Muscle Activity Dependent Expression of hspb11 Figure 6. Knockdown of Hspb11 results in slow muscle myosin disorganization in skeletal muscles. (A, D and G) mmMO-hspb11 injected embryos. (B, E and H) MO(ATG)-hspb11 morphants. (C, F and I) MO(UTR)-hspb11 morphants. (A–C) Phenotypic observations of morpholino injected embryos at 48 hpf. (D–E) Muscle organization determined with birefringence at 72 hpf. (G–I) Slow muscle myosin distribution at 72 hpf shown by immunofluorescence staining with antibody F59. Arrows indicate gaps in fiber distribution. doi:10.1371/journal.pone.0029063.g006 dependent on Hspb11. Moreover, the myofibril organization in superficial muscle cells is affected in the hspb11 morphants, which suggests that Hspb11 is involved in slow muscle myofibril organization and/or maintenance. Discussion By a comparative toxicogenomic approach we demonstrated that the AChE inhibitor APM induced specific responses on the gene expression levels in zebrafish embryos. Other non-AChE inhibiting compounds (DMB and DNP) did not affect the same cluster of genes. Gene set enrichment analyses (GSEA) revealed APM affected genes as enriched in a couple of gene sets involved in neuronal/muscle structure and differentiation. The enrichment of genes within these gene sets particularly after 48 h of exposure might indicate subtle morphological changes caused by APM. This is supported by the birefringence analysis which revealed structural alterations in muscular tissue. It is known that the lack of AChE activity in zebrafish results in an increase of muscular activity and can cause a progressive myopathy [5,8]. We showed that genes of the APM-specific cluster (hspb11, pdlim3b and socs3a) were differently expressed in response to various AChE inhibitors. Pdlim3b, also known as Alp-like, is a structural protein and a member of the PDZ/LIM protein family which are characterized by the presence of both, a PDZ and a LIM domain [27]. Their functions are related to actin anchorage in muscles and non- muscle cells. PDZ/LIM proteins are required for muscle development and maintenance as has been shown for zebrafish pdlim7 [28]. During zebrafish development pdlim3b (alp-like) is expressed in myotomes [27]. Hence, changes in pdlim3b expression in response to AChE inhibitors might be related to early disturbances of muscle structure and differentiation. The compar- ison of the responses to different AChE inhibitors and 4- nitrophenol indicated that socs3a was not induced specifically by AChE inhibitors. Socs3a belongs to the suppressor of cytokine signaling (Socs) protein family. It negatively regulates cytokine signaling in order to prevent excessive cytokine signaling that may disturb the normal homeostasis and cellular functions [29,30]. Hspb11 is a member of the small heat shock proteins (sHSPs) family, which is a subclass of HSPs characterized by their low molecular weight and presence of the conserved a-crystalline domain. Many sHSPs act to prevent or reverse improper protein associations in an ATP independent manner and they are expressed in nearly all species [31,32]. In our microarray experiments, hspb11 was the only significantly induced member of sHSPs in zebrafish embryos exposed to APM. Commonly, the chaperone function, i.e. the support of correct protein folding, is considered as major physiological role of sHSPs and is regarded as the reason for their induction by a variety of stress factors [33]. It has been shown that HSPs are involved in several other processes, such as actin regulation, intermediate filaments assembly, apoptosis signaling or regulation of the cellular redox status [34,35]. Furthermore, various diseases are known to be linked to mutations of sHSPs including cancer, neurodegenerations, and myopathies [36,37]. Up to now, thirteen sHSPs are identified in zebrafish and their developmental and heat shock induced expression patterns have been described. The expression of hspb11 is strongly induced after heat shock and transcripts are found throughout the somites and the hearts [38]. Hspb11 orthologs seem to exist in nearly all vertebrates except mammals. ACh is a neurotransmitter which is released by excitation of motoneurons at the neuromuscular junction. Binding of acetyl- choline to nAChR depolarizes membranes of adjacent muscle cells and induces an intracellular calcium increase. Calcium acts as a messenger linking excitation events at the membrane with downstream effects, like contraction, ATP production and transcription [39,40]. Thus, the observed altered expressions of hspb11 could be mediated via the ACh/nAChR/calcium pathway. In this case, transcriptional up-regulation would primarily be observed in skeletal muscle tissues. An inactivation of nAChR would result in a loss of hspb11 expression and ache mutants should exhibit elevated hspb11 levels as well. Furthermore, it would be likely that compounds, which do not activate nAChR but increase cytosolic calcium levels, induce the expression of hspb11. Indeed, the experimental observations clearly support the anticipated role of nAChR and calcium, i.e. the ache 2/2 mutant exhibited an PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29063