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Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila.

ABSTRACT: Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.

SUBMITTER: Foulk MS

PROVIDER: S-EPMC3964788 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila.

Foulk Michael S MS Waggener John M JM Johnson Janell M JM Yamamoto Yutaka Y Liew Gerald M GM Urnov Fyodor D FD Young Yuki Y Lee Genee G Smith Heidi S HS Gerbi Susan A SA

Chromosoma 20130116 1-2

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, ...[more]

PMID: 23321980

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Project description:Conopomorpha sinensis Bradley (Lepidoptera: Gracilariidae) is the dominant insect pest of litchi (chinensis Sonn.) and longan (Euphoria longan Lour.) fruit trees. Management of this pest species is a challenging task due to its cryptic borer behavior. Controlling C. sinensis at the egg stage is the best alternative strategy to chemical control of C. sinensis adults. However, thorough studies regarding the indirect and sublethal effects of chemicals on the different developmental stages of C. sinensis are insufficient. In this study, the effect of some insecticides was evaluated on C. sinensis eggs. The ovicidal activity of chlorbenzuron, abamectin, chlorantraniliprole, and λ-cyhalothrin was confirmed by morphological observation of the defects in C. sinensis eggs. Moreover, we characterized four essential ecdysone receptor proteins in insects [i.e., two isoform ecdysone receptors (EcR: CsEcRA. CsEcRB) and two isoform ultraspiracle proteins (USP: CsUSP1, CsUSP2)] from C. sinensis eggs. The CsEcRA, CsEcRB, CsUSP1, and CsUSP2 genes consisted of 1521-, 1614-, 1410-, and 1236-bp open reading frames which encoded proteins of 506, 527, 469, and 413 amino acid residues, respectively. Furthermore, the embryonic differential responses of CsEcRs, CsUSPs, and vitellogenin receptor (VgR: CsVgR) to insecticides were evaluated by qRT-PCR. Among the five tested genes, CsVgR and CsUSP1 were the most sensitive to all the tested insecticides, with fold change of the expression diminished by 4.27-8.70 times compared with untreated control insects. The data suggests that these insecticidal compounds regulate the expression of these specific proteins, which might eventually lead to reduced viability of C. sinensis eggs. We present here the first data providing molecular elucidation of ecdysone receptor genes and their differential responses to insecticides in C. sinensis eggs. Together with our previous report of insecticide sublethal effects on two reproduction-related genes in C. sinensis adults, CsVgR and CsUSP1 seem to be appropriate molecular parameters for the evaluation of insecticide impact on C. sinensis. This study exemplifies the potential utility of transcriptional measurement of nuclear receptors as the molecular biomarkers for ecotoxicological evaluations of ovicidal impact of insecticides.

Project description: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.

Dataset Information

Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila.

Publications

Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila.

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