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Keywords:

  • skeletal muscle satellite cells;
  • posthatch muscle growth;
  • pectoralis;
  • Pax7;
  • MyoD;
  • myogenin;
  • myofiber diameter

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. NOTE ADDED IN PROOF
  9. REFERENCES

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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. NOTE ADDED IN PROOF
  9. REFERENCES

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.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. NOTE ADDED IN PROOF
  9. REFERENCES

Pax7-Stained Nuclei Are Situated Underneath the Myofiber Basement Membrane in the Intact Muscle of Growing and Adult Chickens

The expression of Pax7 was investigated in cross-sections from the pectoralis muscle of 9-day-old chickens. Figure 1 depicts merged photomicrographs of a representative microscopic field triple-labeled with an antibody against laminin to highlight the myofiber basement membrane (left and right panels, red), an antibody against Pax7 (left panel, green), and the nuclear stain Hoechst 33258 (right panel, blue). Pax7 clearly stained some of the nuclei present underneath the basement membrane, suggesting that these Pax7+ nuclei are within satellite cells as recently reported in mouse (Seale et al., 2000). Cross-sections from the pectoralis muscle of older chicken (49, 62, and 115 day of age) were also analyzed for the presence and localization of Pax7+ cells. Similar images to those depicted in Figure 1 were observed for the older ages (data not shown) with the exception that myofiber cross-sectional area increases with age (Rosser et al., 2000).

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Figure 1. Pax7 is localized in nuclei under the basal lamina. A cross-section, prepared from pectoralis muscle of a 9-day posthatch chicken, was immunostained for Pax7 (green) and laminin (red). Counterstain was performed with Hoechst 33258 to visualize all nuclei (blue). The image of laminin immunostaining was merged with that of Pax7 (left panel) or with Hoechst (right panel). Scale bar = 20 μm.

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The quantification of Pax7+ cells present in myofibers from the pectoralis muscle of 9- to 115-day-old chickens is further summarized in Table 1. The results demonstrate that the proportion of Pax7+ nuclei to total nuclei surrounded by laminin declines from 20 to 4.8% between days 9 and 115. These values are consistent with the number of satellite cells identified in postnatal and adult murine muscle (Snow, 1977; Hawke and Garry, 2001).

Table 1. Distribution of Pax7+ Cells Out of Total Nuclei Present Underneath the Myofiber Basement Membrane in Cross-sections From Chickens of Different Agesa
 Animal age (days)
94962115
  • a

    Three chickens were studied at each age, analyzing three different fields within two to three serial cross-sections from each pectoralis muscle. To delineate nuclei underneath the myofiber basement membrane, sections were double stained with antibodies against Pax7 and laminin and counterstained with Hoechst 33258 as in Figure 1. Numbers were determined per each myofiber and pooled together for the 200 myofibers analyzed per each muscle sample.

  • b

    Total number of nuclei includes the Pax7+ and Pax7− nuclei associated with each myofiber as delineated by the laminin-stained myofiber periphery. The percentage of Pax7+ nuclei was significantly different (P < 0.01) at every age except between 49 days and 62 days (P > 0.05). From the age of 9 days to 115 days, the frequency of satellite cells decreased significantly (P < 0.0001) by 75%. Before data were analyzed by one-way analysis of variance, they were subjected to an arcsin transformation for percentages and ratios. Analyses of both transformed and untransformed data produced similar results.

Animal numberN1N2N3N1N2N3N1N2N3N1N2N3
Number of Pax7+ nuclei202828323737313338142618
Number of Pax7− nuclei90106105329336296338303331314411395
% Pax7+ nuclei out of total nuclei (average ± SD)b20.04 ± 1.629.57 ± 1.339.41 ± 0.964.86 ± 0.94

Distinct Expression Patterns of Pax7, MyoD, and Myogenin in Cell Cultures From Chicken Muscle

The pattern of Pax7 protein expression in myogenic cultures derived from 9-day-old chickens was analyzed in relation to the expression of MyoD and myogenin by double-immunofluorescence. MyoD protein is expressed by both proliferating and differentiating chicken myoblasts, whereas myogenin protein expression begins as the cells enter differentiation (Yablonka-Reuveni and Paterson, 2001). Hence, the double-immunostaining analysis has allowed us to elucidate whether Pax7 protein expression is modulated during proliferation and differentiation of chicken myoblasts. Figures 2 and 3 depict photomicrographs of parallel cultures fixed on days 3 and 7, respectively, and costained with the antibodies against Pax7 and MyoD or Pax7 and myogenin. Both single-stained and double-stained mononucleated cells were identified when the cultures were stained for Pax7 in conjunction with MyoD or myogenin. In addition, while nuclei in the myotubes stained strongly for MyoD and myogenin, they were mostly negative for Pax7.

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Figure 2. A–B: Representative photomicrographs of day 3 cultures double stained with antibodies against Pax7 and MyoD (A,A′) or Pax7 and myogenin (B,B′). A″,B: Merged images are also depicted. A″′,B″′: Cultures were counterstained with 4′6′-diamino-2-phenylindole (DAPI) to visualize all nuclei. Culture conditions are described in Table 2. Arrows point to double-stained cells in parallel images. Arrowheads point to single-stained cells in parallel images. Scale bar = 50 μm in B″′ (applies to A–B″′).

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Table 2. Distribution of Pax7±/MyoD± and Pax7±/Myogenin± Mononucleated Cells in Cultures From 9-Day-Old Chicken Musclea
DaysTotal number of cells% of total cells% of total cells% nuclei in myotubes
Pax7+/MyoD−Pax7+/MyoD+Pax7−/MyoD+Pax7+/myog−Pax7+/myog+Pax7−/myog+
  • a

    Cells were monitored by double immunofluorescence as described in Figures 2 and 3. The total number of cells is based on the number of DAPI-stained nuclei in single cells and in myotubes. Five to ten arbitrary fields were monitored per culture using a x40 objective, and in all cases, the numbers were averaged per five fields. Studies were repeated three times with similar results.

  • b

    Cultures fixed on days 2–4 were initiated at 4 × 105 cells per 35-mm plates. Cultures fixed on days 7 and 15 were initiated at 1 × 105 cells and 1 × 103 cells, respectively, per 35-mm plates; cultures initiated at the higher density become too crowded for cell counting at the later time points.

231628.1648.426.960.00
366916.1442.9019.137.47
478117.2935.0219.0117.73
796024.0312.8816.2537.53
15b85743.5218.6710.0421.24
228276.953.908.870.00
342154.6310.9319.710.00
487338.756.8323.1814.23
781832.872.8918.0037.31
15103649.230.687.1433.88
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Figure 3. A–B: Representative photomicrographs of day 7 cultures double-stained with antibodies against Pax7 and MyoD (A,A′)or Pax7 and myogenin (B,B′). A″,B: Merged images are also depicted. A″′,B″′: Cultures were counterstained with 4′6′-diamino-2-phenylindole (DAPI) to visualize all nuclei. Culture conditions are described in Table 2. Concave arrows point to double-stained cells in parallel images. Arrowheads point to single-stained cells in parallel images. Straight arrows point to myotubes in parallel images. Scale bar = 50 μm in B″′ (applies to A– B″′).

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The distribution of positive cells in parallel cultures labeled with antibodies against Pax7 and MyoD or Pax7 and myogenin was further quantified. Cultures stained for Pax7 and MyoD were examined for the distribution of the following phenotypic subgroups within mononucleated cells: Pax7+/MyoD−, Pax7+/MyoD+ and Pax7−/MyoD+ along with the number of nuclei in myotubes and total number of cells. A similar distribution analysis was performed when cultures were costained for Pax7 and myogenin. Cultures were analyzed after a shorter (2–4 days) or longer (7 and 15 days) incubation and the data are summarized in Table 2. Day 1 cultures were not immunoquantified in this study, as this was proven problematic due to cell clustering and increased background. As can be calculated based on the data depicted in Table 2, the frequency of all cells with myogenic traits (i.e., the number of single- and double-stained mononucleated cells plus the number of nuclei in myotubes divided by the total number of 4′6′-diamino-2-phenylindole [DAPI] -stained nuclei) ranged between 83% and 90% for days 2 to 4 and between 90% and 93% for days 7 to 15. These high myogenicity values indicate that the cell isolates used in the study were greatly enriched for myogenic cells.

As summarized in Table 2, immunostaining for Pax7 and MyoD showed that most of the cells on day 2 were positive for both Pax7 and MyoD (Pax7+/MyoD+) or just for Pax7 (Pax7+/MyoD−), while a far smaller number of cells were positive only for MyoD (Pax7−/MyoD+). Pax7+/MyoD+ cells remained the predominant cell type through day 4, although their numbers decreased slightly with time, while the number of nuclei in the myotubes increased. However, by day 7, the frequency of Pax7+/MyoD+ cells was reduced along with an increase in the number of cells that fused into myotubes. Myogenin+ cells were also seen by day 2, although their frequency at that time was far lower than that of the MyoD+ cells (Table 2), as expected for myogenic precursors which express MyoD before myogenin. On each culture day analyzed, some of the myogenin+ cells were also positive for Pax7 (Table 2). However, these Pax7+/myogenin+ cells represented only 4.8% of the total Pax7+ cells observed on day 2 (Table 3). The frequency of Pax7+/myogenin+ cells out of the total number of Pax7+ cells increased to approximately 15 to 16% on days 3 and 4 but declined to 8.0 and 1.3% on days 7 and 15, respectively (Table 3). In contrast, Pax7+/MyoD+ cells observed on day 2 comprised 63% of the total Pax7+ cells at this time point, and the frequency of Pax7+/MyoD+ cells compared with total Pax7+ cells was in the range of 67 to 72% on days 3 and 4, declining to 30 and 35% on days 7 and 15, respectively (Table 3). Collectively, the data presented in Tables 2 and 3 suggest that the Pax7+/myogenin+ cells represent an intermediate population and that Pax7 expression is eventually shut off in myogenin+ cells.

Table 3. Frequency of MyoD+ or Myogenin+ Cells Within Mononucleated Pax7+ Cells in Cultures From 9-Day-Old Chicken Musclea
DaysNumber of all Pax7+ cells% of all Pax7+ cells% of all Pax7+ cells
Pax7+/MyoD−Pax7+/MyoD+Pax7+/myog−Pax7+/myog+
  • a

    See footnotes in Table 2.

224236.7763.22
339527.3472.65
440933.0066.99
735565.0934.90
1553369.9830.02
222895.174.82
327683.3316.66
444484.9115.09
729391.918.08
1551798.631.36

To investigate the relationships between the different cell phenotypes, we analyzed myogenic clones derived from the 9-day-old chicken muscle for the presence of Pax7±/MyoD± or Pax7±/myogenin± cells using double immunostaining (Fig. 4) as in the higher density cultures. Clones were analyzed at 7, 11, and 15 days of culture. The same cell phenotypes summarized in Table 2 were identified in the clonal cultures. Nearly all cells within each clone expressed one or both proteins when stained for Pax7 in combination with MyoD or myogenin. Collectively, the clonal analysis demonstrated that individual progenitors can give rise to all the phenotypic combinations discussed in Table 2, indicating a lineal relationship between these cell phenotypes.

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Figure 4. A–B: Representative photomicrographs of day 11 clones double labeled with antibodies against Pax7 and MyoD (A,A′) or Pax7 and myogenin (B,B′). A″,B: Merged images are also depicted. A″′,B″′: Cultures were counterstained with 4′6′-diamino-2-phenylindole (DAPI) to visualize all nuclei. Culture conditions are the same as for the routine cultures depicted in Figures 2 and 3. Concave arrows point to double-stained cells in parallel images. Arrowheads point to single-stained cells in parallel images. Straight arrows point to myotubes in parallel images. Scale bar = 30 μm B″′ (applies to A– B″′).

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Expression of Pax7 and Myogenin During Muscle Growth in Early-Stage Posthatch Chickens

The dynamics of satellite cell proliferation and differentiation during early posthatch days was investigated by studying the kinetics of Pax7 and myogenin protein expression in pectoralis muscle derived from 1-, 3-, and 6-day-old chicks along with an analysis of myofiber growth. The protein expression studies were carried out by both immunofluorescence (Figs. 5, 6) and Western blot analysis (Fig. 7). The latter technique enabled us to quantify the relative expression of these proteins. Both approaches demonstrated the marked variation in the expression patterns of Pax7 and myogenin. The strongest expression of Pax7 was seen on day 1, declining on days 3 and 6 to a similar level (Figs. 5, 7). Myogenin expression was low on day 1 and increased over 30-fold on day 3 (Figs. 5, 7). This elevated myogenin expression was transient and protein levels declined by day 6 to a level similar to that seen on day 1. Notably, Pax7+ and myogenin+ nuclei were randomly scattered throughout the muscle tissue, regardless of chicken age (Figs. 5, 6). The number of Pax7+ or myogenin+ cells per total DAPI-stained nuclei in images of immunolabeled muscle sections was additionally quantified without preselection of fields. The following distribution was found when 3–9 × 103 DAPI-stained nuclei were analyzed per muscle sample: Pax7+ cells were 82.2% (day 1), 14.1% (day 3), 16.5% (day 6); myogenin+ cells were 3.2% (day 1), 43.2% (day 3), 8.1% (day 6). The slightly lower percent value for Pax7+ cells on days 3 and 6 compared with that described in Table 1 for day 9 muscle is likely due to the fact that Pax7+ cell distribution in muscle sections from days 3 and 6 is based on the total number of DAPI-stained nuclei, whereas the day 9 distribution is based strictly on myofiber nuclei. It should be noted that, due to the high background, when staining cross-sections with anti-MyoD, we were unable to investigate the expression of MyoD in vivo by immunofluorescence and cannot presently comment on the possibility of Pax7±/MyoD± populations in vivo.

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Figure 5. Immunofluorescence staining for Pax7 in pectoralis muscle cross-sections prepared from 1-, 3-, and 6-day posthatch chickens. A–C: Sections were immunostained for Pax7 (A–C) and counterstained with 4′6′-diamino-2-phenylindole (DAPI) to visualize all nuclei (A′–C′). Scale bar = 30 μm in C′ (applies to A–C′).

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Figure 6. Immunofluorescence staining for myogenin in pectoralis muscle cross-sections prepared from chicks at 1, 3, and 6 days of age. A–C: Sections were immunostained for myogenin (A–C) and 4′6′-diamino-2-phenylindole (DAPI; A′–C′). Note that sections shown in this figure were prepared in parallel with the sections analyzed in Figure 5. Scale bar = 30 μm in C′ (applies to A–C′).

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Figure 7. Quantification of Pax7 and myogenin protein expression level in pectoral muscle of chickens in the early days posthatch. A: A representative Western blot analysis for Pax7 and myogenin. B,C: Protein bands were quantified by densitometric analysis of Pax7 (B) and myogenin (C) relative to that of α-tubulin. Results are means ± SE; n = 4. Significant differences are marked with asterisks (P < 0.05). Data depicted are representative of five independent experiments.

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Muscle growth was assessed by determining myofiber diameter size in hematoxylin and eosin–stained muscle sections from posthatch chicks. Figure 8 depicts the results of the lesser-diameter analyses performed as described in the Experimental Procedures section. Mean myofiber diameter was 2.91, 4.35, and 8.72 μm for muscle samples from days 1, 3, and 6, respectively. The diameter increase was 1.5-fold from days 1 to 3 and twofold from days 3 to 6. The wide range in myofiber diameter within each of the 3 time points analyzed is likely due to the different developmental ages of primary and secondary myofibers formed during embryogenesis. The variation in myofiber diameter is far less apparent in later posthatch days. Collectively, the kinetics of myofiber diameter increase during early posthatch days was vastly different from the kinetics of Pax7 and myogenin protein expression levels (determined by Western blots) during early posthatch days.

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Figure 8. Histograms of myofiber diameter in the pectoralis muscle of 1-, 3-, and 6-day posthatch chickens. Myofibers are clustered in bin intervals of 0.5 μm. The diameter of individual myofibers was measured in muscle sections from three chicks at each time point (n = 150–275 fibers per chicken and a total of 700 fibers per age group). Because there were no statistical differences between samples from chicks of the same age, myofiber diameters within each age group were pooled and ranked in ascending order within each time point. Data from the various age groups were evaluated for statistical differences using one-way analysis of variance followed by post hoc Tukey test (P < 0.05). Mean ± SD for myofiber diameter (in μm) per each age group: 2.91 ± 0.73 (day 1), 4.35 ± 1.45 (day 3), 8.72 ± 2.25 (day 6). Average diameter for each age group differs significantly from the other two groups (P < 0.0001). Data depicted are representative of three independent experiments.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. NOTE ADDED IN PROOF
  9. REFERENCES

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:

  • I
    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).
  • II
    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, b; Zammit et al., 2002).
  • III
    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.
  • IV
    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.
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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.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. NOTE ADDED IN PROOF
  9. REFERENCES

Animal Procedures

Animal care and experimental procedures were approved by the Animal Welfare Committee of the Faculty of Agriculture at the Hebrew University of Jerusalem, the University of Saskatchewan Committee on Animal Care and Supply (following the Canadian Council on Animal Care Guidelines), and the Institutional Animal Care and Use Committee at the University of Washington (Assurance no. A3464-01, NIH Office of Laboratory Animal Welfare; accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care, International).

Muscle Sampling

Muscle samples were collected from three chickens per each age group to prepare tissue sections for immunofluorescent analysis and for hematoxylin–eosin staining. Chickens ranged in age from 1 to 115 days. To reduce variations between early posthatch chicks, muscle was sampled from animals that hatched within a narrow window of time. Muscle samples were excised from the superficial regions of the proximal half of the left pectoralis major muscle of each chicken. Each sample was approximately 0.5 × 0.5 × 1.2 cm. The long axis of each sample was parallel to the direction of the muscle fibers. For the younger chicks (Cobb strain, 1-, 3-, and 6-day[s] old), the muscle samples were fixed in fresh 4% paraformaldehyde in phosphate-buffered saline (PBS) solution (pH 7.6) for 24 hr. For immunofluorescence, the tissue samples were subjected to a graded series of sucrose solutions as described in Yablonka-Reuveni et al. (1998). The samples, oriented for transverse cross-sectioning, were immersed in Tissue-Tek OCT and then frozen in isopentane cooled by liquid nitrogen. Serial cross-sections (10 μm) were cut at −20°C in a cryostat and placed on glass slides (Superfrost, Menzel-Galzer, Germany). For 9-day and older chickens (White Leghorn), the muscle samples were immediately frozen in Tissue-Tek OCT without prior fixation and sections were further processed as previously described (Rosser et al., 2000).

For hematoxylin–eosin staining, paraformaldehyde-fixed tissue samples from 1-, 3-, and 6-day-old chicks were dehydrated in a graded ethanol series, embedded in paraffin and 5-μm serial sections were cut and placed on Superfrost slides. Sections were deparaffinized in xylene, rehydrated in a graded alcohol series, and stained.

Cell Cultures

The pectoralis muscle from 9-day-old chickens was used to prepare myogenic cultures (Hartley et al., 1992). The method used in the present study for cell isolation is essentially the same as that described in our rat and mouse studies (Kästner et al., 2000; Yablonka-Reuveni, 2004). The whole muscle was harvested into MEM (Eagle's minimal essential medium containing L-glutamine, supplemented with penicillin–streptomycin and nonessential amino acids [Gibco-Invitrogen, Carlsbad, CA]). The muscle was cleaned of connective tissue, cut into 1- to 2-mm3 pieces, and then allowed to settle through MEM in a tissue culture tube. The settling step was repeated three times, resulting in the removal of red blood cells from the muscle preparation. The muscle was then digested with 0.1% Pronase (Calbiochem, San Diego, CA) in a tissue culture incubator (37.5°C, 5% CO2) for 45 min with continuous gentle agitation. The digested muscle was collected by low-speed centrifugation and resuspended in MEM containing 10% horse serum. Single cells were released from the digested muscle by vigorous repetitive triturations, and the suspension was passed through a double-layered lens paper to filter out the larger debris.

Unless otherwise noted, cultures were initiated at a density of 4 × 105 cells per plate in 35-mm dishes coated with 2% gelatin and the medium consisted of MEM containing 10% horse serum and 5% chicken embryo extract (CEE) (Yablonka-Reuveni and Paterson, 2001). CEE was prepared from 10-day-old embryos (using whole embryos) as described in Yablonka-Reuveni (1995) and Shefer and Yablonka-Reuveni (2004). Cells were maintained at 37.5°C in a humidified atmosphere, 95% air, and 5% CO2.

To analyze progeny of individual cells, cultures were initiated at a density of 10 cells per 35-mm plate resulting in clonal growth of the progenitors. Clonal cultures were maintained under the same conditions described above for the higher density cultures. Further details regarding culturing clones from chicken muscle are described in our previous studies (Hartley et al., 1992; Yablonka-Reuveni, 2004).

Primary Antibodies

Antibodies used for immunofluorescence were rabbit polyclonal antibodies against chicken MyoD and chicken myogenin each used at 1:1,000 dilution (Yablonka-Reuveni and Paterson, 2001), a rabbit polyclonal antibody against laminin used at 1:200 (Sigma-Aldrich, St. Louis, MO), and a mouse monoclonal antibody against chicken Pax7 (hybridoma supernatant used at 1:2 dilution or ascites fluid used at 1:4,000 dilution, Developmental Studies Hybridoma Bank, University of Iowa; Kawakami et al., 1997). The antibodies against myogenin and Pax7 were also used in Western blots as described below.

Immunofluorescence of Tissue Sections

Muscle sections from the younger chicks (1-, 3-, and 6-day-old posthatch) were washed with TBS (0.05 M Tris, 0.15 M NaCl, pH 7.4), permeabilized for 15 min with 0.25% Triton X-100 in TBS, and blocked in 5% goat serum in TBS overnight at 4°C. Sections were reacted with the antibodies against Pax7 or myogenin for 1 hr at room temperature followed by overnight at 4°C. After washes in TBS containing 0.05% Tween 20 (TBS-TW20), slides were incubated in Alexa Fluor 488 goat anti-mouse IgG (1:200, Molecular Probes, Eugene, OR) or rhodamine-conjugated goat anti-rabbit IgG (1:250, Jackson, West Grove, PA) for 1 hr at room temperature, and finally washed again in TBS-TW20. Nuclei were detected with DAPI (1 μg/ml dilution in TBS, Sigma-Aldrich). Sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Three arbitrary fields were photographed per each muscle sample by using a ×40 objective and representative images are depicted in the Results section.

For muscle sections from older chickens (9, 49, 62, and 115 days of age), slides were blocked with 5% horse serum and 1% bovine serum albumin in PBS containing 5 mM EDTA. Sections were double-stained with the antibodies against Pax7 and laminin followed by Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG (each at 1:200). Sections were then reacted with Hoechst 33258 (diluted 1:1,500,000 in PBS, Sigma-Aldrich), fixed with 4% formaldehyde and mounted in Geltol (Thermo Shandon, Pittsburgh, PA). To quantify the number of Pax7+ nuclei associated with myofibers, three different fields of view were imaged from each slide using a ×40 objective for the 9-day-old muscle and a ×20 objective for muscles from the older chickens. Fibers (200 per animal) were analyzed, proceeding from the first to the third field of view. All fibers in the first and second fields were analyzed. In the third field of view, additional, adjacent fibers were examined until 200 fibers were studied. The number of Pax7+ nuclei associated with myofibers along with the total number of Hoechst-stained myonuclei were counted for each myofiber separately and then pooled together for the 200 myofibers analyzed.

Immunofluorescence of Cell Cultures

Cultures were fixed with a paraformaldehyde-based fixative (4% paraformaldehyde in 0.1 M sodium phosphate containing 0.03 M sucrose, pH 7.2) for 10 min followed by treatment with TBS containing 0.5% Triton X-100 for 10 min to permeabilize the cells. Cultures were reacted with the paired primary antibodies (anti-Pax7 and anti-MyoD or anti-Pax7 and anti-myogenin) followed by the paired secondary antibodies (Alexa Fluor 568 goat anti-rabbit [1:1,000] and Alexa Fluor 488 goat anti-mouse IgG [1:1,000]) and DAPI staining. All immunolabeling procedures were as described above for cross-sections from younger chicks. Controls for double-labeling specificity of the nuclei included immunostaining omitting the primary antibodies, staining with each of the primary antibodies alone and both secondary antibodies, staining with each of the primary antibodies alone followed by the reciprocal secondary antibody, and immunostaining with the paired primary antibodies followed by each of the secondary antibodies alone. The double-stained cultures were quantified for the number of mononucleated cells stained for one or both antibodies along with the number of nuclei in myotubes and the total number of nuclei (i.e., DAPI-stained nuclei in single cells and myotubes). Images of five to ten arbitrary microscopic fields were analyzed per plate by using a ×40 objective, and the results were pooled for each culture plate.

Myofiber Diameter Analysis

Muscle sections stained with hematoxylin and eosin were used to analyze myofiber diameter. Myofiber diameter was determined by analyzing the lesser-myofiber diameter value. The lesser fiber diameter is defined as the maximum diameter across the lesser aspect of a fiber and has been proven to be an excellent means of determining the actual myofiber diameter when sections through myofibers are transverse (Dubowitz, 1985). At least 10 arbitrary fields in two to three serial sections of each muscle sample were recorded by using a ×40 objective. Myofiber diameter was then determined with Adobe Photoshop software. In each muscle sample, the lesser-fiber diameter was measured for individual myofibers, analyzing 150 to 275 fibers per sample and a total of 700 fibers per age group. A calibrated length was photographed and processed under the same conditions as the histological images to transform the digitized data from pixels to standard length units. Myofibers in muscles from three chicks were quantified for each time point. Because there were no statistically significant differences between chicks, all data for same age chicks were pooled for further analysis.

Western Blot Analysis

Muscle samples were collected from the right half of the pectoralis muscle used in the immunohistochemical analysis and immediately frozen in liquid nitrogen. Extracts were prepared from the frozen muscle sample as previously described (Halevy et al., 2001). Protein content in the extracts was determined using the BCA kit (Pierce, Rockford, IL). Equal amounts of protein per each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters (Bio-Rad, Hercules, CA). Membranes were incubated overnight at 4°C with anti-myogenin and anti-Pax7 primary antibodies, then washed and incubated for 1 hr with horseradish peroxidase–conjugated goat anti-mouse or goat anti-rabbit IgG (Zymed, San Francisco, CA). Proteins were visualized using enhanced chemiluminescence (Pierce). The blots were reprobed with anti α-tubulin. Densitometric analysis was performed on bands using Image Pro-Plus software (Media Cybernetics, Inc., Silver Spring, MD). Protein levels in each lane were normalized to the levels of α-tubulin as an internal standard (Halevy et al., 2001).

Statistics

Data were evaluated by using one-way analysis of variance (ANOVA) followed by Tukey post hoc test to examine statistical differences between means of the various age groups (P < 0.05). When percentages were compared, before data were analyzed by one-way ANOVA, they were subjected to an arcsin transformation for percentages and ratios.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. NOTE ADDED IN PROOF
  9. REFERENCES

We thank Drs. Gabi Shefer and Farrel Robinson (University of Washington) for their valuable comments on the manuscript. O.H., I.R., and Z.Y.R. were funded by a United States-Israel Binational Agricultural Research and Development Fund; B.W.C.R. was funded by a Discover Grant from the Natural Sciences and Engineering Research Council of Canada; and Z.Y.R. was funded by grants from the Cooperative State Research, Education, and Extension Service-United States Department of Agriculture and the National Institute on Aging.

NOTE ADDED IN PROOF

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. NOTE ADDED IN PROOF
  9. REFERENCES

Recent work by Peter Zammit and associates (Zammit et al. [2004] J Cell Biol 166:347–357) is in accordance with our model of satellite cell self-renewal (see Fig. 9, route A). Using isolated adult mouse myofibers to model muscle regeneration, these authors show that Pax7+/MyoD+ satellite cells not only give rise to cells fated to differentiate, but also to Pax7+/MyoD− progeny, presumably destined to replenish the satellite cell pool.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. NOTE ADDED IN PROOF
  9. REFERENCES