Regulation of proliferation and differentiation of myoblasts derived from adult mouse skeletal muscle by specific isoforms of PDGF.

The expression of receptors and the mitogenic response to PDGF by C2 myoblasts, derived from adult mouse skeletal muscle, was investigated. Employing 125I-PDGF binding assays, we showed that the cells exhibit high level binding of PDGF-BB (approximately 165 x 10(3) molecules/cell at saturation) and much lower binding of the PDGF-AA and PDGF-AB (6-12 x 10(3) molecules/cell at saturation). This indicates that the C2 myoblasts express high levels of PDGF receptor beta-subunits and low levels of alpha-subunits. PDGF-BB enhances the proliferation of C2 cells maintained in 2% FCS by about fivefold. PDGF-AB had a moderate effect on cell proliferation (less than twofold) and PDGF-AA had no effect. Inverse effects of PDGF isoforms on the frequency of differentiated myoblasts were observed; the frequency of myosin-positive cells was reduced in the presence of PDGF-BB while PDGF-AA and PDGF-AB had no effect. PDGF may thus act to increase the number of myoblasts that participate in muscle regeneration following muscle trauma by stimulating the proliferation and by inhibiting the differentiation of myogenic cells.

Exercise running and tetracycline as means to enhance skeletal muscle stem cell performance after external fixation.

Prolonged limb immobilization, which is often the outcome of injury and illness, results in the atrophy of skeletal muscles. The basis of muscle atrophy needs to be better understood in order to allow development of effective countermeasures. The present study focused on determining whether skeletal muscle stem cells, satellite cells, are directly affected by long-term immobilization as well as on investigating the potential of pharmacological and physiological avenues to counterbalance atrophy-induced muscle deterioration. We used external fixation (EF), as a clinically relevant model, to gain insights into the relationships between muscle degenerative and regenerative conditions to the myogenic properties and abundance of bona fide satellite cells. Rats were treated with tetracycline (Tet) through the EF period, or exercise trained on a treadmill for 2 weeks after the cessation of the atrophic stimulus. EF induced muscle mass loss; declined expression of the muscle specific regulatory factors (MRFs) Myf5, MyoD, myogenin, and also of satellite cell numbers and myogenic differentiation aptitude. Tet enhanced the expression of MRFs, but did not prevent the decline of the satellite cell pool. After exercise running, however, muscle mass, satellite cell numbers (enumerated through the entire length of myofibers), and myogenic differentiation aptitude (determined by the lineal identity of clonal cultures of satellite cells) were re-gained to levels prior to EF. Together, our results point to Tet and exercise running as promising and relevant approaches for enhancing muscle recovery after atrophy.

Allograft inflammatory factor-1 is expressed by macrophages in injured skeletal muscle and abrogates proliferation and differentiation of satellite cells.

Secretion of regulatory peptides by macrophages in injured skeletal muscle constitutes a pivotal determinator of tissue homeostasis. We analyzed expression of a novel Ca2+- binding peptide expressed by activated macrophages, the allograft inflammatory factor-1 (AIF-1), in rat devascularized skeletal muscle. AIF-1 expression was observed in 94% of all macrophages at the site of the injury 48 hours postdevascularization. The physiological function of AIF-1 in injured skeletal muscle was analyzed using a rat in-vitro model of satellite cell proliferation and differentiation. Addition of AIF-1 to the culture medium resulted in a concentration-dependent and reversible reduction of the total number of cells expressing M-cadherin (p < or = 0.0001), a mediator of the differentiation process of skeletal muscle cells, the proliferation associated PCNA (p < or = 0.0001), and the initiator of muscle differentiation myogenin (p < or = 0.0001). These results provide convincing evidence that activated AIF-1 expressing macrophages constitute the predominant cell type in skeletal muscle 48 hours postinjury, and that AIF-1 regulates reduced proliferation, differentiation, and activation of satellite cells.

MyoD and myogenin expression patterns in cultures of fetal and adult chicken myoblasts.

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.

Vascular smooth muscle cells spontaneously adopt a skeletal muscle phenotype: a unique Myf5(-)/MyoD(+) myogenic program.

Smooth and skeletal muscle tissues are composed of distinct cell types that express related but distinct isoforms of the structural genes used for contraction. These two muscle cell types are also believed to have distinct embryological origins. Nevertheless, the phenomenon of a phenotypic switch from smooth to skeletal muscle has been demonstrated in several in vivo studies. This switch has been minimally analyzed at the cellular level, and the mechanism driving it is unknown. We used immunofluorescence and RT-PCR to demonstrate the expression of the skeletal muscle-specific regulatory genes MyoD and myogenin, and of several skeletal muscle-specific structural genes in cultures of the established rat smooth muscle cell lines PAC1, A10, and A7r5. The skeletal muscle regulatory gene Myf5 was not detected in these three cell lines. We further isolated clonal sublines from PAC1 cultures that homogeneously express smooth muscle characteristics at low density and undergo a coordinated increase in skeletal muscle-specific gene expression at high density. In some of these PAC1 sublines, this process culminates in the high-frequency formation of myotubes. As in the PAC1 parental line, Myf5 was not expressed in the PAC1 sublines. We show that the PAC1 sublines that undergo a more robust transition into the skeletal muscle phenotype also express significantly higher levels of the insulin-like growth factor (IGF1 and IGF2) genes and of FGF receptor 4 (FGFR4) gene. Our results suggest that MyoD expression in itself is not a sufficient condition to promote a coordinated program of skeletal myogenesis in the smooth muscle cells. Insulin administered at a high concentration to PAC1 cell populations with a poor capacity to undergo skeletal muscle differentiation enhances the number of cells displaying the skeletal muscle differentiated phenotype. The findings raise the possibility that the IGF signaling system is involved in the phenotypic switch from smooth to skeletal muscle. The gene expression program described here can now be used to investigate the mechanisms that may underlie the propensity of certain smooth muscle cells to adopt a skeletal muscle identity.(J Histochem Cytochem 48:1173-1193, 2000)

Satellite cells on isolated myofibers from normal and denervated adult rat muscle.

Satellite cells (SCs) in normal adult muscle are quiescent. They can enter the mitotic program when stimulated with growth factors such as basic FGF. Short-term denervation stimulates SC to enter the mitotic cycle in vivo, whereas long-term denervation depletes the SC pool. The molecular basis for the neural influence on SCs has not been established. We studied the phenotype and the proliferative capacity of SCs from muscle that had been denervated before being cultured in vitro. The expression of PCNA, myogenin, and muscle (M)-cadherin in SCs of normal and denervated muscle fibers was examined at the single-cell level by immunolabeling in a culture system of isolated rat muscle fibers with attached SCs. Immediately after plating (Day 0), neither PCNA nor myogenin was present on normal muscle fibers, but we detected an average of 0.5 M-cadherin(+) SCs per muscle fiber. The number of these M-cadherin(+) cells (which are negative for PCNA and myogenin) increased over the time course examined. A larger fraction of cells negative for M-cadherin underwent mitosis and expressed PCNA, followed by myogenin. The kinetics of SCs from muscle fibers denervated for 4 days before culturing were similar to those of normal controls. Denervation from 1 to 32 weeks before plating, however, suppressed PCNA and myogenin expression almost completely. The fraction of M-cadherin(+) (PCNA(-)/myogenin(-)) SCs was decreased after 1 week of denervation, increased above normal after denervation for 4 or 8 weeks, and decreased again after denervation for 16 or 32 weeks. We suggest that the M-cadherin(+) cells are nondividing SCs because they co-express neither PCNA or myogenin, whereas the cells positive for PCNA or myogenin (and negative for M-cadherin) have entered the mitotic cycle. SCs from denervated muscle were different from normal controls when denervated for 1 week or longer. The effect of denervation on the phenotypic modulation of SCs includes resistance to recruitment into the mitotic cycle under the conditions studied here and a robust extension of the nonproliferative compartment. These characteristics of SCs deprived of neural influence may account for the failure of denervated muscle to fully regenerate. (J Histochem Cytochem 47:1375-1383, 1999)

Fibroblast growth factor promotes recruitment of skeletal muscle satellite cells in young and old rats.

Although the role of satellite cells in muscle growth and repair is well recognized, understanding of the molecular events that accompany their activation and proliferation is limited. In this study, we used the single myofiber culture model for comparing the proliferative dynamics of satellite cells from growing (3-week-old), young adult (8- to 10-week-old), and old (9- to 11-month-old) rats. In these fiber cultures, the satellite cells are maintained in their in situ position underneath the fiber basement membrane. We first demonstrate that the cytoplasm of fiber-associated satellite cells can be monitored with an antibody against the extracellular signal regulated kinases 1 and 2 (ERK1 and ERK2), which belong to the mitogen-activated protein kinase (MAPK) superfamily. With this immunocytological marker, we show that the satellite cells from all three age groups first proliferate and express PCNA and MyoD, and subsequently, about 24 hr later, exit the PCNA+/MyoD+ state and become positive for myogenin. For all three age groups, fibroblast growth factor 2 (FGF2) enhances by about twofold the number of satellite cells that are capable of proliferation, as determined by monitoring the number of cells that transit from the MAPK+ phenotype to the PCNA+/MAPK+ or MyoD+/MAPK+ phenotype. Furthermore, contrary to the commonly accepted convention, we show that in the fiber cultures FGF2 does not suppress the subsequent transition of the proliferating cells into the myogenin+ compartment. Although myogenesis of satellite cells from growing, young adult, and old rats follows a similar program, two distinctive features were identified for satellite cells in fiber cultures from the old rats. First, a large number of MAPK+ cells do not appear to enter the MyoD-myogenin expression program. Second, the maximal number of proliferating satellite cells is attained a day later than in cultures from the young adults. This apparent "lag" in proliferation was not affected by hepatocyte growth factor (HGF), which has been implicated in accelerating the first round of satellite cell proliferation. HGF and FGF2 were equally efficient in promoting proliferation of satellite cells in fibers from old rats. Collectively, the investigation suggests that FGF plays a critical role in the recruitment of satellite cells into proliferation.

Gene expression patterns of the fibroblast growth factors and their receptors during myogenesis of rat satellite cells.

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 (FGFR1-FGFR4) 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.

Levels of MyoD protein expression following injury of mdx and normal limb muscle are modified by thyroid hormone.

Thyroid hormone (T3) affects muscle development and muscle regeneration. It also interacts with the muscle regulatory gene MyoD in culture and affects myoblast proliferation. We studied the localization of MyoD protein using a well-characterized polyclonal antibody for immunohistochemistry. Relative numbers of myogenic precursor cells per field were identified by their MyoD expression during muscle regeneration in normal and mdx dystrophic mice, with particular reference to the expression in mononuclear cells and myotubes at various T3 levels. In regeneration by normal muscles, relatively few MyoD+ nuclei per field were present in mononuclear cells of euthyroid and hypothyroid mice. MyoD staining of mononuclear cell nuclei was approximately doubled in fields of regenerating muscles of normal hyperthyroid compared to euthyroid mice, and was observed in precursors that appeared to be aligned before fusion into myotubes. In mdx regenerating muscle, twofold more mononuclear cells positive for MyoD were present in all three treatment groups compared to normal muscles regenerating under the same conditions. Localization was similar to the pattern in normal euthyroid mice. However, in muscles regenerating in hyperthyroid mdx mice, both mononuclear cell nuclei and centrally located nuclei in a subpopulation (about 15%) of new myotubes formed after the crush injury were intensely stained for MyoD protein. The changes observed are consistent with reports on T3-induced alteration of muscle repair, and propose a link between MyoD regulation and the accelerated differentiation during regeneration under high T3 conditions. (J Histochem Cytochem 46:59-67, 1998)

Development and postnatal regulation of adult myoblasts.

The myogenic precursor cells of postnatal and adult skeletal muscle are situated underneath the basement membrane of the myofibers. It is because of their unique positions that these precursor cells are often referred to as satellite cells. Such defined satellite cells can first be detected following the formation of a distinct basement membrane around the fiber, which takes place in late stages of embryogenesis. Like myoblasts found during development, satellite cells can proliferate, differentiate, and fuse into myofibers. However, in the normal, uninjured adult muscle, satellite cells are mitotically quiescent. In recent years several important questions concerning the biology of satellite cells have been asked. One aspect has been the relationship between satellite cells and myoblasts found in the developing muscle: are these myogenic populations identical or different? Another aspect has been the physiological cues that control the quiescent, proliferative, and differentiative states of these myogenic precursors: what are the growth regulators and how do they function? These issues are discussed, referring to previous work by others and further emphasizing our own studies on avian and rodent satellite cells. Collectively, the studies presented indicate that satellite cells represent a distinct myogenic population that becomes dominant in late stages of embryogenesis. Moreover, although satellite cells are already destined to be myogenic precursors, they do not express any of the four known myogenic regulatory genes unless their activation is induced in the animal or in culture. Furthermore, multiple growth factors are important regulators of satellite cell proliferation and differentiation. Our work on the role of one of these growth factors [platelet-derived growth factor (PDGF)] during proliferation of adult myoblasts is further discussed with greater detail and the possibility that PDGF is involved in the transition from fetal to adult myoblasts in late embryogenesis is brought forward.

Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program.

Whereas the understanding of the mechanisms underlying skeletal and cardiac muscle development has been increased dramatically in recent years, the understanding of smooth muscle development is still in its infancy. This paper summarizes studies on the ontogeny of chicken smooth muscle cells in the wall of the aorta and aortic arch-derived arteries. Employing immunocytochemistry with antibodies against smooth muscle contractile and extracellular matrix proteins we trace smooth muscle cell patterning from early development throughout adulthood. Comparing late stage embryos to young and adult chickens we demonstrate, for all the stages analyzed, that the cells in the media of aortic arch-derived arteries and of the thoracic aorta are organized in alternating lamellae. The lamellar cells, but not the interlamellar cells, express smooth muscle specific contractile proteins and are surrounded by basement membrane proteins. This smooth muscle cell organization of lamellar and interlamellar cells is fully acquired by embryonic day 11 (ED 11). We further show that, during earlier stages of embryogenesis (ED3 through ED7), cells expressing smooth muscle proteins appear only in the peri-endothelial region of the aortic and aortic arch wall and are organized as a narrow band of cells that does not demonstrate the lamellar-interlamellar pattern. On ED9, infrequent cells organized in lamellar-interlamellar organization can be detected and their frequency increases by ED10. In addition to changes in cell organization, we show that there is a characteristic sequence of contractile and extracellular matrix protein expression during development of the aortic wall. At ED3 the peri-endothelial band of differentiated smooth muscle cells is already positive for smooth muscle alpha actin (alphaSM-actin) and fibronectin. By the next embryonic day the peri-endothelial cell layer is also positive for smooth muscle myosin light chain kinase (SM-MLCK). Subsequently, by ED5 this peri-endothelial band of differentiated smooth muscle cells is positive for alphaSM-actin, SM-MLCK, SM-calponin, fibronectin, and collagen type IV. However, laminin and desmin (characteristic basement membrane and contractile proteins of smooth muscle) are first seen only at the onset of the lamellar-interlamellar cell organization (ED9 to ED10). We conclude that the development of chicken aortic smooth muscle involves transitions in cell organization and in expression of smooth muscle proteins until the adult-like phenotype is achieved by mid-embryogenesis. This detailed analysis of the ontogeny of chick aortic smooth muscle should provide a sound basis for future studies on the regulatory mechanisms underlying vascular smooth muscle development.

Defining the transcriptional signature of skeletal muscle stem cells.

Satellite cells, the main source of myoblasts in postnatal muscle, are located beneath the myofiber basal lamina. The myogenic potential of satellite cells was initially documented based on their capacity to produce progeny that fused into myotubes. More recently, molecular markers of resident satellite cells were identified, further contributing to defining these cells as myogenic stem cells that produce differentiating progeny and self-renew. Herein, we discuss aspects of the satellite cell transcriptional milieu that have been intensively investigated in our research. We elaborate on the expression patterns of the paired box (Pax) transcription factors Pax3 and Pax7, and on the myogenic regulatory factors myogenic factor 5 (Myf5), myogenic determination factor 1 (MyoD), and myogenin. We also introduce original data on MyoD upregulation in newly activated satellite cells, which precedes the first round of cell proliferation. Such MyoD upregulation occurred even when parent myofibers with their associated satellite cells were exposed to pharmacological inhibitors of hepatocyte growth factor and fibroblast growth factor receptors, which are typically involved in promoting satellite cell proliferation. These observations support the hypothesis that most satellite cells in adult muscle are committed to rapidly entering myogenesis. We also detected expression of serum response factor in resident satellite cells prior to MyoD expression, which may facilitate the rapid upregulation of MyoD. Aspects of satellite cell self-renewal based on the reemergence of cells expressing Pax7, but not MyoD, in myogenic cultures are discussed further herein. We conclude by describing our recent studies using transgenic mice in which satellite cells are traced and isolated based on their expression of green fluorescence protein driven by regulatory elements of the nestin promoter (nestin-green fluorescence protein). This feature provides us with a novel means of studying satellite cell transcriptional signatures, heterogeneity among muscle groups, and the role of the myogenic niche in directing satellite cell self-renewal.

Discrimination of myogenic and nonmyogenic cells from embryonic skeletal muscle by 90 degrees light scattering.

A myogenic cell suspension was isolated from the breast muscles of 10-day-old chicken embryos by trypsin digestion. The cell preparation was subjected to Percoll density centrifugation to reduce the number of fibroblast-like cells present. The Percoll-isolated, predominantly myogenic cell population was then fractionated by flow cytometry using 90 degrees light scattering as the parameter for sorting. Cells exhibiting lower scatter, with a peak of 45 units, produced cultures containing myotubes and gave rise only to myogenic clones. Cells exhibiting higher scatter (120-200 units) produced nonmyogenic cultures and gave rise to nonmyogenic clones. Cells with intermediate light scatter were also detected. The latter produced both myogenic and nonmyogenic clones. The differences in light scatter presumably reflect higher cytoplasmic complexity of the nonmyogenic cells compared with the myogenic cells. Moreover, the differences in light scattering properties of the different cell types offer a means for the isolation of pure populations of myogenic cells directly from the intact muscle.

Patterns of proliferation and differentiation of adult myoblasts define a unique myogenic population.

In here we summarize some of our recent studies on adult chicken myoblasts. We discuss a) myosin isoform expression by the differentiated myoblasts, and b) the role of PDGF during proliferation of the cells. These studies demonstrate that adult myoblasts represent a unique myogenic population (or lineage) which emerges in late development. Furthermore, we discuss possible lineal relationship between adult and somitic/limb bud cells. Unless otherwise noted, the source of myoblasts was chicken pectoralis muscle, and all procedures and antibodies were as described in previous publications (Yablonka-Reuveni, et al., 1987; Yablonka-Reuveni and Seifert, submitted; Hartley, et al., 1991; 1992).

The emergence of the endothelial cell lineage in the chick embryo can be detected by uptake of acetylated low density lipoprotein and the presence of a von Willebrand-like factor.

Vascular endothelial cells from 3- to 10-day-old chicken embryos were identified by the uptake of acetylated low density lipoprotein (Ac-LDL) and the presence of a von Willebrand-like factor. These were determined on cross sections of aortic arches as well as in cell cultures prepared from the arches. To visualize the uptake of Ac-LDL, the fluorescent probe 1,1'-dioctadecyl-1-3,3,3',3'-tetramethyl-indo-carbocyanine perchlorate-Ac-LDL (DiI-Ac-LDL) was used. Following injection of the DiI-Ac-LDL probe into the embryonic heart, the endothelium of the aortic arches became specifically labeled. Also, following the administration of the probe to cell cultures, about 5-10% of the cells became DiI-positive. Indirect immunofluorescence with an antibody against von Willebrand (vW) factor also revealed specific staining of the endothelium of the aortic arches as well as of a subset of cells in cultures from aortic arches. These two histochemical markers were further used to identify the emergence of the endothelial cell lineage in the chicken blastodisc. Cultured cells from embryos incubated in ovo for 16 hr exhibited both uptake of DiI-Ac-LDL and expression of a vW-like factor. The proportion of these cells was about 30% of the total cultured cells and increased to over 50% in cultures of embryos incubated in ovo for 20 hr. However, cells positive for uptake of DiI-Ac-LDL and expression of vW-like factor were lacking in cultures of unincubated eggs or eggs incubated for 6-10 hr. We conclude that the very early endothelial cells in the chick blastodisc are already capable of expressing characteristic properties of vascular endothelium.

Proliferation of chicken myoblasts is regulated by specific isoforms of platelet-derived growth factor: evidence for differences between myoblasts from mid and late stages of embryogenesis.

In the present study we measured the level of PDGF receptor expression by chicken myoblasts and the effect of the three different PDGF isoforms (AA, AB, BB) on DNA synthesis by myoblasts. We examined PDGF receptor expression and function on clonally derived myoblasts in order to eliminate contaminating fibroblasts which are present in myogenic cultures and which bind PDGF. Furthermore, since we have previously shown that fetal myoblasts are replaced with adult myoblasts during late chicken embryogenesis, we compared PDGF receptor expression and function on myoblasts from Embryonic Day 10 (E10, mid development) and from Embryonic Day 19 (E19, late development). We found that all myogenic clones from late embryos (E19) express many receptors for PDGF-BB, far fewer receptors for PDGF-AB, and even fewer, if any, receptors for PDGF-AA. Myoblast clones derived from E10 were more heterogeneous in their PDGF binding pattern ranging from clones similar to E19 clones to clones having very few PDGF binding sites. We also found that both PDGF-AB and PDGF-BB can promote DNA synthesis by clonally derived chicken myoblasts maintained in 2.5% fetal bovine serum whereas PDGF-AA has no detectable effect. Finally, we observed that primary myogenic cultures from E10 and E19 differ strikingly in levels of PDGF binding; E19 cultures bind much more PDGF than do E10 cultures. We conclude that PDGF can enhance the proliferation of chicken myoblasts and that myoblasts responsive to PDGF are more frequent in late than in mid stages of development. We propose that PDGF may be a modulator of myogenesis of adult but not fetal myoblasts.

Long-term maintenance of primary myogenic cultures on a reconstituted basement membrane.

We describe a simple technique for maintaining highly contractile long-term chicken myogenic cultures on Matrigel, a gel composed of basement membrane components extracted from the Engelbreth-Holm-Swarm mouse tumor. Cultures grown on Matrigel consist of three-dimensional multilayers of cylindrical, contracting myotubes which endure for at least 60 d without myotube detachment. A Matrigel substrate increases the initial plating efficiency but does not effect cell proliferation. Large-scale differentiation in cultures maintained on Matrigel is delayed by 1 to 2 d, compared to cultures grown on gelatin-coated dishes. Long-term maintenance on Matrigel also results in increased expression of the neonatal and adult fast myosin heavy chain isoforms. Culturing of cells on a Matrigel substrate could thus facilitate the study of later events of in vitro myogenesis.

Temporal differences in desmin expression between myoblasts from embryonic and adult chicken skeletal muscle.

Desmin expression by myoblasts cultured from embryonic and adult chicken breast muscle was examined employing indirect immunofluorescence. The study was performed in conjunction with [3H]thymidine autoradiography and analysis of skeletal myosin expression in order to determine whether the desmin-expressing cells were terminally differentiated. Following 2 h of labeling with [3H]thymidine, 0.55%, 2.60%, and 15.10% of the cells in mass cultures from 10-day-old embryos, 18-day-old embryos and adults, respectively, incorporated [3H]thymidine and were desmin-positive but did not express skeletal-muscle-specific myosin. Using the same approach we determined that 0.07%, 1.25%, and 7.59% of the mononucleated cells in myogenic clones from 10-day-old embryos, 18-day-old embryos and adults, respectively, were desmin-positive, myosin-negative, [3H]thymidine-positive. We suggest that these desmin-positive, myosin-negative myoblasts are proliferating cells, and we conclude that the progeny of adult myoblasts exhibit more desmin-expressing cells of this type than embryonic myoblasts do.

Temporal expression of regulatory and structural muscle proteins during myogenesis of satellite cells on isolated adult rat fibers.

Myogenic precursors in adult skeletal muscle (satellite cells) are mitotically quiescent but can proliferate in response to a variety of stresses including muscle injury. To gain further understanding of adult myoblasts, we analyzed myogenesis of satellite cells on intact fibers isolated from adult rat muscle. In this culture model, satellite cells are maintained in their in situ position underneath the fiber basement membrane. In the present study patterns of satellite cell proliferation, expression of myogenic regulatory factor proteins, and expression of differentiation-specific, cytoskeletal proteins were determined, via immunohistochemistry of cultured fibers. The temporal appearance and the numbers of cells positive for proliferating cell nuclear antigen (PCNA) or for MyoD were similar, suggesting that MyoD is present in detectable amounts in proliferating but not quiescent satellite cells. Satellite cells positive for myogenin, alpha-smooth muscle actin (alpha SMactin), or developmental sarcomeric myosin (DEVmyosin) appeared following the decline in PCNA and MyoD expression. However, expression of myogenin and alpha SMactin was transient, while DEV-myosin expression was continuously maintained. Moreover, the number of DEVmyosin + cells was only half of the number of myogenin + or alpha SMactin + cells--indicating, perhaps, that only 50% of the satellite cell descendants entered the phase of terminal differentiation. We further determined that the number of proliferating satellite cells can be modulated by basic FGF but the overall schedule of cell cycle entry, proliferation, differentiation, and temporal expression of regulatory and structural proteins was unaffected. We thus conclude that satellite cells conform to a highly coordinated program when undergoing myogenesis at their native position along the muscle fiber.

Skeletal muscle cell populations. Separation and partial characterization of fibroblast-like cells from embryonic tissue using density centrifugation.

Cell suspensions from the breast muscles of 10-day old chicken embryos were separated into non-myogenic, fibroblast-like cell fractions and a mononucleated, myogenic cell fraction by Percoll density centrifugation. Isolated populations were characterized by their morphology in both mass cultures and individual macroscopic clones and by the immunocytochemical detection of skeletal muscle- and smooth muscle-specific proteins in individual cells. Cell populations were also characterized by their protein patterns using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The less dense, non-myogenic cells comprised 16% of the cells. In culture they were predominantly flattened, stellate cells and gave rise to clones lacking myotubes. These fibroblast-like cells were negative for skeletal muscle myosin or muscle type creatine phosphokinase. Less than 0.1% of these cells demonstrated strong fluorescence when stained with anti-desmin or anti-smooth muscle specific actin. This observation suggested that the vast majority of these cells were not related to vascular smooth muscle cells. Also, over 99% of the non-myogenic cells did not display characteristic properties of endothelial cells. The denser myogenic cell fraction comprised over 80% of the cells and in clonal cultures gave rise to about 70% myogenic clones. An additional 30% of clones from this fraction were non-myogenic indicating heterogeneity in this population. We conclude that Percoll centrifugation can be employed for the isolation of myogenic and non-myogenic cell populations directly from the embryonic muscle. Moreover, this procedure allows the direct analysis of cell-specific proteins (e.g., by gel electrophoresis) without the need for cell culturing. The results thus obtained closely reflect the status of the cells in the intact muscle.