Here, we present data indicating that the ecdysone response hierarchy also mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates.     

高流动性蛋白基因过量表达对果蝇发育的影响

HmgD基因编码果蝇高流动性蛋白（High mobility group proteins, HMG）的同源物,它可以参与染色质的组装。目前关于HMGD蛋白在果蝇胚胎发育过程中的作用尚无定论。我们采用UAS-Gal4系统,通过功能获得性突变的方法研究了HmgD基因过量表达对果蝇发育的影响。结果表明：HmgD过量表达对果蝇胚胎期发育的影响较弱,而对后期幼虫的发育具有很大的影响;HmgD过量表达的果蝇胚胎死亡率增高,但这种影响不是很大,因为一部分胚胎仍然能够发育至成体;但是当HmgD在广泛表达的Gal4驱动子（ActGal4）的控制下过量表达时导致子代大量死亡,特别是用4个拷贝的转基因果蝇进行杂交时,后代中的突变型在三龄幼虫末期全部死亡;部分突变型幼虫体内长有黑色素瘤,其血淋巴中的血细胞数量极显著地高于野生型。RT-PCR分析表明,突变幼虫中与血细胞增殖有关的Ras-MAPK途径和Toll途径被异常激活。这些结果显示：HmgD过量表达可能引起染色质结构疏松,激活了特定的转录因子,从而引发了三龄幼虫期异常的转录调控,并导致幼虫死亡。     

Gene transfer to skeletal muscle by site-specific delivery of electroporation and ultrasound

Lipid metabolism drastically changes in response to the environmental factors in metazoans. Lipid is accumulated at the food rich condition, while mobilized in adipocyte tissue in starvation. Such lipid mobilization is also evident during the pupation of the insects. Pupation is induced by metamorphosis hormone, ecdysone via ecdysone receptor (EcR) with lipid mobilization, however, the molecular link of the EcR-mediated signal to the lipid mobilization remains elusive. To address this issue, EcR was genetically knocked-down selectively in 3rd instar larva fat body of Drosophila, corresponding to the adipocyte tissues in mammalians, that contains adipocyte-like cells. In this mutant, lipid accumulation was increased in the fat body. Lipid accumulation was also increased when knocked-down of taiman, which served as the EcR co-activator. Two lipid metabolism regulatory factor, E75B and adipose (adp) as well as cell growth factor, dMyc, were found as EcR target genes in the adipocyte-like cells, and consistently knock-down of these EcR target genes brought phenotypes in lipid accumulation supporting EcR function. These findings suggest that EcR-mediated ecdysone signal is significant in lipid metabolism in insects.     

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.     

Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.     

Regulation of the endocycle/gene amplification switch by Notch and ecdysone signaling

The developmental signals that regulate the switch from genome-wide DNA replication to site-specific amplification remain largely unknown. Drosophila melanogaster epithelial follicle cells, which begin synchronized chorion gene amplification after three rounds of endocycle, provide an excellent model for study of the endocycle/gene amplification (E/A) switch. Here, we report that down-regulation of Notch signaling and activation of ecdysone receptor (EcR) are required for the E/A switch in these cells. Extended Notch activity suppresses EcR activation and prevents exit from the endocycle. Tramtrack (Ttk), a zinc-finger protein essential for the switch, is regulated negatively by Notch and positively by EcR. Ttk overexpression stops endoreplication prematurely and alleviates the endocycle exit defect caused by extended Notch activity or removal of EcR function. Our results reveal a developmental pathway that includes down-regulation of Notch, activation of the EcR, up-regulation of Ttk to execute the E/A switch, and, for the first time, the genetic interaction between Notch and ecdysone signaling in regulation of cell cycle programs and differentiation.     

Molecular chaperones activate the Drosophila ecdysone receptor, an RXR heterodimer

The steroid hormone 20-hydroxyecdysone coordinates the stages of Drosophila development by activating a nuclear receptor heterodimer consisting of the ecdysone receptor, EcR, and the Drosophila RXR receptor, USP. We show that EcR/USP DNA binding activity requires activation by a chaperone heterocomplex like that required for activation of the vertebrate steroid receptors, but not previously shown to be required for activation of RXR heterodimers. Six proteins normally present in the chaperone complex were individually purified and shown to be sufficient for this activation. We also show that two of the six (Hsp90 and Hsc70) are required in vivo for ecdysone receptor activity, and that EcR is the primary target of the chaperone complex.     

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.     

The RXR ortholog USP suppresses early metamorphic processes in Drosophila in the absence of ecdysteroids

The steroid hormone 20-hydroxyecdysone (20E) initiates metamorphosis in insects by signaling through the ecdysone receptor complex, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP). Analysis of usp mutant clones in the wing disc of Drosophila shows that in the absence of USP, early hormone responsive genes such as EcR, DHR3 and E75B fail to up-regulate in response to 20E, but other genes that are normally expressed later, such as (&bgr;)-Ftz-F1 and the Z1 isoform of the Broad-Complex (BRC-Z1), are expressed precociously. Sensory neuron formation and axonal outgrowth, two early metamorphic events, also occur prematurely. In vitro experiments with cultured wing discs showed that BRC-Z1 expression and early metamorphic development are rendered steroid-independent in the usp mutant clones. These results are consistent with a model in which these latter processes are induced by a signal arising during the middle of the last larval stage but suppressed by the unliganded EcR/USP complex. Our observations suggest that silencing by the unliganded EcR/USP receptor and the subsequent release of silencing by moderate steroid levels may play an important role in coordinating early phases of steroid driven development.     

Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation.

The vertebrate retinoid X receptor (RXR) has been implicated in the regulation of multiple hormonal signaling pathways through the formation of heteromeric receptor complexes that bind DNA with high affinity. We now demonstrate that ultraspiracle (usp), a Drosophila RXR homolog, can substitute for RXR in stimulating the DNA binding of receptors for retinoic acid, T3, vitamin D, and peroxisome proliferator activators. These observations led to the search and ultimate identification of the ecdysone receptor (EcR) as a Drosophila partner of usp. Together, usp and EcR bind DNA in a highly cooperative fashion. Cotransfection of both EcR and usp expression vectors is required to render cultured mammalian cells ecdysone responsive. These results implicate usp as an integral component of the functional EcR. By demonstrating that receptor heterodimer formation precedes the divergence of vertebrate and invertebrate lineages, these data underscore a central role for RXR and its homolog usp in the evolution and control of the nuclear receptor-based endocrine system.