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© The Rockefeller University Press,
0021-9525/2001//985 $5.00
The Journal of Cell Biology, Volume 153, Number 5,
, 2001 985-998
Original Article |
The Small Muscle-Specific Protein Csl Modifies Cell Shape and Promotes Myocyte Fusion in an Insulin-like Growth Factor 1–Dependent Manner
r.harvey{at}victorchang.unsw.edu.au
We have isolated a murine cDNA encoding a 9-kD protein, Chisel (Csl), in a screen for transcriptional targets of the cardiac homeodomain factor Nkx2-5. Csl transcripts were detected in atria and ventricles of the heart and in all skeletal muscles and smooth muscles of the stomach and pulmonary veins. Csl protein was distributed throughout the cytoplasm in fetal muscles, although costameric and M-line localization to the muscle cytoskeleton became obvious after further maturation. Targeted disruption of Csl showed no overt muscle phenotype. However, ectopic expression in C2C12 myoblasts induced formation of lamellipodia in which Csl protein became tethered to membrane ruffles. Migration of these cells was retarded in a monolayer wound repair assay. Csl-expressing myoblasts differentiated and fused normally, although in the presence of insulin-like growth factor (IGF)-1 they showed dramatically enhanced fusion, leading to formation of large dysmorphogenic "myosacs." The activities of transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer–binding factor (MEF)2, were also enhanced in an IGF-1 signaling–dependent manner. The dynamic cytoskeletal localization of Csl and its dominant effects on cell shape and behavior and transcription factor activity suggest that Csl plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair.
Key Words: costameres • heart • lamellipodia • Nkx2-5 • skeletal muscle
© 2001 The Rockefeller University Press
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-cardiac actin as direct target genes (Chen and Schwartz 1996; Durocher et al. 1996; Biben et al. 1997; Rivkees et al. 1999; Guo et al. 2000). However, in knockout mice genes encoding the regionally expressed transcription factors Hand1/eHand, Irx4, Msx-2, and N-myc and the actin cross-linking protein SM-22 are also dysregulated (Biben and Harvey 1997; Biben et al. 1997; Tanaka et al. 1999; Bruneau et al. 2000), suggesting a role for Nkx2-5 in the development of patterning information and heart tube morphogenesis. In this paper, we describe the gene Chisel (Csl), which was cloned during a differential screen for genes downregulated in homozygous Nkx2-5 mutant hearts. Csl encodes a protein of 9 kD expressed in the heart, all skeletal muscles, and smooth muscles of the stomach and pulmonary veins. In the heart, Csl transcripts were expressed only in the specialized working myocardium of the atrial and ventricular chambers. Although Csl knockout mice showed no obvious phenotype, over-expression in the C2C12 myogenic cell line led to effects on cytoskeletal dynamics in myoblasts and cell signaling–dependent enhancement of cell fusion in myocytes. The activities of nuclear factor of activated T cells (NFAT) and myocyte enhancer–binding factor (MEF)2 transcription factors were also enhanced in a cell signaling–dependent manner. These data suggest that Csl acts in the signaling network through which the structural, functional, and regulatory states of muscle cells are coordinated.
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allele reverse, 5'-tctgtgcaggctgcagcttc-3'.
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Western Blot Analysis
PVDF membranes (Millipore) were blocked with 0.05% Tween 20, 2% bovine serum albumin, PBS. Csl antibody (1:1,500) was followed by anti–rabbit IgG conjugated to HRP (1:1,000; Silenus). mAbs anti-Hsp 70 (1:1,000; Sigma-Aldrich) and anti-Akt (1:2,000; New England Biolabs, Inc.) were used with anti–mouse IgG–HRP (Silenus). Bands were detected by chemiluminescence (Amersham Pharmacia Biotech).
Cell Culture
Csl cDNA was cloned into pEF FLAG A (a gift from David Huang, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia), pEFBosMyc (a gift from N. Nicola, Walter and Eliza Hall Institute of Medical Research), pEGFP-C1 (CLONTECH Laboratories, Inc.), and a vector carrying a myosin light chain (MLC)1 promoter/enhancer (Musaro and Rosenthal 1999). Activated calcineurin A was cloned into pPGKpuropAv18 (a gift from David Huang). Transfections were performed with lipofectamine (GIBCO BRL) according to instructions. pEF FLAG A was used at 10:1 for puromycin selection where necessary. Two sublines of C2C12 cells were used. FLAG-Csl lines were made from stocks held at the Walter and Eliza Hall Institute of Medical Research and grown as described (Semsarian et al. 1999). Myc-Csl lines were made in cells from the laboratory of H. Blau (Stanford University, Palo Alto, CA) and grown in low glucose DME, 20% FBS, and 0.5% chick embryo extract. In transfections, reporter plasmids were cotransfected with an SV40-LacZ expression plasmid (9:1) for normalization. Luciferase activity was determined using Promega reagents. Flow cytometry was performed on a Becton Dickinson FACSCaliburTM.
Immunofluorescence
C2C12 cells on coverslips were fixed in ice-cold acetone for 15 min or in 4% paraformaldehyde (PFA)/PBS for a minimum of 15 min, followed by 0.5% Triton X-100/PBS for 15 min as appropriate for each antigen. For cryosections, tissue was embedded in OCT compound (Tissue-Tek) and immersed in isopentane cooled almost to the point of solidity using liquid nitrogen. Sections were fixed in ice-cold acetone or 4% PFA/PBS for 15 min. Antibodies were as follows: anti–-actinin mouse mAb (Sigma-Aldrich) (EA-53, 1:100; acetone/PFA fixation); antimyosin heavy chain IIb mouse IgM mAb (supplied by Edna Hardeman, Children's Medical Research Institute, Wentworthville, New South Wales, Australia) (neat; acetone fixation); anti-Csl rabbit polyclonal (1:200; PFA); antimyosin heavy chain mAb (MF20; Developmental Studies Hybridoma Bank) (1:20; acetone); antitalin mAb (Sigma-Aldrich) (1:20; PFA); antimyosin heavy chain I/slow mAb (supplied by Edna Hardeman) (neat; PFA); anti-myc epitope mAb (9E10; Developmental Studies Hybridoma Bank) (1:10; PFA). Confocal images were obtained using a Leica TCS NT system. Photomicrographs were prepared using a Spot-II digital system (Diagnostics Instruments).
Calcineurin Assays
Calcineurin activity was assayed as described (Semsarian et al. 1999) using okadaic acid at 1 µM and calmodulin at 250 nM. Calcineurin activity was calculated as the difference between total phosphatase activity on target peptide (Auspep) with and without 1 µM cyclosporine A (CsA) added to cells 20 min before preparation of extracts or with and without addition of calcineurin autoinhibitory peptide (500 µM) (Calbiochem) to extracts.
Wounding Assays
Parallel 150-µm wounds were made in confluent monolayers using a flame-polished glass needle on a micromanipulator. After 6 h in culture, cells were fixed (4% PFA/PBS) and stained (1% eosin). Wound areas were measured before and after culture using Leica Q500MC software.
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The DNA sequence of Csl showed identity or near identity to numerous mouse, human, rat, and pig ESTs. Using mouse ESTs, primers were designed, and a near full-length cDNA was amplified by reverse transcriptase–PCR from heart RNA, and after cloning multiple isolates were sequenced (Fig. 1 A). BLAST searching revealed that the complete sequence of the human gene spread over five exons was present on two overlapping and fully sequenced cosmid clones derived from the X chromosome (sequence data available from GenBank/EMBL/DDBJ under accession nos. U73508 and U73509; Grieff et al. 1997). Furthermore, the complete rat coding sequence could be assembled from overlapping ESTs. We also isolated and sequenced a Xenopus laevis cDNA (Fig. 1 B) and a partial cDNA from zebrafish (Yelon, D., and D. Stainier, unpublished data). However, searches of the Drosophila melanogaster and Caenorhabditis elegans genomes did not detect Csl-related genes. During the course of this study, Csl was cloned independently from humans as an X-linked cardiac-expressed gene (Patzak et al. 1999). We determined the chromosomal position of mouse Csl by interspecific backcross analysis using a panel of ([C57BL/6J x Mus spretus]F1 x C57BL/6J) mice (Copeland and Jenkins 1991). As in humans (Grieff et al. 1997; Patzak et al. 1999), Csl mapped to the X chromosome and was linked to genes Btk and Figf (data not shown).
Csl proteins were similar in length (85–91 amino acids) with the frog protein being slightly longer than its mammalian counterparts due to the presence of a pentapeptide insertion (Fig. 1 B). Alignments demonstrated strong conservation during vertebrate evolution; mouse Csl is 74% identical to frog Csl and 86 and 92% identical to the human and rat Csl, respectively. No homology was found between Csl and other proteins in public databases.
Csl mRNA Expression in the Embryo and Adult
Most Csl ESTs were derived from heart and skeletal muscle. RNase protection analysis of mRNA samples extracted from a variety of adult mouse tissues showed a strong signal in heart, skeletal muscle, and tongue (Fig. 2 A) with a weaker signal in lung, testes, and large intestine. Northern blot analysis of RNA from the C2C12 myogenic cell line revealed a predominant mRNA species of 1.1–1.2 kb (see Fig. 9 A).
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Csl was also expressed in developing skeletal muscles from E11.5. At E13.5, transcripts were evident in the myotomal compartment of somites and in developing limb, head, and neck muscles in a pattern resembling that of the myogenic regulatory factor gene myogenin (Fig. 2 K).
Creation of Csl Knockout Mice
To assess the function of Csl in muscle development, we generated mutant mice by gene targeting. We designed a conditional targeted allele in which a neomycin resistance cassette was inserted in reverse orientation into the second intron of the Csl gene, and Lox P sites were inserted within the untranslated region of the first coding exon (exon 2) upstream of the predicted initiation codon and downstream of the neomycin cassette (Fig. 3 A). A human placental alkaline phosphatase (hPAP) cDNA was also inserted just downstream of the neomycin cassette such that it replaces exon 2 coding sequences after Cre-mediated deletion. The correctly targeted conditional Csl allele (CslfAP) was detected in 1 in 22 (7/153) neomycin-resistant W9.5 embryonic stem cell colonies. Conditional Csl lines were established from two independently targeted embryonic stem cell clones, and lines carrying the Cre-deleted allele (CslAP) were successfully established after crossing founder chimeras with transgenic mice expressing Cre recombinase in the germline (TgN[CMV-Cre]1Cgn) (Schwenk et al. 1995). Founders were outcrossed onto C57BL/6 mice and progeny were genotyped using PCR (Fig. 3 B). The official designators (Institute for Laboratory Animal Research; http://www4.nas.edu/cls/ilarhome.nsf) for the conditional and Cre-deleted Csl mutant strains are Csltm1Rphand Csltm1.1Rph, respectively.
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mutant mice using an anti-Csl polyclonal antibody revealed no detectable Csl protein (Fig. 4 B), demonstrating that the knockout allele is null. Nevertheless, knockout mice displayed no obvious developmental or structural deficit in heart or skeletal muscle (see Discussion).
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Analysis of the mouse C2C12 myogenic cell line (Blau et al. 1983) using immunofluorescence showed that Csl was expressed in differentiating myocytes and myotubes (Fig. 4, D–G), paralleling Csl mRNA expression (see Fig. 9 A). Fluorescence was eliminated by prior incubation of antibody with excess recombinant Csl protein (Fig. 4 G). Csl was coexpressed with myofilament proteins in both round and bipolar mononuclear myocytes (Fig. 4D and Fig. E; data not shown), indicating activation at the onset of myogenic differentiation and before fusion. Both native Csl (Fig. 4, D–F) and transfected myc epitope–tagged Csl detected with an anti-myc antibody (data not shown) localized predominantly to the cytoplasm in C2C12 myocytes and myotubes.
Detection of Csl Protein in Adult Tissue Sections
Using immunofluorescence, Csl was robustly expressed in muscles of the heart in all chambers and in skeletal muscles (Fig. 5 A). Analysis of sections of Csl knockout animals revealed only background signal, confirming the specificity of the Csl antibody (Fig. 5 B). Furthermore, staining for hPAP activity in Csl knockout mice recapitulated the immunofluorescence patterns (Fig. 5 C). Muscles of the fetal and neonatal stomach were also positive (Fig. 5 D), whereas those of the intestines, bladder, and vasculature were negative. Smooth muscles surrounding pulmonary veins, which are cardiac in character (Millino et al. 2000), also expressed Csl (data not shown), presumably accounting for the RNase protection signal in lungs (Fig. 2 A). Stomach expression was transient, since RNase protection analysis (Fig. 2 A) and immunofluorescence (data not shown) failed to detect Csl transcript or protein in adults. Csl mRNA was also expressed in testes (Fig. 2 A), and in knockout mice the germ cell component was hPAP positive. However, no Csl protein could be detected in these cells (data not shown).
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Intracellular Distribution of Csl Protein
In cardiac and skeletal muscles of the fetus, Csl protein was localized relatively evenly throughout the cell, even in the presence of clearly organized sarcomeres, revealed by double immunofluorescence with -actinin antibody (Fig. 6A and Fig. B). However, a striated pattern of Csl immunolocalization became evident from late fetal to neonatal stages and was the predominant pattern in the adult (Fig. 6D and Fig. J). Confocal microscopic analysis of stretched slow adult skeletal muscles (soleus) revealed that Csl was expressed predominantly in repetitive double stripes (Fig. 6D and Fig. G). Doublets localized to the level of the I-band, flanking the Z-line, the point of attachment of actin thin filaments of adjacent sarcomeres seen as a single line of -actinin in double immunofluorescence (Fig. 6, D–F). This pattern suggests localization to costameres, subsarcolemmal sites of cytoskeletal/membrane adhesion complexes (Pardo et al. 1983; Berthier and Blaineau 1997). Indeed, Csl colocalized with talin (Fig. 6, G–I), a large protein with actin-, vinculin-, integrin-, and dystrophin-binding activities enriched in a variety of matrix–cytoskeleton adhesive complexes, including costameres (Berthier and Blaineau 1997). A weak single stripe of Csl expression was also seen between the strong doublets at the level of the M-line (Fig. 6 C). Unlike -actinin, which is located throughout the myofibril at the level of the Z-line (Fig. 6 L), Csl was present predominantly surrounding or between myofibrils (Fig. 6 K). The honeycomb-like pattern of Csl in transverse sections of fetal myotubes resembled that of the intermediate filament protein desmin, which precisely surrounds the Z-line of myofibrils in a cage-like manner in continuity with costameres at the membrane (Granger and Lazarides 1979). Csl also colocalized with talin in myotendonous junctions (Fig. 6 J).
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Dominant Effects of Csl in C2C12 Cells
We overexpressed Csl in the C2C12 myogenic cell line (Blau et al. 1983) and examined effects on myoblasts and myocyte formation in the presence and absence of insulin-like growth factor (IGF)-1, a known proliferative, viability, promyogenic, hypertrophic, and repair factor for muscle cells (Coolican et al. 1997; Gredinger et al. 1998; Goldspink 1999; Musaro and Rosenthal 1999; Musaro et al. 1999; Semsarian et al. 1999; Lawlor and Rotwein 2000). Prior studies in the rat L6E9 line demonstrated that enforced Csl expression induced formation of large hypertrophied myotubes after 2 d of differentiation in the presence of IGF-1 (Musaro, A., and N. Rosenthal, personal communication). In this study, C2C12 lines stably expressing FLAG-Csl under control of the EF1 promoter were analyzed in detail.
Dominant effects of ectopic FLAG-Csl were first seen at the myoblast stage. In the FC18 Csl-expressing line, myoblasts appeared larger than the F5 vector–only control line due to induction of prominent lamellipodia accompanied by membrane ruffling. To ensure that this was not due to clonal variation or the nature of the epitope tag, we generated further stable lines expressing myc epitope–tagged Csl under control of the EF1 promoter and the myosin light chain-1F promoter/3' enhancer (MLC1), the latter expressed in myocytes but not in myoblasts (Musaro and Rosenthal 1999). An additional control line expressed green fluorescent protein (GFP) under cytomegalovirus promoter control. These new lines were established in a different clonal isolate of C2C12 that underwent more rapid differentiation in low serum (see Materials and Methods). EF1-myc-Csl lines but not parental or control lines showed prominent lamellipodia (Fig. 7A and Fig. B). To quantify the effects, we measured mean cell area using planimetry. Myc-Csl– and FLAG-Csl–expressing myoblasts showed a significant increase in area (Fig. 7 C), most pronounced in the myc-Csl lines ME1 and ME2. The myc-Csl lines MM1 and MM2, which do not express Csl in myoblasts, were the same size as those carrying GFP vector–only or untransfected controls. This effect was due solely to cell spreading on the tissue culture dish, since flow cytometry using forward light scatter showed that the volume of detached Csl-myoblasts was no different from that of controls (Fig. 7D and Fig. E). Immunofluorescence of myc-Csl–expressing myoblasts revealed that a proportion of Csl protein became localized to membrane ruffles at the leading edge of lamellipodia (Fig. 7 G) and to focal adhesions (not shown). Membrane ruffle localization of a fusion protein consisting of GFP linked in frame to the NH2 terminus of Csl was also evident after expression in myoblasts (Fig. 7 H). Despite their prominent lamellipodia, Csl-expressing myoblasts migrated more slowly than controls in a cell monolayer wound repair migration assay (Fig. 7 F).
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-actinin (Fig. 8G and Fig. H; data not shown).
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Using a polyclonal antibody, we detected Csl protein in the heart, all skeletal muscles, and smooth muscles of the stomach and lungs, and this distribution was confirmed by staining for hPAP in Csl knockout mice. The intracellular localization of Csl protein was complex. In embryonic and fetal cardiac and skeletal muscles and in C2C12 myotubes, Csl was present throughout the cytoplasm of the cell, even in the presence of well-formed myofilamentous structures. However, Csl became associated with the sarcomeric cytoskeleton as myofibers matured. This was predominantly in a costameric pattern. Costameres are discrete, rectilinear, subsarcolemmal domains that appear as transverse stripes along the muscle fiber (Pardo et al. 1983; Berthier and Blaineau 1997) originally defined by the expression pattern of vinculin, which aligns with I-band regions of the sarcomere flanking the Z-lines. Costameres contain supramolecular complexes of focal adhesion–type, spectrin-based, and dystrophin-based cytoskeletal membrane adhesion systems that are thought to stabilize the sarcolemma of muscle cells by distributing force to the extracellular matrix during contractions (Berthier and Blaineau 1997). Many of the proteins that localize to or are enriched in costameres are also found in myotendonous and neuromuscular junctions in muscle cells and in focal adhesions and membrane ruffles in nonmuscle cells (Berthier and Blaineau 1997; Brancaccio et al. 1999). In addition to their mechanical functions, the above structures tether many signaling molecules, which control mechanoreception, cytoskeletal remodelling, and other myogenic functions in response to internal and external stimuli (Berthier and Blaineau 1997; Goldspink 1999). The pattern of Csl immunofluorescence in skeletal muscle cells suggests that Csl is localized both within subsarcolemmal costameres and intracellularly in paired rings flanking the Z-lines of myofibrils seen typically as a honeycomb pattern in transverse sections. A lower level of Csl protein was also seen between the I-band doublets overlying the M-line, showing that Csl is not localized exclusively to the costameric lattice. Levels of Csl protein also showed a distinct fiber-type bias in skeletal muscle cells, suggesting modulation by neuronal input and/or load (Goldspink 1999). In cardiocytes, the Csl pattern appeared essentially identical to that in slow skeletal muscle. These complex distributions hint at functions (perhaps multiple) in cytoskeletal/myofilament organization and regulation and/or associated signaling.
We have approached the function of Csl by gene knockout and overexpression experiments. The conditional knockout allele of Csl, which appears to be null, showed no overt developmental phenotype in skeletal muscle or heart, suggesting genetic or functional redundancy and/or a dedicated role in muscle adaptation and regeneration. Analysis of responses to stress, injury, and aging in muscles of knockout mice is underway. Overexpression of Csl in C2C12 myogenic cells induced several dominant effects. In myoblasts, ectopic Csl induced lamellipodia, and Csl protein became localized to leading edge membrane ruffles and focal adhesions. It is well established that cell division and changes in cell shape and behavior, including formation of lamellipodia and membrane ruffling during cell migration, are mediated by actomyosin cytoskeletal dynamics and require the Rho family of small GTPases (Kaibuchi et al. 1999; Bishop and Hall 2000). Formation of lamellipodia seems to be mediated predominantly by Rac1 and stress fibers, and focal adhesion by Rho (Nobes and Hall 1995). These findings suggest that ectopically expressed Csl may engage up- or downstream elements of Rho/Rac-signaling cascades and thus dysregulate actomyosin dynamics. Paradoxically, Csl-expressing cells could not efficiently migrate into cell monolayer wounds. In addition to inducing lamellipodia, Csl may stabilize trailing edge focal adhesions, which normally must disassemble during migration. Whether the effects relate to the normal role of Csl must remain highly speculative at this point, since Csl is not normally expressed in myoblasts. However, there is an intriguing connection between the dominant functions of Csl in myoblasts and its localization to costameres in normal muscle cells, which share many features of other actin membrane adhesion systems such as lamellipodia (Berthier and Blaineau 1997).
During differentiation of C2C12 cells in low serum, Csl overexpression induced formation of large dysmorphogenic myosacs in an IGF-1 signaling–dependent manner. This phenotype did not appear to be due to augmented myogenesis nor was it accompanied by an increase in anabolic metabolism, a component of skeletal muscle hypertrophy. Furthermore, myosac formation was CsA insensitive and therefore independent of the Ca2+/calmodulin–dependent phosphatase calcineurin (Rusnak and Mertz 2000) implicated in skeletal muscle hypertrophy in vitro and in vivo (Dunn et al. 1999; Musaro et al. 1999; Semsarian et al. 1999). Since Csl-induced myosacs contained large numbers of nuclei, we favor the hypothesis that in the C2C12 line, myosacs form through augmentation and dysregulation of myocyte fusion. Myocyte fusion occurs in a complex and highly orchestrated progression involving cell recognition, adhesion, alignment, and membrane fusion (Doberstein et al. 1997; Hirayama et al. 1999). Importantly, induction of promiscuous fusion in C2 cells with Sendai virus or metalloprotease disintegrin overexpression induces a myosac-like phenotype (Yagami-Hiromasa et al. 1995; Hirayama et al. 1999), and expression of dominant negative forms of Rac1 in Drosophila muscle precursors induces promiscuous fusion and formation of myosac-like muscles in situ (Luo et al. 1994). Thus, it is possible that the two dominant effects of overexpressed Csl discussed above share a common mechanism, that is, perturbation of regulated cytoskeletal dynamics. However, Myc-Csl overexpression did not induce myosacs in a subline of C2C12 cells that differentiate 1–2 d in advance of those in which myosacs were observed. Although the reason for this is unclear, it is possible that the rat L6E9 line in which the myosac phenotype was initially observed and the permissive C2C12 line have become less robust with respect to regulation of cytoskeletal remodelling due to adaptations to tissue culture.
In addition to its dominant effects on myoblast and myocyte cell shape and behavior, Csl also augmented the activities of the NFAT and MEF2 families of transcription factors in an IGF-1 signaling–dependent manner at late stages in the culture. For NFAT, this effect was largely CsA insensitive, indicating that it did not involve the calcineurin pathway. The mechanism of this augmentation is unknown, although it highlights the fact that overexpression of Csl can influence intracellular signaling events in addition to structural changes. Since both NFAT and MEF2 regulate the myoglobin promoter, which is selectively expressed in slow fibers (Chin et al. 1998), and since Csl itself is enriched in slow fibers, overexpression of Csl in C2C12 cells may promote a slow fiber phenotype. It is also possible that the activity of NFAT and MEF2 like that of serum response factor, a key transcriptional regulator in muscle cells, is stimulated by increased actin dynamics (Sotiropoulos et al. 1999). Although we acknowledge that overexpression experiments require cautious interpretation, further analysis of the above effects may offer new insights into Csl function in muscle cells and the prominent role played by the cytoskeleton and myofilament and its associated signaling molecules in muscle development, function, and disease.
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This work was supported by the National Health and Medical Research Council, Australia (9937381), the National Institutes of Health National Institute of Aging (RO3 AG14811), and the National Cancer Institute (Department of Health and Human Services). S. Palmer held a Wellcome Trust Travelling Research Fellowship; A. Schindeler held an Australian Postgraduate Award; and T. Yeoh held an NHMRC Postgraduate Medical Scholarship.
Note added in proof: Csl (Smpx) was recently cloned as a gene responsive to stretch in skeletal muscle cells (Kemp, T.J., T.J. Sadusky, M. Simon, R. Brown, M. Eastwood, D.A. Sassoon, and G.R. Coulton. 2001. Identification of a novel stretch-responsive skeletal muscle gene [Smpx]. Genomics. 72:260–271).
Submitted: 2 February 2001
Revised: 5 April 2001
Accepted: 11 April 2001
F. Koentgen's present address is Ozgene, Australian Gene Targeting Center, Nedlands 6009, Australia.
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