Table 1. Mtmr8 is non-cell autonomous for muscle F-actin modeling. Donor genotype Host genotype Result Mtmr8 morphant Wild-type Wild-type Mtmr8 morphant 12/12 Severe defect (100%) 15/15 Wild-type (100%) 10/10 Wild-type (100%) 13/13 Wild-type (100%) 13/13 Severe defect (100%) 12/12 Severe defect (100%) Donor embryos were labeled with fluorescein dextran. At 24 hpf, donor and host embryos were observed under the microscope, and then fixed for phalloidin labeling to determine genotype (as shown in Fig 7C). The phenotype of the transplanted embryos (number and percentage) is reported. doi:10.1371/journal.pone.0004979.t001 differentiation. Shh induces Akt phosphorylation in adult muscle cells and influences the PI3K/Akt pathways in a manner similar to IGF-I [48]. Although an increase in Akt phosphorylation could be detected in response to knockdown of Mtmr8, it was not enough to rescue the expression of Myod and Ptc1. Over-expression of dnPKA mRNA, a downstream of Shh, could reverse the defect induced by Mtmr8 deficient. Previous findings provide a basis for the synergistic role of PI3-kinase/Akt in Hh signaling in embryonic development and Hh-dependent tumors [49]. The PI3K inhibitor LY294002 inhibited migrating neo cells and was able to further inhibit residual dnPKA cell migration. PKA may play an important role in the signaling processes that lead to motility. Either inhibition or hyper-activation of PKA may inhibit cell migration, F-actin polymerization and synthesis [42,50–52]. Figure 8. A hypothesized signal pathway that Mtmr8 regulates zebrafish muscle development. Knockdown of Mtmr8 promotes PI3K/Akt pathway and prevents Hh signaling pathway. The two pathways may interact with each other in regulating F-actin modeling and muscle development. doi:10.1371/journal.pone.0004979.g008 Mtmr8 in Muscle Development Knockdown of Mtmr8 led to hyper-activation of PKA, which caused the reduction expression of Nexilin and F-actin. All these suggested that Mtmr8 and PI3K/Akt play a synergistic role in regulation of Hh signaling in embryonic muscle development. Currently, many potential therapies are evaluated by introduc- ing fluorescently tagged cells into a diseased animal and then following their fate using fluorescence to determine if the introduced cells localize to muscle and participate in the repair process[27]. Mutation or knockdown of several genes in zebrafish have been shown to be the underlying basis of many muscular defects[53–56]. Cell transplantation experiments could confirm that the gene function is required cell-autonomously or non-cell- autonomously within the muscle development, which may be as a model to evaluate the possibility to use cell therapy for human genetic muscle disease during early fetus. Mtmr8 acts in non-cell- autonomous manner during embryos muscle development, which may suggest that cell therapy is not an efficient way to rescue the defect during early embryo development. The effectiveness of cell therapy could also be evaluated in human neuromuscular disorders caused by mutations in other myotubularin family genes. From our studies, we have demonstrated that Mtmr8 have the same function as lipid phosphatases PTEN to dephosphorylate the PI3K products by down-regulating PI3K activity, and to regulate actin modeling and muscle development of zebrafish [57]. However, it is different from tumor suppressor gene, PTEN. Mtmr8 is not only an antagonist of PI3K, but also as a partner to balance the PI3K expression in embryo development regulation of zebrafish. Materials and Methods Maintenance of zebrafish A breeding colony of zebrafish (Danio rerio) were maintained at 28.5uC on a 14 h (hour) light/10 h dark cycle [58]. All embryos were collected by natural spawning and staged according to Kimmel et al. [59]. Kinase inhibitors, LY294002 were dissolved in DMSO at stock concentration of 50 mM, and then diluted to final concentration of 10 mM in embryo media from 10 hpf to 24 hpf, which had no obvious effect on embryos survival, activity and health. Control embryos were treated with the equivalent amount of DMSO solution. RT-PCR and Western blotting Total RNAs were isolated by SV RNA Isolation Reagent (Promega) from different stages and tissues, and the concentration and quality were determined by agarose electrophoresis and spectrophotometer. After treating with DNase I (RNase-free, Promega), the RNAs (about 1 mg) were reverse-transcribed with MMLV (Gibco) at 37uC with oligo(dT)15 primer. b-actin was used as internal control genes. All samples were analyzed in triplicates. Phosphatase activity of zebrafish Mtmr8 and Western blot detection were performed according to previous reports[60,61]. Images of blots were captured with a scanner, and quantitative densitometric analysis was performed using Scion Image. Antisense morpholino and mRNA microinjection Morpholinos were synthesized by GeneTools LLC (Philomath, OR). Following are the sequences for various morpholinos: Mtmr8- MO1 (Splicing antisense), 59-CACCTGCTGACTCAGACC- TACCTTC-39; Mtmr8-MO2 (Splicing antisense), 59-GGCCAA- CATTACCCATGTTTCTTTG-39; Nexilin-MO (Splicing anti- sense), 59-ATAGCCTCTTCATACTTTACCTCTT-39; Standard control MO (Cont MO), 59-CCTCTTACCTCAGTTACAATT- TATA-39. For injection, MOs were injected into fertilized zebrafish PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4979