toxic) compound and 2,4-dinitrophenol (DNP), an oxidative decoupling agent. The test compounds were selected to allow the identification of genes specifically responding to compounds inhibiting acetylcholine esterase. Neurotoxicity is a common MoA among compounds exhibiting an excess toxicity (i.e. ratio of predicted baseline toxicity versus measured toxicity). The organophosphate insecticide and known acetylcholine esterase inhibitor APM [15] exhibits a high acute excess toxicity, i.e. the measured LC50 is 1340fold below the predicted acute toxicity [16]. This clearly supports the specific neurotoxic mode of action. The other compounds were selected as two different non-neurotoxic reference compounds in order to allow the identification of MoA specific gene expression patterns. DNP, an industrial chemical, is a protonionophor or uncoupling agent that disrupts generation of ATP from proton-gradients in the mitochondria [17]. DNP has a moderate excess toxicity of 23. In contrast to APM and DNP the acute fish toxicity of DMB is in the same order of magnitude as the predicted baseline-toxicity. Subsequently, detailed transcriptional analyses of selected genes were applied to link gene expression changes to the MoA. We investigate the expression of the small heat shock protein hspb11 in wildtype, ache and sopfixe mutant embryos in more detail and study its role during development. For the first time, we demonstrate that developmental hspb11 expression is muscle-activity dependent and is indirectly regulated by intracellular calcium levels. Furthermore, Hspb11 is required for slow muscle myofibril organization in the embryo. These results show that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds. Results Toxicogenomic responses to APM exposure are highly specific For transcriptional profiling it was aimed that similar – with respect to toxicity – effect concentrations were used that did not induce severe morphological changes. Based on concentration response curves of zebrafish embryos exposed from 2-50 hours post fertilization (hpf) we selected the modeled LC10 (6 mM for APM, 509 mM for DMB and 14 mM for DNP) as exposure concentrations for the microarray study (Figure S1). Exposures to this effect level did not induce gross morphological changes. However, APM inhibited zebrafish AChE enzymatic activity half- maximally at 0.15 mM and caused an almost complete block of AChE activity at 6 mM (Figure S2). It is known that in ache mutants the lack of AChE activity can cause a progressive myopathy in zebrafish embryos [5,8]. To test whether APM caused similar defects we performed a birefringence analysis of embryos exposed from 12–72 hpf to 6 mM APM (according to [8]). APM treated embryos were immobile and displayed a reduced birefringence compared to controls (Figure 1). This suggests that a prolonged exposure with 6 mM APM caused changes in muscular structures. For the microarray analysis we exposed zebrafish embryos for 24 h (26–50 hpf) and 48 h (2–50 hpf). The different exposure durations were chosen in order to identify potential differences with respect to primary or secondary responses. By hierarchical cluster analysis we identified an APM specific cluster of significantly induced genes after the 24 h as well as the 48 h exposure (Figure S3). The observed differences, however, were more prominent after 24 h of exposure. In order to identify pathways, associated with the transcriptional changes, a gene set enrichment analysis (GSEA) was performed. Some of the identified gene sets in the 24 h and 48 h treatments referred to factors/genes Muscle Activity Dependent Expression of hspb11 Figure 1. Repression of AChE activity with APM resulted in a reduced birefringence. (A) Wild-type zebrafish embryo axial musculature is highly birefringent at 72 hpf. (B) APM [6 mM] exposure from 12–72 hpf resulted in defects in the axial musculature, which are shown by a reduced birefringence at 72 hpf. doi:10.1371/journal.pone.0029063.g001 involved in neuronal and muscle function/differentiation (axon guidance, neurogenesis, voltage gated channel activity, muscle structure and contraction, muscle tissue maintenance and differentiation, supplement Table S1). More gene sets indicating effects specific for neurons and muscles were identified for the 48 h treatment. This may indicate that GSEA revealed (subtle) changes in neuron or muscle structure and differentiation. However, GSEA did not indicate interference with a specific signaling pathway. Transcriptional changes of the zebrafish embryo are AChE-inhibitor concentration-dependent In order to unravel the MoA leading to the expression changes in the APM-specific gene cluster, we selected hspb11 (heat shock protein, alpha-crystallin-related, b11), pdlim3b (PDZ and LIM domain 3b), and socs3a (suppressor of cytokine signaling 3a) for concentration-dependent quantitative expression analysis. The selection of these genes was based on an initial RT-PCR verification experiments (not shown) and the genes covered different potential gene functions, e.g. stress response (hspb11), immuno response (socs3a) and cytoskelatal assembly (pdlim3b). The expression of these genes was gradually increased with elevated APM concentrations. By means of qPCR an about two-fold (pdlim3b), 8-fold (hspb11) and 6-fold (socs3a) maximum induction after 24 h of exposure was observed (Figure 2A). Lowest observed effect concentrations for gene expression changes compared to the control were 0.7 mM and 3 mM of APM. Changes of hspb11 and socs3a gene expression were already detected in 2 h (48 to 50 hpf) APM exposures whereas the induction of hspb11 (approx. 20-fold) exceeded the levels of expression after 24 h of exposure (Figure 2A and B). The expression of hspb11 was also found to be induced by other AChE inhibitors such as propoxur, disulfoton, and galantamine (Figure S4). In contrast, the expression of socs3a was not affected by the disulfoton treatment but also elevated after a treatment with the non-AChE-inhibiting compound 4-nitrophenol (Figure S4D). Pdlim3b was induced by all tested AChE inhibitors and not by 4-nitrophenol. This suggests that hspb11 and pdlim3b up-regulation is a specific response to AChE inhibitors. Due to the strong and sensitive changes in hspb11 expression subsequent functional analyses were focused on hspb11. APM Mediated hspb11 Up-Regulation Depends on nAChR Activity APM-induced hspb11 expression could be mediated by the overstimulation of the nAChR. In order to elucidate the role of nAChR we analyzed hspb11 expression in the zebrafish ache and sopfixe mutant lines. Due to a point mutation in the ache gene, homozygous ache mutant embryos completely lack AChE activity PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29063