Mtmr8 in Muscle Development Figure 2. Targeted knockdown of Mtmr8 using splicing morpholino in zebrafish embryos. (A) Diagram of splicing junction morpholino targeted against Mtmr8 exon-intron boundary. (B) Amino acid sequences of wild type Mtmr8 and Mtmr8-MO1 mediated mis-splicing Mtmr8. Sequencing of the RT-PCR products revealed the mis-splicing transcript leading to a premature stop (asterisk) and causing a truncation in the protein (exon 1 in bold, the other exons in plain, and intron in plain and underlined). (C) RT-PCR detection of Mtmr8 transcript at 24hpf in WT and Mtmr8-MO1 (6 ng) morpholino-injected embryos, comparing cryptic spliced transcript in the morpholino injected embryos to the cDNA PCR products. (D) Live morphology of WT control zebrafish embryo (a) and Mtmr8-MO1 knockdown embryo (b, c) at 24hpf. Injection volume was about 2 nL at 1-cell stage embryos. All scale bars are 100 mm. (E) Statistical data of three independent experiments on Mtmr8 knockdown as well as its overexpression and Mtmr8 mRNA rescue. Results are represented as mean6SD of three separate experiments (60 embryos in each experiment). doi:10.1371/journal.pone.0004979.g002 sitol-3-kinase inhibitor, LY294002[34]. In order to assay the connections between Mtmr8 and PI3-Kinase/AKT pathway during embryo development, we inhibited the PI3K activity with 10 mM LY294002 between 10 hpf and 24 hpf, when transcript of Mtmr8 became restricted to eye field and somites. When treated with 10 mM LY294002, the defects of Mtmr8-MO1 morphant embryos could be further deteriorated (Fig 4B-d), and the Mtmr8- MO2 morphants had severe defects (Fig 4B-e), comparing with Cont morpholino injected morphants which did not alter the wild- type phenotype throughout development 4B-c). In addition, loss of PH/G domain could also produce a significant increase in the relative amount of pAkt compared to Cont MO injected embryos (Fig 4C). The results indicate that the PH/G domain is essential for Mtmr8’s function and that protein encoded by the splice variant may lose part of its functions. Mtmr8 cooperation with PI3K regulates actin filament modeling Mtmr8 is a member of the growing class of dual-specificity phosphatases (DSPs) as PTEN, which controls cell random movement through the Ras, PI3K, PTEN, and F-actin regulatory loop [29]. To examine a possible requirement for Mtmr8 in integrity of the actin cytoskeleton, we used phalloidin-TRITC to stain filamentous actin in embryos at 24 hpf. In almost all the embryos treated with Mtmr8-MO1, we observed obvious defect of the actin cytoskeleton in a proportion of skeletal muscle fibres (Fig 5A-b), compared to the Mtmr8-MO2 morphant (Fig 5A-c) and control embryos (Fig 5A-a). However, when treated with LY294002, the actin cytoskeleton of Mtmr8-MO2 morphant was severely disturbed (Fig 5A-d). Nexilin, a novel actin filament (F-actin)-binding protein, which was strongly expressed at somites at 24hpf [32]. We performed whole-mount in situ hybridization with zebrafish Nexilin riboprobes, and revealed that nexilin was significantly reduced and disorganized in the 24hpf Mtmr8-MO1 morphants (Fig 5B- b), However, the expression of nexilin was not obviously changed in Mtmr8-MO2 morphants (Fig 5B-c) and Cont MO morphants treated with LY294002 (Fig 5B-a). To further understand the function of Nexilin in integrity of the actin cytoskeleton, we performed morpholino to knockdown the zebrafish Nexilin. As shown by RT-PCR, in the control embryos, the amplified PCR product is 382 bp by primers designed from the first and second extrons. However, in the Nexilin- MO morphants, because of inclusion of intron 1, the product is longer than 3 kb, which could not be amplified in one minute (Fig 5C). Embryos injected with the 3 ng Nexilin MO and stained with phalloidin, did show severe skeletal muscle detachments (Fig 5d-b,d), compared to the control (Fig 5d-a,c). Mtmr8 is essential for the hedgehog pathway to regulate muscle development during embryogenesis The Mtmr8 morphants have morphological traits in common with Hedgehog pathway mutants, such as U-shaped somite boundaries. To verify that the defects in Mtmr8 morphants are correlated with the changes in Hh signaling, we examined the expression of myod and patched1 (ptc1), a downstream target gene and receptor of Sonic hedgehog [35,36]. Compared with the control (Fig 6A-a,d), Myod and ptc1 expression is severely reduced and disorganized in Mtmr8 morphants (Fig 6A-b,e). Dominant negative PKA (dnPKA) mRNA, when injected into embryos, results in a broader expression of Hh target genes and a rescue of the Myod and ptc1 expression (Fig 6A-c,f). These results support the conclusion that Mtmr8 is required for Hedgehog signaling in zebrafish development. To study the function of Mtmr8 in slow muscle development, the embryos were then stained with antibody F59, which detects mostly slow myofibrils, although it also reacts weakly with fast Figure 3. Phosphatase activity of zebrafish Mtmr8. Different extracts from Cont MO embryos, Mtmr8 morphants and Mtmr8 morphants co- injected with Mtmr8 mRNA were prepared at 24 hpf (30 embryos/group) and subjected to Western blot detection with anti-Akt antibody or anti- phospho-Akt (pAkt) antibody. The corresponding histograms (right panels) plot the relative Ser(P)473-Akt to total Akt ratio (Relative p-Akt/Akt ratio), determined by band intensities that were analyzed by Scion software, with the ratio in experiment extracts normalized to the ratio determined in Cont MO injected embryo extracts. Results represent mean6SD of three separate experiments. *P,0.05. doi:10.1371/journal.pone.0004979.g003 PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4979