Asymmetric self-renewal and commitment of satellite stem cells in muscle

Satellite cells play a central role in mediating the growth and regeneration of skeletal muscle. However, whether satellite cells are stem cells, committed progenitors, or dedifferentiated myoblasts has remained unclear. Using Myf5-Cre and ROSA26-YFP Cre-reporter alleles, we observed that in vivo 10% of sublaminar Pax7-expressing satellite cells have never expressed Myf5. Moreover, we found that Pax7(+)/Myf5(-) satellite cells gave rise to Pax7(+)/Myf5(+) satellite cells through apical-basal oriented divisions that asymmetrically generated a basal Pax7(+)/Myf5(-) and an apical Pax7(+)/Myf5(+) cells. Prospective isolation and transplantation into muscle revealed that whereas Pax7(+)/Myf5(+) cells exhibited precocious differentiation, Pax7(+)/Myf5(-) cells extensively contributed to the satellite cell reservoir throughout the injected muscle. Therefore, we conclude that satellite cells are a heterogeneous population composed of stem cells and committed progenitors. These results provide critical insights into satellite cell biology and open new avenues for therapeutic treatment of neuromuscular diseases.     

Skeletal muscle progenitor cells and the role of Pax genes

Satellite cells, which lie under the basal lamina of muscle fibres, are marked by the expression of Pax7, and in many muscles of Pax3 also. A pure population of satellite cells, isolated from a Pax3(GFP/+) mouse line by flow cytometry, contribute very efficiently to skeletal muscle regeneration and also self-renew, thus demonstrating their role as muscle stem cells. Pax3/7 regulates the entry of these cells into the myogenic programme via the activation of the myogenic determination gene, MyoD. Pax7 is also essential for the survival of satellite cells. This dual role underlines the importance of ensuring that a tissue stem cell that has lost its myogenic instruction should not be left to run amok, with the potential risk of tissue deregulation and cancer. A somite-derived population of Pax3/Pax7 positive cells is responsible for muscle growth during development and gives rise to the satellite cells of postnatal muscles. In the absence of both Pax3 and Pax7, these cells die or assume other cell fates. Pax3/7 lies genetically upstream of both MyoD and Myf5, which determine the skeletal muscle fate of these cells. To cite this article: M. Buckingham, C. R. Biologies 330 (2007).     

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration

Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.     

Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells

The growth and repair of skeletal muscle after birth depends on satellite cells that are characterized by the expression of Pax7. We show that Pax3, the paralogue of Pax7, is also present in both quiescent and activated satellite cells in many skeletal muscles. Dominant-negative forms of both Pax3 and -7 repress MyoD, but do not interfere with the expression of the other myogenic determination factor, Myf5, which, together with Pax3/7, regulates the myogenic differentiation of these cells. In Pax7 mutants, satellite cells are progressively lost in both Pax3-expressing and -nonexpressing muscles. We show that this is caused by satellite cell death, with effects on the cell cycle. Manipulation of the dominant-negative forms of these factors in satellite cell cultures demonstrates that Pax3 cannot replace the antiapoptotic function of Pax7. These findings underline the importance of cell survival in controlling the stem cell populations of adult tissues and demonstrate a role for upstream factors in this context.     

Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells

Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific NICD(OE) (overexpression of constitutively activated Notch 1 intracellular domain), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICD(OE) upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD(-/-) myoblasts, we further show that NICD(OE) upregulates Pax7 independently of MyoD inhibition. In striking contrast to previous observations, NICD(OE) also inhibits S-phase entry and Ki67 expression and thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effects of NICD(OE) on MyoD and Ki67 but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-Jκ, which binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICD(OE) results in impaired regeneration of skeletal muscles along with increased Pax7(+) mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through direct regulation of Pax7.     

Rb1 gene inactivation expands satellite cell and postnatal myoblast pools

Satellite cells are well known as a postnatal skeletal muscle stem cell reservoir that under injury conditions participate in repair. However, mechanisms controlling satellite cell quiescence and activation are the topic of ongoing inquiry by many laboratories. In this study, we investigated whether loss of the cell cycle regulatory factor, pRb, is associated with the re-entry of quiescent satellite cells into replication and subsequent stem cell expansion. By ablation of Rb1 using a Pax7CreER,Rb1 conditional mouse line, satellite cell number was increased 5-fold over 6 months. Furthermore, myoblasts originating from satellite cells lacking Rb1 were also increased 3-fold over 6 months, while terminal differentiation was greatly diminished. Similarly, Pax7CreER,Rb1 mice exhibited muscle fiber hypotrophy in vivo under steady state conditions as well as a delay of muscle regeneration following cardiotoxin-mediated injury. These results suggest that cell cycle re-entry of quiescent satellite cells is accelerated by lack of Rb1, resulting in the expansion of both satellite cells and their progeny in adolescent muscle. Conversely, that sustained Rb1 loss in the satellite cell lineage causes a deficit of muscle fiber formation. However, we also show that pharmacological inhibition of protein phosphatase 1 activity, which will result in pRb inactivation accelerates satellite cell activation and/or expansion in a transient manner. Together, our results raise the possibility that reversible pRb inactivation in satellite cells and inhibition of protein phosphorylation may provide a new therapeutic tool for muscle atrophy by short term expansion of the muscle stem cells and myoblast pool.     

Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population

In contrast to mammals, salamanders can regenerate complex structures after injury, including entire limbs. A central question is whether the generation of progenitor cells during limb regeneration and mammalian tissue repair occur via separate or overlapping mechanisms. Limb regeneration depends on the formation of a blastema, from which the new appendage develops. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. We describe a multipotent Pax7⁺ satellite cell population located within the skeletal muscle of the salamander limb. We demonstrate that skeletal muscle dedifferentiation involves satellite cell activation and that these cells can contribute to new limb tissues. Activation of salamander satellite cells occurs in an analogous manner to how the mammalian myofiber mobilizes stem cells during skeletal muscle tissue repair. Thus, limb regeneration and mammalian tissue repair share common cellular and molecular programs. Our findings also identify satellite cells as potential targets in promoting mammalian blastema formation.     

An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration

Skeletal muscle tissue provides mechanical force for locomotion of all vertebrate animals. It is prone to damage from acute physical trauma and physiological stress. To cope with this, it possesses a tremendous capacity for rapid and effective repair that is widely held to be accomplished by the satellite cells lying between the muscle fiber plasmalemma and the basement membrane. Cell transplantation and lineage-tracing studies have demonstrated that Pax7-expressing (Pax7(+)) satellite cells can repair damaged muscle tissue repeatedly after several bouts of acute injury. These findings provided evidence that Pax7(+) cells are muscle stem cells. However, stem cells from a variety of other origins are also reported to contribute to myofibers upon engraftment into muscles, questioning whether satellite cells are the only stem cell source for muscle regeneration. Here, we have engineered genetic ablation of Pax7(+) cells to test whether there is any significant contribution to muscle regeneration after acute injury from cells other than this source. We find that such elimination of Pax7(+) cells completely blocks regenerative myogenesis either following injury to the tibialis anterior (TA) muscle or after transplantation of extensor digitorum longus (EDL) muscles into nude mice. As Pax7 is specifically expressed in satellite cells, we conclude that they are essential for acute injury-induced muscle regeneration. It remains to be established whether there is any significant role for stem cells of other origins. The implications of our results for muscle stem cell-based therapy are discussed.     

Elevated satellite cell number in Duchenne muscular dystrophy

The regenerative potential of muscle tissue relies mostly on satellite cells situated between the muscular basal membrane and the sarcolemma. The regeneration of muscle tissue comprises proliferation, the propagation of satellite cells, and their subsequent differentiation with the expression of multiple muscle-specific proteins. However, in Duchenne muscular dystrophy (DMD), regeneration cannot compensate for the loss of muscle tissue. To examine the regenerative potential in DMD, satellite cell nuclei number and markers of differentiation in DMD muscle from various disease states were compared with control muscle. Differentiation of satellite cells is characterized by the helix-loop-helix factor myogenin, which is never co-expressed with Pax7, whereas MyoD1 and Myf5 are co-expressed with Pax7, with Myf5 being present even in muscle of controls. The results indicate that satellite cell number is elevated in DMD in comparison with control muscle, even in advanced stages of dystrophy, suggesting that exhaustion of satellite cells is not the primary cause for failed regeneration. The expression of myogenin is correlated neither with fibrosis nor with age. We suggest variable factors influencing the differentiation of satellite cells in DMD.     

KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors

Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.Subject terms: Differentiation, Muscle stem cells, Epigenetics     

Muscle stem cells and reversible quiescence: The role of sprouty

Quiescence is a critical determinant for sustained stem cell function throughout life. Disruption of cellular quiescence leads to loss of the stem cell pool and impaired tissue repair. In adult skeletal muscle, Pax7⁺ satellite cells (the muscle stem cells) are capable of self-renewal and differentiation in their endogenous environment during repair. In response to muscle injury, Pax7⁺ satellite cells enter the cell cycle; subpopulation returns to quiescence to fully replenish the satellite cell pool while others contribute to myofiber repair. We demonstrate that Sprouty1 (Spry1), an inhibitor of receptor tyrosine kinase signaling is required for the return to quiescence of the self-renewing Pax7⁺ satellite cell pool during repair. The temporal regulation of Spry1 expression during repair and its functional requirement in a subpopulation of cycling Pax7⁺ cells during repair ensure that tissue regeneration and re-establishment of the dormant stem cell pool are coordinated.