eggs at the one-cell stage at a concentration of about 6 ng each embryo. After injection, embryos were incubated at 28.5uC in Embryo Medium [58]. Plasmids pCS2-Mtmr8 and pCS2-dnPKA were linearized for in vitro transcription. Capped sense RNAs were synthesized using SP6 RNA polymerase and the SP6 Cap-Scribe (Roche), following the manufacturer’s instructions, re-suspended in water and injected at a concentration of 100 ng/mL. Whole-mount in situ hybridization and immunohistochemistry Embryos at different stages were collected and pre-treated and fixed as described [57]. Purified plasmids was linearized by selected restriction enzymes and used as templates for in vitro transcription using T7 or Sp6 RNA polymerase to generate DIG- labeled (Roche) sense and anti-sense probes. In situ hybridization was performed as described [62]. F59 antibody was purchased from Developmental Studies Hybridoma Bank (DSHB, University of Iowa, USA), and Prox1 antibody from Abcam (Cambridge, United Kingdom). Zebrafish embryos were fixed overnight in 4% paraformadehyde at 4uC, and then washed in 0.1% Triton in PBS (PBT) and dechorionated. They were then incubated for 1 h in 0.5% Triton in PBS, followed by 5-h incubation in block solution (10% normal goat serum, 1% DMSO, 0.1% Triton in PBS). Embryos were then incubated overnight at 4uC in block solution containing Phalloidin and/or primary antibodies. They were then washed in PBT, and incubated for 5 h at room temperature with secondary antibodies. Antibody and Phalloidin staining of zebrafish embryos were performed as previously described [63]. References 1. Wishart MJ, Dixon JE (2002) PTEN and myotubularin phosphatases: from 3- phosphoinositide dephosphorylation to disease. Trends Cell Biol 12: 579–585. 2. Hunter T (1998) Anti-phosphatases take the stage. Nat Genet 18: 303–305. 3. Denu JM, Stuckey JA, Saper MA, Dixon JE (1996) Form and function in protein dephosphorylation. Cell 87: 361–364. 4. Laporte J, Bedez F, Bolino A, Mandel JL (2003) Myotubularins, a large disease- associated family of cooperating catalytically active and inactive phosphoinosi- tides phosphatases. Hum Mol Genet 12 Spec No 2: R285–292. 5. Fischer EH, Charbonneau H, Tonks NK (1991) Protein tyrosine phosphatases: a diverse family of intracellular and transmembrane enzymes. Science 253: 401–406. 6. Burke TR Jr, Zhang ZY (1998) Protein-tyrosine phosphatases: structure, mechanism, and inhibitor discovery. Biopolymers 47: 225–241. 7. Berger P, Schaffitzel C, Berger I, Ban N, Suter U (2003) Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module. Proc Natl Acad Sci U S A 100: 12177–12182. 8. Lorenzo O, Urbe S, Clague MJ (2006) Systematic analysis of myotubularins: heteromeric interactions, subcellular localisation and endosome related func- tions. J Cell Sci 119: 2953–2959. 9. Tsujita K, Itoh T, Ijuin T, Yamamoto A, Shisheva A, et al. (2004) Myotubularin regulates the function of the late endosome through the gram domain- phosphatidylinositol 3,5-bisphosphate interaction. J Biol Chem 279: 13817–13824. 10. Lorenzo O, Urbe S, Clague MJ (2005) Analysis of phosphoinositide binding domain properties within the myotubularin-related protein MTMR3. J Cell Sci 118: 2005–2012. 11. Galaktionov K, Lee AK, Eckstein J, Draetta G, Meckler J, et al. (1995) CDC25 phosphatases as potential human oncogenes. Science 269: 1575–1577. 12. Li J, Yen C, Liaw D, Podsypanina K, Bose S, et al. (1997) PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275: 1943–1947. 13. Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, et al. (1997) Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet 15: 356–362. 14. Laporte J, Hu LJ, Kretz C, Mandel JL, Kioschis P, et al. (1996) A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast. Nat Genet 13: 175–182. 15. Liaw D, Marsh DJ, Li J, Dahia PL, Wang SI, et al. (1997) Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat Genet 16: 64–67. Mtmr8 in Muscle Development Transplantation Embryos, which were used as donors at later stages, were injected between one-cell to two-cell stages with 2.5% fluorescein dextran (Molecular Probes product) alone or in combination with 6 ng morpholino. Donor and host embryos were dechorionated. The cells were sucked from the prospective mesodermal region of a donor at the sphere stage, and transplanted into the same position of wild-type host embryos at the same stage. Locations of transplanted cells in the hosts were determined by fluorescence stereomicroscopy at the shield stage. Distribution of transplanted cells in the hosts were observed and photographed at 24 hpf postfertilization (hpf) using a Leica fluorescence stereomicroscope. We sucked out the embryos with transplanted cell mainly in the somites, which were fixed at 24 hpf for 2 h at room temperature in 4% paraformaldehyde, and later for phalloidin staining. Statistical analysis Data are presented as mean6SD. The relative expression level of p-Akt in Mtmr8 morphants and control morpholino groups were compared statistically using one-way analysis of variance (AN- OVA), followed by the Tukey’s post hoc test for multiple comparisons. A probability (P) of ,0.05 was statistically considered significant. Author Contributions Conceived and designed the experiments: JM JFG. Performed the experiments: JM ZL. Analyzed the data: JM. Contributed reagents/ materials/analysis tools: JM JFG. Wrote the paper: JM JFG. 16. MacKeigan JP, Murphy LO, Blenis J (2005) Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemore- sistance. Nat Cell Biol 7: 591–600. 17. Chojnowski A, Ravise N, Bachelin C, Depienne C, Ruberg M, et al. (2007) Silencing of the Charcot-Marie-Tooth associated MTMR2 gene decreases proliferation and enhances cell death in primary cultures of Schwann cells. Neurobiol Dis 26: 323–331. 18. Buj-Bello A, Furling D, Tronchere H, Laporte J, Lerouge T, et al. (2002) Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells. Hum Mol Genet 11: 2297–2307. 19. Azzedine H, Bolino A, Taieb T, Birouk N, Di Duca M, et al. (2003) Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie- Tooth disease associated with early-onset glaucoma. Am J Hum Genet 72: 1141–1153. 20. Dang H, Li Z, Skolnik EY, Fares H (2004) Disease-related myotubularins function in endocytic traffic in Caenorhabditis elegans. Mol Biol Cell 15: 189–196. 21. Xue Y, Fares H, Grant B, Li Z, Rose AM, et al. (2003) Genetic analysis of the myotubularin family of phosphatases in Caenorhabditis elegans. J Biol Chem 278: 34380–34386. 22. Rando TA, Blau HM (1994) Primary mouse myoblast purification, character- ization, and transplantation for cell-mediated gene therapy. J Cell Biol 125: 1275–1287. 23. van Deutekom JC, van Ommen GJ (2003) Advances in Duchenne muscular dystrophy gene therapy. Nat Rev Genet 4: 774–783. 24. Gussoni E, Blau HM, Kunkel LM (1997) The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat Med 3: 970–977. 25. Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, et al. (1999) Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401: 390–394. 26. Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D’Antona G, et al. (2003) Cell therapy of alpha-sarcoglycan dystrophic mice through intra- arterial delivery of mesoangioblasts. Science 301: 487–492. 27. Guyon JR, Steffen LS, Howell MH, Pusack TJ, Lawrence C, et al. (2007) Modeling human muscle disease in zebrafish. Biochim Biophys Acta 1772: 205–215. 28. Sasaki AT, Janetopoulos C, Lee S, Charest PG, Takeda K, et al. (2007) G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility. J Cell Biol 178: 185–191. PLoS ONE | www.plosone.org 11 March 2009 | Volume 4 | Issue 3 | e4979