ARTICLE |
Correspondence to: Zipora YablonkaReuveni, Dept. of Biological Structure, Box 357420, U. of Washington, Seattle, WA 98195. E-mail: reuveni@u.washington.edu
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Summary |
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Isolated chicken myoblasts had previously been utilized in many studies aiming at understanding the emergence and regulation of the adult myogenic precursors (satellite cells). However, in recent years only a small number of chicken satellite cell studies have been published compared to the increasing number of studies with rodent satellite cells. In large part this is due to the lack of markers for tracing avian myogenic cells before they become terminally differentiated and express muscle-specific structural proteins. We previously demonstrated that myoblasts isolated from fetal and adult chicken muscle display distinct schedules of myosin heavy-chain isoform expression in culture. We further showed that myoblasts isolated from newly hatched and young chickens already possess the adult myoblast phenotype. In this article, we report on the use of polyclonal antibodies against the chicken myogenic regulatory factor proteins MyoD and myogenin for monitoring fetal and adult chicken myoblasts as they progress from proliferation to differentiation in culture. Fetal-type myoblasts were isolated from 11-day-old embryos and adult-type myoblasts were isolated from 3-week-old chickens. We conclude that fetal myoblasts express both MyoD and myogenin within the first day in culture and rapidly transit into the differentiated myosin-expressing state. In contrast, adult myoblasts are essentially negative for MyoD and myogenin by culture Day 1 and subsequently express first MyoD and then myogenin before expressing sarcomeric myosin. The delayed MyoD-to-myogenin transition in adult myoblasts is accompanied by a lag in the fusion into myotubes, compared to fetal myoblasts. We also report on the use of a commercial antibody against the myocyte enhancer factor 2A (MEF2A) to detect terminally differentiated chicken myoblasts by their MEF2+ nuclei. Collectively, the results support the hypothesis that fetal and adult myoblasts represent different phenotypic populations. The fetal myoblasts may already be destined for terminal differentiation at the time of their isolation, and the adult myoblasts may represent progenitors that reside in an earlier compartment of the myogenic lineage. (J Histochem Cytochem 49:455462, 2001)
Key Words: chicken, fetal myoblasts, adult myoblasts, satellite cells, MyoD, myogenin, MEF2A
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Introduction |
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THROUGHOUT SKELETAL MUSCLE DEVELOPMENT the myogenic precursor cells undergo proliferation and cell cycle withdrawal and differentiation, followed by fusion into multinucleated myofibers. The addition of myogenic cells to existing myofibers continues during the postnatal growth of muscle, allowing the enlargement of myofibers. This process slows as the animal matures, so that little or no myoblast proliferation and fusion into myofibers occurs in the adult. The process of myoblast proliferation and fusion into existing or newly formed myofibers can be initiated in the adult after various kinds of injuries, ranging from major trauma to more subtle stresses such as exercise and stretch. The source of myogenic cells during postnatal muscle growth and regeneration is the satellite cells, situated underneath the myofiber basement membrane. Individual myofibers become encased by a basement membrane during late stages of embryogenesis. It is at that stage that distinction of satellite cells by their morphology and location is first possible (reviewed in
Studies of avian and mammalian myoblasts have indicated that the myogenic precursors present in developing and mature limb and body wall muscles can be divided into distinct populations on the basis of specific morphological and biochemical traits of the cells identified in culture. These myogenic populations were broadly defined as embryonic myoblasts (appear earlier in embryogenesis), fetal myoblasts (appear later in embryogenesis), and adult myoblasts (satellite cells, present in postnatal and adult muscle) (reviewed in
In our previous studies, the distinctions between adult and fetal chicken myoblasts were based on the detection of different schedules of myosin isoform expression during differentiation in cultures of isolated cells. Myoblasts displaying the adult phenotype were already present in the muscle during late stages of embryonic development (
Chicken homologues of all four mammalian MRFs have been described (
Here we report on the use of specific antibodies raised against chicken MyoD and myogenin for the analysis of myogenesis in cultured fetal and adult chicken myoblasts. We also report on the utilization of a commercially available antibody raised against the human MEF2A for tracing nuclei of differentiating myoblasts and myotubes in chicken myogenic cultures. MEF2A is a member of the myocyte enhancer factor 2 family of transcription factors. Members of the MEF2 and MRF families act cooperatively during the activation of muscle-specific genes (
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Materials and Methods |
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Isolation and Culture of Fetal and Adult Chicken Myoblasts
Fetal and adult myoblasts were isolated from the pectoralis muscle of 11-day-old embryos and 3-week-old chickens, respectively. The source of the embryonated eggs and post-hatch chickens (White Leghorn) was detailed previously (
Primary Antibodies
The antibodies against chicken MyoD and myogenin were generated by immunizing rabbits with the hexahistidine (6His)-tagged full-sized chicken proteins made in E. coli. Proteins eluted from NiTA resin in 6.4 M urea/200 mM histidine, pH 7.4, were mixed with adjuvant and injected subdermally every 23 weeks until the titer was satisfactory. The specificity of the antibodies was originally tested by immunoprecipitation of proteins made in the reticulocyte lysate cell-free translation system (
The rabbit polyclonal antibody against MEF2A was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The use of this antibody to detect MEF2A in extracts of C2 cultures via immunoblotting was described in
The monoclonal antibody against sarcomeric myosin (MAb MF20;
Immunolabeling of Cultured Cells
Single- and double-immunofluorescence analyses of methanol-fixed cultures were performed as previously described (
Quantification of Positive Cells
Quantification of the number of cells that fused into myotubes and the number of mononucleated cells reacting with the different antibodies was performed by monitoring a minimum of 10 arbitrary fields per culture plate using a x40 objective. In some instances, indicated in the Results, more than 10 fields were monitored to ensure that at least 500 DAPI-stained nuclei were analyzed per plate. The values of DAPI-stained nuclei included nuclei in mononucleated cells and myotubes. Unless otherwise noted, the frequency of nuclei in myotubes or of mononucleated cells positive for different antibodies was first determined for each plate and then averaged for the parallel plates, as shown in Results. Error values reflect the range of variation between samples when duplicate cultures were analyzed and the SD when triplicate plates were analyzed.
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Results |
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Immunostaining of Cultured Cells with Antibodies Against MyoD, Myogenin, and Myosin
Fig 1 shows micrographs of myogenic cultures from 11-day-old chicken embryos, stained via double immunofluorescence with the polyclonal antibody against MyoD (Fig 1A) or the polyclonal antibody against myogenin (Fig 1B), along with the monoclonal antibody against myosin (Fig 1A' and 1B', respectively). Parallel micrographs of DAPI-stained nuclei are shown in Fig 1A'' and B'', respectively. Micrographs are shown for culture Day 3 when both single cells and multinucleated myotubes are present. The nuclei in some of the mononucleated cells and in all myotubes are positive for MyoD and myogenin.
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Distribution of Cells Expressing MyoD and Myogenin in Fetal Cultures
The quantification of MyoD+ and myogenin+ cells in myogenic cultures from 11-day-old chicken embryos is summarized in Table 1. The cultures, collected every 24 hr, were scored for the total number of cells, the number of cells that fused into myotubes (nuclei in myotubes), and the number of mononucleated cells labeled with each antibody. By Day 1 the cultures were essentially devoid of myotubes and about 9% of the mononucleated cells were already positive for MyoD or myogenin. By Day 2 there was a robust increase in the number of cells that fused into myotubes (2531% of total cells) and this parameter further increased by Day 3 (4756% of total cells). As in Day 1 cultures, the number of mononucleated cells positive for MyoD or myogenin was similar in Day 2 cultures (19% and 18%, respectively) and in Day 3 cultures (11% and 14%, respectively). A double-staining analysis with the MAb against myosin in combination with the anti-MyoD or anti-myogenin Ab demonstrated that most of these MyoD+ or myogenin+ mononucleated cells were negative for myosin (Table 2).
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Distribution of Cells Expressing MyoD and Myogenin in Adult Cultures
The quantification of MyoD+ and myogenin+ cells in myogenic cultures from 3-week-old chickens is summarized in Table 3. The cultures were co-reacted with the anti-MyoD or anti-myogenin Ab along with the MAb against myosin. The immunostaining images of positive cells were identical to those shown in Fig 1 for the fetal cultures. The cultures, collected every 24 hr, were scored for the total number of cells, the number of cells that fused into myotubes, and the number of mononucleated cells that were singly or doubly labeled with the antibodies. By Day 1 the cultures were essentially devoid of myotubes or mononucleated cells positive for any of the antibodies examined. By Day 2 the cultures demonstrated a significant increase in the number of MyoD+ or myogenin+ mononucleated cells. These positive cells were mostly negative for myosin, indicating that they had not yet completed the differentiation process. Furthermore, by culture Day 2 there was a higher number of MyoD+/myosin- cells compared to myogenin+/myosin- cells (37% vs 21%, respectively), indicating that the cells first become positive for MyoD and then for myogenin. The Day 2 cultures were still devoid of myotubes but some myosin+ cells began to emerge, adding up to about 3% of the total cells. By culture Day 3 there was a robust increase in the number of cells that fused into myotubes, but a pool of MyoD+/myosin- or myogenin+/myosin- mononucleated cells was still evident in the Day 3 cultures. The number of all myosin+ mononucleated cells in the Day 3 cultures was low and was similar to that present in Day 2 cultures. As in the fetal cultures, the majority of the MyoD+ or myogenin+ cells in the adult cultures were negative for myosin.
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Distribution of MEF2A+ Cells in Chicken Myogenic Cultures
Fig 2 shows micrographs of myogenic cultures from 11-day-old chicken embryos stained via double immunofluorescence with the polyclonal antibody against MEF2A along with the monoclonal antibody against myosin. Micrographs are shown for culture Day 3 when both single cells and multinucleated myotubes are present. MEF2A+ nuclei were typically seen in myosin+ mononucleated cells and in all myotubes. The double immunostaining images of positive cells in cultures of adult myoblasts were the same as those shown in Fig 2.
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Discussion |
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Generation of antibodies against chicken MyoD and myogenin has enabled us to characterize the adult and fetal myoblasts before they fully differentiated into cells expressing sarcomeric myosin. Compared to cultures of fetal myoblasts that displayed a rapid expression of MyoD and myogenin combined with a rapid fusion into myotubes, adult cultures displayed a lag in MyoD and myogenin expression combined with a delayed fusion into myotubes. Furthermore, the MyoD+ or myogenin+ cells appeared at a similar frequency at each time point analyzed in the fetal cultures, suggesting that the fetal myoblasts express both MRF proteins. On the other hand, in the adult cultures there was a distinct lag in the onset of expression of myogenin compared to MyoD, indicating that the cells first express MyoD and then myogenin. In concordance with earlier studies discussed below, we interpret the findings on the different kinetics of MyoD+ and myogenin+ cells by cultured adult and fetal myoblasts as evidence for the different states of adult and fetal myoblasts along the myogenic lineage.
Clonal analyses of individual chicken myoblasts have suggested that the majority of the fetal myoblasts are in a "committed cell compartment" destined to divide only several times before terminal differentiation. In addition, a small proportion of the myoblasts from this fetal stage was identified as stem cells. These so-called stem cells undergo multiple cell divisions, able to produce self-renewed stem cells along with committed cells (
The present analysis has identified a molecular distinction between fetal and adult chicken myoblasts that is displayed by the cells even before they enter the phase of differentiation and distinct expression of muscle-specific sarcomeric myosins previously reported (
We also demonstrate in the present study that a commercial antibody against human MEF2A protein reacts with differentiated chicken myoblasts. The pattern of MEF2A expression in myosin+ chicken myoblasts and myotubes is identical to its temporal expression pattern in rodent myoblasts (
In conclusion, the availability of suitable antibodies has enabled us to demonstrate that the transition along the MyoDmyogeninMEF2A pathway in adult chicken myoblasts follows the same protein expression program as seen in mouse and rat satellite cells (
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Acknowledgments |
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ZY-R was supported by grants from the National Institutes of Health (AG13798), the Cooperative State Research ServiceUnited States Department of Agriculture (Agreement No. 99-35206-7934) and the United StatesIsrael Binational Agricultural Research and Development Fund (Agreement No. IS-3093-99).
We thank Priscilla Natanson and Anthony Rivera for excellent technical support.
Received for publication November 1, 2000; accepted December 13, 2000.
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