ARTICLE |
Correspondence to: Zipora YablonkaReuveni, Dept. of Biological Structure, Box 357420, University of Washington, Seattle, WA 98195. E-mail: reuveni@u.washington.edu
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Summary |
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Satellite cells are the myogenic precursors in postnatal muscle and are situated beneath the myofiber basement membrane. We previously showed that fibroblast growth factor 2 (FGF2, basic FGF) stimulates a greater number of satellite cells to enter the cell cycle but does not modify the overall schedule of a short proliferative phase and a rapid transition to the differentiated state as the satellite cells undergo myogenesis in isolated myofibers. In this study we investigated whether other members of the FGF family can maintain the proliferative state of the satellite cells in rat myofiber cultures. We show that FGF1, FGF4, and FGF6 (as well as hepatocyte growth factor, HGF) enhance satellite cell proliferation to a similar degree as that seen with FGF2, whereas FGF5 and FGF7 are ineffective. None of the growth factors prolongs the proliferative phase or delays the transition of the satellite cells to the differentiating, myogenin+ state. However, FGF6 retards the rapid exit of the cells from the myogenin+ state that routinely occurs in myofiber cultures. To determine which of the above growth factors might be involved in regulating satellite cells in vivo, we examined their mRNA expression patterns in cultured rat myofibers using RT-PCR. The expression of all growth factors, excluding FGF4, was confirmed. Only FGF6 was expressed at a higher level in the isolated myofibers and not in the connective tissue cells surrounding the myofibers or in satellite cells dissociated away from the muscle. By Western blot analysis, we also demonstrated the presence of FGF6 protein in the skeletal musle tissue. Our studies therefore suggest that the myofibers serve as the main source for the muscle FGF6 in vivo. We also used RT-PCR to analyze the expression patterns of the four tyrosine kinase FGF receptors (FGFR1FGFR4) and of the HGF receptor (c-met) in the myofiber cultures. Depending on the time in culture, expression of all receptors was detected, with FGFR2 and FGFR3 expressed only at a low level. Only FGFR4 was expressed at a higher level in the myofibers but not the connective tissue cell cultures. FGFR4 was also expressed at a higher level in satellite cells compared to the nonmyogenic cells when the two cell populations were released from the muscle tissue and fractionated by Percoll density centrifugation. The unique localization patterns of FGF6 and FGFR4 may reflect specific roles for these members of the FGF signaling complex during myogenesis in adult skeletal muscle. (J Histochem Cytochem 48:10791096, 2000)
Key Words: skeletal muscle, satellite cells, myogenic regulatory factors, MEF2, fibroblast growth factor, FGF receptor, FGF6, FGFR4, HGF, c-met
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Introduction |
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Satellite cells are the myogenic precursors in postnatal and adult muscle. At least some of the satellite cells are mitotically active in the growing muscle, contributing myonuclei to the enlarging fibers. As muscle matures, the addition of myofiber nuclei ceases and the satellite cells become mitotically quiescent. Recruitment of these quiescent precursors occurs in response to overt muscle injury and to more subtle stresses such as stretch, exercise, and muscle hypertrophy. Depending on the degree of injury, the satellite cell progeny may fuse with existing myofibers for localized repair or may fuse with each other to form new fibers when the damage is more extensive (reviewed in
Satellite cells undergoing proliferation and differentiation express the skeletal muscle-specific transcription factors Myf5, MyoD, myogenin, and MRF4. These four myogenic regulatory factors (MRFs) are first expressed during early embryogenesis, when they are involved in the specification of the myogenic lineage (
Cell culture studies have revealed many growth factors and cytokines which can affect proliferation and differentiation of satellite cells (reviewed in
To gain insight into the regulation of satellite cells, we have been studying the dynamics of these cells in cultures of isolated myofibers. In such cultures the satellite cells remain situated in their native position under the basement membrane of the myofibers (
Analyzing adult rat myofibers, we previously showed that satellite cells first become positive for PCNA and MyoD on their activation and entry to the cell cycle. After a short proliferative period, the cells become negative for PCNA and MyoD and enter the differentiated, myogenin+ state (
The short-term effect of FGF2 seen in myofiber cultures was unexpected in view of the commonly held convention that FGF can maintain ongoing proliferation of myoblasts (
A second aspect of the study was to analyze the expression patterns of the FGF receptors (FGFRs) during satellite cell myogenesis. The FGFs transduce their signals to the cell through transmembrane tyrosine kinase receptors (FGFRs), for which four distinct genes have been discovered (FGFR1FGFR4). Further receptor diversity through alternative splicing of pre-mRNAs has been documented for FGFR1FGFR3. Each FGFR variant has a distinct, although sometimes overlapping, profile of affinities to different FGFs (
Along with the analysis of the FGFFGFRs we also conducted studies on the effect and expression of hepatocyte growth factor (HGF) and its transmembrane receptor (c-met) during myogenesis of satellite cells. The c-met receptor was shown to be expressed by proliferating satellite cells, and it has been suggested that HGF has a unique role during recruitment of quiescent satellite cells, shortening the time the cells spend before entering the cell cycle (
Collectively, the present studies on the response of the satellite cells to the different growth factors, along with the gene expression analysis, were expected to enable the determination of which of these growth-promoting agents might play an actual physiological role in the muscle.
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Materials and Methods |
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Animals
Male SpragueDawley rats (B & K Universal; Kent, WA) were used throughout the study. Unless otherwise noted, the rats were 810 weeks old (young adults).
Isolation and Culture of Muscle Fibers
The procedures involved in the isolation and culture of the myofibers have been previously published (
Isolation and Culture of Muscle Connective Tissue Cells
The connective tissue surrounding the myofibers is routinely separated from the myofibers and discarded after the collagenase digestion of the FDB muscle (
Isolation and Culture of Satellite Cells
To focus specifically on satellite cells apart from the whole myofiber, we isolated satellite cells from the combined extensor digitorum longus (EDL) and tibialis anterior (TA) muscles of the rat hindlimbs by Pronase digestion (
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Immunolabeling and Counting of Satellite Cells in Myofiber Cultures
Satellite cells in methanol-fixed myofiber cultures were monitored by immunofluorescence as previously described (
Duplicate culture plates were used at each time point for cell quantification. Positive cells from 30 random fibers per plate were counted, averaged for the duplicate plates, and eventually expressed per 10 fibers. Error bars represent the range of the results within the duplicate plates. Analyses were typically repeated three to five times.
Monitoring Myogenic Cells in Primary Cultures of Muscle-dissociated Cells
Methanol-fixed cultures of muscle-dissociated cells were analyzed by single and double immunofluorescence combined with DAPI staining of nuclei, using published procedures (
Apoptosis Analysis
The possible involvement of programmed cell death (apoptosis) in the myofiber cultures was investigated using the TdT-mediated dUTP nick end-labeling (TUNEL) method, which visualizes apoptotic nuclei (
Analysis of Gene Expression by RT-PCR
Total RNA was isolated using the acid guanidiniumthiocyanatephenolchloroform method (
Fig 2 summarizes the primers used for the study, along with the corresponding RT-PCR products of several rat tissues. Primers were designed to the rat gene sequence with the exception of the primers for Myf5, FGF6, and FGFR3, which were based on the mouse sequence because the rat sequence was not available (
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The analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S6 (rpS6), or H-ras was used as a measure for ubiquitous gene expression in the various experiments. The primers for MyoD, FGFR4, and H-ras span an intron smaller than 1000 bp and the PCR products of these genes were used as control for the absence of genomic DNA. As a further control, we carried out the RT-PCR with H-ras primers at a high cycle (38) omitting the reverse transcriptase.
The PCR products of total RNA isolated from muscle (FDB and EDL/TA) and nonmuscle tissues (brain and liver) that are shown in Fig 2 serve as indicators for the relative level of gene expression in the myofibers and the satellite cell cultures detailed in Results. For example, compared to brain and liver, isolated myofibers express a very low level of FGFR2 and FGFR3 but a significantly higher level of FGF6.
Immunoblotting Analysis of FGF6 Protein
Various tissues collected from 8-week-old rats were frozen in liquid nitrogen, pulverized, and homogenized in 5 volumes of ice-cold buffer. The skeletal muscle tissue was homogenized in a buffer consisting of 100 mM KCl, 20 mM Tris-HCl (pH 7.5), 1 mM DTT, and the protease inhibitors leupeptin (Boehringer Mannheim), soybean trypsin inhibitor (Worthington Biochemical; Freehold, NJ), and aprotinin (Sigma), each at 10 µg/ml, and Pefablock (Boehringer Mannheim) at 240 µg/ml. The homogenate was centrifuged at 16,000 x g for 20 min and the resulting pellet and supernatant fractions were both reacted with Laemmli SDS sample buffer (1 x final concentration;
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Results |
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The Effects of FGFs and HGF on Satellite Cells in Isolated Myofibers
The effects of different FGFs and of HGF on enhancing the number of satellite cells in myofiber cultures was monitored by quantifying the numbers of PCNA+ and myogenin+ nuclei. The results of the investigation are shown in Fig 3. Immunostaining micrographs demonstrating that the PCNA+ and myogenin+ nuclei are within fiber-associated cells whose cytoplasm can be traced with an antibody against the mitogen-activated protein kinases ERK1/ERK2 have been published (
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The effect of FGF1 on the number of PCNA+ cells was examined at concentrations ranging from 0.1 to 5 ng/ml. The maximal increase in PCNA+ cells was observed at 2 ng/ml FGF1 with heparin (Fig 3A). No further increase in PCNA+ cells was seen at higher FGF1 concentrations. Heparin added to the basal medium alone (without FGF1) suppressed the number of PCNA+ cells (Fig 3B). Heparin suppressed cell proliferation even when FGF1 (or the other effective FGFs described below) was added at a low level of 0.1 ng/ml. Such a low level of FGF was sufficient to promote some proliferation in the absence of heparin. These findings indicate that the ratio between the concentration of heparin and the FGFs is critical for achieving maximal proliferation of satellite cells in myofiber cultures.
The effect of FGF2 (2 ng/ml) on the number of PCNA+ satellite cells was minimally enhanced, if at all, by heparin (Fig 3C). We previously found that this concentration of FGF2 produces a maximal effect on the number of PCNA+ satellite cells in myofiber cultures. A higher concentration of FGF2 (up to 10 ng/ml) did not lead to a further increase.
FGF4 (2 ng/ml) also enhanced the number of PCNA+ satellite cells in myofiber cultures. This is shown in Fig 3D, together with the results of parallel plates receiving FGF1 (with heparin) or FGF2. A higher FGF4 concentration (up to 10 ng/ml) did not support a further increase in the number of PCNA+ cells. The effect of FGF4 was not influenced by the addition of heparin (data not shown). Fig 3D also shows that the schedule of transition from the PCNA+ to the myogenin+ state follows a similar pattern in the presence of FGF1, FGF2, or FGF4. The enhancement of PCNA+ cells by these three FGFs is reflected by a similar increase in the number of myogenin+ cells.
The effect of FGF6 on the number of PCNA+ cells was tested at a concentration range between 0.1 and 10 ng/ml. The results obtained with FGF6 at 5 ng/ml (with or without heparin) are shown in Fig 3E. This concentration of FGF6 supported the maximal number of PCNA+ cells. The effect of FGF6 was only minimally (but consistently) enhanced by heparin on culture Days 2 and 3. The results obtained with parallel cultures receiving FGF2 at 2 ng/ml are also included in Fig 3E. FGF6 and FGF2 supported a similar increase in the number of PCNA+ cells.
The effect of HGF (20 ng/ml) on the number of PCNA+ cells is shown in Fig 3F. HGF at 20 and 40 ng/ml led to similar results, whereas HGF at 5 ng/ml supported a slightly smaller number of PCNA+ cells (data not shown). Heparin did not enhance the number of PCNA+ cells beyond that supported by HGF alone (not shown). As seen in Fig 3F, HGF (at 20 ng/ml) and FGF2 (at 2 ng/ml) enhanced the number of PCNA+ cells to a similar degree. Fig 3G shows that these concentrations of HGF and FGF2 also led to a similar increase in the number of myogenin+ cells. The addition of HGF (20 ng/ml) and FGF2 (2 ng/ml) together did not yield a further increase in the number of satellite cells beyond that shown in Fig 3F for each growth factor alone (not shown). Collectively, HGF (like FGF1, FGF2, and FGF4) did not modify the schedule of transition from the PCNA+ to the myogenin+ state.
The effect of FGF6 on the number of myogenin+ cells was examined using 5 ng/ml FGF6 plus heparin (the condition that supported the maximal number of PCNA+ cells). The results are shown in Fig 3H, along with control cultures receiving no additives or supplemented with HGF (20 ng/ml). Typically, the number of myogenin+ cells sharply declines between culture Days 3 and 4 in myofiber cultures receiving basal medium only and in cultures receiving FGF1, FGF2, FGF4, or HGF (Fig 3C, Fig 3D, and Fig 3G; also detailed in our previous publications). As seen in Fig 3H, FGF6 exerted a unique effect on the number of myogenin+ cells, slowing the rapid decline observed in the controls. The addition of FGF6 and HGF together yielded the same kinetics of myogenin+ cells as shown in Fig 3H for FGF6 alone (data not shown).
The effects of FGF5 and FGF7 on the number of PCNA+ cells in myofiber cultures were also examined. Myofibers in basal medium with or without FGF2 were used as controls in these experiments. FGF5 was first tested at a concentration range of 275 ng/ml and then at 100200 ng/ml. FGF7 was tested at 1050 ng/ml. Neither FGF5 nor FGF7 (with or without heparin) was found to enhance the number of PCNA+ cells above that seen in basal medium (not shown).
We further investigated whether the decline in the myogenin+ cells seen by Day 4 in myofiber cultures could be due to apoptosis and whether FGF6 may slow this decline by preventing apoptosis. Cultures were collected every 24 hr for 6 days, starting at time 0, and were analyzed for the presence of apoptotic nuclei using the TUNEL reaction. We concluded that regardless of the absence/presence of FGF2 or FGF6, neither the nuclei of the intact myofibers nor the nuclei of the fiber-associated satellite cells underwent programmed cell death (data not shown).
Expression of FGFs and Their Receptors in Myofiber Cultures
We asked whether the muscle fibers (with their associated satellite cells) express specific FGF or FGFR genes in comparison to the connective tissue cells surrounding the myofibers. Myofibers and muscle connective tissue cells were cultured in parallel under the same conditions used for the studies on the effects of the growth factors. FGF2 (2 ng/ml) was added to the basal medium to support proliferation. Total RNA was isolated from the cultures at different time points and was analyzed by RT-PCR (Fig 4). The RT-PCR products of each gene are shown in paired horizontal lanes; the upper lane shows the analysis of the myofibers (mf) and the lower lane shows the analysis of the connective tissue cells (ctc). Gene expression in the myofibers reflects the overall contribution of both the myofibers and the satellite cells.
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As shown in Fig 4, MyoD, Myf5, myogenin, and MRF4 were detected only in the myofiber cultures. Their expression was upregulated by the first culture day. The highest expression of MyoD seen on Day 1 preceded the highest expression of myogenin seen on Day 3. This pattern agrees with the temporal expression of the MyoD and myogenin proteins by the fiber-associated satellite cells. However, we cannot exclude that at least some, if not most, of the MRF gene expression is contributed by the myofibers themselves. The muscle regulatory/structural genes muscle LIM protein (MLP) (
Depending on the time in culture, expression of FGF1, FGF2, FGF5, FGF7, and HGF was displayed by both the myofibers and connective tissue cells (Fig 4). FGF4 was not analyzed because we could not detect it in preliminary studies. FGF4 was also not detected in the mouse myogenic line MM14 (
Analysis of the FGF and HGF receptor genes showed a unique expression of FGFR4 by the myofibers only. All other receptor genes were expressed by both the cultured myofibers and the connective tissue cells (Fig 4). The expression of all receptor genes, excluding FGFR3, was upregulated in the myofibers by the first culture day. The expression level of FGFR2 and FGFR3 in the myofiber cultures was distinctly low compared to that of brain or liver (shown in Fig 2). The muscle tissue also expressed a very low level of FGFR2 and FGFR3 (Fig 2). The expression level of FGFR2 was far higher in the connective tissue cells than in the myofiber cultures (Fig 4), suggesting that the myofiber FGFR2 might be contributed by remaining connective tissue cells.
The expression of H-ras in myofibers was similar at all time points (Fig 4). In the connective tissue cultures, H-ras was expressed at a lower level at time 0, like the other control genes GAPDH and rpS6 (data not shown). This might be due to the reduced cell activity immediately after the cell isolation. This reduced activity in time 0 connective tissue cells has no impact on the conclusions of the study.
In the investigation shown in Fig 4, the skeletal muscle genes as well as the FGFR4 gene were expressed by the myofibers but not by the connective tissue cells. The connective tissue cells expressed a trace level of these genes when the PCR was carried out at 40 cycles (data not shown). The source of these trace signals is probably the remnant myofibers (and/or infrequent satellite cells) trapped with the connective tissue cells. A trace of FGF6 expression was also seen in the connective tissue cells at a high PCR cycle number. We also investigated whether the medium (basal medium vs the growth medium used for muscle-dissociated cells) or the matrix (Vitrogen vs gelatin) can affect the expression of FGF6 and FGFR4. The same relative higher expression levels in myofibers compared to connective tissue cells were detected for these two genes regardless of the medium or matrix used (data not shown). We also analyzed whether the expression levels of the growth factors and their receptors are changed in myofibers from 1-year-old rats. We concluded that the expression patterns of these genes in the older rats were similar to those shown in Fig 4 for myofibers from 8-week-old rats (data not shown).
Detection of FGF6 Protein in Muscle Tissue
The effect of FGF6 during myogenesis of satellite cells, along with its unique pattern of RNA expression in the myofiber system, led us to further investigate the presence of FGF6 protein in the muscle tissue. Although mRNA transcripts for FGF6 have been detected in regenerating skeletal muscle (
Fig 5 depicts a Western blot analysis of several rat tissues along with recombinant human FGF6 (rhFGF6). Although Lane 1 shows the results with 1 ng rhFGF6, we concluded that the assay allows the detection of as little as 0.05 ng rhFGF6. Fractionation of the FDB muscle extract to soluble and particulate material, which was performed because of the toughness of the tissue, was found to provide a means for FGF6 enrichment. FGF6 was detected in the particulate fraction (Fig 5, Lane 2) but not in the soluble material (data not shown). Testis extract (Fig 5, Lane 4) was included as a positive control for rodent FGF6 on the basis of information provided by Santa Cruz Biotechnologies about FGF6 expression in mouse. The arrows at the sides of the figure point to the migration position of rhFGF6 (left arrow) and rat FGF6-like protein (right arrow). The predicted molecular weight of the rhFGF6 is 19 kD. However, according to the supplier, the rhFGF6 is prone to both N- and C-terminal truncation and contains several proteins ranging from 14 to 16 kD. The molecular weight of the rat FGF6-like protein was found to be about 20 kD. This is in agreement with the 18-kD molecular weight reported for mitogenically active, transgene FGF6 expressed in Chinese hamster ovary cells (
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Distribution of Myogenic Cells in Cultures of Percoll-isolated Cells
To focus specifically on FGFFGFR gene expression in the satellite cells themselves apart from the myofibers, we isolated satellite cells from the rat EDL/TA muscles. The cells were further subjected to Percoll density centrifugation to enrich for nonmyogenic and myogenic cell populations typically present together in preparations of muscle-dissociated cells (
The myogenic potential of the EDL/TA cells was first examined using preparations that were not fractionated by Percoll. Fig 6 depicts immunofluorescence micrographs of Day 5 (Fig 6A) and Day 10 (Fig 6B) cultures of nonfractionated cells reacted with the antibody against myogenin. The frequency of the myogenin+ cells was less than 5% by culture Day 5 but increased to 6080% by culture Day 10. Multinucleated myotubes containing myogenin+ nuclei were also seen at this late stage. MyoD+ cells were present in these cultures by culture Day 1, reaching a frequency of about 5060% by Day 5. A parallel RT-PCR analysis revealed the expression of MyoD by Day 1 and of myogenin by Day 2 in culture (data not shown).
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To determine the distribution of myogenic cells along the Percoll fractions, cultures of Percoll-isolated cells were analyzed by double immunofluorescence with the antibodies against MyoD and MEF2A (both antibodies react with cell nuclei;
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The data in Table 1 demonstrate that the overall frequency of myogenic cells is significantly reduced in the cultures from 2535% and 35% Percoll fractions and is the highest in the culture from the 55% fraction. Table 1 further shows that the emergence of the MEF2A+ phenotype is delayed in cultures from the two bottom Percoll fractions compared to cultures from the three middle fractions. This distinction in the emergence of the differentiated MEF2A+ cells might be due to variations in cell densities by culture Day 8 (summarized in Table 1). Nevertheless, it has permitted the comparison of gene expression by cells at different stages of the myogenic program (see below). Fig 7 further demonstrates that the MyoD+ cells in the bottom 5570% fraction (Fig 7E) are larger than the MyoD+ cells in the other Percoll fractions (Fig 7D). The nature of the cells contributing these larger MyoD+ cells has not been yet resolved. Additional Percoll fractionation studies resulted in cell distributions similar to those shown in Table 1, with the exception that the frequency of myogenic cells in the top fraction ranged between 4 and 12% by culture Day 8 (not shown). This range is probably due to variations in defining the border between the 2535% and 35% Percoll fractions after centrifugation.
The MyoD-/MEF2A- cells in the cultures of the various Percoll fractions do not necessarily represent one common nonmyogenic cell phenotype. These negative cells might consist of authentic connective tissue cells along with fibroblast-like cells that have been derived in the culture from myogenic cells. Cells negative for myogenic traits can even be detected in myogenic clones derived from single myogenic progenitors (
Expression of the FGFs and Their Receptors in Cultures of Percoll-isolated Cells
RT-PCR analysis of the EDL/TA muscle cells cultured before Percoll fractionation demonstrated the expression of FGFs, HGF, and their receptors at various time points (data not shown). To determine more specifically the relative expression of these genes by the different cell populations present in the muscle-dissociated cells, we analyzed gene expression in Percoll-isolated cells. Fig 8 shows RT-PCR analysis of Percoll-isolated cells that were harvested after 5 days in culture. Cells from the bottom Percoll fraction were not included in the present study. Collective analysis of the seven skeletal muscle genes (MyoD, Myf5, myogenin, MRF4, MLP, TnI fast, TnI slow) demonstrates that the cultures recovered from the 40% and 4045% fractions are in a more advance stage of the myogenic program, while the 3540% and 55% fractions are lagging somewhat behind.
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In contrast to the skeletal muscle genes, the expression levels of FGF1, FGF2, FGF7, and HGF decline in the cultures from the denser Percoll fractions, which raises the possibility that the nonmyogenic cells express these genes at higher levels. Compared to myofiber cultures (Fig 4), we could not detect FGF6 with 34 PCR cycles in any of the Percoll fractions. Some FGF6 expression was detected in the cultures from all the Percoll fractions at 38 PCR cycles (data not shown). This indicates that FGF6 is expressed at a far lower level by the myogenic and nonmyogenic cells compared to the myofibers. FGF5 was also barely detectable in these cultures of Percoll-isolated cells, even at 40 PCR cycles (data not shown).
As shown in Fig 8, the expression pattern of FGFR4 differs markedly from that of the other FGFRs and correlates with the degree of myogenicity of the cultures from the different Percoll fractions. In fact, FGFR4 expression level correlates well with the expression of the "early" myogenic markers MyoD and Myf5. The three genes are co-expressed even in fractions that do not yet display increased levels of the "late" differentiation-linked genes. In contrast, FGFR1 expression is reduced as the Percoll concentration increases, suggesting that FGFR1 is elevated in the nonmyogenic cells. The expression of FGFR2 and FGFR3 also does not correlate with the degree of myogenicity, and their detection requires a greater number of PCR cycles compared to the other receptors. The expression level of c-met was also increased in the more myogenic fractions (i.e., 40%, 4055%, and 55% fractions). However, the cultures derived from the top three Percoll fractions (i.e., 2535%, 35%, and 3540%) did not show a distinct gradual increase in the level of c-met, although the degree of myogenicity gradually increased along the gradient (Fig 8).
Fig 9 shows RT-PCR analysis of Percoll-isolated cells harvested immediately after their isolation (Day 0) and after 2 and 4 days in culture. Only the "interface" Percoll fractions were examined. The expression profile of almost all the genes analyzed at Days 2 and 4 was similar to that of Day 5 discussed above. The expression of MyoD in the Day 2 and 4 cultures of the 2535% fraction is in agreement with the range of myogenic cells seen in this fraction using immunofluorescence, as discussed above. FGF2, FGF7, and HGF were expressed at higher levels by cells from the least dense Percoll fractions (2535% and 3540%), with the expression of HGF first seen only by culture Day 4. The detection of FGF5 and FGF6 required a high PCR cycle as in Day 5 cultures (data not shown). Similar to Day 5 cultures, the distribution of both FGFR4 and c-met (but not the other receptor genes) followed a pattern of increased expression in the more myogenic fractions. This was especially apparent for FGFR4.
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Myogenesis was also evident in the Day 0 cells. However, as shown in Fig 9, expression of the housekeeping genes rpS6 and GAPDH varied among the different fractions. This is likely due to the differences in the activity of the cells at the time of their isolation. The expression of the growth factor genes was also significantly reduced in the Day 0 cells compared to the Day 2 and 4 cultures. Nevertheless, the Day 0 cells recovered from the 4055% fraction expressed the four MRFs along with rpS6 and GAPDH, suggesting that at least some of the cells in this fraction were transcriptionally active and underwent myogenesis at the time of cell isolation. Expression of FGFR1, FGFR3, FGFR4, and c-met was also detected in this fraction. Myf5 expression in the Day 0 cells recovered from the 3540% and 5570% fractions indicates the presence of myogenic cells in these fractions as well. The nature of the strong MRF4 signal in the 55-70% fraction has not been further investigated. It might be related to the large MyoD+ cells present in this fraction, as discussed above. The distribution of FGFR4 expression among the Day 0 Percoll fractions is similar to that of Myf5, suggesting that FGFR4 expression follows the degree of expression of the early myogenic gene, as discussed above.
As shown above, FGF5 is expressed in myofiber/connective tissue cultures (prepared from the FDB muscle) but is barely detected in cultures of the isolated satellite cells (prepared from the EDL/TA muscles). A further comparison of isolated satellite cell cultures from the two sources demonstrated similar mRNA expression patterns for the various genes discussed above, except for FGF5. FGF5 was again expressed at a relatively high level only in the cultures from the FDB muscle but not in cultures released from the EDL/TA muscles (data not shown).
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Discussion |
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The investigations described here were carried out to begin defining the FGF signaling pathways that act during myogenesis of skeletal muscle satellite cells. We have taken a comprehensive approach of analyzing the effects of exogenously added growth factors on myogenesis of satellite cells in combination with exploring the gene expression patterns of these growth factors and their receptors in isolated myofibers and satellite cells. The analyses show unique expression patterns for FGF6 and FGFR4 and therefore may hint at specific regulatory roles of these members of the FGF signaling complex during myogenesis in postnatal muscle. We also attempted to conduct parallel immunocytochemical studies to localize the FGF/FGFR proteins themselves in isolated myofibers and satellite cell cultures. However, the various available antibodies did not produce satisfactory signals for immunocytochemistry or did not discriminate among different FGFs/FGFRs.
FGFs and HGF Promote the Proliferation of Satellite Cells to a Similar Extent
Rat satellite cells undergoing proliferation and differentiation in myofiber cultures can be traced by immunostaining of their nuclei with antibodies against PCNA and myogenin (
The Effect and Site of Expression of FGF6 During Myogenesis of Satellite Cells
The mRNA expression studies indicated that FGF6 was expressed in myofiber cultures when the satellite cells enter proliferation. The presence of FGF6 protein in the muscle was also demonstrated by a sensitive Western blotting analysis. This correlation between the FGF6 expression when satellite cells initiate proliferation and the FGF6 effect on enhancing the number of proliferating satellite cells suggests that FGF6 is involved in vivo in the proliferation of satellite cells. Whereas proliferation of satellite cells was also enhanced by other FGFs, only FGF6 was able to slow the rapid decline in myogenin+ cells. By counterstaining the cytoplasm of the fiber-associated satellite cells, we showed that the routinely observed decline in myogenin+ cells is due to transient expression of myogenin but that the cells themselves remain attached to the myofibers (
The unique effect of FGF6 in retarding the decline in myogenin protein expression by satellite cells may reflect a physiological role for FGF6 in regulating the differentiated state of the satellite cells and possibly of the myofibers themselves. Myogenin is a muscle-specific transcription factor for various muscle differentiation target genes (
A number of published studies provide circumstantial evidence for the possible role of FGF6 in the muscle. First, although several FGFs are expressed in the developing muscle, only the expression of FGF6 is restricted to the muscle itself during early embryogenesis (
The distinction of the present study is that it provides a direct insight into the site of expression and the effect of FGF6 in the adult muscle. Future studies on how FGF6 affects satellite cell proliferation and how it may regulate the expression of the differentiation-specific genes can be important for enhancing muscle maintenance. A direct effect of exogenous FGF6 on the myofiber itself would require the presence of appropriate transmembrane receptors at the myofiber surface. Obviously, an mRNA expression analysis such as that reported in the present study cannot distinguish between the contribution of the satellite cells vs the contribution of the myofibers, and can only provide preliminary evidence about the possible presence of FGFRs. We attempted to use commercial antibodies against FGFRs, especially antibodies against FGFR1 and FGFR4, to further approach the subject by immunocytochemistry. However, a comprehensive study of various anti-FGFR antibodies by multiple approaches did not confirm their suitability for immunolocalization studies of rodent cells. Interestingly, an immunohistochemical study of human muscle that utilized human-specific FGFR antibodies documented the expression of FGFR1 and FGFR4 in the plasma membrane of embryonic but not adult myofibers (
Unique Expression and Possible Role of FGFR4 During Myogenesis of Satellite Cells
The mRNA expression studies conducted with isolated myofibers and isolated satellite cells revealed a unique expression pattern for FGFR4 in skeletal muscle. FGFR4 was expressed at relatively higher levels in intact myofibers and in the satellite cells isolated from the muscle compared to the muscle connective tissue cells. A higher level of FGFR4 expression was detected in satellite cells, regardless of the time spent in culture or the degree of differentiation. This suggests that FGFR4 might be expressed by proliferating and differentiating satellite cells. Expression of FGFR1, FGFR2, and FGFR3 was also seen in the myofiber cultures and in cultures of isolated satellite cells. However, only FGFR1 was readily detectable by RT-PCR, whereas the detection of FGFR2 and FGFR3 required a high number of PCR cycles. The analysis of gene expression in enriched myogenic and nonmyogenic cell populations isolated by Percoll density centrifugation has indicated that only FGFR4 expression correlates with the degree of myogenicity. Furthermore, FGF1, FGF2, FGF4, and FGF6 (but not FGF5 and FGF7) enhanced satellite cell proliferation in the myofiber studies. This characteristic profile of effective and ineffective FGFs fits well with the ligand affinities of FGFR4 established by
Previous studies detected the expression of FGFR1 and FGFR4 in developing human and mouse muscles (
It is notable that the mouse MM14 myogenic cells, which have been used extensively to study the role of FGFs during myogenesis, express only FGFR1 but not the other FGFRs (
We propose that in the satellite cell system FGFR1 may regulate ongoing proliferation of myoblasts, acting in a similar manner to its action in other cell systems. In contrast, FGFR4 might be involved in a myogenic-specific pathway. This proposal is supported by our recent studies with satellite cells from the MyoD-/- mouse, in which we demonstrate that the onset of myogenic differentiation and FGFR4 gene expression is similarly delayed (unpublished results). Studies on the FGFR4-/- mutant mouse demonstrated that FGFR4 is dispensable during embryonic development and that the FGFR4-/- mice are healthy and fertile (
In conclusion, our present study provides evidence for the unique expression patterns of FGF6 and FGFR4 during myogenesis in postnatal muscle. To focus further on the functional role of FGF6 and FGFR4 in adult skeletal muscle, we have initiated studies on satellite cells from mutant mice lacking these two components of the FGFFGFR system. The methods developed in the present study for localizing gene expression in myofibers and muscle-dissociated cells can now be utilized for analysis of other growth factors and cytokines that have been considered to be candidate regulators of myogenesis in the postnatal muscle.
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Acknowledgments |
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Supported in parts by grants to ZYR from the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture (agreement no. 95-37206-2356 and 99-35206-7934) and by the National Institutes of Health (AG13798).
We thank Dr David Graves for designing many of the PCR primers used in the study and for his input during the preliminary stage of the investigation.
Received for publication February 8, 2000; accepted March 29, 2000.
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