Cooperation of Mtmr8 with PI3K Regulates Actin Filament Modeling and Muscle Development in Zebrafish Jie Mei, Zhi Li, Jian-Fang Gui* State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of Chinese Academy of Sciences, Wuhan, China Abstract Background: It has been shown that mutations in at least four myotubularin family genes (MTM1, MTMR1, 2 and 13) are causative for human neuromuscular disorders. However, the pathway and regulative mechanism remain unknown. Methodology/Principal Findings: Here, we reported a new role for Mtmr8 in neuromuscular development of zebrafish. Firstly, we cloned and characterized zebrafish Mtmr8, and revealed the expression pattern predominantly in the eye field and somites during early somitogenesis. Using morpholino knockdown, then, we observed that loss-of-function of Mtmr8 led to defects in somitogenesis. Subsequently, the possible underlying mechanism and signal pathway were examined. We first checked the Akt phosphorylation, and observed an increase of Akt phosphorylation in the morphant embryos. Furthermore, we studied the PH/G domain function within Mtmr8. Although the PH/G domain deletion by itself did not result in embryonic defect, addition of PI3K inhibitor LY294002 did give a defective phenotype in the PH/G deletion morphants, indicating that the PH/G domain was essential for Mtmr8’s function. Moreover, we investigated the cooperation of Mtmr8 with PI3K in actin filament modeling and muscle development, and found that both Mtmr8-MO1 and Mtmr8- MO2+LY294002 led to the disorganization of the actin cytoskeleton. In addition, we revealed a possible participation of Mtmr8 in the Hedgehog pathway, and cell transplantation experiments showed that Mtmr8 worked in a non-cell autonomous manner in actin modeling. Conclusion/Significance: The above data indicate that a conserved functional cooperation of Mtmr8 with PI3K regulates actin filament modeling and muscle development in zebrafish, and reveal a possible participation of Mtmr8 in the Hedgehog pathway. Therefore, this work provides a new clue to study the physiological function of MTM family members. Citation: Mei J, Li Z, Gui J-F (2009) Cooperation of Mtmr8 with PI3K Regulates Actin Filament Modeling and Muscle Development in Zebrafish. PLoS ONE 4(3): e4979. doi:10.1371/journal.pone.0004979 Editor: Mikhail V. Blagosklonny, Ordway Research Institute, United States of America Received December 23, 2008; Accepted February 28, 2009; Published March 26, 2009 Copyright: 2009 Mei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Major Basic Research Program (grant No. 6462004CB117401), the Innovation Project of Chinese Academy of Sciences (grant No.KSCX2-YW-N-020), and the Innovation Project of IHB, CAS (grant No. 075A01-1-301). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jfgui@ihb.ac.cn Introduction PTEN (phosphatase and tensin homolog deleted on chromo- some ten) and MTM (myotubularin myopathy) family factors are members of the growing class of dual-specificity phosphatases (DSPs), which can dephosphorylate the products of phosphoino- sitide 3-kinase (PI3K) and antagonize downstream effectors using 3-phosphoinositides as ligands [1,2]. They have been known to contribute to diverse processes that include cellular adhesion, signal transduction, and cell-cycle regulation[3]. Myotubularin- related (MTMR) phosphatases display a conserved active site motif CX5R, and an invariant sequence ‘CSDGWDR’ exists commonly in all the enzymatically active members[4–6]. Eight active members, including MTMR8, have been found in the MTM family. Several studies have further demonstrated that the PH/G domain functions to localize MTM to different subcellular compartments in the cell[7–9], and its deletion leads to activity loss of MTMR3 in vitro[10]. Mutations or altered expression of PTEN or MTM family members have been observed in human cancers[11–13] and in genetic development defects[14,15]. Recent studies using RNA interference have revealed an unexpected role for several MTMs (including MTMR8) in promoting cell proliferation and survival [16,17]. MTM1 is mutated in individuals with X-linked recessive myotubular myopathy [14]. MTM1 is adjacent to MTMR1 on the X chromosome, which plays a role in muscle formation and represents abnormal mRNA splicing in myotonic dystrophy [18]. Function loss of MTMR2 and mutation of MTMR13 causes Charcot–Marie–Tooth disease type 4B1 and 4B2[17,19], a severe demyelinating neuropathy characterized by muscle weakness and sensory loss in the lower extremities beginning in early childhood. In addition, MTMR6 and MTMR9 were identified genetically to be required in Caenorhabditis elegans for endocytosis[20,21]. Recently, the cell-therapy strategies, which use cultured myo- blasts[22] or stem cells, have had notable successes in dystrophic mouse models[23] and DMD muscle[24–26], because transplant- PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4979