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Keywords:

  • muscle;
  • stem cell;
  • satellite cell;
  • chick;
  • zebrafish;
  • Drosophila

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. HETEROGENEITY OF MYOGENIC STEM CELLS
  5. PROPERTIES OF SATELLITE CELLS
  6. UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS
  7. MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES
  8. Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS
  9. PERSPECTIVES
  10. REFERENCES

Stem cells are characterized by their clonal ability both to generate differentiated progeny and to undergo self-renewal. Studies of adult mammalian organs have revealed stem cells in practically every tissue. In the adult skeletal muscle, satellite cells are the primary muscle stem cells, responsible for postnatal muscle growth, hypertrophy, and regeneration. In the past decade, several molecular markers have been found that identify satellite cells in quiescent and activated states. However, despite their prime importance, surprisingly little is known about the biology of satellite cells, as their analysis was for a long time hampered by a lack of genetically amenable experimental models where their properties can be dissected. Here, we review how the embryonic origin of satellite cells was discovered using chick and mouse model systems and discuss how cells from other sources can contribute to muscle regeneration. We present evidence for evolutionarily conserved properties of muscle stem cells and their identification in lower vertebrates and in the fruit fly. In Drosophila, muscle stem cells called adult muscle precursors (AMP) can be identified in embryos and in larvae by persistent expression of a myogenic basic helix–loop–helix factor Twist. AMP cells play a crucial role in the Drosophila life cycle, allowing de novo formation and regeneration of adult musculature during metamorphosis. Based on the premise that AMPs represent satellite-like cells of the fruit fly, important insight into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila. Developmental Dynamics 236:3332–3342, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. HETEROGENEITY OF MYOGENIC STEM CELLS
  5. PROPERTIES OF SATELLITE CELLS
  6. UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS
  7. MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES
  8. Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS
  9. PERSPECTIVES
  10. REFERENCES

Satellite cells and embryonic muscle progenitors have been identified in a broad spectrum of vertebrate species, indicating that they are part of a conserved myogenic pathway. However, despite their primordial and evolutionarily conserved functions (Luo et al., 2005; Wozniak et al., 2005), only few genes that control muscle stem cell specification, quiescence, and entry into the differentiation process have been identified (Luo et al., 2005; Holterman and Rudnicki, 2005; Frock et al., 2006). This work was greatly facilitated by using animal models in which the regulation and function of genes controlling muscle stem cell specification and behavior could be dissected. Here, we discuss recent advances in the field and give examples of how different model organisms have made it possible to determine the embryonic origin of satellite cells. We also describe evidence for muscle stem cells in the fruit fly, Drosophila melanogaster, in which the genetic control of specification and proliferation can be easily dissected.

HETEROGENEITY OF MYOGENIC STEM CELLS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. HETEROGENEITY OF MYOGENIC STEM CELLS
  5. PROPERTIES OF SATELLITE CELLS
  6. UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS
  7. MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES
  8. Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS
  9. PERSPECTIVES
  10. REFERENCES

Adult skeletal muscle possesses a remarkable ability for self-repair in response to tissue injury, suggesting the presence of a stem cell population either resident within muscle or capable of migrating to muscle. Under physiological conditions, the ability of muscle to undergo regeneration is largely attributed to a distinct subpopulation of myogenic cells, termed satellite cells (Mauro, 1961; Sherwood et al., 2004). In addition to satellite cells, alternative sources of muscle stem cells were identified: these sources include side-population (SP) cells, muscle-derived stem cells (MDSC), blood-derived circulating AC133+ cells, and vessel-associated stem cells (mesoangioblasts and pericytes).

The SP cells are present in a broad spectrum of adult tissues, including muscle, and can be isolated by fluorescence-activated cell sorting (FACS) based on their capacity to exclude the Hoechst dye (Asakura et al., 2002). SP cells isolated from muscle (mSP) are unable to undergo myogenic differentiation in vitro, but on intramuscular transplantation, they give rise to both differentiated muscle cells and satellite cells (Gussoni et al., 1999; Asakura et al., 2002). Unlike satellite cells, mSP cells transplanted intravenously are able to migrate from the blood stream into muscle and contribute to the regeneration of diseased muscle (Bachrach et al., 2006).

In addition to satellite cells and mSPs, a distinct stem cell population called MDSCs were purified from the adult skeletal muscle by a culture system of successive preplating combined with FACS sorting (Huard et al., 2003; Cao et al., 2003). MDSCs were found to be heterogenous and to have high self-renewal and proliferation capacities, characteristics usually associated with noncommitted progenitor cells. The late-adhering, Sca-1+/CD34+ MDSCs, when injected into limb muscle or into the circulatory system of mdx mice, were found to contribute to muscle regeneration (Payne et al., 2005). The localization of MDSCs in the adult muscle and their embryonic origin remain unknown.

Moreover, a subpopulation of blood-derived circulating cells expressing a well-characterized hematopoietic marker AC133 (Torrente et al., 2004) were found to undergo myogenic differentiation in vitro (in co-culture with myogenic cells) or in vivo when delivered into the muscles of transgenic scid/mdx mice. After treatment, some injected AC133+ cells localized under the basal lamina of host muscle fibers and expressed satellite cell markers such as M-cadherin and Myf5. A substantial recovery of muscle force was also observed in treated mdx mice. As the AC133+ cells can be easily isolated from the blood, manipulated in vitro, and delivered through the circulation, they seem to represent an attractive tool for future cell therapy applications in muscular dystrophies.

Recently, Dellavalle et al. (2007) reported the isolation of vessel-associated stem cells from the microvasculature of human skeletal muscle. These interstitial cells are similar to previously described mesoangioblasts (De Angelis et al., 1999; Minasi et al., 2002) residing in the embryonic dorsal aorta. However, unlike mesoangioblasts, they do not express endothelial markers, but instead express markers of pericytes such as alkaline phosphatase. Of interest, mesoangioblasts and pericytes can migrate through the vasculature, engraft efficiently in muscles, persist, and contribute to long-term regeneration (Sampaolesi et al., 2003, 2006; Galvez et al., 2006; Dellavalle et al., 2007). These properties make mesoangioblasts and pericytes promising candidates for future cell therapy protocols in muscular dystrophy patients.

PROPERTIES OF SATELLITE CELLS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. HETEROGENEITY OF MYOGENIC STEM CELLS
  5. PROPERTIES OF SATELLITE CELLS
  6. UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS
  7. MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES
  8. Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS
  9. PERSPECTIVES
  10. REFERENCES

Satellite cells are located between the basal lamina and sarcolemma and account for 2–5% of sublaminar nuclei of mature skeletal muscle fibers. Under normal conditions, satellite cells are quiescent, but they become activated in response to muscle damage or disease-induced degeneration. The descendants of activated satellite cells undergo multiple rounds of division before their terminal differentiation and fusion to form multinucleated myotubes.

Molecular Markers

Quiescent satellite cells have been primarily identified by electron microscopy by their morphological characteristics and distinct localization (Mauro, 1961). Several satellite cell markers have also been identified. Among them are paired box transcription factors Pax3 and Pax7 (Seale and Rudnicki, 2000; Relaix et al., 2006); c-met, the receptor for hepatocyte growth factor (HGF; Cornelison and Wold, 1997); myocyte nuclear factor (MNF; Hawke and Garry, 2001; Garry et al., 2000); M-cadherin, a calcium-dependent cell adhesion molecule (Irintchev et al., 1994; Moore and Walsh, 1993); vascular cell adhesion molecule 1 (VCAM1; Rosen et al., 1992); neural cell adhesion molecule (N-CAM; Illa et al., 1992); CD34, a hematopoietic stem cell marker (Beauchamp et al., 2000); and heparan sulfate proteoglycans Syndecan 3 and 4 (Cornelison et al., 2001). It has been clearly demonstrated that activation, proliferation, and differentiation of satellite cells after injury are controlled by members of the myogenic regulatory factor (MRF) family, in a similar manner to muscle precursor cells during embryonic development. The activated satellite cells first express Myf-5 and/or MyoD and finally myogenin and MRF4 as proliferating myoblasts differentiating into multinucleated myotubes (Smith et al., 1994; Cornelison et al., 2000; Kassar-Duchossoy et al., 2004).

The paired box transcription factor Pax7 is the commonly used marker of satellite cells in a wide range of vertebrate species (from fishes to humans). Pax7 was initially thought to be essential for the specification of satellite cells (Seale and Rudnicki, 2000). However, as demonstrated recently (Kuang et al., 2006; Relaix et al., 2006) satellite cells are present in Pax7 mutant mice, but are then gradually lost, suggesting that Pax7 is required for satellite cell survival. Expression of the paralogue of Pax7, Pax3 has also been detected in a subset of satellite cells (Relaix et al., 2006). Because of the major deficit in skeletal muscle in double Pax3/Pax7 mutant mice (Relaix et al., 2005), it has been suggested that Pax3 and Pax7 act together to specify all muscle stem cells of the body and of the limbs.

Quiescent State and Satellite Cell Niche

The study of satellite cell quiescence is particularly difficult because isolating satellite cells for cell culture purposes invariably causes their activation. Quiescent satellite cells express a variety of proteins that have been used for their identification and purification (Hawke and Garry, 2001). However, only a few of these proteins, such as MNF-β (Yang et al., 2000) or Myostatin, a member of the transforming growth factor-β superfamily (McCroskery et al., 2003), appear specific to the quiescent state. An increased density of satellite cells has been observed, associated with the neuromuscular junctions and capillaries (Schmalbruch and Hellhammer, 1977), indicating that these structures may release factors that direct satellite cells to specific muscle locations. The surrounding capillaries may additionally provide progenitor cells (mesoangioblasts, pericytes) that contribute to the satellite cell population. Under normal conditions, satellite cells divide very infrequently and it is believed that the position of the satellite cells beneath the basal lamina of the myofiber play an important role in maintaining their quiescence. In addition, the niche in which the satellite cells reside and in particular the proximity of capillaries and neuromuscular and myotendinous junctions suggest that factors secreted from the adjacent environment may control satellite cell quiescence and activation. Moreover, it has also been suggested that, after activation, the satellite cells may be a source of signaling molecules such as chemoattractants and cytokines known to be involved in muscle regeneration (Sachidanandan et al., 2002).

Mechanisms of Replenishment of Satellite Cells

The number of satellite cells in adult muscle remains relatively constant through repeated bouts of injury and regeneration, thus assuring a sufficient reserve for future needs (Schultz and McCormick, 1994). The capacity of satellite cells to self-renew has been elegantly demonstrated by the transplantation of individual fibers with their associated satellite cells from one mouse strain to another (Collins et al., 2005; Collins and Partridge, 2005). The donor satellite cells were shown to expand to generate new satellite cells in the host, adopting the appropriate anatomical position, expressing the appropriate biochemical markers, and most importantly, mediating subsequent regenerative responses in the host. One of the models proposed to explain self-renewal of satellite cell is based on asymmetric cell division. The mechanism by which asymmetric cell division occurs in satellite cells may be explained by the observation that the plasma membrane associated protein Numb is segregated asymmetrically (Conboy and Rando, 2002). Cells expressing high levels of Numb were found to undergo differentiation, whereas low-level expression of Numb (and constitutively active Notch) resulted in inhibition of differentiation. Of interest, Shinin et al., (2006) by using pulse–chase labeling with bromodeoxyuridine (BrdU) have uncovered that a subset of satellite cells display selective template-DNA strand segregation during mitosis, demonstrating cosegregation of template DNA with the asymmetric cell-fate determinant Numb and expression of the self-renewal marker Pax7 in label-retaining cells. The key role of asymmetric cell division in satellite cell self-renewal is supported by a recent study (Kuang et al., 2007) demonstrating the existence of two subpopulations of Pax7+ satellite cells within adult muscles, based on Myf5 expression. Myf5-, Pax7+ satellite cells extensively contribute to the self-renewal of the stem cell reservoir, whereas Myf5+, Pax7+ cells readily engage in the terminal differentiation program. Myf5-, Pax7+ satellite cells therefore constitute the true adult muscle stem cell, and it was demonstrated that they generate Myf5+, Pax7+ cells through asymmetric cell division. The finding that satellite cells are a heterogeneous population composed of stem cells and committed progenitors provide critical insights into satellite cell biology and might have important implications for treatment of neuromuscular diseases.

Myogenic Stem Cells and Muscle Regeneration

As stated above, the satellite cells are responsible not only for postnatal muscle growth and hypertrophy, but also for regeneration of damaged skeletal muscles. In response to trauma, they proliferate and their progeny exit the cell cycle and undergo terminal differentiation, giving rise to multinucleated muscle fibers (Schultz and McCormick, 1994). In muscular dystrophy conditions, and in particular in Duchenne muscular dystrophy (DMD), the most widespread human dystrophy, the use of satellite and other myogenic stem cells to replace the dystrophic muscle nuclei with normal nuclei has attracted much attention. DMD is a lethal, X chromosome-linked disease caused by mutations in the dystrophin gene that typically result in the disruption of the connection between the sarcomere and the extracellular matrix. Loss of dystrophin initiates myofiber degeneration caused by sarcolemma instability, calcium influxes, muscle overcontraction, and calpain-mediated proteolysis (Emery, 1989; Batchelor and Winder, 2006). Under these conditions, muscle fibers undergo repeated cycles of degeneration and regeneration at high frequency. A DMD-affected organism rapidly exhausts its satellite cell reserves owing to the continual cell cycles, and thereby loses its regenerative capacity (Webster and Blau, 1990). Many researchers have turned to animal models to elucidate the mechanisms leading to muscular diseases; the species used include the dog, cat, mouse, zebrafish, and invertebrates, in which all developmental stages of the disease can be easily assessed (Watchko et al., 2002; Guyon et al., 2007). Most experiments have been performed on the mdx mouse, which has no functional dystrophin in its muscles.

Myoblast transfer therapy (MTT) has been proposed for several years as a potential treatment for DMD. Transplantation experiments with satellite cell-derived myoblasts showed that injected normal muscle precursors could fuse with the pre-existing or regenerating mdx muscle fibers and cause them to induce the synthesis of dystrophin (Partridge et al., 1989). Transplantation of satellite cells isolated from muscles of Pax3-GFP knockin mice into muscles of mdx mice revealed that green fluorescent protein (GFP)-positive cells contributed to both the fiber repair and the muscle satellite cell compartment (Montarras et al., 2005). Also, experiments involving transplantation of whole muscle fibers showed that resident satellite cells were capable of initiating regeneration (Hansen-Smith and Carlson, 1979; Roberts et al., 1989). Grafting isolated myofibers into irradiated mdx-nude mice demonstrated that the fiber-derived satellite cells were self-sufficient as a source of regeneration. Only seven satellite cells associated with one transplanted myofiber were found to generate over 100 new myofibers containing thousands of nuclei (Collins et al., 2005).

However, extrapolation of MTT studies in (small) mice with a far less severe clinical DMD phenotype and pathology to clinical treatment of (large) humans is very difficult. Human trials of MTT started more than 10 years ago, but so far have not been as effective as expected. This finding is mainly because of the reduced survival and low migratory potential of the injected donor myoblasts. To overcome these limitations, a “high-density intramuscular injection” protocol (i.e., a large number of injections per volume of muscle) has been developed for clinical trials (Skuk et al., 2007) and is expected to provide a route for local, muscle type targeted cell therapy. However, several key issues such as the capacity of donor myoblasts to survive, migrate, proliferate, and fuse with the dystrophic muscle need to be addressed to improve the efficacy of MTT.

For all these reasons, the attention has turned to other sources of donor cells and in particular the embryonic vasculature-derived mesoangioblasts (De Angelis et al., 1999; Minasi et al., 2002; Galvez et al., 2006), their adult counterparts, the pericytes (Dellavalle et al., 2007), as well as the circulating AC133+ cells (Torrente et al., 2004) that appear to be the most promising candidates. The mesoangioblasts were found to repopulate and to repair diseased skeletal muscles in dystrophic mice and also in dystrophic golden retriever dogs, which represent a faithful model of human DMD (Sampaolesi et al., 2006). Intra-arterial delivery of wild-type canine mesoangioblasts resulted in an extensive recovery of dystrophin expression and normal muscle morphology and function in some treated individuals. Further trials and more extensive analyses are required before mesoangioblasts and (or) pericytes can be used for therapy of human dystrophic muscles.

Thus, despite the remarkable regeneration capacities of skeletal muscles and the multiple therapeutic strategies already assessed, no effective stem cell-based therapy is yet available to cure dystrophic muscle. For developing successful therapies, one important aspect will be to use a better suited animal model of DMD such as golden retriever dogs instead of mdx mice sharing a similar gene defects but displaying far less severe clinical phenotype and not well adapted body size.

UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. HETEROGENEITY OF MYOGENIC STEM CELLS
  5. PROPERTIES OF SATELLITE CELLS
  6. UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS
  7. MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES
  8. Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS
  9. PERSPECTIVES
  10. REFERENCES

In all vertebrates, skeletal muscles derive from segmented paraxial mesodermal structures termed somites. In birds and mammals, in which somitogenesis has been extensively studied, the newly formed somites are paired epithelial vesicles flanking the neural tube. During development, the ventral part of the somite gives rise to the mesenchymal sclerotome, from which originate the axial skeleton, and the syndetome generating tendons of the body axis. The dorsal part of the somite retains its epithelial structure and becomes the dermomyotome, which itself is further regionalized. Dorsally, the dermomyotome generates the dermal layer of the forming skin of the back, while the cells from the medial region give rise to the deep muscles of the back, and those of the lateral region differentiate into body wall and limb skeletal musculature (Christ and Ordahl, 1995; Stockdale et al., 2000). As elegantly demonstrated in chick and mouse embryos (Gros et al., 2005; Relaix et al., 2005), the dermomyotome is also the source of satellite cells, which are known progenitors of adult skeletal muscles. These findings were made by labeling and by following dermomyotomal cells in living embryos. In chicks, two complementary techniques were used (see Fig. 1): (1) defined somitic areas were electroporated by GFP-expressing vectors and the movements of GFP-positive cells were analyzed using confocal time lapse microscopy; as only part of the electroporated cells express GFP, this method is qualitative rather than quantitative, and (2) quail–chick interspecies grafting of a central portion of the dermomyotome followed by staining for the quail cell perinuclear antigen was applied to detect quail cells and their progeny quantitatively within a chick host. Combining these two approaches (Fig. 1) led to the conclusion that in chick the cell population from the central region of the dermomyotome gives rise to both the embryonic muscle progenitors and to most, if not all, satellite cells (Gros et al., 2005). Similarly, in mice, reporter genes targeted into Pax3 and Pax7 loci were found to label cells that delaminated from the central dermomyotome into the early myotome (Relaix et al., 2005). These cells are maintained as a proliferating population of cells in embryonic and fetal muscles and constitute resident muscle progenitor cells, which become myogenic and ensure muscle formation. By following a stable GFP reporter targeted to Pax3, late in development, these cells were found to adopt satellite cell positions (Relaix et al., 2005), thus supporting the observations made in chick. The same work revealed that, in double Pax3 and Pax7 mutants, muscle development was severely compromised owing to the loss of both embryonic and adult muscle progenitors (Relaix et al., 2006). Embryonic muscle defects were much weaker in Pax3 single mutant mice and practically absent in Pax7 mutants, strongly suggesting that Pax3 and Pax7 play redundant roles in specification and (or) survival of embryonic muscle progenitor cells.

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Figure 1. Identification of somitic precursors of satellite cells in chick and zebrafish embryos. A,B: Schematic representations of (A) in vivo electroporation and (B) chick–quail transplantation experiments revealing a new population of embryonic muscle progenitors required for muscle growth during fetal development and for specification of adult satellite cells. A: Note that electroporation of green fluorescent protein (GFP) expression vector is not quantitative (only a subset of electroporated cells express GFP) but is particularly well adapted to following dermomyotomal cells in vivo. B: In contrast, transplantation of quail dermomyotomal tissue into the chick host embryo allows detection (using quail-specific antibodies) of all quail-derived cells in the chimera. In both experiments, cells originating from the central part of the dermomyotome (DM) were found associated with muscle fibers, occupied satellite cell positions and expressed satellite cell specific marker Pax7. C: Somitic origin of embryonic muscle progenitors and satellite cells in zebrafish. Early somites undergo 90 degrees rotation, shifting Pax7-expressing cells (green) from the anterior to the external somitic compartment. Rotation movements are followed by migration of adaxial cells (blue) originally associated with the notochord (NC). At the end of somitogenesis, the Pax7-positive cells form a thin external cell layer, which appears homologous to the dermomyotome in higher vertebrates and contributes to both the muscle growth (axial muscle fibers and fin muscles) and the population of satellite-like cells. D–F: Satellite cells in lower vertebrates. D: Ultrastructure of satellite cells located between white muscle fibers. Tail fin stage of Esox lucius (Teleostei) 19 days after hatching. E: Ultrastructure of mesenchymal (satellite cells) between red muscle fibers. Acipenser baeri (Chondrostei) at stage 35. F: Pax7-positive cells in Coregonus lavaretus (Teleostei) at tail fin stage, 24 days after hatching. Scale bar = 2.5 μm in D, 6 μm in E, 20μm in F.

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The data reported by Gros et al. (2005) and Relaix et al. (2005) give evidence concerning the origin of satellite cells residing in trunk muscles. However, the origin of satellite cells of head and limb muscles arising from distinct territories remained unknown. Recent work using lineage tracing techniques in chick and mouse embryos has revealed that limb muscle satellite cells derive from migrating hypaxial muscle progenitors (Schienda et al., 2006). The origin of satellite cells associated with head muscles awaits investigation.

MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. HETEROGENEITY OF MYOGENIC STEM CELLS
  5. PROPERTIES OF SATELLITE CELLS
  6. UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS
  7. MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES
  8. Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS
  9. PERSPECTIVES
  10. REFERENCES

In lower vertebrates, the presence of a dermomyotomal compartment of the somite is still controversial and largely debatable. However, the recent identification of the external Pax7-expressing somitic layer in zebrafish (Hollway et al., 2007; Stellabotte et al., 2007; Hammond et al., 2007) and its role in muscle growth strongly suggests that this cell population may correspond to a dermomyotome. Importantly, a similar layer of cells external to the embryonic myotome has been previously detected in several other lower vertebrates such as the lamprey (Nakao, 1977), sturgeon (Devoto et al., 2006), and lungfish (Maurer, 1906). They have been found in diverse teleosts, including the sea bass (Veggetti et al., 1990), herring (Johnston, 1993), and gilthead sea bream (Ramirez-Zarzoza et al., 1995), as well as in the amphibians Xenopus laevis (Hamilton, 1969) and European common frog (Maurer, 1906). In Xenopus laevis, this layer is MyHC-negative and expresses Pax3, Col1a1, and in specific zones XMyf-5 and XMyoD genes (Grimaldi et al., 2004).

To determine the origin of external cells in zebrafish, Hollway et al. (2007) and Stellabotte et al. (2007) applied the vital dye-staining and lineage-tracking technique and showed that they originate from the anterior border of epithelial somites. The anterior somite domain was found to express Pax7 and the chemokine receptor CXCR4. In response to a lateral sdf1a, a chemotactic stimuli, anterior somitic cells migrate toward the lateral part of the somite generating Pax7-positive external cell population. Some of these external cells remain on the external surface and some of them enter the myotome and generate new fast fibers (Fig. 1C). In zebrafish, Hollway et al. (2007) found Pax7-positive cells deep within the myotome. Most of these cells were mitotically inactive and located underneath the basal lamina of individual muscle fibers, strongly suggesting that they are satellite cells (Fig. 1C).

As in zebrafish, in pearlfish somites, Pax7 expression is initiated in a population of cells in the anterolateral domain and subsequently observed at the lateral surface of the somite (Steinbacher et al., 2006). Some of these cells were also found to be myogenin-positive. The external Pax7-positive cells as well as those located deeper in the myotome, on the surface of differentiating fibers were also detected in the sturgeon (Devoto et al., 2006). Of interest, in amphibians, external cells also are the source of myogenic precursors (Grimaldi et al., 2004).

All these studies on nonamniota myogenesis indicate that growth of muscles, as in amniotes, is powered by Pax3- and Pax7-positive somitic progenitors, which later give rise to a satellite cell population. Because of the expression of a similar set of genes, Devoto et al. (2006) suggest that the external layer of cells includes multipotent myogenic precursors and is an ancient and conserved structure, homologous to the amniote dermomyotome. Other authors (Hollway et al., 2007) argue that the external Pax7-positive somitic cells arise by means of the whole somite rotation and, thus, are not equivalent of the amniote dermomyotome.

Whatever their interpretation, these recent findings shed a new light on previous studies describing mechanisms of skeletal muscle development in lower vertebrates and in particular in fishes. It is largely accepted that two processes, hypertrophy (fiber enlargement) and hyperplasy (formation of new muscle fibers) produce muscle growth. During teleost myogenesis two distinct phases of muscle growth, termed “stratified” and “mosaic” hyperplasia, have been identified. During the “stratified” hyperplasia, supplementary fibers are added from discrete germinal zones of the ventral and dorsal extremities of the developing myotome. The “mosaic” hyperplastic growth is seen in the appearance of new fibers of different age (and diameter) throughout the myotome viewed in transverse sections through the embryo (Rescan, 2005). The Pax7-positive muscle precursors identified in zebrafish were found to contribute to muscle growth and are thus expected to be involved in both the stratified and the mosaic hyperplasia. In contrast to birds and mammals, where generally the increase in the number of muscle fibers stops shortly after birth (Goldspink, 1972), in fishes hyperplasia remains important for a long time past the juvenile stage, far into adulthood (Stickland, 1983; Rowlerson and Veggetti, 2001). In the adult fish, the muscle mass increases owing to participation of myogenic stem cells, frequently referred to as myosatellite cells, which are observed outside the muscle fiber basal lamina (Koumans et al., 1993; Johnston and Horne, 1994; Johnston et al., 1998; Fig. 1D,E). New fibers in fishes are assumed to result from recruitment, proliferation, and fusion of satellite cells adjacent to existing fibers (Akster et al., 1995). The morphology, ultrastructure, proliferation, and migration capacity of adult satellite cells in fishes have been extensively studied (Koumans and Akster, 1995; Johnson and Allen, 1995; Devoto et al., 1996; Stoiber and Sänger, 1996). It has been demonstrated that they are able to enter the cell cycle (Alfei et al., 1989, 1993, 1994) and account for both hypertrophy and hyperplasy of muscles (Stickland, 1983; Koumans et al., 1993; Johnston, 2001). However, the origin and molecular characteristics of the myogenic precursors responsible for adult muscle growth in fishes have not been thoroughly investigated. Previous work revealed that the earliest populations of presumptive myogenic cells arose from several zones of the early somite (Vegeetti et al., 1990; Rowlerson and Veggetti, 2001). It has also been suggested that muscle growth becomes powered by mesenchymal cells, which migrate by means of myosepta into myotomes (Stoiber and Sänger, 1996; Kacperczyk and Daczewska, 2005).

As already discussed, satellite cells play an important role in regeneration and this phenomenon has been studied in amphibians, which are able to regenerate their limbs and tail after amputation. The mechanism of new muscle tissue formation during the regeneration is not yet fully understood. In previous experiments on limb and tail regeneration in newts and salamanders, it has been shown that the multinucleate muscle fibers can de-differentiate, giving rise to proliferating muscle precursor cells (Kumar et al., 2000; Echeverri et al., 2001). However, more recent data (Gargioli and Slack, 2004; Morrison et al., 2006; Chen et al., 2006) strongly suggest that, in amphibians (urodeles and Xenopus laevis), satellite cells activation rather than muscle fibers de-differentiation contributes to the proliferating cell progeny population during limb and tail regeneration.

As most studies attempting to identify the origin of satellite cells in lower vertebrates have been performed on the model organism zebrafish, which is a highly derived cyprinid teleost, identification and analysis of similar progenitor cell population in other species will be of interest to gain a better understanding of evolutionarily conserved rules of muscle stem cell specification. On the other hand, several advantages exist that are in favor of zebrafish as a model system. These are relatively quick external embryonic development, transparency of embryos, a large proportion of muscles within the body, access to the gene expression database (zfin.org) and to a large collection of mutants. One of the generated mutations, called sapje, disrupts the zebrafish orthologue of human DMD gene, providing a useful model to study Duchenne muscular dystrophy (Basset et al., 2003).

Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. HETEROGENEITY OF MYOGENIC STEM CELLS
  5. PROPERTIES OF SATELLITE CELLS
  6. UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS
  7. MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES
  8. Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS
  9. PERSPECTIVES
  10. REFERENCES

One way to accelerate understanding of the genetic pathway governing muscle stem cell biology is to apply simple animal models. In the fruit fly Drosophila melanogaster, particularly well suited for genetic analyses, muscle stem cell-like adult muscle precursors (AMPs) characterized by persistent Twi expression (Bate et al., 1991; Currie and Bate, 1991; Fernandes et al., 1991) can be easily identified. These cells, closely associated with multinucleated muscle fibers, stay nondifferentiated during the entire embryonic and the majority of larval life (see Fig. 2). As during all this period AMPs keep expression of Twi known to be specific to nondifferentiated muscle progenitors and then are required for adult muscle formation and regeneration, we propose they represent Drosophila muscle stem cells.

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Figure 2. Adult muscle precursors, the Drosophila muscle stem cells. A: Specification of adult muscle precursors (AMPs): During embryogenesis AMPs, like founder cells (FC), are specified in the somatic mesoderm. At stage 10, embryo cells expressing high levels of Twist give rise to all somatic muscles. Expression of the proneural gene, lethal of scute (l'sc), marks groups of mesodermal cells (promuscular clusters) that have the potential to become muscle progenitors. A process of lateral inhibition, mediated by the neurogenic genes (Notch, Delta), restricts l'sc expression to one cell per cluster. This cell becomes the muscle progenitor, whereas nonselected cells give rise to fusion-competent myoblasts (FCMs). Asymmetric segregation of Numb during division of the progenitor gives rise to pairs of FC or to an FC and an AMP. Each FC will seed the formation of a specific muscle fiber by fusion with FCMs. AMPs do not fuse with FCMs and stay undifferentiated. D: Function of AMPs during myogenesis: AMPs stay quiescent and undifferentiated until the second larval instar. During larval development, AMPs divide actively to produce small pools of myoblasts. During pupal development, most of the larval muscles histolyse. At this time, AMPs begin differentiation and fuse to produce the adult musculature by de novo formation or by regeneration from the persisting larval templates. C: Position of AMPs in an embryonic abdominal segment. Schematic representation of AMPs and muscle fibers in the embryonic abdominal segment. In each hemisegment, six AMPs have been identified. A single ventral cell (v-AMP), a pair of lateral cells (l-AMPs), one dorsal (d-AMP), and two dorsolateral (dl-AMPs) cells. Lateral view of stage 15 embryos stained in left panel with antibodies against Twist (red) and in right panel for green fluorescent protein (GFP; blue) to reveal AMPs and β3-tubuline (green) to reveal muscle fibers. Expression of m6-GFP transgene, a read-out of the Notch pathway is specific to AMP (Lai et al., 2000).

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There are two waves of myogenesis in Drosophila. During embryonic development, the initial wave gives rise to the formation of an array of body wall muscles ensuring mobility of the larva. All larval muscles arise from the mesoderm layer formed by the ventralmost cells of the blastoderm embryo and expressing the basic helix–loop–helix transcription factor Twist (Twi) known to be required for mesoderm specification (Leptin, 1991). Initially uniform, Twi expression becomes modulated so that high and low Twi domains are present in each mesodermal segment (Borkowski et al., 1995; Azpiazu et al., 1996; Riechmann et al., 1997). Mesodermal cells expressing high Twi levels give rise to body wall muscles (Baylies and Bate, 1996), but Twi expression is not maintained in differentiated muscle cells. At the end of larval life, most embryonic muscles are hystolyzed. However, a population of Twi-expressing AMPs (Bate et al., 1991; Currie and Bate, 1991; Fernandes et al., 1991) proliferates and causes a second myogenic wave giving rise to de novo formation and regeneration of the adult fly musculature. There are six AMPs in each embryonic abdominal hemisegment: one ventral (v-AMP), two lateral (l-AMPs), two dorsolateral (dl-AMPs), and one dorsal (d-AMP).

AMPs are produced as sister cells of muscle founder cells (FCs) after the division of muscle progenitors (Ruiz-Gomez and Bate, 1997). Asymmetric segregation of Numb during division of progenitor cells results in the production of two sister cells with different identities. The daughter cell that inherits Numb, and therefore prevents the Notch pathway from functioning, is committed to becoming an FC and assembles an embryonic muscle. Its sister, which does not inherit Numb and which has Notch active signaling, continues to express Twi and postpones differentiation as an AMP cell. For instance, the v-AMP is a sibling cell of the FC of the embryonic ventral acute muscle 3 (VA3) and during pupation it gives rise to the adult ventral muscle. Similarly, the d-AMP is the sibling of the founder of the dorsalmost embryonic muscle, dorsal acute 1 (DA1), and in the adult forms the dorsalmost abdominal muscle. During larval development, AMPs divide actively to produce pools of myoblasts located in the interstices of larval muscles and associated with peripheral nerves (Bate et al., 1991). Finally, during metamorphosis, amplified AMPs contribute to de novo formation and regeneration of adult musculature and as in the embryo, Twi expression declines as differentiation begins. The muscle regeneration process taking place during metamorphosis is particularly well described for a subset of Indirect Flight Muscles, called DLMs, which undergo splitting to form muscle templates to which fuse attracted AMPs (Bernard et al., 2003). The capacities of adult fly musculature to undergo regeneration after localized injury have not yet been tested.

Several observations indicate that AMPs behave like vertebrate muscle stem cells and satellite cells in particular and may, therefore, be their invertebrate homologs. Laser ablation (Farrell and Keshishian, 1999) and transplantation experiments (Roy and VijayRaghavan, 1997) have shown that AMPs are not committed to a particular muscle lineage and the type of muscles they give rise to depends on environmental factors, such as growth factors and signals provided by surrounding tissue. One of the important features of satellite cells is capacity for self-renewal. As shown by laser ablation, the loss of l-AMPs may be compensated for by additional proliferation of AMPs in neighboring segments and subsequent migration to the ablated region (Farrell and Keshishian, 1999). Moreover, the specification of Drosophila AMPs by means of asymmetric Numb segregation is reminiscent of the process of satellite cell renewal in vertebrates. Like satellite cells, AMPs are quiescent during embryonic and early larval life and become activated by environmental factors (Bate et al., 1991). Satellite cells are associated with muscle fibers. Similarly, AMPs are located close to embryonic muscles, which do not histolyse and are likely to control AMP proliferation and (or) differentiation. Thus, AMPs and satellite cells share several features.

To isolate genes that modify the number of AMPs in Drosophila, we performed a gain-of-function screen (Bidet et al., 2003). Functional analyses of candidate genes showed that the receptor tyrosine kinase pathway and in particular the epidermal growth factor (EGF) pathway are required for the specification of a proper number of AMPs (Figeac N. and Jagla K., unpublished data). We observed loss of most AMPs in embryos expressing a dominant-negative form of EGF receptor and supernumerary AMPs when the EGF pathway was up-regulated. Our preliminary data indicate that the increased number of AMPs does not result from an increased proliferation but is instead due to the anti-apoptotic action of the EGF signals. This possibility is supported by the phenotypes observed in embryos overexpressing the inhibitor of apoptosis, DIAP1, as well as in mutants of different inducers of apoptosis. Of interest, a similar anti-apoptotic effect of the EGF pathway on activated satellite cells has been recently reported (Golding et al., 2007). These results provide additional evidence that Drosophila AMPs behave like vertebrate satellite cells and indicate that gaining insight into the genetic control of AMPs specification, proliferation, and survival may help in our understanding of muscle stem cell biology in general.

PERSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. HETEROGENEITY OF MYOGENIC STEM CELLS
  5. PROPERTIES OF SATELLITE CELLS
  6. UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS
  7. MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES
  8. Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS
  9. PERSPECTIVES
  10. REFERENCES

It is largely accepted that satellite cells account for most physiological muscle regenerative potential. Identification of genes that control satellite cell properties is thus of prime importance. The findings discussed here concerning a common origin for the embryonic muscle progenitors and the postnatal satellite cells open new avenues for studying the genetic control of muscle stem cell specification, proliferation and survival, which are of considerable interest in the context of muscular dystrophies. An important issue that needs to be addressed is how a subset of actively proliferating embryonic muscle progenitors becomes a reserve population and gives rise to quiescent adult satellite cells.

Further understanding of the signals and intrinsic genetic determinants regulating the maintenance, activation, and myogenic potential of muscle stem cells is a major challenge for the future. Given the similarities between mammalian, chicken, lower vertebrate, and Drosophila muscle stem cells presented here, the extensive use of simple animal models can offer a way to gain further insight into muscle stem cell biology.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. HETEROGENEITY OF MYOGENIC STEM CELLS
  5. PROPERTIES OF SATELLITE CELLS
  6. UNCOVERING THE EMBRYONIC ORIGIN OF SATELLITE CELLS USING CHICK AND MOUSE MODEL SYSTEMS
  7. MUSCLE STEM CELLS AND MUSCLE GROWTH IN LOWER VERTEBRATES
  8. Drosophila, AN EMERGING MODEL TO STUDY MUSCLE STEM CELLS
  9. PERSPECTIVES
  10. REFERENCES