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Overexpression of Myostatin2 in zebrafish reduces the expression of dystrophin associated protein complex (DAPC) which leads to muscle dystrophy

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O R I G I N A L P A P E R

Overexpression of Myostatin2 in zebrafish reduces the expression

of dystrophin associated protein complex (DAPC) which leads

to muscle dystrophy

Aseervatham Anusha AmaliÆ Cliff Ji-Fan Lin Æ Yi-Hsuan Chen Æ Wei-Lun WangÆ Hong-Yi Gong Æ Ravikumar Deepa Rekha Æ Jenn-Kan LuÆ Thomas T. Chen Æ Jen-Leih Wu

Received: 30 May 2007 / Accepted: 9 April 2008 / Published online: 6 May 2008 Ó National Science Council Taipei 2008

Abstract Myostatin, a member of the TGF-b superfam-ily, is a potent negative regulator of skeletal muscle and growth. Previously, we reported Mstn1 from zebrafish and studied its influence on muscle development. In this study, we identified another form of Myostatin protein which is referred to as Mstn2. The size of Mstn2 cDNA is 1342 bp with 109 and 132 bp of 50 and 30-untranslated regions (UTRs), respectively. The coding region is 1101 bp encoding 367 amino acids. The identity between zebrafish Mstn1 and 2 is 66%. The phylogenetic tree revealed that the Mstn2 is an ancestral form of Mstn1. To study the functional aspects, we overexpressed mstn2 and noticed that embryos became less active and the juveniles with bent

and curved phenotypes when compared to the control. The RT-PCR and in situ hybridization showed concurrent reduction of dystrophin associated protein complex (DAPC). In cryosection and in situ hybridization, we observed the disintegration of somites, lack of transverse myoseptum and loss of muscle integrity due to the failure of muscle attachment in mstn2 overexpressed embryos. Immunohistochemistry and western blot showed that there was a reduction of dystrophin, dystroglycan and sarcogly-can at translational level in overexpressed embryos. Taken together, these results indicate the suitability of zebrafish as an excellent animal model and our data provide the first in vivo evidence of muscle attachment failure by the overexpression of mstn2 and it leads to muscle loss which results in muscle dystrophy that may contribute to Duchenne syndrome and other muscle related diseases. Keywords Myostatin Muscle attachment 

Muscular dystrophy  DAPC  Muscle development

Introduction

A number of genetic factors, hormones and nutritional factors are important for skeletal muscle mass [1]. How-ever, their precise role in the integrated, in vivo regulation of skeletal muscle homeostasis and muscle wasting asso-ciated with chronic illness and aging remains poorly understood. Considerable interest has been focused on the role of mstn [2, 3], a novel regulator of muscle mass generally expressed in skeletal muscle. The mstn (growth and differentiation factor-8) belongs to transforming growth factor-b super family, that plays as a negative growth regulator for skeletal muscle, such that mutations in the mstn gene result in a hypermuscular phenotype in mice A. Anusha Amali and Cliff Ji-Fan Lin contributed equally.

A. A. Amali C. J.-F. Lin  Y.-H. Chen  W.-L. Wang  H.-Y. Gong R. D. Rekha  J.-L. Wu (&)

Laboratory of Marine Molecular Biology and Biotechnology, 301, Institute of Cellular and Organismic Biology, Academia Sinica, 128, Academia Road, Section 2, NanKang, Taipei 11529, Taiwan

e-mail: jlwu@gate.sinica.edu.tw C. J.-F. Lin

Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

W.-L. Wang

Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan

J.-K. Lu

Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan

T. T. Chen

Department of Molecular and Cell Biology and Biotechnology Center, University of Connecticut, Storrs, CT, USA

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and cattle [4, 5]. However, influence of Mstn on muscle phenotypes is still unclear. Homozygous disruption and knockdown of mstn gene in mice [2] and fish [6] result in a significant increase of skeletal muscle mass and a reduction of body fat. The mstn is expressed in cells of the skeletal muscle lineage throughout embryogenesis beginning in the myotome compartment of developing somites [7]. Over-expression of mstn leads to muscle wasting in adult mice [8,9]. Besides, changes in muscle mass due to disease or misuse are often reflected by changes in mstn expression [10,11]. Skeletal muscle mass is lost in genetic disorders such as muscular dystrophy, muscle wasting and aging [12].

Mstn shares several structural similarities to other TGF-b family memTGF-bers. It contains a hydrophoTGF-bic N-terminus that acts as a secretary signal and a conserved RSRR domain that is important for proteolytic processing [5,3,

13]. Cleavage of protein gives rise to an N-terminal latency associated peptide and C-terminal mature signaling pep-tide. The mature signaling peptide binds to the receptor, suggested to be activin type II receptor, to elicit its bio-logical function [14]. Indeed, recombinant Mstn based on the mature signaling portion has been shown to be bio-logically active in repressing the proliferation of cultured myoblast cells [15].

In many aspects mstn in the fish are quite different from that of higher vertebrates. Studies in mice and cattle revealed that mstn mRNA is specifically expressed in developing somites and skeletal muscles [5,4]. Duplication of mstn gene in Atlantic salmon [16] and differential expression of mstn gene in both muscular and non-mus-cular tissues in Asian sea bass [17] have been observed. Recent studies, however, showed that in addition to muscle cells, mstn mRNA also expressed in several other tissues, such as cardiomyocytes, mammary glands and, at a lower level, in adipose tissue [18]. In fish, mstn mRNA was found to be expressed in muscles, eyes, gill filaments, spleen, ovaries, gut, and brain and, to a lesser extent, in testes [19,

6]. Roberts and Goetz [20] reported that mstn mRNA pri-marily expressed in red muscles in brook trout, king mackerel, and yellow perch, but expression in the little tunny is in the white muscles. Two distinct forms of mstn genes have been demonstrated in Atlantic salmon [21], rainbow trout [22], and seabream [23]. Unlike mammals, in fish mstn is expressed in several tissues in addition to the muscle.

To understand the role of mstn in fish skeletal muscle development and growth, here we have cloned and studied the relationship between the mstn1 and mstn2 of zebrafish. To investigate more about the functional aspects, we overexpressed the mstn2 and observed embryos with bent morphology and lower activity [24], an interesting homo-logue of mammalian muscle dystrophy.

Materials and methods Fish culture and maintenance

Routine fish care and maintenance were performed as described [25]. Zebrafish embryos were collected and staged in hours post fertilization (hpf) at standard temper-ature (28.5°C).

Cloning and sequence analysis

We used human GDF-8 (mstn) mRNA sequence (Accession number, NM_005259) as virtual probe to BLAST against zebrafish genomic database (Sanger Center, Zebrafish assembly v2) and found zebrafish genomic clone (zfish41364-181h04.q1c) which was highly homologous to GDF8, containing the recognizable domains shared by TGF-b family, but different from the previously reported zebrafish mstn (Accession number, NM_131019). Accord-ing to this genomic information, we designed gene specific primers and performed the whole tissue of 50 RACE/30 RACE to amplify the mstn2 mRNA sequence. Total RNA was extracted from adult zebrafish using TRIZOL reagent (Invitrogen). First strand cDNA was synthesized using GeneRacer kit (Invitrogen) to obtain a full length cDNA and subjected to PCR. The reverse transcribed sample (2 ll) was used in a 50 ll PCR mixture. The primers for first PCR were GeneRacer 50 Primer and zfmstn2-GSP1 (50-TCA AGA GCA ACC GCA AAG GTC TAC CAC CAT-30). The primers for nested PCR were GeneRacer 50Nested Primer and zfmstn2-GSP2 (50-CTG CAA AGA AAA TTG GGT TGG CCT TGT TG-30). PCR amplification conditions were 35 cycles of 30 s at 94°C, 30 s at 55°C, and 2 min at 72°C. The PCR product was analyzed by 1% agarose gel elec-trophoresis. A discrete PCR product detected was isolated and subcloned into pGEM-T Easy cloning vector (Promega) and subjected to nucleotide sequencing (ABI). To obtain the 30-coding region and 30 untranslated region (30 UTR) sequence further downstream, 30 RACE was performed using the full length cDNA. PCR amplification and sub-cloning procedures were the same as above. Primers for first PCR were GeneRacer 30 Primer and zfmstn2-GSP3 (50 -AGA CAC AGG GAA GCG ATC CAG AC-30). The primers for nested PCR were GeneRacer 30Nested Primer and zfmstn2-GSP4 (50-TTT CTT TGC AGG TCC CTG CTG TAT TCT-30). The nucleotide and the deduced amino acid sequences of the cDNA were analyzed using a BLAST search for sequence homology. Multiple sequence alignment was performed using Pileup program in Unix-based GCG Wisconsin Package and illustrated using GeneDoc program (http://www.psc.edu/biomed/genedoc/). Phylogenetic analysis was conducted using a web-based sequence analysis program (SeqWeb Version2, Accelrys)

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with Jukes-Cantor distance correction method. Neighbor-joining method and branch lengths proportional to distance were used to construct and display the tree, respectively.

To amplify the full-length zebrafish mstn cDNA, a set of sense and antisense primers (50-GGA TCC ACC ATG TTT CTC CTT TTT TAT CTG AG-30), (50-GAA TTC AGA GCA ACC GCA AAG GTC TAC CAC C-30) correspond-ing to the start and stop codon of the open readcorrespond-ing frame (ORF), respectively, were synthesized. A PCR was carried out using the above primers, Platinum Taq DNA polymer-ase (Invitrogen) and zebrafish first-strand cDNA as the template. Amplification conditions were 35 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min and 30 s at 72°C. The PCR products were subjected to 1.5% agarose gel electrophore-sis. The discrete PCR product band, visualized by ethidium bromide staining, was excised from the gel and purified using Gel Extraction Kit (QIAGEN). The purified zebrafish mstn cDNA was subcloned into pcDNA3.1 eukaryotic expression vector at BamHI and EcoRI restriction sites. To verify its authenticity and direction of cloning, the cDNA insert was sequenced (ABI).

RT-PCR analysis

First strand cDNA was synthesized from 5 lg of total RNA using ThermoscriptTMRT-PCR system (Invitrogen). PCR reaction was performed with 2 ll of the RT reaction. The PCR program was as follows: one cycle 94°C, 30 s, 55°C for 30 s, and 72°C for 1 min and performed 30 cycles. The final extension time was 7 min at 72°C. The RT–PCR products were separated by 2% agarose gel electrophoresis. Max was used as an internal control.

Whole mount in situ hybridization

Control and overexpressed embryos at different develop-mental stages were fixed, dechorionated and whole mount in situ hybridization was carried out by using dioxigenin (DIG) labeled riboprobe [25]. By T7 and SP6 RNA poly-merase, the DIG labeled antisense and sense probes were synthesized. We carried out cryosection in 48 hpf embryos. Plasmid construction for overexpression

The full length of mstn2 ORF was amplified and cloned into pcDNA3 eukaryotic expression vector at BamHI and EcoRI restriction sites. The concentration of plasmid was determined by spectrophotometer and 20 lg/ml was injected into embryos between one cell and four cell stages. Immunohistochemistry

Immunohistochemisty was performed by using Anti-dystrophin MANDRA1 (Sigma), Anti-b-dystroglycan

(Novocastra), b-sarcoglycan (Novacastra), anti-rabbit conjugated goat HRP (secondary) was diluted as 1:1,000, 1:100, 1:100, 1:1,000, respectively.

Western blotting

Total protein extract was prepared using lysis buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 12%). For the separation of large proteins, Tris acetate gradient gels were used and transferred onto a nitrocellulose membrane. The membrane was blocked by net buffer at 4°C overnight and the bands were detected using the above antibodies. The membrane was washed, and the activity was detected using an ECL kit according to manufacturer’s protocol.

Fig. 1 Nucleotide and deduced amino acid sequence of the zebrafish mstn2 cDNA. Nucleotides are numbered 50?30. The translation start

codon ATG is boxed and the asterisk indicates the translation stop codon. The putative proteolytic processing site (RSRR) is from R258 to R261. This sequence has deposited in GenBankTM(AY614000)

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Results

Cloning and sequencing of zebrafish mstn2 cDNA Recently, we have isolated mstn1 from zebrafish and demonstrated its function in regulating myogenesis during embryogenesis [6]. However, double muscle phenotype was not prominent as in mammals by the mstn1 mor-pholino knock-down; moreover the tissue-distribution of zebrafish mstn1 is not restricted to the skeletal muscle [6]. So we suspected that another form of mstn may exist in

zebrafish and control the muscle growth as in the mam-malian system. By in-silicon procedure to search potential candidate of Mstn isoforms from zebrafish genomic database, we identified one zebrafish genomic clone (zfish41364-181h04.q1c), which contained highly con-served C-terminal region of TGF-b family protein using human cDNA as virtual probe. We designed gene-specific primer sets according to this candidate sequence and performed 50and 30RACE to obtain the full length of this cDNA using RNA ligase-mediated RACE (RLM-RACE). The size of the cDNA was 1342 bp with 109 and 132 bp

Fig. 2 Comparison of the deduced amino acid sequences of zebrafish Mstn2 and myostatin from mammals and fish species. The nine conserved cysteine residues consisted in TGF-b superfamily motifs are indicated with arrows. The potential proteolytic processing site (RXRR) is also boxed. The shading indicates the amino acids fully conserved among the species compared, and the gaps are shown as dashes (–). Sequences used in this comparison are deduced from

human GDF8 (NM_005259), mouse GDF8 (NM_010834), rat GDF8 (NM_019151), cow GDF8 (AY160688), fugu Mstn1 (AY445321), fugu Mstn2 (AY445322), tilapia Mstn (AF197193), A_salmon Mstn1 (atlantic salmon; AJ297267), A_salmon Mstn2 (AJ344158), R trout Mstn1 (rainbow trout; AF273035), R trout Mstn2 (AF273036), and zMstn (zebrafish; NM_131019)

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of 50 and 30 UTRs, respectively. The coding region is 1101 bp encoding 367 amino acids (Fig.1). By BlastX search, this sequence was found to have high similarity with known gdf8 from various species and with the highest score fit to zebrafish GDF8. This sequence is referred to as zebrafish mstn2. The nucleotide sequence identity between zebrafish mstn1 and mstn2 was only 66.9% which is far lower than other fish species. For example, the nucleotide sequence identity between mstn1 and mstn2 is 94.2%, 93.8% in Atlantic salmon and rain-bow trout, respectively zebrafish mstn2 is not considered to be a duplicated form of mstn because of their low identity at the coding and 30 UTRs.

Amino acid sequence homology and phylogenetic analysis

The zebrafish Mstn2 encodes a polypeptide consisting of 367 amino acids. Like all TGF-b family, amino acid sequence homology analysis of the Mstn1 with Mstn2 from other species contain putative RXRR proteolytic process-ing site and nine conserved cysteine residues (Fig.2). When we compared the zebrafish Mstn1 and Mstn2, the percentage of amino acid identity and similarity were 67.2% and 73.1%, respectively.

The phylogeneic relationship of zebrafish mstn2 with other full length gdf8 available in GenBank is depicted in

Fig. 3 Phylogenetic tree of mstn2. The tree was built with known mstn sequences from the GenBank using the Neighbor-Joining method. Three gdf11 sequences were used as an out group. Sequences, in addition to those in Fig.2, used in construction of this

phylogenetic tree are baboon Mstn (hamadryas baboon; AF019619), goose Mstn (AY448009), turkey Mstn (AF019625), chicken Mstn (AY448007), dog Mstn (AY367768), pig Mstn (AY448008), horse Mstn (AB033541), goat Mstn (AY436347), sheep Mstn (AF019622), B catfish Mstn (blue catfish; AY540 992), C catfish Mstn (channel catfish; AF396747), G_seabreamGDF8 (gilthead seabream; AF258448), G_seabreamGDF8b

(AY046314), S_seabass Mstn (striped sea-bass; AF290910), W_perch Mstn (white perch; AF290911), Whitebass Mstn (AF197194), human GDF11 (NM_005811), mouse GDF11 (XM_125935), rat GDF11 (XM_343148), and zfGDF11-like (zebrafish; AF411599)

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Fig.3. The result showed that zebrafish mstn2 might be the ancestral form of mstn1.

Mstn2 overexpression disrupts early muscle development

To understand whether mstn2 is related to muscle dystro-phy, we overexpressed mstn2. Following injection of mstn2 at one cell stage, embryogenesis proceeds normally. By 24 hpf, however, the embryos appeared to be developmentally delayed. Once the fish hatched out from the chorion (Fig.4), the overexpressed embryos were curved or bent (Fig.4b) and dystrophic compared with controls (Fig.4a). The overexpressed embryos showed less motile and moved in an uncoordinated fashion. The cryosection data also showed distruption of myotomes and spaces in between the muscle fibers (Fig.4d, f) when compared to the controls (Fig.4c).

Down-regulation of dystroglycogen complex (DGC) in zebrafish by overexpression of mstn2

Mstn2 overexpressed zebrafish embryos were analyzed at transcription and translational levels to determine the reduction of DGC (dystrophin, dystroglycan, and sarco-glycan) in the injected embryos. Among the markers, related to muscle dystrophy syndrome such as dystrophin, dystroglycan, sarcoglycan, calpain, caveolin and dysferlin, the expression of dystrophin, dystroglycan, and sarcogly-can were decreased.

RT-PCR results revealed that there was a reduction of dystrophin, dystroglycan and sarcoglycan (Fig.5). The whole mount in situ hybridization of 12 and 96 hpf control embryos, revealed a wide spread distribution of mRNA in

the somite (Fig. 6). In overexpressed embryos, however, we did not observe the strips (V shaped somites) and there was no signal in the somites presumably due to the disor-ganization of cells within the somites.

Immunohistochemical analysis using different antibod-ies showed strong expression of dystrophin, dystroglycan, and sarcoglycan predominantly at the myosepta in the control embryos (Fig.7a, c, e) and no signal could be observed in the injected embryos (Fig.7b, d, f). To confirm the immunohistochemistry results and to investigate the level of protein expression, Western blot analysis was performed. Protein extracts were prepared from uninjected and overexpressed zebrafish embryos, separated by SDS-PAGE, and subsequently transferred onto nitrocellulose membrane and detected. Antibodies showed marked reduction of DGC and the expression was very high in the overexpressed embryos than the control (Fig.7b).

Fig. 4 Zebrafish overexpressed with mstn2 show curved and bent phenotypes. (a) 72 h control embryo, (b) 72 h overexpressed embryos, (c) cryosection of 72 h control embryo, and (d and e) cryosection of 72 h overexpressed embryos

Fig. 5 RT-PCR analysis of control and overexpressed embryos. 1, 3, and 5—12 h, 24 h, and 72 h control embryos; 2, 4, and 6—12 h, 24 h, and 72 h overexpressed embryos

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Discussion

Here we report the identification and characterization of zebrafish mstn2. In mammals, myostatin plays a major role in muscle development. Though mstn has been cloned in different vertebrates, it is not known if mstn2 has the same function in these animals as it does in higher vertebrates. The results of the present study clearly demonstrate several major differences between mstn expression in fish and other vertebrates. Comparison of nucleotide and amino acids showed very low percentage of similarity and identity. Phylogenetic tree analysis revealed that the zebrafish mstn2 is an ancestral form. These results conclude that these two genes are derived from two different genes. In mice [5], cattle [26], and zebrafish [6], mutation or knockdown in the mstn gene showed a marked increase in body, muscle mass and growth. Inhibition of the mstn gene product is predicted to increase muscle mass and improve disease phenotype in a variety of primary and secondary myopathies [27]. Duch-enne muscular dystrophy (DMD), the most common lethal muscle wasting disease is a result of an absence of muscle dystrophin. Although this disorder causes a rather uniform

pattern of muscle wasting, affected patients display phe-notypic variability. We hypothesized that genetic variation in myostatin is a modifier of the DMD phenotype [28]. The inhibition of mstn in vivo improves the dystrophic syn-drome phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD) [29]. Transgenic expression of myostatin inhibitor follistatin increases the skeletal muscle and dystrophic pathology in mdx mice [30]. Blockade of endogenous Mstn by using intraperitoneal injections of anti-MSTN antibodies, for three months resulted in an increased body weight, muscle mass, muscle size and absolute muscle strength in mdx mouse along with a significant decrease in muscle degeneration and concentration of creatine kinase [27]. The more surprizing observation was the leakage of Mck into the serum, dropped to near wild-type levels in the anti-myostatin treated mice [31, 32]. It was proved that overexpression of mstn in mice leads to muscle loss [8,9]. To study more on the functional aspects of mstn1 and 2, we knocked down mstn1 [6] and overexpressed mstn2 by CMV promoter. We observed the overexpressed embryos become less active, bent, and curved in the trunk region. The phe-notypic difference is very much similar to muscle dystrophy Fig. 6 Whole mount in situ

hybridization of control and overexpressed embryos. Embryos hybridized with dystrophin (a–d), dystroglycan (e–h), and sarcoglycan (i–l). (a, e, i) mstn2 overexpressed 12 h embryos, (b, f, j) control 12 h embryos, (c, g, k) mstn2 overexpressed 96 h embryos, and (d, h, l) control 96 h embryos

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syndrome phenotype. To confirm the muscle dystrophy, we used different marker genes related to muscle dystrophy and checked at RNA and protein level (Figs.6 and 7). The results showed decreased expression of dystrophin, dys-troglycan, sarcoglycan. Our result was supported in the muscle dystrophy syndrome phenotype showed absence of dystrophin, dystroglycan, and abnormally high plasma and muscle creatine kinase [33, 34]. In vertebrate skeletal muscle, the DAPE is thought to link the extracellular matrix to the intracellular cytoskeleton [35]. Orthologs of proteins in the human DAPC have been identified in zebrafish [36–

38]. Morpholino experiments directed against dystrophin and b-dystroglycan mRNA show both specific reduction of the dystrophin protein and repression of DAPE proteins [24,

38,39]. In the mstn2 overexpressed embryos, the pheno-typic difference shared to varying extents with those found

in zebrafish due to morpholino based translational knock down of dystrophin [38] and dystroglycan [24]. Myostatin mutations and pharmacological strategies increase muscle mass in vivo, suggesting that interruption of myostatin expression may be useful in diseases characterized by muscle wasting, such as the muscular dystrophies. It has been demonstrated in !-sarcoglycan-deficient mouse model of limb-girdle muscular dystrophy (LGMD) 2C by anti-body-mediated myostatin blockade in vivo, led to increased fiber size, muscle mass and absolute force [40]. Further-more, systemic gene delivery of myostatin propeptide (MRPO), a natural inhibitor of myostatin, could enhance body-wide skeletal muscle growth [41].

To determine precisely the cellular basis for muscle degeneration, we continuously monitored mutant somites by cryosection. We observed that the disintegration of Fig. 7 Immunohistochemical

detection of control and overexpressed embryos by using Mandra1 (Anti-dystrophin antibody), anti-dystroglycan antibody, and sarcoglycan antibody. (a, c, and e) Control-96 h embryos, (b, d, and f) Mstn2 overexpressed 96 h embryos (b). Decreased (dystrophin, dystroglycan, and sarcoglycan) expression of and overexpressed embryos. Protein extracts from the 96 hpf injected with approximately 20 ng of mstn2 plasmid and were separated by electrophoresis, transferred to a nitrocellulose membrane and analyzed by westernblotting. OE—Mstn2 overexpressed 96 h embryos. (g) Anti-dystrophin antibody, (h) anti-dystroglycan antibody (i) sarcoglycan antibody and (j) b actin antibody

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somites, lack of transverse myoseptum and loss of muscle integrity due to muscle attachment failure were observed in the mstn2 overexpressed embryos. The disruption is char-acterized by cheveron shaped myosepta yielding to U-shaped somites [42]. The transverse section revealed that the cellular appearance of muscles in overexpressed embryos was less organized than the control embryos. The mode of muscle cell loss in overexpressed embryos is similar to the loss of muscle in human congenital muscular dystrophy in several ways. Through histological studies, we have shown that the overexpression of mstn2 leads to loss or mislocalization of somites with a general disruption of muscle integrity of the muscle that may be due to the loss of dystroglycan, dystrophin and sarcoglycan because they are important for muscle integrity [43, 32]. These phenotypes are very similar to those observed in muscle degeneration that occurs in human suffering from muscular dystrophy. Translation blocking morpholinos targeted to a zebrafish dystrophin was recently reported to cause an uncharacterized disorganization of the somites similar to that seen in degenerating sap mutant embryos, and reduced levels of the DAPC component b-sarcoglycan, although no specific defect in muscle fiber integrity was reported [24]. There are several features of zebrafish embryogenesis and genetics that make it well suited for the study of muscular dystrophy. Zebrafish embryonic skeletal muscle is simply organized with single myotubes extending across each somite and attached at either end to the ECM of the transverse myoseptum. Since dystrophin and dystroglycan are both expressed at early stages of zebrafish development [37], it is possible that the loss of these proteins could destabilize muscle fibers interaction at this location during critical stage of development [24].

Our data provide the first in vivo evidence of muscle attachment failure by the overexpression of mstn2 which leads to muscle loss and muscle dystrophy that may con-tribute to Duchenne and other muscle diseases. Collectively, these results provide the first in vivo genetic evidence that mstn is not only important for myogenesis but if it is high it will collapse the vertebrate muscle attachments, as has been found in mdx mice, but also for their stability, and that failure of these attachments can lead to a progressive muscular dystrophy. Finally mstn2 over-expression therefore provides a model for the novel pathological mechanism of muscle attachment failure that could well contribute to some of the many different human muscular dystrophies or congenital myopathies.

References

1. Florini JR, Ewton DZ, Coolican SA (1996) Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17(5):481–517

2. Lee SJ, McPherron AC (1999) Myostatin and the control of skeletal muscle mass. Curr Opin Genet Dev 9(5):604–607 3. Sharma M, Langley B, Bass J, Kambadur R (2001) Myostatin in

muscle growth and repair. Exerc Sport Sci Rev 29(4):155–158 4. Kambadur R, Sharma M, Smith TP, Bass JJ (1997) Mutations in

myostatin (GDF8) in double-muscled Belgian Blue and Pied-montese cattle. Genome Res 7(9):910–916

5. McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387(6628):83–90

6. Amali AA, Lin CJ, Chen YH, Wang WL, Gong HY, Lee CY, Ko YL, Lu JK, Her GM, Chen TT, Wu JL (2004) Up-regulation of muscle-specific transcription factors during embryonic somito-genesis of zebrafish (Danio rerio) by knock-down of myostatin-1. Dev Dyn 229(4):847–856

7. McPherron AC, Lee SJ (2002) Suppression of body fat accu-mulation in myostatin deficient mice. J Clin Invest 109(5):595– 601

8. Zimmers TA, Davies MV, Koniaris LG, Haynes P, Esquela AF, Tomkinson KN, McPherron AC, Wolfman NM, Lee SJ (2002) Induction of cachexia in mice by systemically administered myostatin. Science 296(5572):1486–1488

9. Reisz-Porszasz S, Bhasin S, Artaza JN, Shen R, Sinha-Hikim I, Hogue A, Fielder TJ, Gonzalez-Cadavid NF (2003) Lower skel-etal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin. Am J Physiol 285(4):E876–E888 10. Lalani R, Bhasin S, Byhower F, Tarnuzzer R, Grant M, Shen R,

Asa S, Ezzat S, Gonzalez-Cadavid NF (2000) Myostatin and insulin-like growth factor-I and -II expression in the muscle of rats exposed to the microgravity environment of the NeuroLab space shuttle flight. J Endocrinol 167(3):417–428

11. Wehling M, Cai B, Tidball JG (2000) Modulation of myostatin expression during modified muscle use. FASEB J 14(1):103–110 12. Tsuchida K (2006) The role of myostatin and bone morphoge-netic proteins in muscular disorders. Expert Opin Biol Ther 6(2):147–154

13. Lee SJ (2008) Genetic analysis of the role of proteolysis in the activation of latent myostatin. PLoS ONE 3(2):e1628

14. Lee SJ, McPherron AC (2001) Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci USA 98(16):9306–9311 15. Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, Kambadur R (2000) Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem 275(51):40235–40243

16. Ostbye TK, Wetten OF, Tooming-Klunderud A, Jakobsen KS, Yafe A, Etzioni S, Moen T, Andersen O (2007) Myostatin (MSTN) gene duplications in Atlantic salmon (Salmo salar): evidence for different selective pressure on teleost MSTN1 and -2. Gene 403(1–2):159–69

17. De Santis C, Evans BS, Smith-Keune C, Jerry DR (2008) Molecular characterization, tissue expression and sequence vari-ability of the barramundi (Lates calcarifer) myostatin gene. BMC Genomics 9(1):82

18. Sharma M, Kambadur R, Matthews KG, Somers WG, Devlin GP, Conaglen JV, Fowke PJ, Bass JJ (1999) Myostatin, a trans-forming growth factor-beta super family member, is expressed in heart muscle and is upregulated in cardiomyocyte after infarct. J Cell Physiol 180(1):1–9

19. Maccatrozzo L, Bargelloni L, Radaelli G, Mascarello F, Patar-nello T (2001a) Characterization of the myostatin gene in the gilthead seabream (Sparus aurata): sequence, genomic structure, and expression pattern. Mar Biotechnol 3(3):224–230

20. Roberts SB, Goetz FW (2001) Differential skeletal muscle expression of myostatin across teleost species, and the isolat evidence for different selective pressure on teleost MSTN1 and -2. Gene 403(1–2):159–69

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21. Ostbye TK, Galloway TF, Nielsen C, Gabestad I, Bardal T, Andersen O (2001) The two myostatin genes of Atlantic salmon (Salmo salar) are expressed in a variety of tissues. Eur J Biochem 268(20):5249–5257

22. Rescan PY, Jutel I, Ralliere C (2001) Two myostatin genes are differentially expressed in myotomal muscles of the trout (On-corhynchus mykiss). J Exp Biol 204(20):3523–3529

23. Maccatrozzo L, Bargelloni L, Cardazzo B, Rizzo G, Patarnello T (2001b) A novel second myostatin gene is present in teleost fish. FEBS lett 509(1):36–40

24. Guyon JR, Mosley AN, Zhou Y, O’Brien KF, Sheng X, Chiang K, Davidson AJ, Volinski JM, Zon LI, Kunkel LM (2003) The dystrophin associated protein complex in zebrafish. Hum Mol Genet 12(6):601–615

25. Westerfield M (1995) The zebrafish book: a guide for the labo-ratory use of zebrafish (Danio rerio), 2nd edn. Eugene, University of Oregon Press

26. Grobet L, Martin LJ, Poncelet D, Pirottin D, Brouwers B, Riquet J, Schoeberlein A, Dunner S, Menissier F, Massabanda J, Fries R, Hanset R, Georges M (1997) A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet 17(1):71–74

27. Bogdanovich S, Krag TO, Barton ER, Morris LD, Whittemore LA, Ahima RS, Khurana TS (2002) Functional improvement of dystrophic muscle by myostatin blockade. Nature 420(6914):418–421

28. Nishiyama A, Takeshima Y, Saiki K, Narukage A, Oyazato Y, Yagi M, Matsuo M (2007) Two novel missense mutations in the myostatin gene identified in Japanese patients with Duchenne muscular dystrophy. BMC Med Genet 12(8):19

29. Torres LF, Duchen LW (1987) The mutant mdx: inherited myopathy in the mouse. Morphological studies of nerves, mus-cles and end-plates. Brain 110(2):269–299

30. Nakatani M, Takehara Y, Sugino H, Matsumoto M, Hashimoto O, Hasegawa Y, Murakami T, Uezumi A, Takeda S, Noji S, Sunada Y, Tsuchida K (2008) Transgenic expression of a myo-statin inhibitor derived from follimyo-statin increases skeletal muscle mass and ameliorates dystrophic pathology in mdx mice. FASEB J 22(2):477–87

31. Zammit PS, Partridge TA (2002) Sizing up muscular dystrophy. Nat Med 8(12):1355–1356

32. Collins CA, Morgan JE (2003) Duchenne’s muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategies. Int J Exp Pathol 84(4):165–172

33. Bulfield G, Siller WG, Wight PA, Moore KJ (1984) X chromo-some-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci USA 81(4):1189–1192

34. Khurana TS, Davies KE (2003) Pharmacological strategies for muscular dystrophy. Nat Rev 2(5):379–390

35. Sakurai IM, Yoshida M, Imamura M, Davies KE, Ozawa E (2004) ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan. Hum Mol Genet 13(7):693–702

36. Chambers SP, Dodd A, Overall R, Sirey T, Lam LT, Morris GE, Love DR (2001) Dystrophin in adult zebrafish muscle. Biochem Biophys Res Commun 286(3):478–483

37. Bolanos-Jimenez F, Bordais A, Behra M, Strahle U, Sahel J, Rendon A (2001) Dystrophin and Dp71, two products of the DMD gene, show a different pattern of expression during embryonic development in zebrafish. Mech Dev 102(1–2):239–241 38. Parsons MJ, Campos I, Hirst EM, Stemple DL (2002) Removal of

dystroglycan causes severe muscular dystrophy in zebrafish embryos. Development 129(14):3505–3512

39. Bassett DI, Bryson-Richardson RJ, Daggett DF, Gautier P, Keenan DG, Currie PD (2003) Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo. Development 130(23):5851–5860

40. Bogdanovich S, McNally EM, Khurana TS (2008) Myostatin blockade improves function but not histopathology in a murine model of limb-girdle muscular dystrophy 2C. Muscle Nerve 37(3):308–16

41. Qiao C, Li J, Jiang J, Zhu X, Wang B, Li J, Xiao X (2008) Myostatin propeptide gene delivery by adeno-associated virus serotype 8 vectors enhances muscle growth and ameliorates dystrophic phenotypes in mdx mice. Hum Gene Ther 19(3):241– 254. doi:10.1089/hum.2007.159

42. Dodd A, Chambers SP, Love DR (2004) Short interfering RNA-mediated gene targeting in the zebrafish. FEBS lett 561(1–3):89–93 43. Grady RM, Teng H, Nichol MC, Cunningham JC, Wilkinson RS, Sanes JR (1997) Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dys-trophy. Cell 90(4):729–738

數據

Fig. 1 Nucleotide and deduced amino acid sequence of the zebrafish mstn2 cDNA. Nucleotides are numbered 5 0 ?3 0
Fig. 2 Comparison of the deduced amino acid sequences of zebrafish Mstn2 and myostatin from mammals and fish species
Fig. 3 Phylogenetic tree of mstn2. The tree was built with known mstn sequences from the GenBank using the  Neighbor-Joining method
Fig. 4 Zebrafish overexpressed with mstn2 show curved and bent phenotypes. (a) 72 h control embryo, (b) 72 h overexpressed embryos, (c) cryosection of 72 h control embryo, and (d and e) cryosection of 72 h overexpressed embryos

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