The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis

We have studied the role of Notch-1 and its antagonist Numb in the activation of satellite cells during postnatal myogenesis. Activation of Notch-1 promoted the proliferation of myogenic precursor cells expressing the premyoblast marker Pax3. Attenuation of Notch signaling by increases in Numb expression led to the commitment of progenitor cells to the myoblast cell fate and the expression of myogenic regulatory factors, desmin, and Pax7. In many intermediate progenitor cells, Numb was localized asymmetrically in actively dividing cells, suggesting an asymmetric cell division and divergent cell fates of daughter cells. The results indicate that satellite cell activation results in a heterogeneous population of precursor cells with respect to Notch-1 activity and that the balance between Notch-1 and Numb controls cellular homeostasis and cell fate determination.     

The regulation of Notch signaling in muscle stem cell activation and postnatal myogenesis

The Notch signaling pathway is an evolutionarily conserved pathway that is critical for tissue morphogenesis during development, but is also involved in tissue maintenance and repair in the adult. In skeletal muscle, regulation of Notch signaling is involved in somitogenesis, muscle development, and the proliferation and cell fate determination of muscle stems cells during regeneration. During each of these processes, the spatial and temporal control of Notch signaling is essential for proper tissue formation. That control is mediated by a series of regulatory proteins and protein complexes that enhance or inhibit Notch signaling by regulating protein processing, localization, activity, and stability. In this review, we focus on the regulation of Notch signaling during postnatal muscle regeneration when muscle stem cells ("satellite cells") must activate, proliferate, progress along a myogenic lineage pathway, and ultimately differentiate to form new muscle. We review the regulators of Notch signaling, such as Numb and Deltex, that have documented roles in myogenesis as well as other regulators that may play a role in modulating Notch signaling during satellite cell activation and postnatal myogenesis.     

Single-fiber isolation and maintenance of satellite cell quiescence.

The activity of satellite cells during myogenesis, development, or skeletal muscle regeneration is strongly modelled using cultures of single muscle fibers. However, there are variations in reported features of gene or protein expression as examined with single-fiber cultures. Here, we examined the potential differences in activation of satellite cells on normal mouse muscle fibers produced during a standard isolation protocol, with or without agitation during collagenase digestion. Activation was detected in satellite cells on fibers after 24 and 48 h of culture in basal growth medium using immunodetection of the incorporation of bromodeoxyuridine (BrdU) into DNA and quantification of the number of BrdU-positive cells per fiber. After 24 and 48 h in culture under nonactivating conditions, the number of activated (BrdU+) satellite cells was greater on fibers that had received gentle agitation during collagenase digestion than on those that were subject to digestion without agitation during isolation. The findings are interpreted to mean that at least some of the variation among published reports may derive from the application of various methods of fiber isolation. The information should be useful for maintaining satellite cell quiescence during studies of the regulatory steps that lead to satellite cell activation.     

Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation

Endogenous gut-derived bacterial lipopolysaccharides have been implicated as important cofactors in the pathogenesis of liver injury. However, the molecular mechanisms by which lipopolysaccharides exert their effect are not entirely clear. Recent studies have pointed to proinflammatory cytokines such as tumor necrosis factor-alpha as mediators of hepatocyte injury. Within the liver, Kupffer cells are major sources of proinflammatory cytokines that are produced in response to lipopolysaccharides. This review will focus on three important molecular components of the pathway by which lipopolysaccharides activate Kupffer cells: CD14, Toll-like receptor 4, and lipopolysaccharide binding protein. Within the liver, lipopolysaccharides bind to lipopolysaccharide binding protein, which then facilitates its transfer to membrane CD14 on the surface of Kupffer cells. Signaling of lipopolysaccharide through CD14 is mediated by the downstream receptor Toll-like receptor 4 and results in activation of Kupffer cells. The role played by these molecules in liver injury will be examined.     

Temporal appearance of satellite cells during myogenesis.

In this study, differences between fetal and adult myoblasts in clonal and high density culture have been used to determine when adult myoblasts can first be detected during avian development. The results indicate that avian adult myoblasts are apparent as a distinct population of myoblasts during the midfetal stage of development. Three different criteria were used to differentiate fetal and adult myoblasts and demonstrate when adult myoblasts become a major proportion of the myoblast population: (1) differences in slow myosin heavy chain 1 (MHC1) isoform expression, (2) initiation of DNA synthetic activity, and (3) average myoblast length. Fetal chicken (ED10-12) pectoralis muscle (PM) myoblasts form myotubes that express slow MHC1 after prolonged culture, while adult chicken PM myoblasts do not. Fetal avian myoblasts were active in DNA synthesis and large when first isolated, reaching peak rates of synthesis by 24 hr in culture, while adult myoblasts were inactive in DNA synthesis and small when first isolated, only reaching peak rates of DNA synthesis and size at 3 days of incubation. A dramatic decrease in the percentage of muscle colonies with fibers that expressed slow MHC1 was observed between the midfetal stage and hatching in the chicken, along with a corresponding decrease in myoblast DNA synthetic activity and average length during this same period in both the chicken and the quail. Myoblast activity and average length increased again 3-4 days posthatch and a small transient increase in the number of slow MHC1-expressing clones was also associated with the massive growth of muscle that occurs in the neonatal bird. We conclude that adult myoblasts are present as a distinct population of myoblasts at least as early as the midfetal stages of avian development.     

Characterization of myogenesis from adult satellite cells cultured in vitro

We describe several characteristics of in vitro myogenesis from adult skeletal muscle satellite cells from the rat and several amphibian species. The timing of cell proliferation and fusion into myotubes was determined, and in urodeles, myogenesis from satellite cells was clearly demonstrated for the first time. Growth factors are known to stimulate satellite cell proliferation. Acidic FGF mRNA was present in rat satellite cells during proliferation but it was not detected in myotubes. Fibronectin was synthesized in satellite cells during proliferation and expelled into the extracellular medium when the myotubes differentiated. We suggest that fibronectin plays a part in the formation of myotubes, as this process was inhibited by anti-fibronectin IgG. Adult satellite cells might differ from fetal myoblasts since they were observed to exhibit the opposite response to a phorbol ester (TPA) to that of the myoblasts. We therefore examined the possibility that the different levels of protein kinase C activity and different phorbol ester binding characteristics in the two cell types account for these opposite responses. Our results suggest that the difference is not connected with the phorbol ester receptor but might be caused by events subsequent to protein kinase C activation. Localized extracellular proteolytic activity might have a role in cell mobilization and/or fusion when satellite cells are activated. We showed that the content of plasminogen activators, chiefly urokinase, was larger in tissues from slow twitch muscles which regenerate more rapidly than fast muscles. The urokinase level rose sharply in cultures when cells fused into myotubes, and was twice as high in slow muscle cells as in fast ones. We also found that, in vitro, slow muscle satellite cells displayed greater myogenicity, but that phorbol ester inhibited their mitosis and myogenicity. We conclude that satellite cells acquire characteristics which differentiate them from myoblasts and correspond to the fast and slow muscles from which they originate.     

Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche

Satellite cells are situated beneath the basal lamina that surrounds each myofiber and function as myogenic precursors for muscle growth and repair. The source of satellite cell renewal is controversial and has been suggested to be a separate circulating or interstitial stem cell population. Here, we transplant single intact myofibers into radiation-ablated muscles and demonstrate that satellite cells are self-sufficient as a source of regeneration. As few as seven satellite cells associated with one transplanted myofiber can generate over 100 new myofibers containing thousands of myonuclei. Moreover, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. Following experimental injury, these cells proliferate extensively and regenerate large compact clusters of myofibers. Thus, within a normally stable tissue, the satellite cell exhibits archetypal stem cell properties and is competent to form the basal origin of adult muscle regeneration.     

Adiponectin receptors activation performs dual effects on regulating myogenesis and adipogenesis of young and aged muscle satellite cells

ObjectivesSkeletal muscle mass and function deteriorate with ageing. Adiponectin receptors (APNrs), mainly activated by adiponectin, participate in various physiological activities and have varying signalling pathways at different ages. This study aimed to explore whether discrepant performance exists in APNr activation regulating young and aged muscle satellite cells (MUSCs) and whether age‐related muscle dysfunction could be alleviated upon APNr activation.MethodsThe gastrocnemius muscle phenotype was observed in male mice aged 2 and 18 months. An APNr agonist (AdipoRon) was used in vitro and in vivo to investigate the changes in cell biological behaviours and whether muscle dysfunction could be retarded after APNr activation.ResultsAged mice exhibited decreased muscle mass and increased fat infiltration. APNr activation inhibited C2C12 cells and young MUSCs (YMUSCs) proliferation but showed no obvious effect on aged MUSCs (AMUSCs). Moreover, APNr activation inhibited the migration of both YMUSCs and AMUSCs. Interestingly, APNr activation hampered the myogenic differentiation but advanced the adipogenic differentiation of YMUSCs, yet exact opposite results were presented in AMUSCs. It was demonstrated that Wnt and PI3K signalling pathways may mediate the phenotypic differences. Furthermore, in vivo experiments verified that APNr activation ameliorated age‐related muscle atrophy and excessive fat infiltration.ConclusionsAPNr activation exerted dual effects on the regulation of myogenesis and adipogenesis of YMUSCs and AMUSCs and rescued age‐related skeletal muscle dysfunction.

Adiponectin receptor (APNr) activation had different effects on myogenic and adipogenic differentiation between aged and young skeletal muscle satellite cells (MUSCs), owing to the different reactions of PI3K and Wnt signalling pathways in young and aged states. Activation of APNr could promote the myogenic differentiation of aged MUSCs and inhibit their adipogenic differentiation, and further improve age‐related skeletal muscle atrophy and reduce fat accumulation.     

Stem cells and niches: mechanisms that promote stem cell maintenance throughout life

Niches are local tissue microenvironments that maintain and regulate stem cells. Long-predicted from mammalian studies, these structures have recently been characterized within several invertebrate tissues using methods that reliably identify individual stem cells and their functional requirements. Although similar single-cell resolution has usually not been achieved in mammalian tissues, principles likely to govern the behavior of niches in diverse organisms are emerging. Considerable progress has been made in elucidating how the microenvironment promotes stem cell maintenance. Mechanisms of stem cell maintenance are key to the regulation of homeostasis and likely contribute to aging and tumorigenesis when altered during adulthood.     

Biosynthesis of laminin and fibronectin by rat satellite cells during myogenesis in vitro

The biosynthesis of fibronectin and laminin was studied in satellite cells cultured from adult rat muscles before (day 4) and after fusion and formation of myotubes (day 14) using (35S) methionine as a tracer. The kinetics of incorporation into immunoprecipitable fibronectin and laminin were recorded at intervals from 1.5 to 24 hours of incubation with the tracer from the cells, the pericellular matrix and the culture medium. The rate of synthesis of fibronectin and laminin expressed as dpm/microgram DNA were constant from the mononucleated cell to the myotube state. Both glycoproteins were detected in the cells and in the pericellular matrix. When the results were expressed as the percentage of incorporation into total protein, major changes could be observed in the early phase of the kinetic studies in the cells and the pericellular matrix. Both showed an increase from the mononucleated myoblast to myotube, suggesting that an increasing fraction of total protein biosynthesis is directed towards these two extracellular matrix glycoproteins. At the same time, there was a decrease in the secretion into the medium of freshly synthesized radiolabeled fibronectin and laminin. Our results confirm the synthesis of varying ratios of both extracellular matrix macromolecules by undifferentiated mononucleated myogenic cells as well as myotubes.     

Stem cell and niche development in the postnatal rat testis

Adult tissue stem cells self-renew and differentiate in a way that exactly meets the biological demand of the dependent tissue. We evaluated spermatogonial stem cell (SSC) activity in the developing rat testis and the quality and accessibility of the stem cell niche in wild type, and two busulfan-treated models of rat pup recipient testes using an SSC transplantation technique as a functional assay. While our results revealed a 69-fold increase in stem cell activity during rat testis development from neonate to adult, only moderate changes in SSC concentration were observed, and stem cells from neonate, pup, and adult donor testes produce spermatogenic colonies of similar size. Analysis of the stem cell niche in recipient rat testes demonstrated that pup testes support high levels of donor stem cell engraftment when endogenous germ cells are removed or compromised by busulfan treatment. Fertility was established when rat pup donor testis cells were transplanted into fetal- or pup-busulfan-treated recipient rat pup testes, and the donor genotype was transmitted to subsequent generations. These results provide insight into stem cell/niche interactions in the rat testis and demonstrate that techniques originally developed in mice can be extended to other species for regenerative medicine and germline modification.