Clin Genet 2000: 57: 1625 Printed in Ireland.

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Developmental Biology: Frontiers for Clinical Genetics

Section Editor: Roderick R McInnes e-mail: mcinnes@sickkids.on.ca

The molecular regulation of myogenesis

Sabourin LA, Rudnicki MA. The molecular regulation of myogenesis. Clin Genet 2000: 57: 16 25. Munksgaard, 1999 Over the past years, several studies have unraveled important mechanisms by which the four myogenic regulatory factors (MRFs: MyoD, Myf-5, myogenin, and MRF4) control the specication and the differentiation of the muscle lineage. Early experiments led to the hypothesis that these factors were redundant and could functionally replace one another. However, recent experiments using in 6i6o and in 6itro models have demonstrated that in fact different aspects of the myogenic program are controlled by different factors in 6i6o, suggesting that these factors play distinct roles during myogenesis. The activity of the MRFs during proliferation and differentiation of muscle precursor cells has clearly been demonstrated to be dependent on specic cell-cycle control mechanisms as well as distinct interactions with other regulatory molecules, such as the ubiquitously expressed E proteins and several other transcription factors. Furthermore, the observation that the MRFs can recruit chromatin remodeling proteins has shed some light on the mechanisms by which the MRFs activate gene expression. Recently, a functional role for MyoD during satellite cell activation and muscle repair has been identied in 6i6o, which cannot be substituted for by the other MRFs. This has put forward the hypothesis that these factors also play specic biological roles following muscle injury and repair.

Luc A Sabourin and Michael A Rudnicki

Institute for Molecular Biology and Biotechnology, MOBIX, McMaster University, Hamilton, Ontario, Canada

Corresponding author: Michael A Rudnicki, McMaster University, 1280 Main St. West, Life Sciences Rm 437, Hamilton, Ontario L8S 4K1, Canada. Tel: + 1 905 5259140 (ext: 27424); e-mail: rudnicki@mcmaster.ca Received 8 October 1999, revised and accepted for publication 14 October 1999

The myogenic regulatory factors (MRFs) are part of a superfamily of basic helix-loop-helix (bHLH) transcription factors including c-myc and acheatescute (13). The MRF subfamily consists of MyoD (Myf-3) (4), Myf-5 (5), myogenin (Myf-1) (6), and MRF4 (Myf-6/Herculin) (7 9). Twelve years ago, the MyoD gene was rst isolated from subtractive hybridization procedures using myoblast-specic cDNA libraries (4). The MyoD cDNA was identied by virtue of its ability to convert broblasts into myogenic cells. Subsequently, low stringency library screens uncovered three more MRFs all capable of inducing myogenic conversion when overexpressed in a vast number of nonmuscle cell lines. The MRF proteins contain a conserved basic DNA-binding domain essential for sequence-specic DNA binding and a helix-loop-helix motif required for heterodimerization. Each of the MRFs has been shown to heterodimerize in 6itro and in 6i6o with E proteins and to bind DNA in a sequence-specic manner at sites known as E-boxes (CANNTG). This DNA motif
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is present in the promoters of many skeletal muscle-specic genes and mediates gene activation in an MRF-dependent manner (13, 1012).
Expression of the myogenic factors during embryogenesis

The genes encoding the four MRFs have been shown to be expressed in a temporally distinct pattern. As determined by in situ hybridization, activation of Myf-5 occurs rst in the rostral somites of the mouse around 8 days postcoitum (dpc) and is down-regulated after day 14 (13). The activation of myogenin is observed at 8.5 dpc followed by MyoD at about 10.5 dpc along with markers of terminal differentiation (14). MRF4 is expressed transiently between days 9 and 12 and repressed until after birth (15). In the limb bud, Myf-5 is expressed transiently between days 10 and 12, followed by co-expression of myogenin and MyoD after day 10.5 and MRF4 after day 16 (1315). The specic promoter elements that gov-

The molecular regulation of myogenesis

ern the temporo-spatial expression of the MRFs have yet to be fully dened. However, several studies have identied specic enhancer elements for MyoD and myogenin (16 23). Induction of the myogenin gene has been shown to be dependent on an E-box and a myocyte enhancer factor-2 element for proper expression in the somites and limbs of the developing mouse, suggesting that its expression is MRFdependent, in part (17, 23). The expression of the MyoD gene during embryogenesis has been found to be regulated by at least two distinct enhancers. A rst element, located 5 kb upstream of the core promoter region, directs MyoD expression during the terminal differentiation of myogenic precursor cells into myotubes and myobers (18, 22). A second enhancer, found 20 kb upstream of the MyoD start site, has been demonstrated to direct the expression of a LacZ reporter gene during embryogenesis in specic somitic subdomains (19 21). The superposition of the expression patterns generated by both enhancers results in a pattern that is indistinguishable from the endogenous MyoD gene. The control of MyoD expression through the 20-kb element appears to be E-box-independent (24). In addition, demethylation of the MyoD locus has been demonstrated to play an important role during its activation de no6o (25). Interestingly, Tajbakhsh and co-workers have shown that expression of MyoD is dependent on either Myf-5 or Pax-3 (a paired-box domain protein), demonstrating that MyoD acts downstream of these genes during myogenesis (26) (and reviewed in (27)). In an independent study, ectopic expression of the Pax-3 gene in embryonic tissues was shown to induce the expression of MyoD and Myf-5 (28). In contrast, overexpression of Pax-3 in cultured myoblasts inhibits terminal differentiation in 6itro and this phenomenon appears to be dependent on Pax-3 DNA-binding activity (29). Further dissection of the MyoD promoter will likely uncover other enhancer elements important for the MyoD gene activation.
Targeted inactivation of the MRF genes

defect and results in viable and fertile mice but is unable to fully compensate for the absence of Myf-5 during myogenic determination (32, 33). However, mice decient for both MyoD and Myf-5 die at birth owing to a complete absence of skeletal myoblasts and muscle (34). Mice lacking myogenin display a normal number of myoblasts but die at birth because of an absence of myobers (35, 36). In contrast, inactivation of MRF4 results in viable mice with apparently normal muscles but with a fourfold increase in myogenin expression (3739). Taken together, these experiments have then dened two groups of MRFs. The primary MRFs, MyoD and Myf-5, appear to be required for myogenic determination, whereas the secondary MRFs, myogenin and MRF4, are required downstream of MyoD and Myf-5 as differentiation factors (Fig. 1). In addition, these studies have demonstrated that some MRFs can substitute for one another without affecting overall muscle development, suggesting the existence of potential redundancy among the MRFs (40). Recently, the use of MyoD-lacZ transgenic mice bred into the MyoD- or Myf-5-decient backgrounds has been used to address potential redundancy (41). MyoD-/- mice display normal epaxial (paraspinal and intercostal) muscle development, whereas hypaxial (limb and abdominal wall) development is delayed by 2.5 days (Fig. 2). In contrast, Myf-5-/- embryos exhibit normal muscle development in the limb buds and branchial arches, and markedly delayed development of epaxial muscles (41). Furthermore, normal migration of Pax-3-expressing cells into the limb buds and subsequent induction of Myf-5 in myogenic precursors are observed, suggesting that Myf-5 expression in the limb is insufcient for the normal progression of myogenic development (41). These observations suggest that MyoD and Myf-5 play distinct roles during the formation of epaxial and hypaxial muscles and argue against the existence of redundancy among the MRFs.

Gene targeting experiments have provided much insight into the functions of the MRFs in 6i6o. The introduction of null mutations in the four MRFs into the germline of mice has demonstrated the existence of a hierarchical relationship among the MRFs. Inactivation of MyoD in mice results in an apparently normal muscle phenotype with a fourfold increase in Myf-5 expression (30). Similarly, Myf-5-decient mice display normal skeletal muscles but die perinatally because of severe rib defects (31). Interestingly, introduction of the myogenin cDNA into the Myf-5 locus is able to rescue the rib

Fig. 1. Targeted inactivation of the MRFs has dened two groups of factors. The primary MRFs, MyoD and Myf-5, are required at the determination step for commitment of the proliferating somitic cells to the myogenic lineage. The committed cells (myoblasts) can proliferate and further differentiate into myocytes and mature into myobers under the action of the secondary MRFs, myogenin and MRF4.

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Fig. 2. Myogenic cell lineages. The temporal expression pattern of a MyoD-LacZ transgene in different null background shows that the 5-kb enhancer of MyoD, expressed in differentiated myocytes, is activated in the epaxial lineage (blue) in the absence of MyoD and in the hypaxial domain (red) in the absence of Myf-5. MyoD-/- mice display normal epaxial muscle development, whereas hypaxial development is delayed by 2.5 days, suggesting a specic role for Myf-5 in the establishment of the epaxial musculature. In contrast, Myf-5-/- embryos exhibit normal muscle development in the limb buds and branchial arches, and markedly delayed development of epaxial muscles, demonstrating a functional role for MyoD in the establishment of the hypaxial musculature.

Regulatory mechanisms of MRF activity

In 6itro differentiation systems have provided further into the regulation of MRFs activity and the relationships between growth and differentiation. Several studies have demonstrated that the MRFs can efciently heterodimerize with products of the E2-2 (ITF2) and E2-5 genes (E12, E47, and ITF1) (4244) (see Fig. 3 and reviewed in (2)). These heterodimers activate muscle-specic transcription of E-box-containing muscle gene promoters (44). However, it is not clear whether specic heterodimers play distinct biological roles. The MRFs have been shown to be negatively regulated by the HLH protein Id, which lacks a basic DNA-binding domain (45, 46). The Id factors, encoded by at least four different genes (Id1, Id2, Id3, and Id4), act in a dominant negative manner by heterodimerizing with E proteins preventing their association with the MRFs and subsequent muscle-specic gene activation (46) (Fig. 3). Similarly, MyoD activity has been shown to be inhibited in 6itro by the murine twist (mTwist) protein (47). The mTwist protein is also thought to sequester E proteins, preventing MRF-E protein heterodimer formation. In addition to Id and mTwist proteins, MyoD has recently been shown to be negatively regulated by dimerization with Mist1, a novel bHLH factor that lacks a transactivation domain (48). The resulting heterodimer does not bind E-box-containing promoters. The activity of the MRFs is also tightly coupled to the cell cycle (reviewed in (49, 50)). For example, the hypophosphorylated form of the retinoblastoma protein (Rb) has been demon18

strated to associate with MyoD and to be required for efcient transactivation of E-box-containing muscle-specic promoters (51). The Rb protein is also necessary to maintain the differentiated phenotype of cultured myotubes. In addition, induction of differentiation in cultured myoblasts results in up-regulation of the cell-cycle inhibitors p21 (WAF-1, Cip1) and p16 (5254). The p21 gene has been demonstrated to bear E-box in its upstream regulatory regions and is activated by MyoD overexpression in transient assays (53). Supporting these observations, high expression of p21 has been correlated with the activation of the myogenin gene during embryogenesis (55). The cell-cycle MRF connection is further substantiated by the observation that overexpression of cyclin D1, a G1-S cyclin and a Cdk4 activator, inhibits MyoD activity and subsequent transactivation of E-boxcontaining reporter genes (56, 57). Furthermore, MyoD activity has been demonstrated to be downregulated by direct interaction with the C-terminal domain of Cdk4 (58) (Fig. 3). This interaction appears to require the cyclin D1-dependent nuclear targeting of Cdk4. The activity of the MRFs is further coupled to cellular proliferation by the negative regulatory effect of the AP1 heterodimer Fos/c-Jun. The proto-oncogene, c-Jun, directly binds MyoD and inhibits its activity (59, 60). Furthermore, upon differentiation of cultured myoblasts in 6itro, the c-Fos promoter is down-regulated by MyoD through an E-box within the c-Fos promoter (61). Various other oncogenes such as c-myc, N-ras, and Ha-ras also inhibit muscle differentiation in 6itro, suggesting

The molecular regulation of myogenesis

that growth-promoting factors negatively regulate the MRFs (6266). In addition, viral oncogenes such as adenovirus E1a inhibit differentiation and MRF activity by direct physical interaction (67, 68). MRF activity is also potentially negatively regulated through phosphorylation. For example, myogenin is phosphorylated directly by protein kinase C (PKC) in 6itro and in 6i6o (69). Fibroblast growth factor (FGF) treatment or PKC overexpression results in threonine-87 phosphorylation in the DNA-binding domain and a loss of DNA-binding activity. Similarly, protein kinase A negatively regulates myogenin through an indirect mechanism (70). Recently, the mitogen-activated protein kinase (MAPK) pathway has been shown to be activated in differentiating muscle cells and to positively regulate the expression and activity of the MyoD protein (71). However, in contrast, Bennet and co-workers have demonstrated that upon mitogen withdrawal from C2C12 myoblasts, the MAPK p42Erk2 is inactivated concomitant with up-regulation of muscle-specic genes (72). Supporting this, they showed that overexpression of MAPK phosphatase-1 inhibited p42Erk2 activity and was sufcient to relieve the inhibitory effects of mitogens on muscle-specic gene expression. Similarly, continuous activation of MEK, a MAP kinase kinase, is detrimental to insulin-like growth factor-1- (IGF-1) or FGF-2-induced myogenesis (73). Recently, the activity of stress-activated protein kinase 2 (SAPK2/p38) has also been demonstrated to be important for the terminal differentiation of C2C12 myoblasts (74). Whether different components of the MAPK pathway play distinct biological roles

during growth or differentiation remains to be elucidated.

Regulation of transcription through MRF/co-factor interactions

In recent years, attempts at identifying the mechanisms underlying MRF functions have uncovered a number of MRF-binding proteins and co-factors. Interestingly, these co-activators are known to play important roles in chromatin remodeling, RNA polymerase II functions, or are transcription factors themselves. Among the co-activators, MyoD has been shown to interact directly with p300/CBP (7578). This interaction was demonstrated both in 6i6o and in 6itro and appeared to be required for the terminal differentiation of cultured myoblasts. This interaction occurs through the carboxyl cysteine/histidine-rich (C/H3) domain of p300 and increases the ability of MyoD to transactivate an E-box-containing reporter construct (76, 78). Active gene expression is associated with histone acetylation and loss of histone/DNA interaction. Interestingly, in addition to p300/CBP, MyoD interacts with the histone acetyltransferase PCAF in a multiprotein complex also containing p300/CBP (78). Disruption of this complex by anti-PCAF antibody microinjection inhibits muscle differentiation, indicating that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program. In a separate study, Gerber and co-workers have demonstrated that a cysteine-histidine-rich region of MyoD, upstream of the basic DNA-

Fig. 3. The activity of the MyoD family is coupled to cell-cycle control. In proliferating myoblasts, activated cyclin-dependent kinases (Cdk4) inhibit MyoD activity through direct interaction. Expression of Id proteins precludes the formation of E protein-MRFs heterodimers. Upon differentiation, withdrawal from the cell cycle is maintained by a positive feedback loop in which high p21 and Rb expression prevents re-entry into the cell cycle and the MRF-E protein complex is activated. Green arrows denote positive, whereas blunt red arrows denote negative regulatory relationships.

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binding domain, is necessary for chromatin remodeling and gene activation by MyoD (79). Although no known muscle diseases have been associated with genetic alterations in any of the MRFs (80), there is evidence that mutations in co-factors for the MRFs are contributing to the pathogenesis of rhabdomyosarcomas. Such genetic modications include the amplication of MDM2, for which overexpression has been shown to inhibit myoblast differentiation (81). In addition, heterokaryon formation studies have revealed that rhabdomyosarcomas are decient for an unknown factor required for MyoD activity (82). In addition to these factors, MyoD also interacts with components of the transcriptional machinery. Recently, the TATA-binding protein TFIID has been identied as a novel MyoD-binding protein and found to stabilize the binding of MyoD to its consensus binding site (83). Furthermore, MyoD has been observed to facilitate the association of TFIIB with the preinitiation complex subsequent to DNA binding. Interaction of the MRF-E12 dimers with muscle LIM protein also results in increased DNA-binding activity and stimulation of myogenesis (84). Transcription factors such as MEF2-C, a member of the MEF2 family of transcription factors, has also been demonstrated to interact directly and synergize with the MyoD-E12 heterodimer but not with either protein alone (reviewed in (8587)). Similarly, serum response factor, a MEF2-related protein, has been shown to bind and enhance the activity of MRFs-E12 heterodimers (88).
Muscle regeneration and satellite cell function: role of MyoD

Satellite cells, the stem cells of adult skeletal muscles, reside beneath the basal lamina of adult skeletal muscle closely juxtaposed against the muscle bers (89). Satellite cells arise around 17 dpc during mouse embryogenesis and are believed to represent a unique myoblast lineage. Satellite cells mediate the postnatal growth of muscle and contribute for the most part to the formation of the adult muscle mass (89). Satellite cells make up 27% of the nuclei associated with a particular myober. This proportion varies with age and a particular muscle group. Satellite cells are normally mitotically quiescent but are activated and re-enter the cell cycle in response to stress induced by weight-bearing exercise or trauma, including injury (89 91). The daughter cells of the activated satellite cells, called myogenic precursor cells (mpcs), undergo multiple rounds of division prior to fusion with the existing 20

or new myobers. Satellite cells appear to form a population of stem cells that are biologically and biochemically distinct from their descendant mpcs (89, 92). The total number of quiescent satellite cells in adult muscle remains relatively constant over multiple cycles of degeneration and regeneration, suggesting that self-renewal in the satellite cell compartment maintains a population of quiescent cells (89). However, the numbers and proliferative potential of satellite cells become progressively reduced in muscle diseases presenting with muscular atrophy, such as Duchenne muscular dystrophy (DMD), which is likely due to high levels of ongoing regeneration (93, 94). The essential role played by satellite cells in muscle regeneration, muscle hypertrophy, and postnatal muscle growth has been demonstrated extensively (89, 92, 95). However, the molecular mechanisms underlying the activation and function of myogenic stem cells are still unclear. As determined by polymerase chain reaction analysis, quiescent satellite cells display no detectable levels of either of the four MRFs (96). Upon injury and activation, MyoD is rapidly up-regulated within 12 h. This up-regulation of MyoD occurs prior to the expression of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation. The expression of myogenin occurs last during the time associated with fusion and differentiation (97, 98). Analysis of gene expression by reverse transcription-polymerase chain reaction of individual satellite cells following their activation in intact muscle bers (96) substantiates that quiescent satellite cells express no detectable MRFs but do express the c-met receptor tyrosine kinase (the receptor for hepatocyte growth factor). Activated satellite cells rst express either Myf-5 or MyoD. Subsequently, both factors are co-expressed during the proliferative phase. Following proliferation, myogenin and MRF4 are expressed in cells entering the terminal differentiation program. The absence of MRF mRNA in satellite cells prior to activation suggests that satellite cells represent a stem cell lineage that is distinct from myoblasts. Furthermore, the de no6o induction of Myf-5 and MyoD transcription implies that inductive signals are involved, analogous to those that occur during embryogenesis (27, 99). The role of MyoD in satellite cell function has been investigated by interbreeding MyoD-/- mice (30) with mdx mice. The mdx mouse carries a loss of function point mutation in the X-linked dystrophin gene, and thus is an animal model for human Duchenne and Becker muscular dystrophy (93). The mdx mice display a high regenerative capacity leading to muscle hypertrophy, making it

The molecular regulation of myogenesis

an attractive model to investigate the role of the MRFs during muscle regeneration. Studies have shown that the compound mutant mice (mdx :MyoD-/-) exhibit markedly increased penetrance of the mdx phenotype characterized by muscle atrophy and increased myopathy leading to premature death (100). By 3 5 months of age, mdx:MyoD -/- mice develop a profound dorsal ventral curvature of the spine similar to the lordosis and kyphosis of patients with DMD. Interestingly, unlike mdx mice, mdx :MyoD-/- animals also display severe cardiomyopathy, a hallmark of DMD patients (101). Muscle regeneration is severely impaired in MyoD-/- mice and is characterized by an almost complete absence of proliferative myogenic precursor cells as determined by 3H-thymidine incorporation or immunohistochemistry with antibody reactive to PCNA (100). However, electron microscopic examination of MyoD-decient muscle reveals the presence of morphologically normal satellite cells. However, cell counts show that their relative abundance is increased by 1.8-fold in MyoD-/- muscle and 13-fold in mdx :MyoD-/- muscle. These data suggest that up-regulation of MyoD is required for satellite cells to enter the mpc proliferative phase prior to terminal differentiation. In the absence of MyoD, myogenic stem cells undergo several rounds of division and return to a quiescent state rather than progressing through the developmental program. Taken together, these experiments strongly support the hypothesis that satellite cells form a stem cell compartment that is the source of myogenic precursor cells (100). To gain insight into the regeneration decit of MyoD-/- muscle, satellite cell-derived primary cultures from adult MyoD-/- hind limb muscle were generated and analyzed for proliferative and differentiation potential. Low passage MyoD-/- myogenic cells exhibit a broblast-like morphology distinct from the bipolar morphology of wildtype myoblasts (102). Myogenic cells lacking MyoD express c-met (96, 103), but do not express desmin, an intermediate lament protein typically expressed in myoblasts in 6itro and in 6i6o (104). Following the induction of differentiation in 6itro, wildtype myoblasts undergo cell-cycle arrest and fuse into multinucleated myotubes, whereas MyoD-/- cells continue to proliferate and yield reduced numbers of predominantly mononuclear myocytes after several days in differentiation medium (102, 105). In addition, the expression of differentiation-specic markers is drastically reduced or absent in MyoD-/cells. As for MyoD-/- muscle tissue, MyoD-/- myoblast cultures display a fourfold increase in Myf-5 mRNA expression, suggesting that overexpression

Fig. 4. Role of MyoD in satellite cell function. Upon activation, quiescent satellite cells, expressing c-met, rst express Myf-5 or MyoD before co-expressing both and progressing through the developmental program. In the absence of MyoD, satellite cells appear to exhibit a propensity for self-renewal rather than progression through the differentiation program. Expression of Myf-5 alone may allow self-renewal either before returning to quiescence (yellow arrow) or up-regulating MyoD and formation of proliferative mpcs (white arrows).

of Myf-5 cannot alleviate the differentiation defect imparted by the inactivation of MyoD in these cells. Furthermore, culture mixing experiments using LacZ -marked MyoD-/- cells has demonstrated that the MyoD-/- cellular phenotype is cell autonomous. Interestingly, expression of IGF -1 is markedly increased in MyoD -/- myogenic cells cultured under differentiation conditions, suggesting that MyoD normally negatively regulates IGF-1 expression in primary myogenic cells. One possibility is that IGF1 promotes proliferation and inhibits differentiation of MyoD-/- myoblasts via an autocrine loop. In addition, the expression of M-cadherin is markedly reduced in MyoD-/- myogenic cells and a requirement for M-cadherin has been reported for cell-cycle withdrawal and myoblast fusion (106, 107). Taken together, these results suggest that MyoD-/myogenic cells represent an intermediate stage in the satellite cell activation pathway downstream of the quiescent state but upstream of the mpc compartment (102) (see Fig. 4). Since Myf-5-/- mice die perinatally (31), a denitive role for Myf-5 in satellite cell activation has yet to be determined.
Future work

It is now well established that the MRFs constitute a group of four bHLH transcription factors that play a pivotal role during the specication and differentiation of muscle cells. To date, several signaling pathways that regulate MRF activity have been identied. However, it will be of interest to identify additional regulatory components such as kinases, phosphatases, and other transducers that are directly controlling the activity of the MRFs under growth and differentiation condi21

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tions. Of great interest will be the identication of the regulatory elements and the factors involved in the control of the de no6o MRF gene expression during embryogenesis and satellite cell activation. This is especially important for the induction of MyoD and Myf-5 as these determination factors are likely to be subject to different regulatory mechanisms, whether the cells are in a context of embryogenesis or muscle regeneration. The activity of the MyoD protein has been demonstrated to be modulated by its interaction with various nuclear co-factors and transactivators. The identication of additional interacting co-factors or RNA polymerase II-associated components will provide further insights into our understanding of the molecular mechanisms underlying tissue-specic gene expression. In addition, the identication of specic target genes for which the expression is regulated by one or more MRFs will be valuable for understanding the distinct biological roles played by each of the MRFs. Finally, the specic functions of each of the MRFs during satellite cell activation and muscle regeneration remain to be determined. The use of MyoD-decient and MRF4-/- mice will prove useful in elucidating their role during satellite cell activation but a major difculty lies in the generation of viable animals bearing mutations for the other two MRFs. The establishment of conditional mutant lines may circumvent the viability problems encountered with the Myf-5-/- or myogenin-null mice. Alternatively, the use of knockins, as demonstrated by Wang and co-workers (33), may be helpful in this regard.

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