Satellite cells express distinct patterns of myogenic proteins in immature skeletal muscle (original) (raw)
Related papers
Animal Science Journal, 2002
Myogenic regulatory factors (MRFs) are important in the control of skeletal muscle development. To understand myogenic regulation by MRFs in bovine adult muscle cells, their expressions, namely that of Myf5, MyoD, myogenin, and MRF4 in the biceps femoris muscle (BF) and in the satellite cell culture, were analyzed by RT-PCR. In the BF, all four MRFs were expressed and in particular, myogenin and MRF4 were strongly expressed, whereas Myf5 was faintly expressed. The satellite cells prepared from the BF expressed Myf5, but only a trace of MyoD, at day 9 of culture. During the growth of the cells to day 14, the MyoD and myogenin expressions gradually increased, and that of MyoD expression reached its maximum at the confluence of the culture. After induction of myogenic differentiation by a serum-free medium at day 14, Myf5 expression gradually decreased, and the up-regulated expression of MyoD was suppressed, whereas myogenin expression continued to increase sharply. Following the myogenin expression, MRF4 also drastically increased toward the myotube formation of the cells. When huge myotubes were formed at day 18, Myf5 was expressed at a low level, whereas the MyoD expression remained at a moderate level.
Developmental Biology, 1994
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 differentiationspecific, 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, α-smooth muscle actin (αSMactin), or developmental sarcomeric myosin (DEVmyosin) appeared following the decline in PCNA and MyoD expression. However, expression of myogenin and αSMactin was transient, while DEVmyosin expression was continuously maintained. Moreover, the number of DEVmyosin+ cells was only half of the number of myogenin+ or αSMactin+ 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.
Muscle satellite cell-specific genes identified by genetic profiling of MyoD-deficient myogenic cell
Developmental Biology, 2004
Satellite cells are committed myogenic progenitors that give rise to proliferating myoblasts during postnatal growth and repair of skeletal muscle. To identify genes expressed at different developmental stages in the satellite cell myogenic program, representational difference analysis of cDNAs was employed to identify more than 50 unique mRNAs expressed in wild-type myoblasts and MyoD À/À myogenic cells. Novel expression patterns for several genes, such as Pax7, Asb5, IgSF4, and Hoxc10, were identified that were expressed in both quiescent and activated satellite cells. Several previously uncharacterized genes that represent putative MyoD target genes were also identified, including Pw1, Dapk2, Sytl2, and NLRR1. Importantly, many genes such as IgSF4, Neuritin, and Klra18 that were expressed exclusively in MyoD À/À myoblasts were also expressed by satellite cells in undamaged muscle in vivo but were not expressed by primary myoblasts. These data are consistent with a biological role for activated satellite cells that induce Myf5 but not MyoD. Lastly, additional endothelial and hematopoietic markers were identified supporting a nonsomitic developmental origin of the satellite cell myogenic lineage.
Journal of Muscle Research and Cell Motility, 1995
We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch musde; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous rnyotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.
Cell differentiation, 1980
Satellite cells were isolated from skeletal muscles of adult normal and dystrophic mice (C57/6J/dy strain) by sequential digestion of tissue fragments with collagenase, hyaluronidase and trypsin. These cells exhibit in culture similar behaviour to that of embryonic myoblasts, undergoing an initial duplicative period lasting about 2--3 days, followed by a shorter phase (1--2 days) of rapid cell fusion. During the duplicative phase most of the satellite cells appear round-shaped, whereas embryonic myoblasts appear typically spindle-shaped: both cell types actively incorporate [3H]thymidine. During the subsequent days of culture an increasing number of satellite cells becomes spindle-shaped; afterwards the cells contact each other and fuse into multinucleated myotubes. The majority of spindle-shaped satellite cells is unable to incorporate [3H]thymidine, thus behaving as post-mitotic cells. Concomitantly with satellite cell fusion, an increase of about 80-fold of creatine phosphokinase...
Differentiation, 1994
Cells of the embryonic mesenchymal cell line C3HlOT1/2 have revealed the potential that the four regulatory factors belonging to the MyoD family have to activate myogenesis. In the present study we have further investigated the myogenic phenotype of C3H10T1/2 cells stably transfected with either Myf5, MyoD, myogenin or MRF4 cDNAs. We have studied the influence of each transfected cDNA on expression of the four endogenous muscle regulatory genes and on the ability of these embryonic myogenic derivatives to express adult muscle genes. No trace of endogenous transcripts distinct from the exogenous one was found in any of the four converted populations at the myoblast stage. This indicates that cross-activation within the MyoD family does not occur at the myoblast stage in these cells. Similarly, evidence was obtained that auto-or cross-activation of the MyfS gene occurs neither at the myoblast stage nor at the myotube stage and that no autoactivation of the MRF4 gene occurs. Our results together with previous observations indicate that in C3H 10T1/2 myogenic derivatives: (1) Autoactivation at the myoblast stage is restricted to MyoD (2) Expression from each cDNA alone is sufficient to establish and maintain the myoblast phenotype (3) The endogenous MyfS gene is not mobilized. We have also observed that endogenous transcripts for MyoD and myogenin begin to accumulate at the onset of differentiation in the four myogenic derivatives, whereas accumulation of endogenous MRF4 transcripts starts after myotubes have formed and occurs at a much lower level (100-to 500-fold lower) than in differentiated cultures of myosatellite cells. However, neither this low level of MRF4 transcripts nor higher levels from the transfected MRF4 cDNA affected (prevented or stimulated) the accumulation of dystrophin transcripts or of adult muscle-gene transcripts (e.g., myosin heavy chain IIB, acetylcholine receptor &-subunit and M form of aldolase A), which occurred at similar levels in the four myogenic derivatives: Thus, despite the fact that MRF4 gene ex
Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells
Basic Cell Culture Protocols
Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.
Expression of MyoD1 coincides with terminal differentiation in determined but inducible muscle cells
EMBO Journal, 1989
We have examined the expression of MyoD1, a potential determination factor of myogenic cells, in permissive and inducible C2 myoblasts. These two types of myoblasts exhibit distinct requirements to undergo terminal differentiation. Unlike permissive cells, inducible cells fail to differentiate in the presence of growth medium plus fetal calf serum and require insulin to undergo terminal differentiation. We show that while expression of MyoD1 is constitutive in permissive cells, no trace of MyoD1 transcripts is found in inducible cells at the myoblast stage. In these cells, however, expression of MyoD1 accompanies differentiation. This indicates that MyoD1 may not be required for the maintenance of the myoblast phenotype, and could act as an effector of terminal differentiation in already determined muscle cells. Our results provide new evidence that permissive and inducible cells represent two distinct stages of the progression of determined muscle cells toward terminal differentiat...