Reduced Mobility of Fibroblast Growth Factor (FGF)-Deficient Myoblasts Might Contribute to...

MOLECULAR AND CELLULAR BIOLOGY, Sept. 2003, p. 6037–6048 Vol. 23, No. 170270-7306/03/$08.00�0 DOI: 10.1128/MCB.23.17.6037–6048.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Reduced Mobility of Fibroblast Growth Factor (FGF)-DeficientMyoblasts Might Contribute to Dystrophic Changes in the

Musculature of FGF2/FGF6/mdx Triple-Mutant MicePetra Neuhaus,1 Svetlana Oustanina,1 Tomasz Loch,1 Marcus Kruger,1 Eva Bober,1

Rosanna Dono,2 Rolf Zeller,2 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 cellproliferation, 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 haveexplored the consequences of the loss of multiple FGFs (FGF2 and FGF6 in particular) for muscle regenerationin mdx mice, which serve as a model for chronic muscle damage. We show that the combined loss of FGF2 andFGF6 leads to severe dystrophic changes in the musculature. We found that FGF6 mutant myoblasts haddecreased migration ability in vivo, whereas wild-type myoblasts migrated normally in a FGF6 mutantenvironment after transplantation of genetically labeled myoblasts from FGF6 mutants in wild-type mice andvice versa. In addition, retrovirus-mediated expression of dominant-negative versions of Ras and Ral led to areduced migration of transplanted myoblasts in vivo. We propose that FGFs are critical components of themuscle regeneration machinery that enhance skeletal muscle regeneration, probably by stimulation of musclestem cell migration.

Skeletal muscle development and regeneration during em-bryonic and adult life consists of proliferation, migration, anddifferentiation of myogenic stem cells. Important componentsof these processes are growth factors, such as fibroblast growthfactors (FGFs), bone morphogenetic proteins, and Wnts, thathave been implicated in such diverse functions as inductionand maintenance of myogenesis (27), stimulation of growthand differentiation of primary myoblasts (16, 25), restriction ofskeletal muscle growth (31), and skeletal muscle regeneration(17, 18, 40). During adult life, regeneration of skeletal muscledepends on satellite cells, which are located underneath thebasal lamina of myofibers. It is generally assumed that satellitecells are muscle stem cells maintained in a quiescent state andare activated in response to various physiological and patho-physiological requirements (reviewed in reference 42). Al-though the embryonic origin of satellite cells is still not under-stood, 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-derivedmyogenic precursor cells (14) can migrate extensively and crossthe basal lamina of myofibers (24, 50). Upon transplantation,implanted myoblasts fuse with existing myofibers but also re-main viable as muscle precursor cells and contribute to hostmyofiber regeneration (50, 53). Thus far, the nature of thesignals that direct the migration of muscle precursor cells dur-ing skeletal muscle regeneration has remained largely un-known. However, during development, several signals havebeen identified that direct the migration of muscle cells. For

example, emigration of muscle precursor cells from the somiteinto the limb bud is dependent on hepatocyte growth factor(HGF) signaling (4, 23), and FGF receptor-mediated FGFsignaling sustains myoblast migration to limb buds from thesomite (25). In addition, FGF2 and FGF4 have been shown tostimulate migration of mouse embryonic limb myogenic cells invitro and in vivo in chick embryos (25, 51). FGFs have alsoemerged as key mediators of cell migration in vivo in Drosoph-ila melanogaster and Caenorhabditis elegans development, con-firming the importance of molecules that were initially identi-fied and studied in cell culture (35).

Previously, we have described a role for FGF6 in skeletalmuscle regeneration (17). We found that regeneration of skel-etal muscles in FGF6 and FGF6/mdx mutants is impaired; thisis probably due to the reduced accumulation of MyoD acti-vated muscle precursor cells during regeneration. In addition,it has been reported that targeted transgene delivery of FGF2and FGF6 genes led to an enhancement of skeletal musclerepair. FGF gene-treated wounds showed on average a 20-foldincrease of regenerating myotubes expressing the markerCD56 versus untreated controls (12). Nevertheless, a signifi-cant degree of regeneration was observed in FGF6 and FGF6/mdx mice, indicating that additional pathways exist that act inparallel to FGF6-mediated cell signaling. Since a number ofother FGF molecules are expressed in skeletal muscle, includ-ing FGF2 (18), FGF5 (21), and FGF7 (30), we wanted toexplore whether these family members contribute to skeletalmuscle regeneration and can compensate for the absence ofFGF6. In addition, we wanted to learn more about the mech-anisms by which FGFs affect regeneration and to elucidate thedownstream events that are activated by FGF receptor-medi-ated signaling in muscle precursor cells.

* Corresponding author. Mailing address: Institute of PhysiologicalChemistry, University of Halle-Wittenberg, Hollystr. 1, 06097 Halle,Germany. Phone: 49-0-345-557-3813. Fax: 49-0-345-557-3811. E-mail:[email protected].

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We generated several double and triple mutants of FGFsand dystrophin and performed transplantation experimentswith genetically labeled FGF6-deficient myoblasts. We showthat the combined loss of FGF2 and FGF6 leads to a severeincrease of dystrophic changes in the musculature of mdx miceand that FGF6�/� myoblasts have a reduced migration abilityin vivo. Using dominant-negative retroviruses for Ras and Ral,we demonstrate that interference with the Ras-signaling path-way reduces the mobility of transplated myoblasts in vivo. Wepropose that, based on our in vivo and in vitro data, FGFssupport the migration of myoblasts, probably by signaling viathe Ras/Ral pathway, resulting in an accumulation of activatedmyogenic precursor cells at sites where these cells are neededfor efficient skeletal muscle regeneration.

MATERIALS AND METHODS

Origin of mouse mutants, induction of muscle regeneration, and immunohis-tochemical analysis. Studies describing the generation of MyLC1/3-lacZ (26),FGF2 (11), FGF5 (48), FGF6 (17), and FGF7 (20) mutant mice have beenpublished. mdx mice were bred on a C57BL/6 background for �10 generationsin our laboratory. Induced damage of skeletal muscle was done as describedpreviously (17). Intravenous injection with Evans blue dye (EBD) was performedas described earlier (45). To count the number of satellite cells, myotubes wereisolated as described previously (3) and stained with an antibody against CD34.For each genotype, three individual mice were investigated. Positive cells werecounted 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 bydigestion with a mixture of proteolytic enzymes (39). For transplantation exper-iments cells were collected, resuspended in phosphate-buffered saline, and in-jected (at 1 � 105 to 2 � 105 cells in 25 �l) into regenerating tibialis interiormuscles of 4-month-old recipient mice 24 h after freeze-crush injury. The posi-tion of the injection site after various regenerations was determined by therelative position of the injection site in respect to the absolute size and the originof the muscle. To express dominant-negative versions of Ras (H-RasS17N) and Ral(RALS28N) in myoblasts, mutant cDNAs were cloned into the retrovirus plasmidpMSCVneo and transfected together with a gag-pol expression construct inecotropic 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 inthe presence of 5 �g of Polybrene/ml. At 112 days after transplantation themusculus tibialis anterior was removed, fixed, embedded in OCT, and seriallysectioned by using a cryomicrotome. Staining for �-galactosidase activity wasaccomplished as described previously (41).

Quantitative RT-PCR and Northern blot analysis. Isolation of RNA andNorthern blot analysis was done by using established procedures that have beendescribed previously (5). Reverse transcription-PCR (RT-PCR) analysis wasessentially done as described previously (27). RNA was treated with RNase-freeDNase 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, deoxynucleosidetriphosphates at 200 �M each, primers at 500 nM each, Taq polymerase at 0.05U/�l, 5 �l of cDNA, and 0.625 �l of Sybr Green I (at a 1:40,000 dilution oforiginal stock). The following primer pairs were used LacZ (5�-CCGACGGCACGCTGATTGAAG-3� and 5�-ATACTGCACCGGGCGGGAAGG, AT-3�)and HPRT (5�-GCTGGTGAAAAGGACCTCT-3� and 5�-CACAGGACTAGAACACCTG, C-3�). Quantitative RT-PCR was achieved by cDNA amplificationin the presence of the DNA-binding dye SYBR Green I. Relative quantitation ofmyostatin and LacZ expression was done by using the comparative Ct method.The Ct value was defined as the cycle number at which the PCR amplificationgraph passed a threshold, which by default is defined as 10 times the meanstandard deviation of fluorescence in all wells over the baseline cycles. For eachexperiment, the amount of targets and endogenous reference (HPRT [hypoxan-thine phosphoribosyltransferase]) was determined from the standard curve.Next, the target values were normalized to the endogenous reference, assumingthat HPRT expression was identical in the different samples. The relative amountof 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.

Migration assay. Migration assays were performed in a Neuroprobe standard48-well chemotaxis chamber (NeuroProbe, Inc., Gaithersburg, Md.) with Neu-roprobe polycarbonate filters (8-�m pore size), which have been coated withfibronectin (Invitrogen, Inc.). Migration assays were performed by adding serumfree Dulbecco modified Eagle medium supplemented with FGF2, FGF6, HGF,and insulin-like growth factor 1 (IGF-1; R&D Systems, Inc.) at different con-centrations into the bottom wells of the chamber. Subsequently, 105 cells sus-pended 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 removedwith the aid of a wiper (Neuroprobe). The filters were inverted, fixed with 70%ethanol, and reacted with an anti-desmin antibody (Sigma, Inc.). Bound anti-bodies were visualized with a Vectastain ABC staining kit (Vector Laboratories)by using diaminobenzidine as substrate. The number of desmin-positive cells wascounted in randomly chosen viewing fields and calculated as the total number ofdesmin-positive cells per square millimeter.

RESULTS

Generation of FGF double- and triple-mutant mice. Previ-ously, we have described that FGF6�/� mutant mice show askeletal muscle regeneration defect with fibrosis and myotubedegeneration after freeze-crush injury and that FGF6/mdx mu-tant mice are characterized by enhanced dystrophic changes.Despite these changes, we still detected a substantial amountof regenerating myotubes in the skeletal muscles of FG6/mdxmice, as indicated by the presence of myotubes with variablediameters and centrally located nuclei.

Since several FGFs other than FGF6 are expressed in skel-etal muscle, we decided to analyze the consequences of thelack of additional FGFs on skeletal muscle regeneration. In thecourse 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 ofthese double and triple mutants were viable and fertile, al-though some of them showed a reduced breeding performanceand life span. Skeletal muscles of mutant mice were analyzedafter freeze-crush injury by a routine histology regimen com-prising of hematoxylin-eosin and trichrome staining, as well asimmunohistochemical staining for MyoD and myogenin-posi-tive cells (data not shown). None of these mice showed anadditional skeletal muscle phenotype or an enhanced regener-ation defect, except for FGF2/FGF6 and FGF2/FGF6/mdxmutant mice. FGF5 mutant mice, which are characterized byan increased activity of their hair follicles, appeared to haveeven longer hairs on a FGF7 and FGF6 mutant background,although this phenomenon was not investigated in detail. Nofurther additional phenotype was evident.

FGF2/FGF6/mdx triple mutants show enhanced dystrophicchanges in the skeletal musculature. Similar to FGF6/mdxmice FGF2/FGF6/mdx triple mutant mice displayed nomarked clinical signs of severe dystrophy at up to 6 months ofage except for a dorsal-ventral curvature of the spine. In ad-dition, triple mutants showed a palpable stiffness of their mus-culature, which was particularly evident in the pelvic and theshoulder girdle.

Macroscopic inspection of skinned mutants revealed thepresence of patches of pale tissue within several muscles (Fig.1, compare panels A and B). Upon histological analysis, itbecame apparent that these areas contained a high degree ofconnective tissue (Fig. 1H). The remaining myotubes showedhuge differences in caliber size, and numerous necrotic fiberswere present (see arrows in Fig. 1H). In general, pathological

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changes in most skeletal muscles were more severe than in mdxand FGF6/mdx mice (Fig. 1E and F). No significant enhance-ment of the mdx dystrophic phenotype was obvious in FGF2/mdx double-mutant mice (Fig. 1G). FGF2 mice, which sufferfrom abnormalities in the cytoarchitecture of the neocortex,skin wound healing defects, and autonomic dysfunction, didnot show any changes in the musculature, a finding similar tothat observed with FGF6 mutant mice (Fig. 1C).

To further substantiate dystrophic changes of skeletal mus-cle, 9-month-old FGF2/FGF6/mdx triple-mutant, mdx, andwild-type mice were intravenously injected with EBD (45).EBD, a low-molecular-weight diazo dye, is taken up by musclefibers with a focal breakdown of the plasmalemma, an initialevent in muscle cell necrosis (52). Individual muscle fibers thathave taken up the dye show a bright red emission upon fluo-rescent microscope analysis (Fig. 2 G to I and M to O). EBD-injected wild-type mice did not show an uptake of the dye in

skeletal muscle fibers (Fig. 2A, D, G, J, and M), whereas inmdx mice the dye was incorporated to varying degrees intodifferent skeletal muscles, preferentially those in the hindlimbs. In agreement with previous findings (45), affected mus-cles in mdx mice were not stained homogeneously but insteaddisplayed blue streaks that represented damaged muscle fibers(Fig. 2B, E, H, K, and N). Significantly, FGF2/FGF6/mdx miceshowed a much more extensive uptake of EBD into skeletalmuscle 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 accumu-lation was observed between different animals and differentmuscles, probably reflecting various environmental influences.

Changes in the expression of regulatory factors in FGFmutant mice are partially compensated for during regenera-tion. We next wanted to characterize the expression of regu-latory factors and markers of cell proliferation and differenti-

FIG. 1. Enhanced dystrophic changes in the musculature of FGF2�/�/FGF6�/�/mdx mice. (A and B) Macroscopic views of skinned 12-week-old mdx (A) and FGF2�/�/FGF6�/�/mdx (B) mice showing pale patches of tissue within the muscle fibers (arrows). (C to H) Hematoxylin-eosin-stained paraffin sections of the M. latissimus dorsi of different mutant mouse strains. FGF2�/� (C) and FGF2�/�/FGF6�/� (D) mutants do notshow dystrophic muscle fibers, whereas mdx (E), FGF6�/�/mdx (F), and FGF2�/�/mdx (G) mutants display dystrophic changes of the muscle fibersat a comparable severity. The most severe phenotype is evident in FGF2�/�/FGF6�/�/mdx mutants (H).

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ation in mutant muscle tissue. Northern blot analysis revealedthat expression of the muscle regulatory factors MyoD andmyogenin, which are markers for activated satellite cells, wasbelow the detection limit in the diaphragms of wild-type mice

and FGF2 and FGF6 mutant mice but strongly upregulated inmdx mice (Fig. 3). In the diaphragms of FGF2/FGF6/mdx andFGF6/mdx mutant mice, however, the expression was lowerthan in mdx mice, indicating a reduced presence of activated

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 dyeuptake (C). (D to O) Cryosections of M. gluteus (D to I) and diaphragma (J to O) of injected mice. Pictures were obtained by using differentialinterference contrast (DIC) optics (D to F and J to L) or fluorescence illumination (G to I and M to O). The enhanced dystrophic phenotype ofFGF2�/�/FGF6�/�/mdx mice is apparent under DIC illumination in the M. gluteus muscle (F) and in the diaphragm (L). The increased uptakeof EBD in dystrophic muscle fibers of triple mutant mice leads to strong fluorescence in virtually all muscle fibers. In mdx mice only a minorityof myofibers show a strong EBD fluorescence (H and N). No EBD uptake was observed in wild-type control animals (G and M).



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satellite cells. MRF4, which is the predominant myogenic fac-tor in adult muscle tissues, showed only slight changes in itsexpression level. During the course of muscle regeneration,embryonic MyHC isoforms are reexpressed, indicating thepresence of newly formed myotubes. In the diaphragms of mdxmice, high levels of the embryonic and neonatal MyHC iso-forms were found; these levels decreased in FGF6/mdx andFGF6/FGF2/mdx mutant mice, indicating a reduced formationof new myotubes. In contrast, expression of the cell cycle reg-ulatory gene c-myc was not greatly diminished in FGF6/mdxand FGF6/FGF2/mdx mutant mice compared to mdx mice(Fig. 3).

In addition to stimulatory growth factors, negative regula-tors of muscle growth are believed to control muscle develop-ment and regeneration. We therefore investigated the expres-sion of myostatin, which is a member of the transforminggrowth factor � superfamily and a key negative regulator ofskeletal muscle growth in muscles of mutant mice by quanti-

tative PCR. We found that the expression of myostatin wasupregulated in muscles of FGF2 and FGF6 mutant mice butstrongly downregulated in mdx mice, a finding most likelyindicating the need for increased myoblast proliferation toreplace damaged muscle fibers (data not shown).

The lack of FGF2 and FGF6 does not cause a significantreduction of the number of skeletal muscle satellite cells. Inorder to analyze whether the enhanced dystrophic changes inthe skeletal musculature of FGF2/FGF6/mdx triple mutantswere due to a reduction of the amount of satellite cells, wedetermined the number of CD34� satellite cells in wild-type,mdx mutant, and FGF2/FGF6/mdx triple-mutant mice. Previ-ous experiments had revealed that the number of CD34� cellslocated on myotubes strictly correlated with the number ofsatellite cells identified by transmission electron microscopy (S.Oustanina et al., unpublished results) and is a faithful molec-ular marker for satellite cells (1). As shown in Fig. 4, myotubesof FGF2/FGF6/mdx triple-mutant mice contained virtually thesame number of satellite cells in relation to myotube nuclei(3.05%) as did mdx mutant mice (3.1%). Although the numberof satellite cells was slightly reduced compared to wild-typecontrol animals (3.56%), this reduction is minor and statisti-cally not significant. In addition, no significant differences inthe growth and differentiation kinetics of myogenic cells wereobserved when satellite cells derived from isolated myotubeswere placed in culture and compared to cells derived fromwild-type and mdx mutant control animals (data not shown).From these experiments we concluded that a reduction of thenumber and differentiation ability of FGF2/FGF6/mdx satellitecells could not account for the enhanced dystrophic changes inthe skeletal musculature of triple-mutant mice.

Reduced migration of FGF6 mutant myoblasts in vivo.FGFs are known to regulate growth and differentiation ofprimary, secondary, and adult myoblasts. In addition, they havebeen shown to stimulate migration of myogenic cells in miceand other organisms. The established role of FGFs as regula-tors of cell migration (reviewed in reference 38) and the abilityof muscle satellite cells to migrate extensively (24, 50) led us tostudy the migration of FGF6 mutant muscle precursor cells invivo in mice. To generate a suitable genetic label that wouldallow us to identify transplanted muscle cells, we crossed FGF6mutants with transgenic MyLC-1/3-LacZ mice (26). MyLC-1/3-LacZ mice show a specific expression of the reporter gene instriated muscles and can be used to track transplanted cells inrecipient mice (14). Both strains were bred on a C57BL/6background to allow isogenic transplantation without necessityfor immunosuppressive drugs.

Next, we isolated satellite cells from MyLC-1/3-LacZ andMyLC-1/3-LacZ/FGF6�/� mice (1 � 105 to 2 � 105 cells) andtransplanted them back into the M. tibialis anterior of eitherC57BL/6 wild-type or FGF6�/� mice. Shortly before trans-plantation, the same muscle was artificially damaged by freeze-crush injury to promote regeneration and myoblast recruit-ment. At 112 days after transplantation, muscles wereremoved, sectioned, and subjected to LacZ staining (wild-typeto wild-type transplantations [n 3]; wild-type to FGF6�/�

transplantations [n 3]; FGF6�/� to wild-type transplanta-tions [n 4]). As shown in Fig. 5, no significant differences inthe amount of LacZ-positive cells were noted close to theimplantation sites when wild-type cells were transferred to

FIG. 3. Northern blot analysis of FGF/mdx mutant mice. Genesthat are characteristic for muscle cell regeneration (MyoD, myogenin,emb MyHC, and neonatal MyHC) are significantly upregulated in mdxmice, 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 elevatedexpression of c-myc, reflecting an increase of cellular proliferation.GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as aloading control.



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FGF6�/� (Fig. 5B), FGF6�/� cells were transferred to wild-type hosts (Fig. 5E), or wild-type cells were transferred towild-type hosts (Fig. 5H). However, when sections were com-pared that were taken ca. 3 mm apart from the injection siteeither toward the previous injury (“proximal to injury”) or inthe opposite direction (“distal to injury”), considerable differ-ences were noted between the different strains. Significantlyfewer LacZ-positive cells were present on matched sectionswhen FGF6�/� cells were transplanted into C57BL/6 hosts(Fig. 5D and F) compared to wild-type cells transplanted intowild-type (Fig. G and I) or FGF6�/� hosts (Fig. 5A and C). Nomajor differences were found between transplantations of wild-type cells into FGF6�/� and wild-type hosts (Fig. 5, comparepanels A and C to panels G and I). It is interesting that wefound only few differences in the migration pattern of trans-planted cells proximal or distal to the lesions. This might beexplained by a similar activation of the distal aspects of dam-aged fibers compared to the damaged site itself.

Inhibition of Ras-signaling interferes with skeletal myoblastmigration in vivo. FGF receptor-mediated signaling is achievedvia different intracellular signaling pathways, including the Raspathway, Src-family tyrosine kinases, phosphoinositide 3-ki-nase (PI3K), and the PLC pathway (8). Interestingly, the Raspathway has recently be linked to the migratory ability of

C2C12 myoblasts in vitro in response to FGF2, HGF, andIGF-1 (46), indicating that the Ras-Ral pathway is essential forthe migration of muscle progenitor cells. To evaluate whetheractivation of the Ras-Ral pathway is crucial for migration ofskeletal myoblasts in vivo, we generated murine stem cell virus-based retroviruses expressing dominant-negative versions ofRas (H-RasS17N) and Ral (RALS28N) and infected MyLC-1/3-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 anteriormuscle of C57BL/6 mice. As shown in Fig. 6, no significantdifferences in the number of LacZ-positive cells were notedclose to the implantation sites between hosts that have receivedmyoblasts infected with a control virus expressing green fluo-rescent protein (GFP) and hosts that have received MyLC-1/3-LacZ myoblasts infected either with a dominant-negativeversion of Ras (H-RasS17N) or Ral (RALS28N).

On the other hand, when sections were compared that weretaken from regions ca. 3 mm apart from the implantation siteeither toward the previously applied injury (Fig. 6C, F, and I)or at the opposite side (Fig. 6A, D, and G), a different pictureemerged. Significantly fewer myoblasts were found that havebeen infected either with the dominant-negative Ras (H-

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) Myotubeswere isolated from the M. flexor digitorum brevis muscles of different mouse strains and stained with an antibody to CD34. Positive cells werecounted 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.



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RasS17N) or Ral (RALS28N) retrovirus compared to a con-trol virus expressing GFP. As in the previous set of transplan-tation experiments, more cells were found in the vicinity of thepreceding injury, indicating that not all signals that led to aguided migration of myoblasts to a site of muscle regenerationwere inhibited by the dominant-negative Ras and Ral mole-cules.

To further support the idea that the reduced presence oftransplanted FGF6�/�, Ras (H-RasS17N), and Ral (RALS28N)infected cells at a distance from the injection site is due to areduced migration capability and not to a reduced prolifera-tion and/or differentiation of muscle precursor cell, we ana-lyzed the absolute number of MyLC-1/3-LacZ-expressing cellsafter transplantation into different strains. Since it is virtuallyimpossible to count all cells, particularly in the immediatevicinity of the transplantation site, we used quantitative RT-PCR with LacZ mRNA from the whole muscle. As shown inFig. 7, similar values for LacZ expression were found in com-plete muscles of wild-type mice that received MyLC-1/3-LacZand MyLC-1/3-LacZ/FGF6�/� cells and in muscles ofFGF6�/� mice that received MyLC-1/3-LacZ cells (Fig. 7A).Similar results were obtained when muscles were investigatedthat had received myoblasts infected with a mock retrovirus orwith viruses expressing Ras (H-RasS17N) and Ral (RALS28N)(Fig. 7D). Likewise, no significant differences in the expressionlevel of LacZ mRNA were found when tissue samples were

derived from the area close to the transplantation site (Fig. 7Cand F). In contrast, when the area around the transplantationsite was intentionally removed and the rest of the muscle wasanalyzed by RT-PCR, a significant reduction of LacZ mRNAwas 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 myoblastsinfected with Ras (H-RasS17N) and Ral (RALS28N) retrovi-ruses compared to cells infected with a control virus (Fig. 7E).

Since LacZ mRNA is present only in differentiated myo-tubes derived from transplanted myoblasts, the level of LacZmRNA is an indicator of both proliferation and differentiation.Based on the virtually identical LacZ expression levels in totalmuscles, we concluded that proliferation and/or differentiationof muscle precursor cells was not greatly affected by the lack ofFGF6 or interference of Ras/Ral signaling. Because we nor-malized the PCRs within each triple group, our analysis onlyallows a comparison of wild-type and mutant samples withinone group. Small differences in the number of transplantedcells close to the injection site, which might have been expecteddue to a better (or worse) migration of cells away from theinjection site would have escaped our attention due to the factthat the bulk of the cells remains located close to the injectionsite.

Chemotactic effects of FGFs and reduced mobility of FGF2/FGF6 mutant myoblasts in vitro. Since our in vivo studies

FIG. 7. FGF mutant and Ras/Ral dominant-negative (Dn) infected myoblasts have no overt proliferation defect. Quantitative real-time PCRand semiquantitative control reactions of transplanted LacZ mRNA-positive cells. (A and D) Quantitative RT-PCR analyses demonstrate nosignificant differences in LacZ expression after transplantation of wild-type, FGF mutant, and Ras/Ral Dn infected myoblasts into complete hostmuscles. (B and E) A decrease in LacZ expressing muscle cells was only evident when the tissue around the injected site was removed. PCRs werenormalized within each triple group; hence, the expression between different groups cannot be compared. The numbers are shown in relation tothe number of LacZ expression in wild-type hosts (FGF6�/� cells) or in relation to the number of mock-transfected cells (myoblasts expressingthe dominant-negative Ras and Ral isoforms).



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suggested that FGF2 and FGF6 are important regulators ofmyoblast migration, we next investigated the migratory abilityof satellite cell-derived myoblasts in a modified Boyden Cham-ber assay. To exclude nonmyogenic cells that might contami-nate our satellite cell preparations, we identified migrant myo-blasts by immunohistochemical staining with an antibodyagainst the intermediate filament desmin at the end of a 6-hincubation period.

We observed an increase in adult myoblast migration afteraddition of FGF2, FGF6, HGF, and IGF-1 at concentrationsof 1 to 10 ng/ml of medium. Interestingly, a rather high basallevel of cell migration was observed when cells were derivedfrom wild-type mice, although exogenously added FGF2,FGF6, HGF, and IGF-1 strongly stimulated migration. In con-trast, the basal level of migration across the filter dropped toabout a third when myoblasts were isolated from FGF2/FGF6double-mutant animals (Fig. 8). Satellite cells from FGF6 mu-tant mice also showed a reduced migratory ability, but it wasless pronounced than that of FGF2/FGF6 double-mutant cells(data not shown). The addition of FGF2 and FGF6 stimulatedthe migration of myoblasts, although the degree of migrationwas lower than that of the matched wild-type controls. Whenthe concentrations of FGF2 and FGF6 were raised to 50 ng/ml,the extent of migration increased significantly, although thelevel observed with wild-type myoblasts was never reached(Fig. 8). In contrast, HGF and IGF-1, which were the mostpotent inducers of satellite cell migration in our assay, stimu-lated migration of satellite cells isolated from FGF2/FGF6mutants to the same extent as wild-type cells, thus excludingthe possibility that satellite cells from FGF2/FGF6 mice weredefective in a more general manner (Fig. 8).

DISCUSSION

Redundancies and specificities of FGFs for muscle develop-ment and repair. FGFs play important roles in a wide varietyof 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 expressionduring embryonic development, yielded only minor pheno-types compared to dramatic effects observed after mutation ofthe FGF4 (arrest of morula development [13]), FGF8 (defectsin gastrulation, cardiac, craniofacial, forebrain, midbrain, andcerebellar development [34]), and FGF10 (arrest of lung andlimb bud development [44]) genes. A common explanation forsuch findings is redundancy of functions, which might lead tocompensation of severe defects. We therefore expected thatthe combined inactivation of three different members of theFGF family (i.e., FGF5, FGF6, and FGF7), which are all ex-pressed during somite formation and muscle development,would generate strong developmental defects. Surprisingly,this was not the case, suggesting that either FGF5, FGF6, orFGF7, although coexpressed in somites and skeletal muscle donot have a significant overlap of function or that additionalmembers of the family, such as FGF2, compensate for thecombined loss of three growth factor genes.

Variations in the expression of compensating FGF genes indifferent genetic backgrounds (10) might also have contributedto the failure to detect a major role of FGF6 for muscleregeneration as shown by Fiore et al. (15). Another possibleexplanation for differences in the phenotype is the presence orabsence of additional modifier genes or the different design ofthe targeted mutation, which might lead to considerable dif-ferences in the observed phenotypes (49). In addition, even ina uniform genetic background, stochastic variations in the ex-pression of a regulatory circuit can result in dramatic differ-ences in phenotypic consequences (29).

Among the FGF family members FGF2 is particularly inter-esting with respect to muscle regeneration (28). FGF2 is apotent stimulator of the proliferation and fusion of myoblastsin vitro and enhances muscle regeneration in vivo (33). Like-wise, transgene delivery of FGF2 and FGF6 genes led to astrong enhancement of skeletal muscle repair (12), furtheremphasizing the importance of FGF2 and FGF6 for musclerepair.

FIG. 8. Reduced migration of FGF2�/�/FGF6�/�/mdx myoblasts in vitro. FGF2 and FGF6 stimulate the migration of satellite cell-derivedwild-type myoblasts in a modified Boyden Chamber assay. Migration of wild-type myoblasts were stimulated at a concentration of 1 ng of FGF/mlof medium and increased further at 10 ng of FGF/ml of medium. Reduced migration of cells derived from FGF2�/�/FGF6�/� mice can partiallybe overcome when the FGF concentrations in the medium were increased to 50 ng/ml.



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FGFs contribute to various cellular events, including pro-liferation and migration. FGFs were mainly connected withthe regulation of proliferation and the inhibition of differen-tiation. Several lines of evidence suggest that their role isstrongly dependent on the cellular environment (38). In high-density cultures FGFs stimulate differentiation, which is prob-ably due to an intrinsic downregulation of FGF receptors (37),whereas in low-density cultures FGFs strongly repress terminaldifferentiation (6). In vivo, the various activities of FGFs aresubject to additional regulatory input, including the differentialexpression of various heparan sulfate glycosaminoglycans thatare required for signaling from their respective receptors (7).

Regulation of cell migration by FGFs has been documentedin several organisms and for different cell types (reviewed byMontell [35]). In the mouse, FGF2 and FGF4 have been shownto stimulate the migration of embryonic limb myogenic cellsand of C2C12 myoblasts in vitro (46, 51).

We have demonstrated that the mobility of FGF6-deficientmyoblasts is significantly reduced in vivo. The finding thatmyoblasts with a repressed Ras/Ral pathway also showed astrong reduction of migration supports our conclusion thatFGF signaling is crucial for myoblast migration and that thelack of FGFs leads to a reduced accumulation of activatedmyoblasts in regions with increased demand for muscle stemcells for tissue repair. In vitro, the reduced ability of FGF2 andFGF6 mutant myoblasts to migrate can be rescued to someextent by the addition of supraphysiological concentrations ofeither FGF2 or FGF6, indicating that both growth factorsmight partially compensate for each other. This partial com-pensation might also explain why the phenotype of the FGF2/FGF6 double-mutant mice is stronger than the FGF6 mutantalone and why no muscle phenotype was observed in FGF2mutant mice.

In principle, some of our data might also be explained by adecrease in myoblast proliferation and/or differentiation. Ourassay system relies on the detection of MyLC-LacZ, which isswitched on in differentiated muscle cells. If either prolifera-tion of myoblasts drops or the differentiation of muscle pre-cursor cells is altered due to the lack of FGFs or the expressionof dominant-negative Ras and Ral mutants, we might alsocount different numbers of MyLC-LacZ-positive cells. To ruleout this possibility, we have performed real-time quantitativeRT-PCR for MyLC-LacZ mRNA to compare the absolutenumbers of MyLC-LacZ-positive cells between engineeredmyoblasts and wild-type controls. Since we did not find signif-icant differences between these samples, we can rule out thatlack of FGF6 or inhibition of Ras/Ral signaling results in amajor alteration of the proliferation and differentiation ofmyoblasts.

The Ras/Ral pathway appears to transmit migratory signalselicited by FGFs in vivo. Ras is involved in the regulation ofnumerous cellular events, namely, cell proliferation, inhibitionof differentiation, and migration (see reference 9 for a recentreview). Ras can be activated by several growth factors thathave been shown to control skeletal muscle cell development,differentiation, and migration, including IGFs, HGFs, andFGFs (46). IGF-1 is known to stimulate myoblast migration invitro (46) and retains (in transgenic mice) the proliferativeresponse to muscle injury characteristic of younger animals(36). HGF has been demonstrated to be important for limb

muscle precursor cell migration in vivo (4, 23) and for C2C12myoblast (46) and muscle satellite cell (2) migration in vitro.Thus, it seems likely that, in addition to FGFs, other growthfactors or cellular signals such as Ca2� increase (9) lead to anactivation of Ras and subsequently of Ras downstream targetssuch as Ral, Raf, and PI3K (9). Our finding that inhibition ofRas signaling by a dominant-negative Ras mutant results in astronger inhibition of migration compared to the inactivationof FGF6 supports the view that several different growth factorsfunnel into the activation of Ras and stimulate migration. Ex-pression of a dominant-negative Ral mutant inhibited migra-tion in vivo to virtually the same extent as inhibition of Ras. Itseems therefore reasonable to assume that Ral mediates acti-vation of the cellular migration machinery in response to Rassignaling. At the moment it is hard to decide whether Ral issolely responsible for activation of migration. Although we didnot observe an inhibition of migration when myoblasts weretreated with LY294002, a specific inhibitor of PI3K, andU0126, an inhibitor of MEK (data not show), before trans-plantation, it is difficult to deduce from these negative resultsthat PI3K is not involved in migration of myoblasts in vivosince myoblasts may recover from this treatment and reactivatethe pathway once the drug is removed and the cells are trans-planted into host muscle.

Migration is an important element of stem cell-mediatedtissue repair. The importance of migration for tissue repair islong known. Most injuries require the infiltration of damagedtissue with various types of cells, which actively migrate intothe target area. The concept that muscle precursor cells, whichcontribute to regenerating muscle in a region of muscle dam-age, are not all locally derived but are also recruited fromexogenous sources, including adjacent muscles, has been putforward by several labs (24, 50). However, the mechanisms bywhich muscle precursor cells achieve this task have remainedenigmatic so far. The necessity to learn more about this prob-lem has become even more eminent with the unexpected find-ing that bone marrow-derived cells migrate into areas of in-duced muscle degeneration and participate in the regenerationof the damaged fibers (14) and that Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulationinto host muscle tissues (47). Our demonstration that FGFsmediated signaling, most likely via the Ras/Ral pathway, willfacilitate the understanding of this process and might help todesign strategies for improved availability of stem cell-derivedorganotypic cells in disease-related muscle frailty.

ACKNOWLEDGMENTS

The excellent technical assistance of S. Kruger, Y. Pooch, and U.Ziese is gratefully acknowledged. We are indebted to E. Fuchs (Uni-versity of Chicago) for supplying FGF7 mutant mice, to H. Thoenen(MPI f. Psychiatrie, Martinsried, Germany) for the donation of FGF5mutant mice, and to M. Buckingham and R. Kelly (Institut Pasteur,Paris, France) for MyLC1/3-lacZ mice. We also thank H. Koide (To-kyo Institute of Technology, Tokyo, Japan) for supplying Ras and Ralmutants.

This work was supported by the Deutsche Forschungsgemeinschaft,the “Fonds der Chemischen Industrie,” and the Wilhelm-Roux Pro-gram for Research of the Martin Luther University.

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