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Transcriptional Response of Zebrafish Embryos Exposed to Neurotoxic Compounds Reveals a Muscle Activity Dependent hspb11 Expression

ABSTRACT: Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of muscular activity. Whether AChE inhibitors and related neurotoxic compound also provoke specific changes in gene transcription patterns that allow to establish a mechanistic link useful for diagnostic purposes has, however, yet not been investigated. Therefore, as a proof of principle, we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive gene in response to AChE inhibitors. Comparison of expression in wild-type, ache and fixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels led to an hspb11 transcriptional up-regulation which suggest that elevated intracellular calcium levels and the resulting increase in muscular activity triggering hspb11 up-regulation. During early zebrafish development, hspb11 was specifically expressed in muscle pioneers and morpholino-knockdown resulted in defects in slow muscle myosin organization. These findings demonstrate that a combination of transcriptome and functional analyzes in the zebrafish embryo is able to correlate gene expression changes to the mechanism of action of neurotoxic compounds.

ORGANISM(S): Danio rerio

PROVIDER: GSE27680 | GEO | 2011/12/01

SECONDARY ACCESSION(S): PRJNA138173

REPOSITORIES: GEO

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Project description:Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of muscular activity. Whether AChE inhibitors and related neurotoxic compound also provoke specific changes in gene transcription patterns that allow to establish a mechanistic link useful for diagnostic purposes has, however, yet not been investigated. Therefore, as a proof of principle, we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive gene in response to AChE inhibitors. Comparison of expression in wild-type, ache and fixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels led to an hspb11 transcriptional up-regulation which suggest that elevated intracellular calcium levels and the resulting increase in muscular activity triggering hspb11 up-regulation. During early zebrafish development, hspb11 was specifically expressed in muscle pioneers and morpholino-knockdown resulted in defects in slow muscle myosin organization. These findings demonstrate that a combination of transcriptome and functional analyzes in the zebrafish embryo is able to correlate gene expression changes to the mechanism of action of neurotoxic compounds. Zebrafish embryos were exposed for 24 h and 48 h (26-50 and 2-50 hpf) to 5,83 µM of azinphos-methyl (APM), 509 µM of 1,4-dimethoxybenzene (DMB) and 14,2 µM 2,4-dinitrophenol (DNP), the respective LC10 of each compound, in order to determine mode of action specific responding genes, or not exposed and parallel treated as controls. 50 zebrafish embryos were used per treatment, and 4 biological independent replicates were analysed, respectively.

Project description:Domoic acid (DA) is a neuroexcitatory amino acid that is naturally produced by some marine diatom species of the genus Pseudo-nitzschia. Ingestion of DA-contaminated seafood by humans results in a severe neurotoxic disease known as amnesic shellfish poisoning (ASP). Clinical signs of ASP include seizures and neuronal damage from activation of AMPA and kainate receptors. However, the impacts of DA exposure at levels below those known to induce outward signs of neurobehavioral exicitotoxicity have not been well characterized. To further understand the mechanisms of neurotoxic injury associated with DA exposure, we examined the transcriptome of whole brains from zebrafish (Danio rerio) receiving intracoelomic (IC) DA at both symptomatic and asymptomatic doses. A majority of zebrafish exposed to high-dose DA (1.2 µg DA/g) exhibited clinical signs of neuroexcitotoxicity (EC50 of 0.86 µg DA/g) within 5 to 20 minutes of IC injection. All zebrafish receiving low-dose DA (0.47 µg DA/g) or vehicle only maintained normal behavior. Microarray analysis of symptomatic and asymptomatic exposures collectively yielded 306 differentially expressed genes (1.5-fold, p = 0.05) predominately represented by signal transduction, ion transport, and transcription factor functional categories. Transcriptional profiles were suggestive of neuronal apoptosis following an overwhelming of protective adaptive pathways. Further, potential molecular biomarkers of neuropathic injury, including Nrdg4, were identified and may be relevant to DA exposure levels below that causing neurobehavioral injury. Our results validate zebrafish as a vertebrate model to study mechanisms of DA neurotoxicity and provide a basis for identifying pathways of DA-induced injury as well as biomarkers of asymptomatic and symptomatic DA exposure levels. Keywords: neurotoxic disease To further understand the mechanisms of neurotoxic injury associated with DA exposure, we examined the transcriptome of whole brains from zebrafish (Danio rerio) receiving intracoelomic (IC) DA at both symptomatic and asymptomatic doses. A majority of zebrafish exposed to high-dose DA (1.2 µg DA/g) exhibited clinical signs of neuroexcitotoxicity (EC50 of 0.86 µg DA/g) within 5 to 20 minutes of IC injection. All zebrafish receiving low-dose DA (0.47 µg DA/g) or vehicle only maintained normal behavior. Microarray analysis of symptomatic and asymptomatic exposures collectively yielded 306 differentially expressed genes (1.5-fold, p = 0.05) predominately represented by signal transduction, ion transport, and transcription factor functional categories. All animal studies were carried out under approved IACUC protocols at the University of Washington.

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Transcriptional Response of Zebrafish Embryos Exposed to Neurotoxic Compounds Reveals a Muscle Activity Dependent hspb11 Expression

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