MOLECULAR AND CELLULAR BIOLOGY, Sept. 2003, p. 6037–6048 0270-7306/03/$08.00⫹0 DOI: 10.1128/MCB.23.17.6037–6048.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 23, No. 17
Reduced Mobility of Fibroblast Growth Factor (FGF)-Deficient Myoblasts Might Contribute to Dystrophic Changes in the Musculature of FGF2/FGF6/mdx Triple-Mutant Mice Petra Neuhaus,1 Svetlana Oustanina,1 Tomasz Loch,1 Marcus Kru ¨ger,1 Eva Bober,1 2 2 Rosanna Dono, Rolf Zeller, and Thomas Braun1* Institute of Physiological Chemistry, University of Halle-Wittenberg, 06097 Halle, Germany,1 and Department of Developmental Biology, University of Utrecht, 3584 Utrecht, The Netherlands2 Received 13 December 2002/Returned for modification 20 February 2003/Accepted 22 May 2003
Development and regeneration of muscle tissue is a highly organized, multistep process that requires cell proliferation, migration, differentiation, and maturation. Previous data implicate fibroblast growth factors (FGFs) as critical regulators of these processes, although their precise role in vivo is still not clear. We have explored the consequences of the loss of multiple FGFs (FGF2 and FGF6 in particular) for muscle regeneration in mdx mice, which serve as a model for chronic muscle damage. We show that the combined loss of FGF2 and FGF6 leads to severe dystrophic changes in the musculature. We found that FGF6 mutant myoblasts had decreased migration ability in vivo, whereas wild-type myoblasts migrated normally in a FGF6 mutant environment after transplantation of genetically labeled myoblasts from FGF6 mutants in wild-type mice and vice versa. In addition, retrovirus-mediated expression of dominant-negative versions of Ras and Ral led to a reduced migration of transplanted myoblasts in vivo. We propose that FGFs are critical components of the muscle regeneration machinery that enhance skeletal muscle regeneration, probably by stimulation of muscle stem cell migration. example, emigration of muscle precursor cells from the somite into the limb bud is dependent on hepatocyte growth factor (HGF) signaling (4, 23), and FGF receptor-mediated FGF signaling sustains myoblast migration to limb buds from the somite (25). In addition, FGF2 and FGF4 have been shown to stimulate migration of mouse embryonic limb myogenic cells in vitro and in vivo in chick embryos (25, 51). FGFs have also emerged as key mediators of cell migration in vivo in Drosophila melanogaster and Caenorhabditis elegans development, confirming the importance of molecules that were initially identified and studied in cell culture (35). Previously, we have described a role for FGF6 in skeletal muscle regeneration (17). We found that regeneration of skeletal muscles in FGF6 and FGF6/mdx mutants is impaired; this is probably due to the reduced accumulation of MyoD activated muscle precursor cells during regeneration. In addition, it has been reported that targeted transgene delivery of FGF2 and FGF6 genes led to an enhancement of skeletal muscle repair. FGF gene-treated wounds showed on average a 20-fold increase of regenerating myotubes expressing the marker CD56 versus untreated controls (12). Nevertheless, a significant degree of regeneration was observed in FGF6 and FGF6/ mdx mice, indicating that additional pathways exist that act in parallel to FGF6-mediated cell signaling. Since a number of other FGF molecules are expressed in skeletal muscle, including FGF2 (18), FGF5 (21), and FGF7 (30), we wanted to explore whether these family members contribute to skeletal muscle regeneration and can compensate for the absence of FGF6. In addition, we wanted to learn more about the mechanisms by which FGFs affect regeneration and to elucidate the downstream events that are activated by FGF receptor-mediated signaling in muscle precursor cells.
Skeletal muscle development and regeneration during embryonic and adult life consists of proliferation, migration, and differentiation of myogenic stem cells. Important components of these processes are growth factors, such as fibroblast growth factors (FGFs), bone morphogenetic proteins, and Wnts, that have been implicated in such diverse functions as induction and maintenance of myogenesis (27), stimulation of growth and differentiation of primary myoblasts (16, 25), restriction of skeletal muscle growth (31), and skeletal muscle regeneration (17, 18, 40). During adult life, regeneration of skeletal muscle depends on satellite cells, which are located underneath the basal lamina of myofibers. It is generally assumed that satellite cells are muscle stem cells maintained in a quiescent state and are activated in response to various physiological and pathophysiological requirements (reviewed in reference 42). Although the embryonic origin of satellite cells is still not understood, it is known that molecules such as MyoD (32), Pax7 (43), and MNF (19) regulate satellite cell formation and function. Satellite cell-derived myoblasts and bone marrow-derived myogenic precursor cells (14) can migrate extensively and cross the basal lamina of myofibers (24, 50). Upon transplantation, implanted myoblasts fuse with existing myofibers but also remain viable as muscle precursor cells and contribute to host myofiber regeneration (50, 53). Thus far, the nature of the signals that direct the migration of muscle precursor cells during skeletal muscle regeneration has remained largely unknown. However, during development, several signals have been identified that direct the migration of muscle cells. For * Corresponding author. Mailing address: Institute of Physiological Chemistry, University of Halle-Wittenberg, Hollystr. 1, 06097 Halle, Germany. Phone: 49-0-345-557-3813. Fax: 49-0-345-557-3811. E-mail:
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We generated several double and triple mutants of FGFs and dystrophin and performed transplantation experiments with genetically labeled FGF6-deficient myoblasts. We show that the combined loss of FGF2 and FGF6 leads to a severe increase of dystrophic changes in the musculature of mdx mice and that FGF6⫺/⫺ myoblasts have a reduced migration ability in vivo. Using dominant-negative retroviruses for Ras and Ral, we demonstrate that interference with the Ras-signaling pathway reduces the mobility of transplated myoblasts in vivo. We propose that, based on our in vivo and in vitro data, FGFs support the migration of myoblasts, probably by signaling via the Ras/Ral pathway, resulting in an accumulation of activated myogenic precursor cells at sites where these cells are needed for efficient skeletal muscle regeneration.
MOL. CELL. BIOL. Migration assay. Migration assays were performed in a Neuroprobe standard 48-well chemotaxis chamber (NeuroProbe, Inc., Gaithersburg, Md.) with Neuroprobe polycarbonate filters (8-m pore size), which have been coated with fibronectin (Invitrogen, Inc.). Migration assays were performed by adding serum free Dulbecco modified Eagle medium supplemented with FGF2, FGF6, HGF, and insulin-like growth factor 1 (IGF-1; R&D Systems, Inc.) at different concentrations into the bottom wells of the chamber. Subsequently, 105 cells suspended in Dulbecco modified Eagle medium were loaded into the upper well. After 6 h of incubation, cells from the upper surface of each filter were removed with the aid of a wiper (Neuroprobe). The filters were inverted, fixed with 70% ethanol, and reacted with an anti-desmin antibody (Sigma, Inc.). Bound antibodies were visualized with a Vectastain ABC staining kit (Vector Laboratories) by using diaminobenzidine as substrate. The number of desmin-positive cells was counted in randomly chosen viewing fields and calculated as the total number of desmin-positive cells per square millimeter.
RESULTS MATERIALS AND METHODS Origin of mouse mutants, induction of muscle regeneration, and immunohistochemical analysis. Studies describing the generation of MyLC1/3-lacZ (26), FGF2 (11), FGF5 (48), FGF6 (17), and FGF7 (20) mutant mice have been published. mdx mice were bred on a C57BL/6 background for ⬎10 generations in our laboratory. Induced damage of skeletal muscle was done as described previously (17). Intravenous injection with Evans blue dye (EBD) was performed as described earlier (45). To count the number of satellite cells, myotubes were isolated as described previously (3) and stained with an antibody against CD34. For each genotype, three individual mice were investigated. Positive cells were counted in relation to myotube nuclei. Transplantation experiments, construction of viruses, and LacZ staining. Satellite cells were purified from skeletal muscle of 4- to 6-week-old mice by digestion with a mixture of proteolytic enzymes (39). For transplantation experiments cells were collected, resuspended in phosphate-buffered saline, and injected (at 1 ⫻ 105 to 2 ⫻ 105 cells in 25 l) into regenerating tibialis interior muscles of 4-month-old recipient mice 24 h after freeze-crush injury. The position of the injection site after various regenerations was determined by the relative position of the injection site in respect to the absolute size and the origin of the muscle. To express dominant-negative versions of Ras (H-RasS17N) and Ral (RALS28N) in myoblasts, mutant cDNAs were cloned into the retrovirus plasmid pMSCVneo and transfected together with a gag-pol expression construct in ecotropic Phoenix packaging cell lines to obtain virus titers in the range of 5 ⫻ 105 to 1 ⫻ 106/ml. Myoblasts were infected at a multiplicity of infection of 3 in the presence of 5 g of Polybrene/ml. At 112 days after transplantation the musculus tibialis anterior was removed, fixed, embedded in OCT, and serially sectioned by using a cryomicrotome. Staining for -galactosidase activity was accomplished as described previously (41). Quantitative RT-PCR and Northern blot analysis. Isolation of RNA and Northern blot analysis was done by using established procedures that have been described previously (5). Reverse transcription-PCR (RT-PCR) analysis was essentially done as described previously (27). RNA was treated with RNase-free DNase and reverse transcribed by using Expand reverse transcriptase (Roche). PCRs were performed in 25 l of reaction mix containing 1⫻ TaqPol buffer (Eppendorf), 1⫻ Enhancer (Eppendorf), 1.5 mM MgCl2, deoxynucleoside triphosphates at 200 M each, primers at 500 nM each, Taq polymerase at 0.05 U/l, 5 l of cDNA, and 0.625 l of Sybr Green I (at a 1:40,000 dilution of original stock). The following primer pairs were used LacZ (5⬘-CCGACGGCA CGCTGATTGAAG-3⬘ and 5⬘-ATACTGCACCGGGCGGGAAGG, AT-3⬘) and HPRT (5⬘-GCTGGTGAAAAGGACCTCT-3⬘ and 5⬘-CACAGGACTAGA ACACCTG, C-3⬘). Quantitative RT-PCR was achieved by cDNA amplification in the presence of the DNA-binding dye SYBR Green I. Relative quantitation of myostatin and LacZ expression was done by using the comparative Ct method. The Ct value was defined as the cycle number at which the PCR amplification graph passed a threshold, which by default is defined as 10 times the mean standard deviation of fluorescence in all wells over the baseline cycles. For each experiment, the amount of targets and endogenous reference (HPRT [hypoxanthine phosphoribosyltransferase]) was determined from the standard curve. Next, the target values were normalized to the endogenous reference, assuming that HPRT expression was identical in the different samples. The relative amount of LacZ mRNA was calculated by using the formula: 2⫺⌬⌬Ct, where ⌬⌬Ct ⫽ [Cttarget ⫺ CtHPRT]mutant ⫺ [Cttarget ⫺ CtHPRT]wild type. For wild-type samples, ⌬⌬Ct equals zero and 20 equals one. For mutant mice, the value of 2⫺⌬⌬Ct indicates the fold change in gene expression relative to the wild-type control.
Generation of FGF double- and triple-mutant mice. Previously, we have described that FGF6⫺/⫺ mutant mice show a skeletal muscle regeneration defect with fibrosis and myotube degeneration after freeze-crush injury and that FGF6/mdx mutant mice are characterized by enhanced dystrophic changes. Despite these changes, we still detected a substantial amount of regenerating myotubes in the skeletal muscles of FG6/mdx mice, as indicated by the presence of myotubes with variable diameters and centrally located nuclei. Since several FGFs other than FGF6 are expressed in skeletal muscle, we decided to analyze the consequences of the lack of additional FGFs on skeletal muscle regeneration. In the course of the present study, we generated FGF5/FGF6, FGF5/ FGF6/mdx, FGF6/FGF7, FGF5/FGF6/FGF7, FGF6/FGF7/ mdx, FGF2/FGF6, and FGF2/FGF6/mdx mutant mice. All of these double and triple mutants were viable and fertile, although some of them showed a reduced breeding performance and life span. Skeletal muscles of mutant mice were analyzed after freeze-crush injury by a routine histology regimen comprising of hematoxylin-eosin and trichrome staining, as well as immunohistochemical staining for MyoD and myogenin-positive cells (data not shown). None of these mice showed an additional skeletal muscle phenotype or an enhanced regeneration defect, except for FGF2/FGF6 and FGF2/FGF6/mdx mutant mice. FGF5 mutant mice, which are characterized by an increased activity of their hair follicles, appeared to have even longer hairs on a FGF7 and FGF6 mutant background, although this phenomenon was not investigated in detail. No further additional phenotype was evident. FGF2/FGF6/mdx triple mutants show enhanced dystrophic changes in the skeletal musculature. Similar to FGF6/mdx mice FGF2/FGF6/mdx triple mutant mice displayed no marked clinical signs of severe dystrophy at up to 6 months of age except for a dorsal-ventral curvature of the spine. In addition, triple mutants showed a palpable stiffness of their musculature, which was particularly evident in the pelvic and the shoulder girdle. Macroscopic inspection of skinned mutants revealed the presence of patches of pale tissue within several muscles (Fig. 1, compare panels A and B). Upon histological analysis, it became apparent that these areas contained a high degree of connective tissue (Fig. 1H). The remaining myotubes showed huge differences in caliber size, and numerous necrotic fibers were present (see arrows in Fig. 1H). In general, pathological
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FIG. 1. Enhanced dystrophic changes in the musculature of FGF2⫺/⫺/FGF6⫺/⫺/mdx mice. (A and B) Macroscopic views of skinned 12-weekold mdx (A) and FGF2⫺/⫺/FGF6⫺/⫺/mdx (B) mice showing pale patches of tissue within the muscle fibers (arrows). (C to H) Hematoxylin-eosinstained paraffin sections of the M. latissimus dorsi of different mutant mouse strains. FGF2⫺/⫺ (C) and FGF2⫺/⫺/FGF6⫺/⫺ (D) mutants do not show dystrophic muscle fibers, whereas mdx (E), FGF6⫺/⫺/mdx (F), and FGF2⫺/⫺/mdx (G) mutants display dystrophic changes of the muscle fibers at a comparable severity. The most severe phenotype is evident in FGF2⫺/⫺/FGF6⫺/⫺/mdx mutants (H).
changes in most skeletal muscles were more severe than in mdx and FGF6/mdx mice (Fig. 1E and F). No significant enhancement of the mdx dystrophic phenotype was obvious in FGF2/ mdx double-mutant mice (Fig. 1G). FGF2 mice, which suffer from abnormalities in the cytoarchitecture of the neocortex, skin wound healing defects, and autonomic dysfunction, did not show any changes in the musculature, a finding similar to that observed with FGF6 mutant mice (Fig. 1C). To further substantiate dystrophic changes of skeletal muscle, 9-month-old FGF2/FGF6/mdx triple-mutant, mdx, and wild-type mice were intravenously injected with EBD (45). EBD, a low-molecular-weight diazo dye, is taken up by muscle fibers with a focal breakdown of the plasmalemma, an initial event in muscle cell necrosis (52). Individual muscle fibers that have taken up the dye show a bright red emission upon fluorescent microscope analysis (Fig. 2 G to I and M to O). EBDinjected wild-type mice did not show an uptake of the dye in
skeletal muscle fibers (Fig. 2A, D, G, J, and M), whereas in mdx mice the dye was incorporated to varying degrees into different skeletal muscles, preferentially those in the hind limbs. In agreement with previous findings (45), affected muscles in mdx mice were not stained homogeneously but instead displayed blue streaks that represented damaged muscle fibers (Fig. 2B, E, H, K, and N). Significantly, FGF2/FGF6/mdx mice showed a much more extensive uptake of EBD into skeletal muscle fibers, rendering some muscles completely blue (Fig. 2C, F, I, L, and O). However, it should be pointed out that, similar to the findings in mdx mice, variability in dye accumulation was observed between different animals and different muscles, probably reflecting various environmental influences. Changes in the expression of regulatory factors in FGF mutant mice are partially compensated for during regeneration. We next wanted to characterize the expression of regulatory factors and markers of cell proliferation and differenti-
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FIG. 2. Enhanced EBD uptake into myofibers of FGF2⫺/⫺/FGF6⫺/⫺/mdx mice. (A to C) Macroscopic views of the pelvic girdle of wild-type (WT) (A), mdx (B), and FGF2⫺/⫺/FGF6⫺/⫺/mdx (C) mice after the intravenous injection of EBD. Triple mutants show the highest degree of dye uptake (C). (D to O) Cryosections of M. gluteus (D to I) and diaphragma (J to O) of injected mice. Pictures were obtained by using differential interference contrast (DIC) optics (D to F and J to L) or fluorescence illumination (G to I and M to O). The enhanced dystrophic phenotype of FGF2⫺/⫺/FGF6⫺/⫺/mdx mice is apparent under DIC illumination in the M. gluteus muscle (F) and in the diaphragm (L). The increased uptake of EBD in dystrophic muscle fibers of triple mutant mice leads to strong fluorescence in virtually all muscle fibers. In mdx mice only a minority of myofibers show a strong EBD fluorescence (H and N). No EBD uptake was observed in wild-type control animals (G and M).
ation in mutant muscle tissue. Northern blot analysis revealed that expression of the muscle regulatory factors MyoD and myogenin, which are markers for activated satellite cells, was below the detection limit in the diaphragms of wild-type mice
and FGF2 and FGF6 mutant mice but strongly upregulated in mdx mice (Fig. 3). In the diaphragms of FGF2/FGF6/mdx and FGF6/mdx mutant mice, however, the expression was lower than in mdx mice, indicating a reduced presence of activated
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FIG. 3. Northern blot analysis of FGF/mdx mutant mice. Genes that are characteristic for muscle cell regeneration (MyoD, myogenin, emb MyHC, and neonatal MyHC) are significantly upregulated in mdx mice, but are expressed close to wild-type levels in FGF2⫺/⫺, FGF6⫺/⫺, FGF6⫺/⫺/mdx, and FGF2⫺/⫺/FGF6⫺/⫺/mdx mice. mdx, FGF6⫺/⫺/mdx, and FGF2⫺/⫺/FGF6⫺/⫺/mdx mice show an elevated expression of c-myc, reflecting an increase of cellular proliferation. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control.
satellite cells. MRF4, which is the predominant myogenic factor in adult muscle tissues, showed only slight changes in its expression level. During the course of muscle regeneration, embryonic MyHC isoforms are reexpressed, indicating the presence of newly formed myotubes. In the diaphragms of mdx mice, high levels of the embryonic and neonatal MyHC isoforms were found; these levels decreased in FGF6/mdx and FGF6/FGF2/mdx mutant mice, indicating a reduced formation of new myotubes. In contrast, expression of the cell cycle regulatory gene c-myc was not greatly diminished in FGF6/mdx and FGF6/FGF2/mdx mutant mice compared to mdx mice (Fig. 3). In addition to stimulatory growth factors, negative regulators of muscle growth are believed to control muscle development and regeneration. We therefore investigated the expression of myostatin, which is a member of the transforming growth factor  superfamily and a key negative regulator of skeletal muscle growth in muscles of mutant mice by quanti-
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tative PCR. We found that the expression of myostatin was upregulated in muscles of FGF2 and FGF6 mutant mice but strongly downregulated in mdx mice, a finding most likely indicating the need for increased myoblast proliferation to replace damaged muscle fibers (data not shown). The lack of FGF2 and FGF6 does not cause a significant reduction of the number of skeletal muscle satellite cells. In order to analyze whether the enhanced dystrophic changes in the skeletal musculature of FGF2/FGF6/mdx triple mutants were due to a reduction of the amount of satellite cells, we determined the number of CD34⫹ satellite cells in wild-type, mdx mutant, and FGF2/FGF6/mdx triple-mutant mice. Previous experiments had revealed that the number of CD34⫹ cells located on myotubes strictly correlated with the number of satellite cells identified by transmission electron microscopy (S. Oustanina et al., unpublished results) and is a faithful molecular marker for satellite cells (1). As shown in Fig. 4, myotubes of FGF2/FGF6/mdx triple-mutant mice contained virtually the same number of satellite cells in relation to myotube nuclei (3.05%) as did mdx mutant mice (3.1%). Although the number of satellite cells was slightly reduced compared to wild-type control animals (3.56%), this reduction is minor and statistically not significant. In addition, no significant differences in the growth and differentiation kinetics of myogenic cells were observed when satellite cells derived from isolated myotubes were placed in culture and compared to cells derived from wild-type and mdx mutant control animals (data not shown). From these experiments we concluded that a reduction of the number and differentiation ability of FGF2/FGF6/mdx satellite cells could not account for the enhanced dystrophic changes in the skeletal musculature of triple-mutant mice. Reduced migration of FGF6 mutant myoblasts in vivo. FGFs are known to regulate growth and differentiation of primary, secondary, and adult myoblasts. In addition, they have been shown to stimulate migration of myogenic cells in mice and other organisms. The established role of FGFs as regulators of cell migration (reviewed in reference 38) and the ability of muscle satellite cells to migrate extensively (24, 50) led us to study the migration of FGF6 mutant muscle precursor cells in vivo in mice. To generate a suitable genetic label that would allow us to identify transplanted muscle cells, we crossed FGF6 mutants with transgenic MyLC-1/3-LacZ mice (26). MyLC-1/ 3-LacZ mice show a specific expression of the reporter gene in striated muscles and can be used to track transplanted cells in recipient mice (14). Both strains were bred on a C57BL/6 background to allow isogenic transplantation without necessity for immunosuppressive drugs. Next, we isolated satellite cells from MyLC-1/3-LacZ and MyLC-1/3-LacZ/FGF6⫺/⫺ mice (1 ⫻ 105 to 2 ⫻ 105 cells) and transplanted them back into the M. tibialis anterior of either C57BL/6 wild-type or FGF6⫺/⫺ mice. Shortly before transplantation, the same muscle was artificially damaged by freezecrush injury to promote regeneration and myoblast recruitment. At 112 days after transplantation, muscles were removed, sectioned, and subjected to LacZ staining (wild-type to wild-type transplantations [n ⫽ 3]; wild-type to FGF6⫺/⫺ transplantations [n ⫽ 3]; FGF6⫺/⫺ to wild-type transplantations [n ⫽ 4]). As shown in Fig. 5, no significant differences in the amount of LacZ-positive cells were noted close to the implantation sites when wild-type cells were transferred to
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FIG. 4. The lack of FGF2 and FGF6 does not cause a significant reduction of the numbers of skeletal muscle satellite cells. (A and B) Quantitative assessment of CD34-positive satellite cells derived from wild-type, mdx, and FGF2/FGF6/mdx triple-knockout mice. (C) Myotubes were isolated from the M. flexor digitorum brevis muscles of different mouse strains and stained with an antibody to CD34. Positive cells were counted in relation to myotube nuclei. No significant differences in the number of satellite cells between different mutant strains were detected. The arrows in panel C indicate CD34-positive cells.
FGF6⫺/⫺ (Fig. 5B), FGF6⫺/⫺ cells were transferred to wildtype hosts (Fig. 5E), or wild-type cells were transferred to wild-type hosts (Fig. 5H). However, when sections were compared that were taken ca. 3 mm apart from the injection site either toward the previous injury (“proximal to injury”) or in the opposite direction (“distal to injury”), considerable differences were noted between the different strains. Significantly fewer LacZ-positive cells were present on matched sections when FGF6⫺/⫺ cells were transplanted into C57BL/6 hosts (Fig. 5D and F) compared to wild-type cells transplanted into wild-type (Fig. G and I) or FGF6⫺/⫺ hosts (Fig. 5A and C). No major differences were found between transplantations of wildtype cells into FGF6⫺/⫺ and wild-type hosts (Fig. 5, compare panels A and C to panels G and I). It is interesting that we found only few differences in the migration pattern of transplanted cells proximal or distal to the lesions. This might be explained by a similar activation of the distal aspects of damaged fibers compared to the damaged site itself. Inhibition of Ras-signaling interferes with skeletal myoblast migration in vivo. FGF receptor-mediated signaling is achieved via different intracellular signaling pathways, including the Ras pathway, Src-family tyrosine kinases, phosphoinositide 3-kinase (PI3K), and the PLC pathway (8). Interestingly, the Ras pathway has recently be linked to the migratory ability of
C2C12 myoblasts in vitro in response to FGF2, HGF, and IGF-1 (46), indicating that the Ras-Ral pathway is essential for the migration of muscle progenitor cells. To evaluate whether activation of the Ras-Ral pathway is crucial for migration of skeletal myoblasts in vivo, we generated murine stem cell virusbased retroviruses expressing dominant-negative versions of Ras (H-RasS17N) and Ral (RALS28N) and infected MyLC1/3-LacZ myoblasts before transplantation in C57BL/6 hosts. Using the same experimental setup as described for MyLC-1/ 3-LacZ/FGF6⫺/⫺ cells above, approximately 1 ⫻ 105 to 2 ⫻ 105 infected myoblasts were injected into the M. tibialis anterior muscle of C57BL/6 mice. As shown in Fig. 6, no significant differences in the number of LacZ-positive cells were noted close to the implantation sites between hosts that have received myoblasts infected with a control virus expressing green fluorescent protein (GFP) and hosts that have received MyLC-1/ 3-LacZ myoblasts infected either with a dominant-negative version of Ras (H-RasS17N) or Ral (RALS28N). On the other hand, when sections were compared that were taken from regions ca. 3 mm apart from the implantation site either toward the previously applied injury (Fig. 6C, F, and I) or at the opposite side (Fig. 6A, D, and G), a different picture emerged. Significantly fewer myoblasts were found that have been infected either with the dominant-negative Ras (H-
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FIG. 5. Decreased migration ability of genetically labeled FGF6⫺/⫺ myoblasts in vivo after transplantation in isogenic hosts. FGF6⫺/⫺ myoblasts do not migrate as efficiently as wild-type myoblasts after transplantation into injured M. tibialis. (A to I) LacZ-stained cryosections close to the injection site (B, E, and H), 3 mm distal (A, D, and G), and 3 mm proximal (C, F, and I) to the site of injury. The chart represents the average of three independent transplantation experiments. The inlet in the chart depicts the experimental setup. A significant decrease in the number of transplanted myoblasts was detected proximal or distal to the injury site when FGF6⫺/⫺ myoblasts were transplanted into wild-type hosts (D to F) in comparison to transplanted wild-type myoblasts in either wild-type hosts (G to I) or FGF6⫺/⫺ hosts (A to C).
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FIG. 6. Inhibition of Ras/Ral signaling interferes with myoblast migration in vivo. (A to I) Myoblasts infected with retroviruses expressing dominant-negative isoforms of Ras (H-Ras S17N) (D to F) or Ral (ral S28N) (G to I) show reduced migration after transplantation into injured M. tibialis muscle of wild-type mice in comparison to cells infected by a retrovirus expressing GFP (A to C). The chart represents the average of three independent transplantation experiments. A significant decrease in the number of transplanted myoblasts expressing either H-Ras S17N or Ral S28N was detected proximal or distal to the injury site.
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FIG. 7. FGF mutant and Ras/Ral dominant-negative (Dn) infected myoblasts have no overt proliferation defect. Quantitative real-time PCR and semiquantitative control reactions of transplanted LacZ mRNA-positive cells. (A and D) Quantitative RT-PCR analyses demonstrate no significant differences in LacZ expression after transplantation of wild-type, FGF mutant, and Ras/Ral Dn infected myoblasts into complete host muscles. (B and E) A decrease in LacZ expressing muscle cells was only evident when the tissue around the injected site was removed. PCRs were normalized within each triple group; hence, the expression between different groups cannot be compared. The numbers are shown in relation to the number of LacZ expression in wild-type hosts (FGF6⫺/⫺ cells) or in relation to the number of mock-transfected cells (myoblasts expressing the dominant-negative Ras and Ral isoforms).
RasS17N) or Ral (RALS28N) retrovirus compared to a control virus expressing GFP. As in the previous set of transplantation experiments, more cells were found in the vicinity of the preceding injury, indicating that not all signals that led to a guided migration of myoblasts to a site of muscle regeneration were inhibited by the dominant-negative Ras and Ral molecules. To further support the idea that the reduced presence of transplanted FGF6⫺/⫺, Ras (H-RasS17N), and Ral (RALS28N) infected cells at a distance from the injection site is due to a reduced migration capability and not to a reduced proliferation and/or differentiation of muscle precursor cell, we analyzed the absolute number of MyLC-1/3-LacZ-expressing cells after transplantation into different strains. Since it is virtually impossible to count all cells, particularly in the immediate vicinity of the transplantation site, we used quantitative RTPCR with LacZ mRNA from the whole muscle. As shown in Fig. 7, similar values for LacZ expression were found in complete muscles of wild-type mice that received MyLC-1/3-LacZ and MyLC-1/3-LacZ/FGF6⫺/⫺ cells and in muscles of FGF6⫺/⫺ mice that received MyLC-1/3-LacZ cells (Fig. 7A). Similar results were obtained when muscles were investigated that had received myoblasts infected with a mock retrovirus or with viruses expressing Ras (H-RasS17N) and Ral (RALS28N) (Fig. 7D). Likewise, no significant differences in the expression level of LacZ mRNA were found when tissue samples were
derived from the area close to the transplantation site (Fig. 7C and F). In contrast, when the area around the transplantation site was intentionally removed and the rest of the muscle was analyzed by RT-PCR, a significant reduction of LacZ mRNA was detected in a wild-type host that received MyLC-1/3-LacZ/ FGF6⫺/⫺ cells compared to MyLC-1/3-LacZ transplanted cells (Fig. 7B), as well as in wild-type hosts that received myoblasts infected with Ras (H-RasS17N) and Ral (RALS28N) retroviruses compared to cells infected with a control virus (Fig. 7E). Since LacZ mRNA is present only in differentiated myotubes derived from transplanted myoblasts, the level of LacZ mRNA is an indicator of both proliferation and differentiation. Based on the virtually identical LacZ expression levels in total muscles, we concluded that proliferation and/or differentiation of muscle precursor cells was not greatly affected by the lack of FGF6 or interference of Ras/Ral signaling. Because we normalized the PCRs within each triple group, our analysis only allows a comparison of wild-type and mutant samples within one group. Small differences in the number of transplanted cells close to the injection site, which might have been expected due to a better (or worse) migration of cells away from the injection site would have escaped our attention due to the fact that the bulk of the cells remains located close to the injection site. Chemotactic effects of FGFs and reduced mobility of FGF2/ FGF6 mutant myoblasts in vitro. Since our in vivo studies
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FIG. 8. Reduced migration of FGF2⫺/⫺/FGF6⫺/⫺/mdx myoblasts in vitro. FGF2 and FGF6 stimulate the migration of satellite cell-derived wild-type myoblasts in a modified Boyden Chamber assay. Migration of wild-type myoblasts were stimulated at a concentration of 1 ng of FGF/ml of medium and increased further at 10 ng of FGF/ml of medium. Reduced migration of cells derived from FGF2⫺/⫺/FGF6⫺/⫺ mice can partially be overcome when the FGF concentrations in the medium were increased to 50 ng/ml.
suggested that FGF2 and FGF6 are important regulators of myoblast migration, we next investigated the migratory ability of satellite cell-derived myoblasts in a modified Boyden Chamber assay. To exclude nonmyogenic cells that might contaminate our satellite cell preparations, we identified migrant myoblasts by immunohistochemical staining with an antibody against the intermediate filament desmin at the end of a 6-h incubation period. We observed an increase in adult myoblast migration after addition of FGF2, FGF6, HGF, and IGF-1 at concentrations of 1 to 10 ng/ml of medium. Interestingly, a rather high basal level of cell migration was observed when cells were derived from wild-type mice, although exogenously added FGF2, FGF6, HGF, and IGF-1 strongly stimulated migration. In contrast, the basal level of migration across the filter dropped to about a third when myoblasts were isolated from FGF2/FGF6 double-mutant animals (Fig. 8). Satellite cells from FGF6 mutant mice also showed a reduced migratory ability, but it was less pronounced than that of FGF2/FGF6 double-mutant cells (data not shown). The addition of FGF2 and FGF6 stimulated the migration of myoblasts, although the degree of migration was lower than that of the matched wild-type controls. When the concentrations of FGF2 and FGF6 were raised to 50 ng/ml, the extent of migration increased significantly, although the level observed with wild-type myoblasts was never reached (Fig. 8). In contrast, HGF and IGF-1, which were the most potent inducers of satellite cell migration in our assay, stimulated migration of satellite cells isolated from FGF2/FGF6 mutants to the same extent as wild-type cells, thus excluding the possibility that satellite cells from FGF2/FGF6 mice were defective in a more general manner (Fig. 8). DISCUSSION Redundancies and specificities of FGFs for muscle development and repair. FGFs play important roles in a wide variety of biological processes, including control of cell differentiation, organ growth, and migration. It was therefore surprising that
the inactivation of certain FGF genes, such as FGF5 (22), FGF6 (17), and FGF7 (20), despite their specific expression during embryonic development, yielded only minor phenotypes compared to dramatic effects observed after mutation of the FGF4 (arrest of morula development [13]), FGF8 (defects in gastrulation, cardiac, craniofacial, forebrain, midbrain, and cerebellar development [34]), and FGF10 (arrest of lung and limb bud development [44]) genes. A common explanation for such findings is redundancy of functions, which might lead to compensation of severe defects. We therefore expected that the combined inactivation of three different members of the FGF family (i.e., FGF5, FGF6, and FGF7), which are all expressed during somite formation and muscle development, would generate strong developmental defects. Surprisingly, this was not the case, suggesting that either FGF5, FGF6, or FGF7, although coexpressed in somites and skeletal muscle do not have a significant overlap of function or that additional members of the family, such as FGF2, compensate for the combined loss of three growth factor genes. Variations in the expression of compensating FGF genes in different genetic backgrounds (10) might also have contributed to the failure to detect a major role of FGF6 for muscle regeneration as shown by Fiore et al. (15). Another possible explanation for differences in the phenotype is the presence or absence of additional modifier genes or the different design of the targeted mutation, which might lead to considerable differences in the observed phenotypes (49). In addition, even in a uniform genetic background, stochastic variations in the expression of a regulatory circuit can result in dramatic differences in phenotypic consequences (29). Among the FGF family members FGF2 is particularly interesting with respect to muscle regeneration (28). FGF2 is a potent stimulator of the proliferation and fusion of myoblasts in vitro and enhances muscle regeneration in vivo (33). Likewise, transgene delivery of FGF2 and FGF6 genes led to a strong enhancement of skeletal muscle repair (12), further emphasizing the importance of FGF2 and FGF6 for muscle repair.
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FGFs contribute to various cellular events, including proliferation and migration. FGFs were mainly connected with the regulation of proliferation and the inhibition of differentiation. Several lines of evidence suggest that their role is strongly dependent on the cellular environment (38). In highdensity cultures FGFs stimulate differentiation, which is probably due to an intrinsic downregulation of FGF receptors (37), whereas in low-density cultures FGFs strongly repress terminal differentiation (6). In vivo, the various activities of FGFs are subject to additional regulatory input, including the differential expression of various heparan sulfate glycosaminoglycans that are required for signaling from their respective receptors (7). Regulation of cell migration by FGFs has been documented in several organisms and for different cell types (reviewed by Montell [35]). In the mouse, FGF2 and FGF4 have been shown to stimulate the migration of embryonic limb myogenic cells and of C2C12 myoblasts in vitro (46, 51). We have demonstrated that the mobility of FGF6-deficient myoblasts is significantly reduced in vivo. The finding that myoblasts with a repressed Ras/Ral pathway also showed a strong reduction of migration supports our conclusion that FGF signaling is crucial for myoblast migration and that the lack of FGFs leads to a reduced accumulation of activated myoblasts in regions with increased demand for muscle stem cells for tissue repair. In vitro, the reduced ability of FGF2 and FGF6 mutant myoblasts to migrate can be rescued to some extent by the addition of supraphysiological concentrations of either FGF2 or FGF6, indicating that both growth factors might partially compensate for each other. This partial compensation might also explain why the phenotype of the FGF2/ FGF6 double-mutant mice is stronger than the FGF6 mutant alone and why no muscle phenotype was observed in FGF2 mutant mice. In principle, some of our data might also be explained by a decrease in myoblast proliferation and/or differentiation. Our assay system relies on the detection of MyLC-LacZ, which is switched on in differentiated muscle cells. If either proliferation of myoblasts drops or the differentiation of muscle precursor cells is altered due to the lack of FGFs or the expression of dominant-negative Ras and Ral mutants, we might also count different numbers of MyLC-LacZ-positive cells. To rule out this possibility, we have performed real-time quantitative RT-PCR for MyLC-LacZ mRNA to compare the absolute numbers of MyLC-LacZ-positive cells between engineered myoblasts and wild-type controls. Since we did not find significant differences between these samples, we can rule out that lack of FGF6 or inhibition of Ras/Ral signaling results in a major alteration of the proliferation and differentiation of myoblasts. The Ras/Ral pathway appears to transmit migratory signals elicited by FGFs in vivo. Ras is involved in the regulation of numerous cellular events, namely, cell proliferation, inhibition of differentiation, and migration (see reference 9 for a recent review). Ras can be activated by several growth factors that have been shown to control skeletal muscle cell development, differentiation, and migration, including IGFs, HGFs, and FGFs (46). IGF-1 is known to stimulate myoblast migration in vitro (46) and retains (in transgenic mice) the proliferative response to muscle injury characteristic of younger animals (36). HGF has been demonstrated to be important for limb
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muscle precursor cell migration in vivo (4, 23) and for C2C12 myoblast (46) and muscle satellite cell (2) migration in vitro. Thus, it seems likely that, in addition to FGFs, other growth factors or cellular signals such as Ca2⫹ increase (9) lead to an activation of Ras and subsequently of Ras downstream targets such as Ral, Raf, and PI3K (9). Our finding that inhibition of Ras signaling by a dominant-negative Ras mutant results in a stronger inhibition of migration compared to the inactivation of FGF6 supports the view that several different growth factors funnel into the activation of Ras and stimulate migration. Expression of a dominant-negative Ral mutant inhibited migration in vivo to virtually the same extent as inhibition of Ras. It seems therefore reasonable to assume that Ral mediates activation of the cellular migration machinery in response to Ras signaling. At the moment it is hard to decide whether Ral is solely responsible for activation of migration. Although we did not observe an inhibition of migration when myoblasts were treated with LY294002, a specific inhibitor of PI3K, and U0126, an inhibitor of MEK (data not show), before transplantation, it is difficult to deduce from these negative results that PI3K is not involved in migration of myoblasts in vivo since myoblasts may recover from this treatment and reactivate the pathway once the drug is removed and the cells are transplanted into host muscle. Migration is an important element of stem cell-mediated tissue repair. The importance of migration for tissue repair is long known. Most injuries require the infiltration of damaged tissue with various types of cells, which actively migrate into the target area. The concept that muscle precursor cells, which contribute to regenerating muscle in a region of muscle damage, are not all locally derived but are also recruited from exogenous sources, including adjacent muscles, has been put forward by several labs (24, 50). However, the mechanisms by which muscle precursor cells achieve this task have remained enigmatic so far. The necessity to learn more about this problem has become even more eminent with the unexpected finding that bone marrow-derived cells migrate into areas of induced muscle degeneration and participate in the regeneration of the damaged fibers (14) and that Sca-1, CD34 musclederived stem cells were able to migrate from the circulation into host muscle tissues (47). Our demonstration that FGFs mediated signaling, most likely via the Ras/Ral pathway, will facilitate the understanding of this process and might help to design strategies for improved availability of stem cell-derived organotypic cells in disease-related muscle frailty. ACKNOWLEDGMENTS The excellent technical assistance of S. Kru ¨ger, Y. Pooch, and U. Ziese is gratefully acknowledged. We are indebted to E. Fuchs (University of Chicago) for supplying FGF7 mutant mice, to H. Thoenen (MPI f. Psychiatrie, Martinsried, Germany) for the donation of FGF5 mutant mice, and to M. Buckingham and R. Kelly (Institut Pasteur, Paris, France) for MyLC1/3-lacZ mice. We also thank H. Koide (Tokyo Institute of Technology, Tokyo, Japan) for supplying Ras and Ral mutants. This work was supported by the Deutsche Forschungsgemeinschaft, the “Fonds der Chemischen Industrie,” and the Wilhelm-Roux Program for Research of the Martin Luther University. REFERENCES 1. Beauchamp, J. R., L. Heslop, D. S. Yu, S. Tajbakhsh, R. G. Kelly, A. Wernig, M. E. Buckingham, T. A. Partridge, and P. S. Zammit. 2000. Expression of
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