ing whole cells have the potential to correct the symptoms of disease although each has its inherent disadvantages[27]. PI3K is conserved across eukaryotic organisms and regulates many facets of pathways involving cellular growth, survival, metabolism, vehicle trafficking, and chemotaxis[28]. The path- ways controlling random movement parallel those of the amplification step of chemotaxis that is controlled through a regulatory loop containing Ras, PI3K, PTEN, and F-actin[29]. The initial activation of Ras and PI3K is independent of F-actin polymerization. However, F-actin polymerization is essential for amplifying the signal and stabilizing the leading edge in neutrophils and Dictyostelium [30,31]. In Dictyostelium, F-actin recruits additional PI3K to the newly forming leading edge, enhancing the PIP3 response and downstream effector function [29,30]. Cells with decreased PI3K activity exhibit a decrease in the second peak of RacB activation and F-actin polymerization, which has been linked to pseudopod extension. To reveal the biological functions of MTM family members, we identified Mtmr8 from the model animal zebrafish. Based on its expression pattern predominantly in the eye field and somites during early somitogenesis, we analyzed its physiological roles in embryo development by morpholino-mediated knockdown. We firstly showed that loss-of-function of Mtmr8 led to defects in somitogenesis, and further examined the possible underlying mechanism and the PH/G domain function. Moreover, we investigated the cooperation of Mtmr8 with PI3K in actin filament modeling and muscle development, and revealed a possible participation of Mtmr8 in the Hedgehog pathway. The findings revealed a new role of Mtmr8 and its functional mechanism in neuromuscular development. Results Molecular characterization and expression pattern of Mtmr8 in zebrafish embryos The complete ORF for the Mtmr8 encodes polypeptides of 632 amino acids, which contains 14 exons and 13 introns (identical to human MTMR8). An amino acid sequence alignment of zebrafish, chicken and human Mtmr8 polypeptides is shown in Fig. 1A. Zebrafish Mtmr8 exhibit 63% and 64% identity, and 80% and 78% similarity, with human and chicken Mtmr8 respectively, and higher identities exist in the Myotub-related and PTPc_DSPc motifs (amino acids 155–263 and 264–432 of zebrafish Mtmr8). The high homology implies that the zebrafish Mtmr8 may have the same functions as in human. Whole-mount in situ hybridization was used to analyze the expression pattern of Mtmr8 during zebrafish embryogenesis. The expression distribution of Mtmr8 was same to the result reported by Thisse B and C [32]. As shown in Fig. 1B, Mtmr8 mRNA is expressed in prechordal plate and eye field at 50% epiboly (Fig. 1B- a). Between 1–13 somites, Mtmr8 transcript becomes restricted to eye field and somites (Fig 1B-b). At 19 somites, Mtmr8 expression is shown in the eye, telencephalon and ventral mesoderm (Fig 1B-c). At 24hpf, Mtmr8 is expressed predominantly in the eye and vasculature (Fig 1B-d). Later, Mtmr8 is expressed in the vasculature at 48hpf (Fig 1B-e). Targeted knockdown of zebrafish Mtmr8 impaired embryo development To determine the physiological effect of Mtmr8, we undertook loss-of-function experiments in zebrafish by using morpholinos. Firstly, we designed a splice junction morpholino targeted against the first coding exon-intron boundary to evaluate the knockdown effect and efficacy in vivo. As shown in Fig. 2A and Fig. 2B, the Mtmr8 in Muscle Development exon-intron morpholino (MO1) introduces intron 1 (about 5 kb) into the altered transcript, and includes a premature termination codon. Because the altered transcript is more than 5kb in its sequence, it could not be amplified from the morpholino-injected embryos in one minute by RT-PCR (Fig. 2C). In the control embryos, the amplified PCR product is 310 bp by the same primers designed from the first and third extrons (Fig. 2C). And, the altered transcript sequence was further amplified and verified by bi-directional DNA sequencing. Microscope observation indicated that the morpholino-mediated Mtmr8 knockdown resulted in dramatic phenotypic abnormalities in somitogenesis (Fig 2D). At 24hpf, control experiments in which embryos were injected with Cont morpholino did not alter the wild-type phenotype throughout zebrafish development (Fig 2D-a), whereas the Mtmr8-MO1 morphants exhibited dose-dependent effect on embryos and resulted in mild and severe phenotype defects. In mild defects, morpholino-injected embryos showed delayed development, shorter and curled trunk and tail compared to wild-type or to control MO-injected embryos (Fig. 2D-b). In severe defects, the morphants were marked by small heads, abnormal tail fins, and U-shaped somites (Fig. 2D-c). Fig. 2E shows the percentages of normal, mild and severe defect embryos under different injection doses of morpholino. Moreover, an increasing percentage of normal or mild defect embryos was observed when the embryos were co-injected with 100 pg capped Mtmr8 RNA and 6 ng Mtmr8 MO1 (Fig. 2E). However, the defects could not be reduced when co-injected with 100 pg of capped GFP RNA and Mtmr8 MO1 (Data not shown). In addition, overexpression of zebrafish Mtmr8 by injection of capped RNA (100 pg) did not cause a visible phenotype. We used 100 pg doses in all the gain-of- function experiments described below if not indicated otherwise. Role of Mtmr8 in regulating PI3K/Akt signaling pathway Recent studies indicated that several MTMs might control PI3K/Akt activation by virtue of its ability to dephosphorylate PIP3[33]. However, there was no any direct report about the PIP3 lipid phosphatase activity of Mtmr8. Because knockdown of Mtmr8 expression gave distinct developmental phenotypes, we asked whether the PIP3 lipid phosphatase activity of Mtmr8 was conserved in the development of zebrafish. To confirm it, we compared the level of phosphorylated Akt (pAkt) to total Akt (Akt) in whole fish lysates prepared from wild type zebrafish and Mtmr8 morphant embryos. As shown in Fig 3, knockdown of Mtmr8 expression produces a significant increase in the relative amount of pAkt compared to Cont MO injected embryos, whereas the elevated level of p-Akt could be reduced by coinjection with Mtmr8 mRNA. These results indicate that zebrafish Mtmr8 exhibits PIP3 lipid phosphatase activity and functions to negatively regulate the PI3-Kinase/AKT pathway. Mtmr8 PH/G domain plays a critical role with PI3K in regulating zebrafish embryogenesis PH/G domain plays an important role in the activity of MTMR family [10]. To delete the PH/G domain of zebrafish Mtmr8, we designed a splice junction morpholino targeted against the second coding exon-intron boundary. In the Mtmr8-MO2 morphants, the targeted exon was eliminated, which encodes partial of the PH/G domain, as shown by a smaller PCR transcript which was verified by bi-directional DNA sequencing (Fig 4A). However, unlike Mtmr8-MO1 morphants (Fig 4B-a), the Mtmr8-MO2 morphants display no obvious defects, except mild abnormal in the head (Fig 4B-b). Intriguingly, previous studies showed that the defects resulted by homozygous single or double mutants of ptena2/2 and ptenb2/2, were rescued by treatment with the phosphatidylino- PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4979