Transcriptional Response of Zebrafish Embryos Exposed to Neurotoxic Compounds Reveals a Muscle Activity Dependent hspb11 Expression Nils Klu¨ ver1*, Lixin Yang 2 , Wibke Busch1, Katja Scheffler1¤, Patrick Renner1, Uwe Stra¨ hle2, Stefan Scholz1 1 Department of Bioanalytical Ecotoxicology, UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany, 2 Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany Abstract Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore 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 unspecific 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 induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sopfixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds. Citation: Kl u¨ver N, Yang L, Busch W, Scheffler K, Renner P, et al. (2011) Transcriptional Response of Zebrafish Embryos Exposed to Neurotoxic Compounds Reveals a Muscle Activity Dependent hspb11 Expression. PLoS ONE 6(12): e29063. doi:10.1371/journal.pone.0029063 Editor: Henry H. Roehl, University of Sheffield, United Kingdom Received July 22, 2011; Accepted November 20, 2011; Published December 19, 2011 Copyright: 2011 Kl u¨ver et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Federal Ministry of Education and Research (BMBF) GenDarT2 [PTJ-BIO/0315190] and by the European Commission Integrating Project ZF Health [HEALTH-F4-2010-242048]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nils.kluever@ufz.de ¤ Current address: Department for Stem Cell Biology and Applied Cell Techniques, University of Leipzig, Leipzig, Germany Introduction Understanding of chemical-induced biological responses and their underlying pathways is indispensable to evaluate the impact of chemicals on organisms. Toxicogenomic analysis is an effective approach to better understand these pathways and their related adverse effects [1–3]. A major step forward is the identification of chemical-specific gene expression signatures which could be used to assign mechanisms of action (MoA) to non-characterized or unknown compounds [4]. Changes in gene expression by exposure to chemicals can be directly inferred from binding of a compound to a transcription factor. Prominent examples are the aryl hydrocarbon or estrogen receptors. However, many compounds, e.g. neurotoxic chemicals, may not interfere directly with a transcription factor but affect gene expression patterns indirectly through a complex signaling cascade. In the present study, we examined the toxicogenomic response to azinphos-methyl (APM), an AChE-inhibitor, using zebrafish embryos as a model. AChE catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) and is important for the cholinergic neurotransmission. Furthermore, non-cholinergic functions of AChE have been described in neuronal development [5,6]. AChE is the target of many toxins like insecticides, chemical weapons, and snake venoms [7]. Prolonged AChE inhibition or complete lack of AChE results in an accumulation of ACh and overstimulation of the muscle, leading to spasms and myopathy- like phenotypes [5,8,9]. The zebrafish embryo has been selected as model for the following reasons: (i) Toxicogenomic approaches in the zebrafish embryo have recently shown that chemicals can provoke specific and sensitive gene expression changes [10–12]. (ii) It has been described that AChE is required for neuronal and muscular development in zebrafish embryos and no other ACh- hydrolyzing enzymes are present in this organism [5,13,14]. (iii) Appropriate mutant strains that either lack AChE (ache) or nAChR (sopfixe) activity are available to support functional analyses. First, we compared changes in gene expression patterns provoked by APM with those of two non-AChE inhibiting compounds, 1,4-dimethoxybenzene (DMB), a narcotic (baseline PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29063