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The paired-box transcription factor Pax7 plays a critical role in the specification of satellite cells in mouse skeletal muscle. In the present study, the position and number of Pax7-expressing cells found in muscles of growing and adult chickens confirm the presence of this protein in avian satellite cells. The expression pattern of Pax7 protein, along with the muscle regulatory proteins MyoD and myogenin, was additionally elucidated in myogenic cultures and in whole muscle from posthatch chickens. In cultures progressing from proliferation to differentiation, the expression of Pax7 in MyoD+ cells declined as the cells began expressing myogenin, suggesting Pax7 as an early marker for proliferating myoblasts. At all time points, some Pax7+ cells were negative for MyoD, resembling the reserve cell phenotype. Clonal analysis of muscle cell preparations demonstrated that single progenitors can give rise to both differentiating and reserve cells. In muscle tissues, Pax7 protein expression was the strongest by 1 day posthatch, declining on days 3 and 6 to a similar level. In contrast, myogenin expression peaked on day 3 and then dramatically declined. This finding was accompanied by a robust growth in fiber diameter between day 3 and 6. The distinctions in Pax7 and myogenin expression patterns, both in culture and in vivo, indicate that while some of the myoblasts differentiate and fuse into myofibers during early stages of posthatch growth, others retain their reserve cell capacity. Developmental Dynamics 231:489–502, 2004. © 2004 Wiley-Liss, Inc.
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Skeletal muscle fibers are formed during embryogenesis and continue to enlarge postnatally until the mature size has been reached. This postnatal myofiber growth entails an increase in myofiber protein accretion and in the number of myofiber nuclei (reviewed by Allen et al., 1979; Edgreton and Roy, 1991). The primary source of these additional myofiber nuclei are the satellite cells, myogenic stem cells situated on the surface of the myofiber between the myofiber plasmalemma and its covering basement membrane (Mauro, 1961; Hawke and Garry, 2001; Zammit and Beauchamp, 2001).
Satellite cells are first detected in muscles during late stages of fetal development, when the myofiber basement membrane is formed (Yablonka-Reuveni, 1995). At birth, skeletal muscle nuclei consist of a high percentage of proliferating satellite cells. However, at the end of the growth phase, the number of satellite cells decreases to less than 5% of total muscle fiber nuclei and at this stage, most of the satellite cells become quiescent (Hawke and Garry, 2001). The quiescent satellite cells can rapidly enter the cell cycle in response to various muscular stresses ranging from work overload to major muscle trauma. Depending on the degree of repair required, these activated satellite cells undergo either limited or multiple rounds of cell division, followed by withdrawal from the cell cycle and fusion into existing or newly formed fibers (Grounds and Yablonka-Reuveni, 1993; Bischoff, 1994).
Proliferation and differentiation of myoblasts is regulated by the muscle-specific basic helix-loop-helix (bHLH) family of transcription factors (reviewed in Weintraub, 1993; Ludolph and Konieczny, 1995; Naya and Olson, 1999). Upon satellite cell activation, the muscle-specific transcription factors are expressed in a sequential pattern with Myf5 and MyoD being expressed in the proliferating progeny followed by myogenin expression as the cells enter differentiation (Smith et al., 1994; Yablonka-Reuveni and Rivera, 1994; Cornelison and Wold, 1997; Cooper et al., 1999; Yablonka-Reuveni et al., 1999a; Kästner et al., 2000; Yablonka-Reuveni and Paterson, 2001). Additional studies have suggested that Myf5 is already expressed in quiescent satellite cells but likely at a lower level than in proliferating satellite cells (Cooper et al., 1999; Beauchamp et al., 2000).
Pax7, paired-box containing transcription factor, has been shown to play a pivotal role in the formation of adult mouse skeletal muscle (Seale et al., 2000). Pax7 is expressed by quiescent satellite cells in the normal muscle, but skeletal muscle of Pax7-/- mice lacks satellite cells and cells cultured from Pax7-/- muscles are unable to undergo myogenesis (Seale et al., 2000). It was further shown that Pax7 expression is higher during myoblast proliferation and declines in correlation with differentiation in mouse myogenic cultures (Asakura et al., 2000; Seale et al., 2000). However, the pattern of Pax7 expression during the early phase of postnatal growth, and its interplay with transcription factors regulating differentiation has not been elucidated.
Using specific antibodies raised against chicken MyoD, myogenin, and Pax7, we monitored the expression pattern of these proteins in myogenic cultures and in whole muscle from posthatch chickens. We report that these proteins exhibit different patterns of expression, with the expression of Pax7 declining as the cells move into the myogenin-expressing state, while the expression of MyoD is sustained. Furthermore, within the Pax7+ cell population, some cells are positive for MyoD and others are not; the latter cells could represent reserve myogenic progenitors. Our additional studies on the expression of Pax7 protein in muscle tissues provide evidence that it is an early marker of myogenesis during posthatch muscle growth and that its expression is maintained by satellite cells in adult chicken muscle.
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This study accentuates Pax7 as a marker for muscle precursor cells during myogenesis in chickens. First, we show that Pax7+ cells are present in the intact muscle underneath the myofiber basement membrane. Second, by using cell culture and in vivo approaches, we conclude that Pax7+ cells represent mostly cells in the pre-myogenin state.
Double immunostaining of muscle sections with antibodies to Pax7 and laminin localized the position of the Pax7+ cells underneath the myofiber basement membrane. The frequencies of the Pax7+ cells identified in the intact muscle at different ages corroborate with earlier reports on the declining number of satellite cells identified in postnatal muscle by ultrastructural means (Snow, 1977; Hawke and Garry, 2001). Previously, Pax7 RNA transcripts have been detected in the satellite cell position in mouse muscle (Seale et al., 2000). Hence, we infer that the Pax7+ nuclei present in chicken muscle at early and advanced ages represent satellite cells.
The cell culture analysis described in the present study sheds light on the phase(s) at which Pax7 is expressed along the myogenic program in early posthatch as discussed below:
Pax7+/MyoD− cells were present at all time points analyzed, and their number increased in advanced cultures (i.e., day 7 and 15). In parallel with the increase in Pax7+/MyoD− cells, the frequency of Pax7+/MyoD+ cells declined and a large number of cells transited into the myogenin+ state and/or fused into myotubes in advanced cultures. We propose that the Pax7−/MyoD+ cells found at all time points represent cells that have transited into the Pax7−/myogenin+ state (i.e., Pax7−/MyoD+/myogenin+ cells). This proposal is supported by the similar frequency of Pax7−/MyoD+ and Pax7−/myogenin+ cells within each time point (present study) and by the observation that differentiated chicken myoblasts express both MyoD and myogenin when maintained in standard culture conditions (Yablonka-Reuveni and Paterson, 2001
Because most Pax7+ cells were negative for myogenin at all time points analyzed, we deduce that the Pax7+/MyoD− cells are negative for both MyoD and myogenin. Collectively, in accordance with the expression patterns of Pax7, MyoD, and myogenin in adult mouse muscle for which (a) quiescent satellite cells express Pax7 but not MyoD or myogenin and (b) proliferating satellite cells express Pax7 and MyoD but not myogenin (Yablonka-Reuveni et al., 1999a
; Seale et al., 2000
), we propose that the Pax7+/MyoD− cells identified in the present chicken study represent reserve myoblasts. The Pax7+/MyoD− cells could be progeny of proliferating Pax7+/MyoD+ progenitors and/or represent Pax7+/MyoD+ cells that have shut off MyoD expression (see Fig. 9
). The alternative explanation that Pax7+/MyoD− cells are derived by ongoing proliferation of Pax7+/MyoD− cells is not likely in view of the well-established MyoD+ phenotype of proliferating satellite cells (Yablonka-Reuveni and Rivera, 1994
; Yablonka-Reuveni et al., 1999a
; Zammit et al., 2002
Pax7 and myogenin expression appeared to be mutually exclusive throughout the cell culture analysis. However, at all time points, a small number of cells exhibited a Pax7+/myogenin+ phenotype. This phenomenon raises the possibility that the Pax7+/myogenin+ cells represent an intermediate population facing the onset of differentiation and that Pax7 expression is subsequently shut off as the myogenin+ cells fully differentiate. The Pax7+/myogenin+ cells may retain their capability to proliferate under the appropriate conditions while Pax7−/myogenin+ cells may have undergone terminal differentiation. This scenario is in accordance with previous studies indicating that cells retain their capacity to undergo proliferation at the onset of myogenin expression (Andres and Walsh, 1996
). Further studies are required to establish the nature of the Pax7+/myogenin+ cells and whether there is an inhibitory feedback loop between Pax7 and myogenin.
The clonal analysis demonstrates that individual progenitors can give rise to all the aforementioned phenotypic combinations (Pax7+/MyoD− and Pax7±/MyoD+ or Pax7+/myognein− and Pax7±/myogenin+). Hence, the clonal studies show a lineal relationship between the different cell phenotypes, indicating that individual progenitors give rise to all cell phenotypes. This lineal relationship is further discussed in connection with the scheme depicted in Figure 9
. Progenitors founding the clones could be either Pax7−/MyoD+ cells that have entered the proliferative state upon their isolation from the intact tissue and/or Pax7+/MyoD+ cells that have been already activated in vivo.
Figure 9. A model depicting satellite cell dynamics during myogenesis in early posthatch muscle development. Quiescent satellite cells expressing Pax7 only, are driven to the cell cycle during muscle growth. A,B: Fully proliferating cells express both Pax7 and MyoD and undergo either (A) stochastic on/off gene switch in separate cells or (B) asymmetric divisions, leading to subsequent differentiation or return to the reserve pool. The “decision” to undergo differentiation is accompanied by the induction of myogenin expression and those cells that express Pax7 and MyoD as well as myogenin are probably at the turning point to differentiation. Cells will differentiate (black arrows) when myogenin expression increases and Pax7 levels are decreasing and eventually will be shut off all together. However, under specific signals, cells undergoing differentiation may go back into the cell cycle to their Pax7+/MyoD+ position (dashed arrows).
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Pax7 and myogenin expression patterns are also strikingly different during early posthatch muscle growth in vivo. Pax7 expression was highest on day 1 posthatch and declined on day 3 in contrast to a dramatic induction of myogenin on day 3. This suggests that, by day 3, a substantial part of the myoblasts have undergone differentiation. By day 6 myogenin levels decreased dramatically, while Pax7 levels remained similar to that seen on day 3. We suggest that the marked reduction in myogenin levels on day 6 is due to a decline in the number of cells which have the capacity to differentiate. This induction and decline in myogenin expression has been observed previously in our studies using Northern blot analysis of chicken muscle tissue, whereby myogenin mRNA level peaked after MyoD level at 3 days posthatch (Halevy et al., 1996). Taken together, the in vivo expression studies described here suggest that a pool of reserve and/or proliferating satellite cells remains present at comparable levels on days 3 and 6 posthatch, while the robust phase of myoblast differentiation, which produces myoblasts that fuse with the enlarging myofiber, peaks on day 3. This in vivo expression pattern is similar to that observed in the cell culture studies where the frequency of total myogenin+ or Pax7+/myogenin+ mononucleated cells first rises then declines, while the number of nuclei in the myotubes continues to rise (Tables 2, 3). Also, the frequency of total Pax7+ mononucleated cells fluctuates only slightly during later days in culture (Tables 2, 3).
In view of our results in both cell cultures and muscle tissues, we propose the scheme shown in Figure 9 for satellite cell dynamics during myogenesis in early posthatch muscle development. In this model, we suggest that quiescent Pax7+ cells are driven to the cell cycle by external signals and that the process is accompanied by the induction of MyoD protein as previously reported in rodents (Yablonka-Reuveni and Rivera, 1994; Cooper et al., 1999; Yablonka-Reuveni et al., 1999a, b). After several divisions, cells expressing both Pax7 and MyoD undergo asymmetric divisions (or a stochastic on/off gene switch in separate cells), both of which can lead to subsequent differentiation or cells may return to the reserve pool of myogenic progenitors (Fig. 9, solid arrows). Asymmetric division has been postulated to be one of the processes by which satellite cells retain their steady-state numbers throughout adult life; one daughter cell becomes a progenitor cell, while the other is committed to differentiate into muscle (Moss and Leblond, 1971; Quinn et al., 1988; McGeachie and Grounds, 1995; Shultz, 1996). A recent study has further proposed a possible molecular basis for asymmetric cell division during myogenesis of mouse satellite cells (Conboy and Rando, 2002). However, it is unlikely that asymmetric cell division was maintained, because all cells eventually differentiated. Alternatively, a stochastic gene switch in a single cell can dictate whether this cell will differentiate or return to the reserve pool; the combination of on/off switches of many genes, and of events that are affected by external or internal signals and occur with different probabilities, eventually leads to either process (Dennis and Charbord, 2002; Paldi, 2003). Future studies are required to determine whether either or both asymmetric cell divisions and stochastic on/off switches of gene expression are involved in replenishing the satellite cell reserve pool.
In the model presented in Figure 9, the cell's “decision” to undergo differentiation is marked by the expression of myogenin and the disappearance of Pax7 expression while MyoD is still expressed. One study has suggested that myogenic cells expressing myogenin still retain their capability to synthesize DNA, and they become postmitotic only when the cyclin-dependent kinase p21 is induced (Andres and Walsh, 1996). In accordance with this study, we suggest that the differentiation process can be further fine tuned with an early phase in which cells expressing myogenin along with Pax7 and MyoD are at the turning point for differentiation; under specific signals, these cells may go back to the cell cycle, to their Pax7+/MyoD+ position (Fig. 9, dashed arrows).
It may well be that some of the reserve cells depicted in the scheme shown in Figure 9 express Myf5. Recent studies have suggested that quiescent satellite cells may indeed express Myf5, although the expression level is likely far reduced compared with proliferating cells (Cooper et al., 1999; Beauchamp et al., 2000). However, in the absence of appropriate reagents to detect chicken Myf5 protein, we are unable at present to discern the relevance of Myf5 expression by chicken satellite cells and their proliferating or differentiating progeny.
Another aspect of this research was the analysis of fiber growth during early posthatch. The robust growth in fiber diameter observed by day 6 follows the peak of myogenin expression on day 3. This pattern matches that of myogenin expression in a regenerating mouse muscle, which reflects differentiating myoblasts and disappears in mature myofibers (Garrett and Anderson, 1995). However, the different kinetics of Pax7 and myogenin expression implicates several parallel processes during posthatch muscle development. In accordance with our studies in culture, we suggest that, while a subset of the satellite cells is in a proliferative state, another subset undergoes differentiation and in parallel, a third subset returns to the reserve pool. The ratio between these subpopulations changes during the first days posthatch and dictates the rate of myofiber growth. Thus, the measurement of fiber diameter represents overall muscle growth, which is a result of cell proliferation and hypertrophy.
Does Pax7 expression mark the emergence of satellite cells in the chicken? Previous studies identified differences between myoblasts from adult and fetal chicken muscle based on the characteristics of the isolated myoblasts in culture. These studies further indicated that the adult-type myoblasts emerge during late stages of fetal development (Hartley et al., 1991, 1992; Feldman and Stockdale, 1992; Stockdale, 1992; Yablonka-Reuveni, 1995). The emergence of the adult-type myoblasts (as seen in cell culture analysis) likely reflects the onset of satellite cell formation in late stage chicken embryos when myofibers become fully surrounded by a basement membrane (Yablonka-Reuveni, 1995). In the mouse, Pax7 is important for the formation of satellite cells and postnatal muscle growth but not for prenatal myogenesis, i.e., embryonic muscle development is not affected in mice lacking Pax7 (Seale et al., 2000). In view of this mouse study and our identification of fetal and adult-type myoblasts in chicken, it was attractive to consider the possibility that Pax7 expression in chicken muscle is specific to adult-type myoblasts while fetal-type myoblasts do not express Pax7. However, studying myoblasts isolated from the pectoralis muscle of 10-day-old chicken embryos, we concluded that fetal myoblasts, like adult myoblasts, express Pax7 (data not shown). Thus, it is probable that Pax7 expression cannot be used as a means of distinguishing between the different myoblast populations identified during muscle histogenesis in embryonic and posthatch chickens.
In summary, this study shows that Pax7 is an early marker for chicken satellite cells. Different from MyoD whose expression marks both proliferating and differentiating satellite cells, Pax7 expression primarily marks satellite cells and their proliferative progeny. The distinctions in Pax7, MyoD, and myogenin expression patterns observed in the present study suggest a dynamic process of myogenesis during early posthatch chicken growth whereby myoblasts proliferate, differentiate, and fuse into fibers as well as maintain the pool of reserve myogenic progenitors.