Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4^+/−Six5^−/− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.     

Decorin expression in quiescent myogenic cells

Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells.     

alpha7 integrin expressing human fetal myogenic progenitors have stem cell-like properties and are capable of osteogenic differentiation

During muscle development, precursor cells fuse to form myofibers. Following injury in adult muscle, quiescent satellite cells become activated to regenerate muscle in a fashion similar to fetal development. Recent studies indicate that murine skeletal myoblasts can differentiate along multiple cell lineages including the osteoblastic pathway. However, little is known about the multipotency of human myogenic cells. Here, we isolate myogenic precursor cells from human fetal and adult muscle by sorting for the laminin-binding alpha7 integrin and demonstrate their differentiation potential and alteration in adhesive behavior. The alpha7-positive human fetal progenitors were efficient at forming myotubes and a majority expressed known muscle markers including M-cadherin and c-Met, but were heterogeneous for desmin and MyoD expression. To test their pluripotent differentiation potential, enriched populations of alpha7-positive fetal cells were subjected to inductive protocols. Although the myoblasts appeared committed to a muscle lineage, they could be converted to differentiate along the osteoblastic pathway in the presence of BMP-2. Interestingly, osteogenic cells showed altered adhesion and migratory activity that reflected growth factor-induced changes in integrin expression. These results indicate that alpha7-expressing fetal myoblasts are capable of differentiation to osteoblast lineage with a coordinated switch in integrin profiles and may represent a mechanism that promotes homing and recruitment of myogenic stem cells for tissue repair and remodeling.     

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.     

Megf10 regulates the progression of the satellite cell myogenic program

We identify here the multiple epidermal growth factor repeat transmembrane protein Megf10 as a quiescent satellite cell marker that is also expressed in skeletal myoblasts but not in differentiated myofibers. Retroviral expression of Megf10 in myoblasts results in enhanced proliferation and inhibited differentiation. Infected myoblasts that fail to differentiate undergo cell cycle arrest and can reenter the cell cycle upon serum restimulation. Moreover, experimental modulations of Megf10 alter the expression levels of Pax7 and the myogenic regulatory factors. In contrast, Megf10 silencing in activated satellite cells on individual fibers or in cultured myoblasts results in a dramatic reduction in the cell number, caused by myogenin activation and precocious differentiation as well as a depletion of the self-renewing Pax7⁺/MyoD⁻ population. Additionally, Megf10 silencing in MyoD^−/− myoblasts results in down-regulation of Notch signaling components. We conclude that Megf10 represents a novel transmembrane protein that impinges on Notch signaling to regulate the satellite cell population balance between proliferation and differentiation.     

Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro

Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.     

Estrogen influences satellite cell activation and proliferation following downhill running in rats.

To investigate the influence of estrogen on postexercise muscle repair processes, we examined the effects of estrogen supplementation (0.25-mg pellet) on numbers of myofibers positive for markers of total, activated, and proliferating satellite cells in rat skeletal muscles 72 h following downhill running. Ovariectomized female rats (n = 44) were divided into four groups (n = 11 per group): sham (no estrogen) controls (SC); sham, exercised (SE); estrogen-supplemented controls (EC); and estrogen-supplemented, exercised (EE). After 8 days of estrogen exposure, animals were exposed to 90 min of treadmill running at 17 m/min (-13.5 degrees ). Seventy-two hours later, soleus and white vastus muscles were removed and immunostained for total [paired box homeotic gene 7 (Pax7)], [activated myogenic differentiation factor D (MyoD)], and proliferating [5-bromo-2'-deoxyuridine (BrdU)] satellite cells. beta-Glucuronidase activity was increased (P < 0.05) in both muscles following exercise; however, the postexercise elevations in enzyme activity were attenuated in the EE group compared with the SE group in the soleus (P < 0.05). Immunohistochemical analysis revealed that exercised groups displayed increased numbers of myofibers containing total, activated, and proliferating satellite cells compared with control groups (P < 0.05). Furthermore, greater numbers of fibers positive for markers of total, activated, and proliferating satellite cells were observed postexercise in EE animals compared with SE animals for both muscles (P < 0.05). The results demonstrate that estrogen may potentially influence post-damage repair of skeletal muscle through activation of satellite cells.     

Pannexin 1 and Pannexin 3 Channels Regulate Skeletal Muscle Myoblast Proliferation and Differentiation

Pannexins constitute a family of three glycoproteins (Panx1, -2, and -3) forming single membrane channels. Recent work demonstrated that Panx1 is expressed in skeletal muscle and involved in the potentiation of contraction. However, Panxs functions in skeletal muscle cell differentiation, and proliferation had yet to be assessed. We show here that Panx1 and Panx3, but not Panx2, are present in human and rodent skeletal muscle, and their various species are differentially expressed in fetal versus adult human skeletal muscle tissue. Panx1 levels were very low in undifferentiated human primary skeletal muscle cells and myoblasts (HSMM) but increased drastically during differentiation and became the main Panx expressed in differentiated cells. Using HSMM, we found that Panx1 expression promotes this process, whereas it was impaired in the presence of probenecid or carbenoxolone. As for Panx3, its lower molecular weight species were prominent in adult skeletal muscle but very low in the fetal tissue and in undifferentiated skeletal muscle cells and myoblasts. Its overexpression (∼43-kDa species) induced HSMM differentiation and also inhibited their proliferation. On the other hand, a ∼70-kDa immunoreactive species of Panx3, likely glycosylated, sialylated, and phosphorylated, was highly expressed in proliferative myoblasts but strikingly down-regulated during their differentiation. Reduction of its endogenous expression using two Panx3 shRNAs significantly inhibited HSMM proliferation without triggering their differentiation. In summary, our results demonstrate that Panx1 and Panx3 are co-expressed in human skeletal muscle myoblasts and play a pivotal role in dictating the proliferation and differentiation status of these cells.     

Isolation,Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.     

Teashirt 3 expression in the chick embryo reveals a remarkable association with tendon development

Drosophila teashirt (tsh) is involved in the patterning of the trunk identity together with the Hox genes. In addition, it is also a player in the Wingless and the Hedgehog pathways. In birds and mammals, three Tshz genes are identified and the expression patterns for mouse Tshz1 and Tshz2 have been reported during embryogenesis. Recently, we showed that all three mouse Tshz genes can rescue the Drosophila tsh loss-of-function phenotype, indicating that the function of the teashirt genes has been conserved during evolution. Here we describe the expression pattern of chick TSHZ3 during embryogenesis. Chick TSHZ3 is expressed in several tissues including mesodermal derivatives, the central and peripheral nervous systems. Emphasis is laid on the dynamic expression occurring in regions of the somites and limbs where tendons develop. We show that TSHZ3 is activated in the somites by FGF8, a known inducer of the tendon marker SCX.