Table of contents

Role of bHLH proteins in muscle specification vs. differentiation

The myogenic basic helix-loop-helix (bHLH) proteins regulate both skeletal muscle specification and differentiation: MyoD and Myf5 establish the muscle lineage, whereas myogenin mediates differentiation. MyoD is more efficient than myogenin at initiating the expression of skeletal muscle genes, and the molecular basis for this difference has been investigated. A conserved amphipathic alpha-helix in the carboxy terminus of the myogenic bHLH proteins has distinct activities in MyoD and myogenin: the MyoD helix facilitates the initiation of endogenous gene expression, whereas the myogenin helix functions as a general transcriptional activation domain. Thus, the alternate use of a similar motif for gene initiation and activation provides a molecular basis for the distinction between specification and differentiation within the myogenic bHLH gene family (Bergstrom, 2001).

Together, these experiments demonstrate that MyoD and myogenin share a structurally conserved carboxy-terminal alpha-helical motif that performs a distinct function in each protein. In the specification gene, MyoD, helix III is necessary for the efficient initiation of the expression of at least a subset of endogenous skeletal muscle genes, but it does not have significant function as a classical activation domain. In myogenin, the differentiation gene, helix III acts as a general transcriptional activation domain but cannot facilitate the initiation of skeletal muscle gene expression. This structural motif appears to be a major distinguishing feature between the activities of MyoD and myogenin, since substitution of this motif into myogenin converts it into a protein with activity similar to MyoD and Myf5. The fact that an additional substitution of the MyoD histidine-rich region is necessary for the full activity of the chimeric transcription factor suggests that the helix III activity might be further facilitated or regulated by interactions that are dependent on this region. The mechanism by which the MyoD helix III increases the efficiency of endogenous gene initiation remains unknown. This region might be necessary for the activation of a subset of skeletal muscle promoters, perhaps overcoming promoter-specific negative regulators. Since the transiently transfected desmin promoter does not require these domains for activation, yet these domains are required for the full activation of the endogenous desmin gene, it seems unlikely that simple cis regulatory sequences are sufficient to account for the dependence of the endogenous skeletal muscle genes on these domains. The histidine- and cysteine-rich region is necessary for MyoD-mediated chromatin remodeling, and it is possible that helix III also contributes to chromatin remodeling at skeletal muscle gene loci (Bergstrom, 2001).

The development and differentiation of distinct cell types is achieved through the sequential expression of subsets of genes; yet, the molecular mechanisms that temporally pattern gene expression remain largely unknown. In skeletal myogenesis, gene expression is initiated by MyoD and includes the expression of specific Mef2 isoforms and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. p38 activity facilitates MyoD and Mef2 binding at a subset of late-activated promoters, and the binding of Mef2D recruits Pol II. Most importantly, expression of late-activated genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that a MyoD-mediated feed-forward circuit temporally patterns gene expression (Penn, 2004).

Temporally patterned gene expression in a complex program of cell differentiation is achieved through a feed-forward mechanism. MyoD initiates the expression of specific Mef2 isoforms and activates the p38 MAPK pathway. p38 activity facilitates MyoD and Mef2 binding at genes expressed late in the myogenic program, and the binding of Mef2D recruits Pol II and correlates with the transcription of these genes. Most importantly, expression of some late-stage genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that the timing of expression is programmed by an intrinsic delay while Mef2 isoforms and p38 activity accumulate, and substantiating the role of a transcriptional feed-forward circuit in temporally patterning gene expression. Because p38 and Mef2D cooperate with MyoD to regulate only a subset of late-stage genes, it is likely that additional sets of genes might require other MyoD-regulated intermediate factors (Penn, 2004).

This study suggests two distinct roles of p38 kinase: (1) as a rate limiting factor in the binding of Mef2 and MyoD, and (2) in facilitating phosphorylation and progression of Pol II. The role of p38 in facilitating the binding of MyoD and Mef2 is likely to be through an effect on chromatin, since it does not alter the binding of these factors in gel-shift assays, and the recent demonstration that the p38 pathway targets the SWI/SNF complex to muscle loci through an interaction with MyoD might account for its effect on factor binding, although other mechanisms, such as histone phosphorylation, might also effect factor binding. The role of p38 in facilitating Pol II phosphorylation and progression is likely to be through the phosphorylation of Mef2D, because prior studies have shown that p38 phosphorylation of the Mef2 activation domain greatly potentiates the transcriptional activity of Mef2. This study shows that the Mef2D isoform is rate limiting for transcription at a subset of late promoters. This suggests that the Mef2D isoform has promoter-specific activities and that the relative abundance of Mef2 isoforms determines which subsets of promoters are actively transcribed (Penn, 2004).

Satellite cells are myogenic precursors responsible for skeletal muscle regeneration. Satellite cells are absent in the Pax-7-/- mouse, suggesting that this transcription factor is crucial for satellite cell specification. Analysis of Pax-7 expression in activated satellite cells unexpectedly revealed substantial heterogeneity within individual clones. Further analyses show that Pax-7 and myogenin expression are mutually exclusive during differentiation, where Pax-7 appears to be up-regulated in cells that escape differentiation and exit the cell cycle, suggesting a regulatory relationship between these two transcription factors. Indeed, overexpression of Pax-7 down-regulates MyoD, prevents myogenin induction, and blocks MyoD-induced myogenic conversion of 10T1/2 cells. Overexpression of Pax-7 also promotes cell cycle exit even in proliferation conditions. Together, these results suggest that Pax-7 may play a crucial role in allowing activated satellite cells to reacquire a quiescent, undifferentiated state. These data support the concept that satellite cell self-renewal may be a primary mechanism for replenishment of the satellite cell compartment during skeletal muscle regeneration (Olguin, 2004).

A working model is presented for the role of Pax-7 in satellite cell physiology. Mitotically quiescent satellite cells express a subset of characteristic proteins including the markers syndecan-3, syndecan-4, and c-met but are heterogeneous for Pax-7 protein. Upon activation, satellite cells proliferate and up-regulate MyoD. Proliferating myoblasts that are positive for both Pax-7 and MyoD behave as a heterogeneous population where a small fraction of cells are prone to precocious differentiation, inducing myogenin and losing Pax-7 expression, while a small number retain precursor characteristics. Co-expression of Pax-7 and MyoD might be required to retain myoblasts in a proliferative state and prevent premature differentiation. As the myogenic program proceeds, MyoD family transcription factors are up-regulated and Pax-7 is down-regulated in cells committed to differentiation. A small number of precursor cells up-regulate Pax-7 and down-regulate MyoD, exit the cell cycle, and form a new satellite cell pool. Additional events might be involved to evade apoptosis and acquire the final satellite cell position beneath the basal lamina of regenerated muscle fibers (Olguin, 2004).

In skeletal muscle development, the myogenic regulatory factors myf5 and myoD play redundant roles in the specification and maintenance of myoblasts, whereas myf6 has a downstream role in differentiating myocytes and myofibers. It is not clear whether the redundancy between myf5 and myoD is within the same cell lineage or between distinct lineages. Using lineage tracing and conditional cell ablation in mice, this study demonstrates the existence of two distinct lineages in myogenesis: a myf5 lineage and a myf5-independent lineage. Ablating the myf5 lineage is compatible with myogenesis sustained by myf5-independent, myoD-expressing myoblasts, whereas ablation of the myf6 lineage leads to an absence of all differentiated myofibers, although early myogenesis appears to be unaffected. It was also demonstrated the existence of a significant myf5 lineage within ribs that has an important role in rib development, suggested by severe rib defects upon ablating the myf5 lineage (Haldar, 2008).

Rhabdomyosarcomas are characterized by expression of myogenic specification genes, such as MyoD and/or Myf5, and some muscle structural genes in a population of cells that continues to replicate. Because MyoD is sufficient to induce terminal differentiation in a variety of cell types, attempts were made to determine the molecular mechanisms that prevent MyoD activity in human embryonal rhabdomyosarcoma cells. This study shows that a combination of inhibitory Musculin:E-protein complexes and a novel splice form of E2A compete with MyoD for the generation of active full-length E-protein:MyoD heterodimers. A forced heterodimer between MyoD and the full-length E12 robustly restores differentiation in rhabdomyosarcoma cells and broadly suppresses multiple inhibitory pathways. These studies indicate that rhabdomyosarcomas represent an arrested progress through a normal transitional state that is regulated by the relative abundance of heterodimers between MyoD and the full-length E2A proteins. The demonstration that multiple inhibitory mechanisms can be suppressed and myogenic differentiation can be induced in the RD rhabdomyosarcomas by increasing the abundance of MyoD:E-protein heterodimers suggests a central integrating function that can be targeted to force differentiation in muscle cancer cells (Yang, 2009).

Ascl2 inhibits myogenesis by antagonizing the transcriptional activity of myogenic regulatory factors

Myogenic regulatory factors (MRFs), including Myf5, MyoD and Myog (see Drosophila Nautilus), are muscle-specific transcription factors that orchestrate myogenesis. Although MRFs are essential for myogenic commitment and differentiation, timely repression of their activity is necessary for the self-renewal and maintenance of muscle stem cells (satellite cells). This study defines Ascl2 (see Drosophila Achaete) as a novel inhibitor of MRFs. During mouse development, Ascl2 is transiently detected in a subpopulation of Pax7+ MyoD+ progenitors (myoblasts) that become Pax7+ MyoD- satellite cells prior to birth, but is not detectable in postnatal satellite cells. Ascl2 knockout in embryonic myoblasts decreases both the number of Pax7+ (see Drosophila Paired) cells and the proportion of Pax7+ MyoD- cells. Conversely, overexpression of Ascl2 inhibits the proliferation and differentiation of cultured myoblasts and impairs the regeneration of injured muscles. Ascl2 competes with MRFs for binding to E-boxes in the promoters of muscle genes, without activating gene transcription. Ascl2 also forms heterodimers with classical E-proteins to sequester their transcriptional activity on MRF genes. Accordingly, MyoD or Myog expression rescues myogenic differentiation despite Ascl2 overexpression. Ascl2 expression is regulated by Notch signaling, a key governor of satellite cell self-renewal. These data demonstrate that Ascl2 inhibits myogenic differentiation by targeting MRFs and facilitates the generation of postnatal satellite cells (Wang, 2017).

Upstream repression and activation of myogenesis

FGFs are likely to act to delay differentiation of myoblasts in the early development of limbs. Differentiation of muscle and cartilage within developing vertebrate limbs occurs in a proximodistal progression. To investigate the cues responsible for regulating muscle pattern, mouse myoblasts were implanted into early chick wings prior to endogenous chick muscle differentiation. Fetal myogenic cells originating from transgenic mice carrying a lacZ reporter are readily detected in vivo after implantation and their state of differentiation determined with species-specific antibodies to MyoD and myosin heavy chain. When mouse myogenic cells are implanted at the growing tip of early stage 21 limbs MyoD expression is suppressed and little differentiation of the mouse cells is detected initially. At later stages ectopically implanted mouse cells come to lie within muscle masses, re-express MyoD and differentiate in parallel with differentiating chick myoblasts. However, if mouse cells are implanted either proximally at stage 21 or into the limb tip at stage 24, situations in which mouse cells encounter endogenous differentiating chick myoblasts earlier, MyoD suppression is not detected and a higher proportion of mouse cells differentiate. Mouse cells that remain distal to endogenous differentiating myogenic cells are more likely to remain undifferentiated than myoblasts that lie within differentiated chick muscle. Undifferentiated distal mouse cells are still capable of differentiating if explanted in vitro, suggesting that myoblast differentiation is inhibited in vivo. In vitro, MyoD is suppressed in primary mouse myoblasts by the addition of FGF2 and FGF4 to the culture media. Taken together, these data suggest that the inhibition of myogenic differentiation in the distal limb involves MyoD suppression in myoblasts, possibly through an FGF-like activity (Robson, 1996).

While wingless expression in early Drosophila development is ectodermal, and ectodermal Wingless is required for mesodermal expression of nautilus, in Xenopus embryos the prospective mesoderm expresses wingless homolog Xwnt-8. Xenopus mesoderm is induced initially with domains of dorsal and ventral fate, then further patterned to generate somitic mesoderm by signals from the gastrula organizer. Expression of a dominant-negative Xwnt-8 (dnXwnt-8) inhibits embryonic responses to Wnt signaling in a cell-nonautonomous fashion. By expressing dnXwnt-8 in Xenopus, a requirement can be established for Wnt signaling in localized expression in prospective mesoderm of XMyoDa (Drosophila homolog: nautilus) and Xenopus-posterior (Xpo). XPO is a novel protein that is normally widely expressed early but later becomes restricted to the ventrolateral marginal zone by the mid-gastrula stage. Because ectopic expression of functional Xwnt-8 in the dorsal marginal zone of the gastrula induces ectopic XMyoDa and Xpo, both gain-of-function and loss-of-function experiments support a model in which endogenous Xwnt-8 functions to induce expression of genes involved in specification of ventral and somitic mesoderm. It thus appears that the inductive effect of Wingless on nautilus in Drosophila is conserved in Xenopus, although the Wingless homolog is expressed in mesoderm and not in ectoderm (Hoppler, 1996).

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Transient exposure of embryos or explants to lithium, which mimics Wnt/ß-catenin signaling, has been used as a tool to regulate the activation of this pathway at different times and places during early development. Activation of Wnt/ß-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/ß-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. Zygotic Wnt/ß-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/ß-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells (Shi, 2002).

To understand how the skeletal muscle lineage is induced during vertebrate embryogenesis, an attempt has been made to identify the regulatory molecules that mediate induction of the myogenic regulatory factors MyoD and Myf-5. Either signals from the overlying ectoderm or Wnt and Sonic hedgehog signals can induce somitic expression of the paired box transcription factors, Pax-3 (Drosophila homolog: Paired) and Pax-7, concomitant with expression of Myf-5 and prior to that of MyoD. Infection of embryonic tissues in vitro with a retrovirus encoding Pax-3 is sufficient to induce expression of MyoD, Myf-5, and myogenin in the neural tube as well as in both paraxial and lateral plate mesoderm in the absence of inducing tissues. Together, these findings imply that Pax-3 may mediate activation of MyoD and Myf-5 in response to muscle-inducing signals from either the axial tissues or overlying ectoderm and identify Pax-3 as a key regulator of somitic myogenesis (Maroto, 1997).

The splotch (Pax3) mouse mutant serves as a model for developmental defects of several types, including defective migration of dermomyotomal cells to form the limb musculature. Abnormalities of the ribs, neural arches, and acromion are described in Sp2H homozygous embryos, indicating a widespread dependence of lateral somite development on Pax3 function. Moreover, the intercostal and body wall muscles, derivatives of the ventrolateral myotome, are also abnormal in Sp2H homozygotes. Pax3 is expressed in the dermomyotome, but not in either the sclerotome or the myotome, raising the possibility that Pax3-dependent inductive influences from the dermomyotome are necessary for early specification of lateral sclerotome and myotome. Support for this idea comes from analysis of gene expression markers of lateral sclerotome (tenascin-C and scleraxis) and myotome (myogenin, MyoD, and Myf5). All exhibit ventrally truncated domains of expression in Sp2H homozygotes, potentially accounting for the rib and intercostal muscle truncations. In contrast, the medial sclerotomal marker Pax1 is expressed normally in mutant embryos, arguing that Pax3 is not required for development of the medial sclerotome. Most of the somitic markers show ectopic expression in anteroposterior and mediolateral dimensions, suggesting a loss of definition of somite boundaries in splotch and explaining the rib and muscle fusions. An exception is Myf5, which is not ectopically expressed in Sp2H homozygotes, consistent with the suggestion that Pax3 and Myf5 function in different pathways of skeletal myogenesis. PDGFalpha and its receptor are candidates for mediating signaling between myotome and sclerotome. Both genes are misexpressed in Sp2H embryos, suggesting that PDGFalpha/PDGFRalpha may function downstream of Pax3, accounting for the close similarities between the splotch and Patch mutant phenotypes. These findings point to additional regulatory functions for the Pax3 transcription factor, apart from those already demonstrated for development of the neural tube, neural crest, and dermomyotome (Henderson, 1999).

Activation of myogenesis in newly formed somites is dependent on signals derived from neighboring tissues, namely axial structures (neural tube and notochord) and dorsal ectoderm. In explants of paraxial mesoderm from mouse embryos, axial structures preferentially activate myogenesis through a Myf5-dependent pathway, while dorsal ectoderm is preferentially activated through a MyoD-dependent pathway. Cells expressing Wnt1 will preferentially activate Myf5 while cells expressing Wnt7a will preferentially activate MyoD. Wnt1 is expressed in the dorsal neural tube and Wnt7a in dorsal ectoderm in the early embryo, therefore both can potentially act in vivo to activate Myf5 and MyoD, respectively. Wnt4, Wnt5a and Wnt6 exert an intermediate effect activating both Myf5 and MyoD equivalently in paraxial mesoderm. Sonic Hedgehog synergises with both Wnt1 and Wnt7a in explants from E8.5 paraxial mesoderm but not in explants from E9.5 embryos. Signaling through different myogenic pathways may explain the rescue of muscle formation in Myf5 null embryos, which do not form an early myotome but later develop both epaxial and hypaxial musculature. Explants of unsegmented paraxial mesoderm contain myogenic precursors capable of expressing MyoD in response to signaling from a neural tube isolated from E10.5 embryos, the developmental stage when MyoD is present throughout the embryo. Myogenic cells cannot activate MyoD in response to signaling from a less mature neural tube. Together these data suggest that different Wnt molecules can activate myogenesis through different pathways such that commitment of myogenic precursors is precisely regulated in space and time to achieve the correct pattern of skeletal muscle development (Tajbakhsh, 1998).

Signals originating from tissues surrounding somites are involved in mediolateral and dorsoventral patterning of somites and in the differentiation of the myotome. Wnt-1 and Wnt-3a, which encode members of the Wnt family of cystein-rich secreted signaling molecules, are coexpressed at the dorsal midline of the developing neural tube, an area adjacent to the dorsomedial portion of the somite. Several lines of evidence indicate that Wnt-1 and Wnt-3a have the ability to induce the development of the medial and dorsal portion of somites, as well as to induce myogenesis. To address whether these Wnt signalings are really essential for the development of somites during normal embryogenesis, the development of somites was investigated in mouse embryos lacking both Wnt-1 and Wnt-3a. The medial compartment of the dermomyotome is not formed and the expression of a lateral dermomyotome marker gene, Sim-1, is expanded more medially in the absence of these Wnt signalings. In addition, the expression of a myogenic gene, Myf-5, is decreased at 9.5 days post coitum whereas the level of expression of a number of myogenic genes, in particular MyoD, in the later stage appear normal. These results indicate that Wnt-1 and Wnt-3a signalings actually regulate the formation of the medial compartment of the dermomyotome and the early part of myogenesis. Differential regulation of Myf-5 and MyoD suggests that a second unknown signal from the surface ectoderm or from the dorsal neural tube in later stage embryos is sufficient for the activation of Myf-5 and MyoD expression and the progression of myotome development. Thus, there is a functional redundancy in myogenesis between inducing signals (Ikeya, 1998).

Myogenic regulatory factors (MRFs) comprise a family of transcription factors that when expressed in a cell reflects the commitment of that cell to a myogenic fate before any cytological sign of muscle differentiation is detectable. Myogenic cells in limb skeletal muscles originate from the lateral half of the somites. Cells that migrate away from the lateral part of the somites to the limb bud do not initially express any member of the MRF family. Expression of MRFs in the muscle precursor cells starts after the migration process is completed. The extracellular signals involved in activating the myogenic program in muscle precursor cells in the in vivo limb are not known. Sonic Hedgehog (SHH) expressed in the posterior part of the limb bud (the zone of polarizing activity) could be involved in the differentiation of muscle precursor cells in the limb. Retrovirally overexpressed SHH in the limb bud first induces the extension of the expression domain of the Pax-3 gene, then that of the MyoD gene, and finally that of the myosin protein. This leads to a hypertrophy of the muscles in vivo. Addition of SHH to primary cultures of myoblasts results in an increase in the proportion of myoblasts that incorporate bromodeoxyuridine, resulting in an increase in the number of myotubes. These data show that SHH is able to activate myogenesis in vivo and in vitro in already committed myoblasts and suggest that the stimulation of the myogenic programme by SHH involves activation of cell proliferation. It is suggested that SHH may activate Pax-3 expression, which in turn activates MyoD (Duprez, 1998).

Signals from the neural tube, notochord, and surface ectoderm promote somitic myogenesis. Somitic myogenesis is under negative regulation as well; BMP signaling serves to inhibit the activation of MyoD and Myf5 in Pax3-expressing cells. BMP-4 is highly expressed in both the dorsal-neural tube and lateral plate mesoderm; when ectopically expressed, between the axial (nerve cord) and paraxial (lateral plate mesoderm) tissues, BMP-4 can block somitic expression of MyoD. BMP antagonist Noggin is expressed within the dorsomedial lip of the dermomyotome, where Pax3-expressing cells first initiate the expression of MyoD and Myf5 to give rise to myotomal cells in the medial somite. Consistent with the expression of Noggin in dorsomedial dermomyotomal cells that lie adjacent to the dorsal neural tube, coculture of somites with fibroblasts programmed to secrete Wnt1 (which is expressed in dorsal neural tube) can induce somitic Noggin expression. Ectopic expression of Noggin lateral to the somite dramatically expands MyoD expression into the lateral regions of the somite, represses Pax3 expression in this tissue, and induces formation of a lateral myotome. Together, these findings indicate that the timing and location of myogenesis within the somite are controlled by relative levels of BMP activity and localized expression of a BMP antagonist (Reshef, 1998).

p202 is a primarily nuclear, interferon-inducible murine protein that is encoded by the Ifi 202 gene. Overexpression of p202 in transfected cells retards cell proliferation. p202 modulates the pattern of gene expression by inhibiting the activity of various transcription factors, including NF-kappaB, c-Fos, c-Jun, E2F-1, and p53. p202 is constitutively expressed in mouse skeletal muscle and the levels of p202 mRNA and p202 greatly increase during the differentiation of cultured C2C12 myoblasts to myotubes. When overexpressed in transfected myoblasts, p202 inhibits the expression of one muscle protein (MyoD) without affecting the expression of a second one (myogenin). Thus, the decrease in the level of MyoD (but not of myogenin) during muscle differentiation may be the consequence of the increase in p202 level. Overexpressed p202 also inhibits the transcriptional activity of both MyoD and myogenin. This inhibition is correlated with an interaction of p202 with both proteins, as well as the inhibition by p202 of the sequence-specific binding of both proteins to DNA. This inhibition of the expression of MyoD and of the transcriptional activity of MyoD and myogenin may account for the inhibition of the induction of myoblast differentiation by premature overexpression of p202 (Datta, 1998).

Mutations in neurogenic genes result in an expression of nautilus, and a muscle precursor hyperplasia (too many cells) (Corbin, 1991). Ectopic expression of the intracellular domain of mNotch, the mouse homolog of Notch) functions as a constitutively activated repressor of myogenesis both in cultured cells and frog embyros. The mNotch intracellular domain contains a nuclear localization signal and localizes to the nucleus. Removal of the nuclear localization signal reduces nuclear localization and diminishes the inhibition of myogenesis caused by Myf-5 or MyoD. The target for Notch inhibition seems to be the bHLH region of MyoD, as MyoD derivatives missing the N terminus, the C terminus and the C/H region (residues 63-98) are all inhibited. However, mNotch does not affect the MyoD activation domain, but may inhibit a co-factor required for MyoD activation (Kopan, 1994).

When a proliferating myoblast culture is induced to differentiate by deprivation of serum in the medium, a significant proportion of cells escape from terminal differentiation, while the rest of the cells differentiate. Using C2C12 mouse myoblast cells, this heterogeneity observed upon differentiation was investigated with an emphasis on the myogenic regulatory factors. The differentiating part of the cell population follows a series of well-described events, including expression of myogenin, p21(WAF1), and contractile proteins, and permanent withdrawal from the cell cycle and cell fusion, whereas the rest of the cells do not initiate any of these events. Interestingly, the latter cells show an undetectable or greatly reduced level of MyoD and Myf-5 expression, which is originally expressed in the undifferentiated proliferating myoblasts. When these undifferentiated cells are isolated and returned to the growth conditions, they progress through the cell cycle and regain MyoD expression. These cells demonstrate identical features with the original culture on the deprivation of serum. They produced both MyoD-positive differentiating and MyoD-negative undifferentiated populations once again. Thus the undifferentiated cells in the serum-deprived culture have been designated 'reserve cells'. Upon serum deprivation, MyoD expression rapidly decreases as a result of down-regulation in approximately 50% of the cells. After this heterogenization, MyoD positive cells express myogenin, which is the earliest known event of terminal differentiation and marks irreversible commitment to this, while MyoD-negative cells do not differentiate and became the reserve cells. Ectopic expression of MyoD converts the reserve cells to differentiating cells, indicating that down-regulation of MyoD is a causal event in the formation of reserve cells (Yoshida, 1998). Pax-3 (splotch), Myf-5 (targeted with nlacZ), and splotch/Myf-5 homozygous mutant mice were analyzed to investigate the roles that these genes play in programming skeletal myogenesis. In splotch and Myf-5 mutant embryos, myogenic progenitor cell perturbations and early muscle defects are distinct. Remarkably, splotch/Myf-5 double homozygotes have a dramatic phenotype not seen in the individual mutants: body muscles are absent. MyoD does not rescue this double mutant phenotype since activation of this gene proves to be dependent on either Pax-3 or Myf-5. Therefore, Pax-3 and Myf-5 define two distinct myogenic pathways, and MyoD acts genetically downstream of these genes for myogenesis in the body. This genetic hierarchy does not appear to operate for head muscle formation (Tajbakhsh, 1997).

Myogenic regulatory factors (MRF) of the MyoD family regulate the skeletal muscle differentiation program. Non-muscle cells transfected with exogenous MRF either are converted to the myogenic lineage or fail to express the muscle phenotype, depending on the cell type analysed. MRF-induced myogenic conversion of NIH3T3 cells results in an incomplete reprogramming of these cells. Transfected cells withdraw from the cell cycle and undergo biochemical differentiation but, surprisingly, terminally differentiated myocytes absolutely fail to fuse into multinucleated myotubes. Analysis of muscle regulatory and structural gene expression fails to provide an explanation for the fusion defectiveness. However, myogenic derivatives of NIH3T3 cells are unable to accumulate the transcripts encoding muscle-specific isoforms of the integrin subunit ss1D and the transcription factor MEF2D1b2, that depend on muscle-specific alternative splicing. These results suggest that the fusion into myotubes is under a distinct genetic control that might depend, at least partially, on differential splicing (Russo, 1998).

Bcl-2 expression in skeletal muscle cells identifies an early stage of the myogenic pathway, inhibits apoptosis, and promotes clonal expansion. Bcl-2 expression is limited to a small proportion of the mononucleate cells in muscle cell cultures, ranging from approximately 1%-4% of neonatal and adult mouse muscle cells to approximately 5%-15% of the cells from the C2C12 muscle cell line. In rapidly growing cultures, some of the Bcl-2-positive cells coexpress markers of early stages of myogenesis, including desmin, MyoD, and Myf-5. In contrast, Bcl-2 is not expressed in multinucleate myotubes or in those mononucleate myoblasts that express markers of middle or late stages of myogenesis, such as myogenin, muscle regulatory factor 4 (MRF4), and myosin. The small subset of Bcl-2-positive C2C12 cells appear to resist staurosporine-induced apoptosis. Furthermore, though myogenic cells from genetically Bcl-2-null mice form myotubes normally, the muscle colonies produced by cloned Bcl-2-null cells contain only about half as many cells as the colonies produced by cells from wild-type mice. This result suggests that, during clonal expansion from a muscle progenitor cell, the number of progeny obtained is greater when Bcl-2 is expressed (Dominov, 1998).

In vertebrates, skeletal muscle is derived from progenitor cell populations located in the epithelial dermomyotome compartment of the each somite. These cells become committed to the myogenic lineage upon delamination from the dorsomedial and dorsolateral lips of the dermomyotome and entry into the myotome or dispersal into the periphery. Paraxis is a developmentally regulated basic-helix-loop-helix transcription factor that is required to direct and maintain the epithelial characteristic of the dermomyotome. Therefore, it was hypothesized that Paraxis acts as an important regulator of early events in myogenesis. Expression of the muscle-specific myogenin-lacZ transgene was used to examine the formation of the myotome in the paraxis-/- background. Two distinct types of defects were observed that mirror the different origins of myoblasts in the myotome. In the medial myotome, where the expression of the myogenic factor Myf5 is required for commitment of myoblasts, the migration pattern of committed myoblasts is altered in the absence of Paraxis. In contrast, in the lateral myotome and migratory somitic cells, which require the expression of MyoD, expression of the myogenin-lacZ transgene is delayed by several days. This delay correlates with an absence of MyoD expression in these regions, indicating that Paraxis is required for commitment of cells from the dorsolateral dermomyotome to the myogenic lineage. In paraxis-/-/myf-/- neonates, dramatic losses are observed in the epaxial and hypaxial trunk muscles that are proximal to the vertebrae in the compound mutant, but not those at the ventral midline or the non-segmented muscles of the limb and tongue. In this genetic background, myoblasts derived from the medial (epaxial) myotome are not present to compensate for deficiencies of the lateral (hypaxial) myotome. These data demonstrate that Paraxis is an important regulator of a subset of the myogenic progenitor cells from the dorsolateral dermomyotome that are fated to form the non-migratory hypaxial muscles (Wilson-Rawls, 1999).

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development -- namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. In chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. Ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes one another's effects on MyoD. Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose one another's trancriptional actions on MyoD. The Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, it is proposed that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. These results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells (Bendall, 1999).

MyoD inhibits cell proliferation and promotes muscle differentiation. A paradoxical feature of rhabdomyosarcoma (RMS), a tumor arising from muscle precursors, is the block of the differentiation program and the deregulated proliferation despite MyoD expression. A deficiency in RMS of a factor required for MyoD activity has been implicated by previous studies. p38 MAP kinase (MAPK) activation, which is essential for muscle differentiation, is deficient in RMS cells. Enforced induction of p38 MAPK by an activated MAPK kinase 6 (MKK6EE) restores MyoD function and enhances MEF2 activity in RMS deficient for p38 MAPK activation, leading to growth arrest and terminal differentiation. Stress and cytokines can activate the p38 MAPK in RMS cells, however, these stimuli do not promote differentiation, possibly because they activate p38 MAPK only transiently and they also activate JNK, which can antagonize differentiation. The data indicate that p38 MAPK stimulates MyoD activity by a mechanism that does not involves the interaction with the p38 MAPK substrate MEF2. Moreover, a p38 MAPK-dependent activation of MyoD through direct phosphorylation could not be detected. Thus, p38 MAPK might indirectly activate MyoD by targeting bHLH-interacting proteins or cofactors like p300 and PCAF It is concluded that selective and sustained p38 MAPK activation, which is distinct from the stress-activated response, is required for differentiation and can be disrupted in human tumors (Puri, 2000).

The mechanisms by which pluripotent embryonic cells generate unipotent tissue progenitor cells during development are unknown. Molecular/genetic experiments in cultured cells have led to the hypothesis that the product of a single member of the MyoD gene family (MDF) is necessary and sufficient to establish the positive aspects of the determined state of myogenic precursor cells: i.e., the ability to initiate and maintain the differentiated state. Embryonic cell type determination also involves negative regulation, such as the restriction of developmental potential for alternative cell types, that is not directly addressed by the MDF model. In the experiments reported here, phenotypic restriction in myogenic precursor cells is assayed by an in vivo 'notochord challenge' to evaluate their potential to 'choose' between two alternative cell fate endpoints: cartilage and muscle. The notochord challenge assay used here to demonstrate the loss of cartilage potential in precursor cells of the limb muscles is fundamentally different from previous assays of limb muscle precursor cell specification in vitro: (1) the assay is designed to challenge muscle phenotype commitment with cartilage-inducing signals in the native embryonic environment, using embryonic signals that are known to affect somite cell specification; (2) two differentiated cell type endpoints ('choices'), muscle and cartilage, are scored simultaneously; (3) the assay is well-defined anatomically, allowing detailed analysis of the morphogenetic potential of the implanted cells (Williams, 2000).

Two separate myogenic precursor cell populations were found to be phenotypically restricted while expressing the Pax3 gene and prior to MDF gene activation. Therefore, while MDF family members act positively during myogenic differentiation, the process of phenotypic restriction (the negative aspect of cell specification) requires cellular and molecular events and interactions that precede MDF expression in myogenic precursor cells. The qualities of muscle formed by the determined myogenic precursor cells in these experiments further indicate that their developmental potential is intermediate between that of myoblastic stem cells taken from fetal or adult tissue (which lack mitotic and morphogenetic potential when tested in vivo) and embryonic stem cells (which are multipotent). It is hypothesized that such embryonic myogenic progenitor cells represent a distinct class of determined embryonic cell, one that is responsible for both tissue growth and tissue morphogenesis (Williams, 2000).

Sonic hedgehog (Shh) is a secreted signaling molecule for tissue patterning and stem cell specification in vertebrate embryos. Shh mediates both long-range and short-range signaling responses in embryonic tissues through the activation and repression of target genes by its Gli transcription factor effectors. Despite the well-established functions of Shh signaling in development and human disease, developmental target genes of Gli regulation are virtually unknown. The role of Shh signaling has been examined in the control of Myf5, a skeletal muscle regulatory gene for specification of muscle stem cells in vertebrate embryos. Shh is required for Myf5 expression in the specification of dorsal somite epaxial muscle progenitors. However, these studies did not distinguish whether Myf5 is a direct target of Gli regulation through long-range Shh signaling, or alternatively, whether Myf5 regulation is a secondary response to Shh signaling. To address this question, transgenic analysis with lacZ reporter genes was used to characterize an Myf5 transcription enhancer that controls the activation of Myf5 expression in the somite epaxial muscle progenitors in mouse embryos. This Myf5 epaxial somite (ES) enhancer is Shh-dependent, as shown by its complete inactivity in somites of homozygous Shh mutant embryos, and by its reduced activity in heterozygous Shh mutant embryos. Furthermore, Shh and downstream Shh signal transducers specifically induce ES enhancer/luciferase reporters in Shh-responsive 3T3 cells. A Gli-binding site located within the ES enhancer is required for enhancer activation by Shh signaling in transfected 3T3 cells and in epaxial somite progenitors in transgenic embryos. These findings establish that Myf5 is a direct target of long-range Shh signaling through positive regulation by Gli transcription factors, providing evidence that Shh signaling has a direct inductive function in cell lineage specification (Gustafsson, 2002).

In vertebrates, despite the evidence that extrinsic factors induce myogenesis in naive mesoderm, other experiments argue that the initiation of the myogenic program may take place independent of these factors. To resolve this discrepancy, this issue was re-addressed using short-term in vivo microsurgery and culture experiments in chick. The results show that the initial expression of the muscle-specific markers Myf5 and MyoD is regulated in a mesoderm-autonomous fashion. The reception of a Wnt signal is required for MyoD, but not Myf5 expression; however, the source of the Wnt signal is intrinsic to the mesoderm. Gain- and loss-of-function experiments indicate that Wnt5b, which is expressed in the presomitic mesoderm, represent the MyoD-activating cue. Despite Wnt5b expression in the presomitic mesoderm, MyoD is not expressed in this tissue: these experiments demonstrate that this is due to a Bmp inhibitory signal that prevents the premature expression of MyoD before somites form. These results indicate that myogenesis is a multistep process that is initiated prior to somite formation in a mesoderm-autonomous fashion: as somites form, influences from adjacent tissues are likely to be required for maintenance and patterning of early muscles (Linker, 2003).

In this report, an antisense RNA strategy has shown that disruption of ALP expression affects the expression of the muscle transcription factors myogenin and MyoD, resulting in the inhibition of muscle differentiation. Introduction of a MyoD expression construct into ALP-antisense cells is sufficient to restore the capacity of the cells to differentiate, illustrating that ALP function occurs upstream of MyoD. It is known that MyoD is under the control of serum response factor (SRF), a transcriptional regulator whose activity is modulated by actin dynamics. A dramatic reduction of actin filament bundles is observed in ALP-antisense cells and treatment of these cells with the actin-stabilizing drug jasplakinolide stimulates SRF activity and restores the capacity of the cells to differentiate. Furthermore, it was shown that modulation of ALP expression influences SRF activity, the level of its coactivator, MAL, and muscle differentiation. Collectively, these results suggest a critical role of ALP on muscle differentiation, likely via cytoskeletal regulation of SRF (Pomies, 2007).

Brown fat can increase energy expenditure and protect against obesity through a specialized program of uncoupled respiration. This study shows, by in vivo fate mapping, that brown, but not white, fat cells arise from precursors that express Myf5, a gene previously thought to be expressed only in the myogenic lineage. The transcriptional regulator PRDM16 (PRD1-BF1-RIZ1 homologous domain containing 16; homolog of Drosophila Hamlet) controls a bidirectional cell fate switch between skeletal myoblasts and brown fat cells. Loss of PRDM16 from brown fat precursors causes a loss of brown fat characteristics and promotes muscle differentiation. Conversely, ectopic expression of PRDM16 in myoblasts induces their differentiation into brown fat cells. PRDM16 stimulates brown adipogenesis by binding to PPAR-gamma (peroxisome-proliferator-activated receptor-gamma) and activating its transcriptional function. Finally, Prdm16-deficient brown fat displays an abnormal morphology, reduced thermogenic gene expression and elevated expression of muscle-specific genes. Taken together, these data indicate that PRDM16 specifies the brown fat lineage from a progenitor that expresses myoblast markers and is not involved in white adipogenesis (Seale, 2008).

Myf cascade and control of rib specification

Hox genes are essential for the patterning of the axial skeleton. Hox group 10 has been shown to specify the lumbar domain by setting a rib-inhibiting program in the presomitic mesoderm (PSM). Mice have been produced with ribs in every vertebra by ectopically expressing Hox group 6 in the PSM, indicating that Hox genes are also able to specify the thoracic domain. The information provided by Hox genes to specify rib-containing and rib-less areas is first interpreted in the myotome through the regional-specific control of Myf5 and Myf6 expression. This information is then transmitted to the sclerotome by a system that includes FGF and PDGF signaling to produce vertebrae with or without ribs at different axial levels. These findings offer a new perspective of how Hox genes produce global patterns in the axial skeleton and support a redundant nonmyogenic role of Myf5 and Myf6 in rib formation (Vinagre, 2010).

Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice

Kruppel-like factor 5 (Klf5) (see Drosophila dar1) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. This study shows that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation (see myogenesis in Drosophila). During muscle regeneration after injury (cardiotoxin injection), Klf5 is induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impairs muscle regeneration, and myotube formation is suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppresses induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD (see Drosophila nau) and Mef2 (see Drosophila Mef2), and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment is greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2 (Hayashi, 2016).

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