Dataset Information

A versatile ?C31 based reporter system for measuring AP-1 and Nrf2 signaling in Drosophila and in tissue culture.

ABSTRACT: This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ?C31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined integration sites furthermore enable the experimenter to compare the activity of different reporters in one organism. The reporter constructs have a modular design to facilitate the combination of promoter elements (synthetic transcription factor binding sites or natural regulatory sequences), reporter genes (eGFP, or DsRed.T4), and genomic integration sites. The system was used to analyze and compare the activity and signal response profiles of two stress inducible transcription factors, AP-1 and Nrf2. To complement the transgenic reporter fly lines, tissue culture assays were developed in which the same synthetic ARE and TRE elements control the expression of firefly luciferase.

SUBMITTER: Chatterjee N

PROVIDER: S-EPMC3324472 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A versatile ΦC31 based reporter system for measuring AP-1 and Nrf2 signaling in Drosophila and in tissue culture.

Chatterjee Nirmalya N Bohmann Dirk D

PloS one 20120411 4

This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ΦC31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined ...[more]

PMID: 22509270

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Project description:Methylmercury (MeHg) can elicit cognitive and motor deficits due to its developmental neuro- and myotoxic properties. While previous work has demonstrated that Nrf2 antioxidant signaling protects from MeHg toxicity, in vivo tissue-specific studies are lacking. In Drosophila, MeHg exposure shows greatest developmental toxicity in the pupal stage resulting in failed eclosion (emergence of adults) and an accompanying 'myosphere' phenotype in indirect flight muscles (IFMs). To delineate tissue-specific contributions to MeHg-induced motor deficits, we investigated the potential of Nrf2 signaling in either muscles or neurons to moderate MeHg toxicity. Larva were exposed to various concentrations of MeHg (0-20 µM in food) in combination with genetic modulation of the Nrf2 homolog cap-n-collar C (CncC), or its negative regulator Keap1. Eclosion behavior was evaluated in parallel with the morphology of two muscle groups, the thoracic IFMs and the abdominal dorsal internal oblique muscles (DIOMs). CncC signaling activity was reported with an antioxidant response element construct (ARE-GFP). We observed that DIOMs are distinguished by elevated endogenous ARE-GFP expression, which is only transiently seen in the IFMs. Dose-dependent MeHg reductions in eclosion behavior parallel formation of myospheres in the DIOMs and IFMs, while also increasing ARE-GFP expression in the DIOMs. Modulating CncC signaling via muscle-specific Keap1 knockdown and upregulation gives a rescue and exacerbation, respectively, of MeHg effects on eclosion and myospheres. Interestingly, muscle-specific CncC upregulation and knockdown both induce lethality. In contrast, neuron-specific upregulation of CncC, as well as Keap1 knockdown, rescued MeHg effects on eclosion and myospheres. Our findings indicate that enhanced CncC signaling localized to either muscles or neurons is sufficient to rescue muscle development and neuromuscular function from a MeHg insult. Additionally, there may be distinct roles for CncC signaling in myo-morphogenesis.

Dataset Information

A versatile ?C31 based reporter system for measuring AP-1 and Nrf2 signaling in Drosophila and in tissue culture.

Publications

A versatile ΦC31 based reporter system for measuring AP-1 and Nrf2 signaling in Drosophila and in tissue culture.

Similar Datasets

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