Figure 2. APM concentration dependent induction of hspb11, pdlim3b and socs3a in zebrafish embryos. APM exposures for (A) 24 h (26–50 hpf) and (B) 2 h (48–50 hpf). Concentrations are given in mM. Bars represent the relative gene expression as fold change of the respective untreated control as mean 6 standard deviation of three independent replicate exposures. Control = ctrl. * P,0.05. doi:10.1371/journal.pone.0029063.g002 [5]. The sopfixe mutant harbors a point mutation in the d-subunit of the muscular nAChR, encoded by the gene chrnd, and, hence, lacks a functional nAChR. The d-subunit is expressed in all striated muscles in embryonic and early larval stages [18]. Homozygous (2/2) forms of both mutant embryos show severe impairment of motility but are viable up to 5 days post fertilization (dpf). Interestingly, the expression of hspb11 was strongly increased in 48 hpf ache 2/2 and repressed in sopfixe 2/2 mutant embryos (Figure 3A). Furthermore, exposure of sopfixe 2/2 with APM (24– 48 hpf) did not result in further induction of hspb11 expression, whereas sopfixe siblings exposed to APM showed a 9-fold up- regulation (Figure 3B). These data indicate that hspb11 induction by APM is dependent on a functional nAChR. Modulators of Cytosolic Calcium Levels Induce hspb11 Expression ACh triggers muscular contraction by depolarizing muscle cells and subsequent transient release of calcium from the sarcoplas- matic reticulum. Calcium is not only the principal signaling molecule for muscle contraction, it is also required for normal development of muscles. It is hypothesized that excess activation of AChR by unhydrolyzed ACh results in a large influx of calcium ions in muscle cells. Indirect evidence for the role of calcium ions is provided by studies that demonstrate a rescue of myopathy (provoked by exposure to AChE inhibitors) by calcium channel blockers [19–21]. Hence, we concluded that hspb11 induction by AChE inhibition might be a result of increased cytosolic calcium levels and increased muscular contractions mediated by overstim- ulation of the nAChR. Hence, non-AChE inhibiting compounds that lead to an increase in cytosolic calcium should increase hspb11 mRNA abundance. Therefore, we exposed zebrafish with thapsigargin and caffeine. Thapsigargin is a specific blocker of Muscle Activity Dependent Expression of hspb11 Figure 3. APM mediated hspb11 induction depends on nAChR activity and increased intracellular calcium levels. (A) hspb11 expression analysis in ache and sopfixe2/2 zebrafish mutant embryos at 48 hpf. (B) hspb11 expression in sopfixe mutants and siblings (+/? = heterozygous and wildtype) after APM (6 mM) exposure (26–50 hpf). (C) 1 mM Thapsigargin (TG) and 2 mM caffeine (CAF) induce hspb11 expression in zebrafish embryos (exposure period 48–50 hpf). Bars represent the relative gene expression as fold change of the respective untreated control as mean 6 standard deviation of three independent replicate exposures Control = ctrl. * P,0.05. doi:10.1371/journal.pone.0029063.g003 the sarco/endoplasmic reticulum calcium-ATPase (SERCA) [22]. Caffeine represents an activator of ryanodine receptors (RyRs), a major mediator of calcium-induced calcium release in adaxial muscles [23]. Exposure of wildtype embryos with either thapsi- gargin [1 mM] for 2 h (48–50 hpf) or caffeine [2 mM] for 24 h (26–50 hpf) resulted in an increased hspb11 gene expression. Thapsigargin induced hspb11 mRNA expression 11-fold and caffeine 23-fold (Figure 3C). Hspb11 Is Expressed in Muscle Pioneers and Shows a Muscle-activity Dependent Expression In order to determine the function of hspb11, we first analyzed the spatiotemporal hspb11 expression. At 18 hpf hspb11 is expressed in the adaxial cells (i.e. somitic cells next to the notochord, Figure 4A–A0). At 24 hpf hspb11 mRNA expression is restricted to muscle pioneers (Figure 4B and B9). To elucidate whether the hspb11 transcripts are exclusively expressed at these early stages in muscle pioneers, we screened the hspb11 expression pattern in smu 2/2 mutant embryos. smu mutants are defective in smoothened - a mediator of hedgehog signals - and lack muscle pioneers [24]. In smu 2/2 embryos hspb11 expression was lost (Figure S5A and B) indicating that hspb11 is specifically expressed in muscle pioneers. During later development hspb11 expression was restricted to the notochord (Figure 4C). Interestingly, this hspb11 expression pattern changed in APM exposed embryos and in ache2/2 mutant embryos where transcripts were also detected in the myotomes of the trunk in addition to the notochord (Figure 4D and E). To test whether the developmental hspb11 expression is dependent on nAChR activity we investigated the hspb11 expression pattern of sopfixe mutants. Of a single cross of heterozygote parents we used 30 embryos at 18 hpf for WISH. In 8 embryos, we did not detect any hspb11 expression pattern in muscle pioneers whereas 22 embryos showed a wildtype hspb11 expression pattern, as expected for a recessive trait inherited in a mendelian fashion (Figure 4F and H). Further, at 24 hpf the sopfixe 2/2 embryos lack hspb11 expression pattern whereas sopfixe+/? embryos have a wildtype hspb11 expression pattern (Figure 4G and I). To exclude that the lack of hspb11 expression pattern is caused PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29063