Mtmr8 in Muscle Development inhibitor, LY294002. Each picture represents typical results out of three separate experiments (30 embryos in each experiment). All scale bars are 100 mm. (C) Immunoblot of different extracts from Cont MO embryos and Mtmr8-MO2 morphants at 24 hpf (30 embryos/group). Relative p-Akt/Akt ratio was 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.g004 myofibrils. While slow fibrils in Cont MO embryos were well organized with striation at 24 hpf (Fig 6B-a), fibrils of Mtmr8 morpholino-injected embryos were shorter and reduced (Fig 6B- b). When injecting Mtmr8 morpholino together with dnPKA mRNA, we detected myofibrils increased and longer than those of Mtmr8 morphants (Fig 6B-c). Labeling with anti-Prox1, a slow muscle nuclear marker, shows that the numbers of muscle pioneer and slow muscle cells are reduced in Mtmr8 morphant embryos (Fig 6B-e) compared to control embryos (Fig 6B-d), which is rescued by coinjection of dnPKA mRNA (Fig 6B-f). These findings indicate that the expression of Mtmr8 is dependent on Hh signaling. Mtmr8 acts non-cell autonomously to F-actin modeling Previous studies have demonstrated that the transplanted labeled cells at the prospective mesodermal region (as shown in Fig 7A) will mainly differentiated into several regions including the neural tube, brain, and somites at 24 hpf when the muscle cells are easily distinguished from other cell types morphologically [37]. To determine whether Mtmr8 acts in a cell autonomous or non-cell autonomous manner in muscle development, reciprocal cell transplantation experiments were carried out between wild-type and Mtmr8 morphant embryos. We transplanted donor fluoresce- in-labeled cells into age-matched host embryos at 4 hpf. At 24 hpf, the phenotype of the embryos were observed under the microscope. The embryos with transplanted cell in the somites (Fig 7B) were chosen for phalloidin staining. In wild-type embryos, cells transplanted from Mtmr8 morphant embryos could not change wild-type phenotype and F-actin modeling (Fig 7C-a, b, Table 1). In the reciprocal experiment, when wild-type donor cells were implanted into a Mtmr8 morphant host, the phenotype was consistent with Mtmr8 morphant, the U-shaped somites were still existed and the signal of F-actin was disorganized (Fig 7C-c, d, Table 1). We conclude that Mtmr8 protein acts non-cell autonomously within the actin assembly to promote slow fibres development. Discussion Recent studies clearly showed that myotubularin specifically dephosphorylates phosphatidylinositol 3-monophosphate (PI3P). The action of myotubularin on PI3P levels may implicate two parallel pathways by acting both as a protein phosphatase decreasing PI3P level by down-regulating PI3K activity and, a lipid phosphatase directly degrading PI3P than PI4P in vivo[38]. Such a dual activity has also been suggested for PTEN[39]. Mutations in MTMR family proteins are associated with the human neuromuscular disorders X-linked myotubular myopathy (myotubularin and MTMR1) and type 4B Charcot-Marie-Tooth neuropathy (MTMR2 and MTMR13). These diseases arise from impaired development and/or maturation of skeletal muscle cells and myelinating Schwann cells, respectively. Although a signifi- cant body of evidence has linked MTMR to endocytic membrane trafficking events, their role in neuromuscular development is currently unclear. Our findings in zebrafish demonstrated that Mtmr8 is an essential component, together with PI3K, nexilin and F-actin, of the positive-feedback cycle that maintains the normal development of muscle. According to previous studies[40–42] and our current studies, a diagram is depicted to explain Mtmr8 function and the relationship between Mtmr8 and the hedgehog pathway (Fig 8). Until now, because of no available means to prevent myotubular myopathy, our findings for the first time provided a new clue to study MTM family related disease in vivo. In this study, we presented evidence that the PH/G domain is indeed a function domain of Mtmr8 that contributes to the development of zebrafish. Deletion of the PH/G domain could result in increasing the relative amount of pAkt (Fig. 4C). Akt is activated by various growth factors and hormones such as IGF-1 and insulin. Activation occurs at the plasma membrane after PI3K-dependent generation of PIP3. Thereby, Akt is recruited to the plasma membrane via its PH (pleckstrin homology) domain and is activated by phosphorylation at Thr308 by PDK1 followed by phosphorylation at Ser473 by the mTOR complex[43]. Previous studies have demonstrated that the PH/G domain in a number of different MTMs bind phosphoinositides and also function to localize MTMs to different subcellular compartments in the cell[7–9]. All these indicated that Mtmr8 could inhibit PI3K/Akt activation through its functional PH/G domain, loss of which results in apoptotic signaling and cell death. Moreover, identifying the specific binding partners of the PH/G domains on the MTMs will provide important clues to the specific functions regulated by other MTMs as well as the mechanisms whereby loss of some MTMs lead to disease. Recently studies demonstrated the existence of PI3K-dependent and -independent pathways for F-actin polymerization during chemoattractant-stimulated lamella extension in the human neutrophil. One pathway is dependent on PI3Kc activation and downstream is dependent on PKCd and Akt/PKB. This pathway controls the formation of 70% to 80% of the F-actin in the lamella region [44]. In vitro studies, activation of PI3K activity alone is sufficient to remodel actin filaments to increase cell migration through the activation of Akt and p70S6K1 in CEF cells [45]. However, in vivo, the F-actin polymerization and modeling is also Mtmr8-dependent. Although the PI3K/Akt was activated in Mtmr8 knocked embryos, it was not enough to model actin filaments alone. Nexilin is a F-actin binding protein and mediates cell motility, over-expression of which promoted cell migration and adhesion[46]. However, the mechanism of its function is not clear. In Mtmr8 deficient embryos, the expression of Nexilin was reduced, which induced the defect of F-actin modeling. Inhibition of PI3K with LY294002 did not alter the initial formation of these F-actin- rich cup structures at the plasma membrane but it did prevent Akt/PKB recruitment to these cups and their subsequent fusion into the large rings characteristic of normal[47]. However, the effects of inhibiting PI3K on the embryo cytoskeleton are not well characterized. Low dose of LY294002 didn’t obviously affect F- actin, whereas it severely disturbed the modeling of F-actin in Mtmr8 morphant and PH/G losing embryos. These indicated that the increasing of pAkt is a very important recovery mechanism for Mtmr8 deficient, although it could not completely rescue the defects. The model about the mechanisms underlying the Hh signalling pathway is clearly shown by Masai et al.[41]. And Sonic hedgehog (Shh) has been reported to act as a mitogen and survival factor for muscle satellite cells. PI3K/Akt pathway is essential for Shh’s actions and directly involved in adult muscle cell proliferation and PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4979