elevated hspb11 expression in muscle tissue similar as in embryos exposed to APM. In sopfixe2/2 mutants, which do not express a functional nAChR, the hspb11 developmental expression pattern was completely lost and an induction of hspb11 with APM was not possible. This data and the repressed transcription of hspb11 by MS-222 provided evidences that developmental hspb11 expression is muscle activity dependent. Modulation of intracellular calcium levels with thapsigargin or caffeine, which most likely affect the whole zebrafish embryo and is not restricted to muscles, resulted in hspb11 induction. Calcium dependent signal transduction cascades play important roles in the control of skeletal muscle gene expression in mammals and increased calcium levels result in, for instance, suppression of gene expression of myogenin and nAChR [41]. Likewise, in ache 2/2 mutant zebrafish embryos the sub-cellular localization of nAChRs is disturbed [5]. In our study, however, we did not detect down- regulation of muscle-specific genes such as nAChR or myogenin. It has been shown in zebrafish that acetylcholine and calcium signaling are required for the organization of slow muscle myofibrils [42]. Additionally, zebrafish mutants that exhibit disturbed calcium regulation in muscle tissues, such as ache, sopfixe or acc, showed disarrayed muscle fibers [5,18,43]. In our study, we showed that hspb11 expression is modulated in ache and sopfixe mutants. A continuous elevation of calcium levels and subse- quently enhanced calcium-activated protease activity has been shown to cause myofibre degeneration [20,44]. This suggests that the correct regulation of cytosolic calcium transients is important for hspb11 gene expression and myofibril organization and Hspb11 may compensate for calcium-induced protein degradation. We demonstrate that down-regulation of Hspb11 has no effect on muscle pioneer development, which is supported by the eng2a expression. Furthermore, morpholino knockdown of Hspb11 alters the distribution of slow muscle myosin. Hence, Hspb11 is important for myofibril organization and/or maintenance. Hspb11 may play a critical role in folding and assembly of various sarcomeric proteins during myofibrillogenesis and it remains to identify Hspb11 client proteins. The transcriptional regulation of hspb11 can be mediated via specific transcription factors, e.g. Creb, Nfat, Nf-kB, which are important components of intracellular signaling pathways and are regulated by calcium [45,46]. By analyzing sequences 5 kb upstream of the hspb11 transcriptional start site, several putative binding sites of these transcription factors are present (27 for Creb, 12 for Nfat, and 5 for Nf-kB, respectively, data not shown). However, the precise mechanism of the hspb11 transcriptional regulation remains to be further investigated. In summary, the combination of comparative toxicogenomics and functional analysis has led to the identification of hspb11 as a possible marker for interference with neuromuscular signaling and/or calcium signaling. Further research need to address how elevated calcium levels and hspb11 expression are linked. Materials and Methods Ethics Statement All zebrafish husbandry and experimental procedures were performed in accordance with the German animal protection standards and were approved by the Government of Saxony, Landesdirektion Leipzig, Germany (Aktenzeichen 75-9185.64). Based on the Guidelines on the protection of experimental animals by the Council of Europe, Directive 2010/63/EU, which allows zebrafish embryos to be used up to the moment of independent feeding (approximately 5 days after fertilization). Because embryos used here were no more than 4 days old, no license is required by Muscle Activity Dependent Expression of hspb11 Council of Europe (1986), Directive 2010/63/EU or the local authority. Fish maintenance A wildtype zebrafish strain (WiK) was obtained from the Max Planck Institute of Developmental Biology (Tu¨ bingen, Germany) and cultured at 26uC according to standard procedures [47]. Mutant lines achesb55, sopfixe and smub577 have been described previously and were kept at the Institute of Toxicology and Genetics, Karlsruhe [5,18,48]. Chemical exposure experiments Embryos were exposed from 2–50 hpf, 26–50 hpf, 48–50 hpf and/or 12 hpf (5-somite stage) to 72 hpf in 100 ml crystallization dishes covered with watchmaker glasses with the chemicals. The following chemicals were used: APM (azinphos-methyl, CAS#86- 50-0, purity 98,5%, Fluka), DMB (1,4-dimethoxybenzene, CAS#150-78-7, purity 99%, Sigma-Aldrich), 2,4-DNP (2,4- dinitrophenol, CAS#51-28-5, purity 99%, Fluka), GAL (galanta- mine hydrobromide, CAS#1953-04-4, purity 98%, Sigma), propoxur (CAS#114-26-1, purity 99,9%, Fluka), disulfoton (CAS#298-04-4, purity 99%, Fluka), 4-nitrophenol (CAS#100- 02-7, Fluka), thapsigargin (CAS#67526-95-8, purity $98%, Sigma), caffeine (CAS#58-08-2, purity $99%, Fluka) and MS- 222 (Ethyl 3-aminobenzoate methanesulfonate, CAS#886-86-2, purity 98%, Sigma-Aldrich). Stock solutions of the chemicals were prepared either in exposure medium (294.0 mg/L CaCl2*2H2O, 123.3 mg/L MgSO4*7H2O, 64.7 mg/L NaHCO3, 5.7 mg/L KCl; according to ISO guideline 15088 or DMSO (thapsigargin)). Exposure concentrations were prepared by dilution of stock solutions. In case of thapsigargin DMSO concentration in exposure and control media was adjusted to 0.01%. Exposure concentrations of APM, DMB and DNP were analyzed by GC- MS by the TZW (Technologie Zentrum Wasser, Karlsruhe, Germany). After 48 h of exposure 90% (APM, 5.23 mmol/L), 75% (DMB, 379 mmol/L) or 74% (DNP, 10.6 mmol/L), respectively, of the nominal concentrations could be detected. Microarray analysis RNA was extracted from 50 embryos for each treatment (controls and embryos exposed to the LC10, 4 independent biological replicates each). We used the 4644 K Agilent D. rerio oligo microarrays (Amadid #015064, Agilent, Bo¨blingen, Ger- many). The synthesis/labeling of cRNA with Cy3 (Agilent Low RNA Input Linear Amplification Kit) and hybridizations with the microarray slides were performed according to the manufacturer’s instructions. Slides were scanned with an Agilent DNA Microarray Scanner. Fluorescent intensities of individual microarray spots were extracted using the Agilent Feature Extraction software (Version 9.1). Log2-transformed fluorescent intensities were quantile normalized and used to calculate the ratio for each treatment versus the mean fluorescent value of the respective control. Analysis of statistical significance with SAM [49], false discovery rate adjusted to 0, p,0.03) and hierarchical clustering were performed with these ratios and the TMEV software package version 4.3 [www.tm4.org] [50]. The microarray data is MIAME compliant, and raw and normalized data have been submitted to the Gene Expression Omnibus (GEO) database [www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc = GSE27680]. For the identification of biological functions and pathways associated with the changes in gene expression caused by the APM treatment, Gene Set Enrichment Analyses (GSEA) [www. broadinstitute.org/gsea/index.jsp] [51,52] was performed. Since PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29063