by a loss of muscle pioneers in sopfixe2/2 embryos, we analyzed the eng2a expression in sopfixe2/2 embryos. It is known that muscle pioneers highly express engrailed1 and engrailed2 genes [25]. In all embryos of a cross of heterozygous parents, we detected muscle pioneer specific eng2a expression at 18 hpf (Figure S5C). These altered hspb11 expression patterns led us to ask if the hspb11 expression is muscle-activity dependent and can be repressed by blocking the muscle contraction with the anesthetic MS-222, a voltage-gated Na+ channel blocker, in order to prevent action potentials. Treatment with MS-222 [0.5 mM] for a 6 h (18 hpf to 24 hpf) resulted in a significant hspb11 repression (Figure 5A) and the wildtype hspb11 expression pattern was lost (Figure 5B and C). This suggests that the developmental hspb11 gene expression is muscle activity dependent. Hspb11 is Involved in Slow Muscle Myosin Organization and/or Maintenance To examine the developmental function of Hspb11, we performed a morpholino-mediated knockdown of Hspb11. Therefore we used two translation-blocking morpholinos, MO(ATG)-hspb11 and MO(59UTR)-hspb11, and a mismatch control morpholino (mmMO-hspb11) that harbors five mismatches compared to the MO(ATG)-hspb11 were used. To avoid interfer- ence with nonspecifically induced apoptosis a morpholino directed against p53 was co-injected [26]. To prove that the injected hspb11 morpholinos are specific to hspb11 mRNA we performed additional control experiments. First, we injected a gfp mRNAs that contains either the morpholino binding site for MO(ATG)- hspb11 or MO(59UTR)-hspb11 upstream of the GFP coding sequence and we detected a bright GFP fluorescence (Figure S6). Coinjection of gfp mRNA with either MO(ATG)-hspb11 or MO(59UTR)-hspb11 strongly reduced the GFP fluorescence (Figure S6). Further, coinjection with the designed mmMO-hspb11 and gfp mRNA did not reduce the GFP fluorescence (Figure S6). Both MO(ATG)-hspb11 and MO(59UTR)-hspb11 morphants showed a ventral body curvature. In MO(ATG)-hspb11 and MO(59UTR)-hspb11 injected embryos, 82% (n = 79, 2 replicates) and 73% (n = 114, 2 replicates) showed a morphant phenotype, respectively. The MO(59UTR)-hspb11 phenotype was much Muscle Activity Dependent Expression of hspb11 Figure 5. Blocking the skeletal muscle contraction with MS-222 inhibits developmental hspb11 expression. (A) qPCR hspb11 expression analysis. (B) Wildtype hspb11 expression pattern at 24 hpf. (C) MS222 treatment results in the loss of hspb11 expression pattern at 24 hpf. doi:10.1371/journal.pone.0029063.g005 stronger and mmMO-hspb11 control injected embryos showed no effects (Figure 6A–C). Additionally, we tried to rescue this morphant phenotype by coinjection of a hspb11 mRNA that is not targeted by MO(ATG)-hspb11 and MO(59UTR)-hspb11. Coinjec- tion of the hspb11 mRNA with either MO(ATG)-hspb11 or MO(59UTR)-hspb11 reduced the ventral body curvature pheno- types to 53% (n = 98, 2 replicates) and 51% (n = 137, 2 replicates), respectively. In MO(ATG)-hspb11, MO(59UTR)-hspb11 and mmMO-hspb11 injected embryos, we detected a wildtype eng2a expression pattern in muscle pioneers at 18 hpf (Figure S5D and E). Furthermore, knockdown of Hspb11 did not lead to a decrease in birefringence (Figure 6D–F). However, 80% (n = 10) of hspb11 morphants showed a disruption in slow muscle myosin distribution. Gaps were formed between slow myofibers in knockdown embryos (Figure 6G–I). There are no other impairments, such as extra long intersomitic myofibers, detached myofibers or defective myosepta. This indicates that the development of muscle pioneers is not Figure 4. Developmental hspb11 expression pattern in wildtype, APM exposed, ache and sopfixe mutant embryos. Expression pattern of hspb11 at 18 hpf were detected in developing adaxial musculature (A–A0). During later developmental stages, at 24 hpf, hspb11 mRNA transcripts were restricted to muscle pioneers (B and B9) and became only expressed in the notochord at 50 hpf (C). (A, B) lateral view, (A9) dorsal view and (A0 and B9) cross sections. Increased expression pattern in axial musculature was observed in APM exposed (D) and homozygous (2/2) ache mutant embryos (E). Hspb11 expression in homozygous sopfixe2/2 embryos (F and G) and heterozygous/wildtype (+/?) siblings (H and I). doi:10.1371/journal.pone.0029063.g004 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29063