Cell Tissue Res (1999) 296:199–212

© Springer-Verlag 1999

INVITED ARTICLE

Beate Brand-Saberi · Bodo Christ

Genetic and epigenetic control of muscle development in vertebrates

Received: 23 September 1998 / Accepted: 18 November 1998

Abstract The skeletal body muscle of vertebrates is derived from segmentally arranged mesodermal structures, the somites. Only the dorsal epithelial half of the somite, the dermomyotome, gives rise to muscle cells during normal development. Head muscle takes its origin from the somites, the unsegmented paraxial head mesoderm and the prechordal mesoderm. Some muscle precursor cells, for instance those for limb and tongue muscle, migrate over considerable distances before differentiating at their target sites. In recent years, our understanding of the molecular events underlying myogenesis has increased considerably. Muscle differentiation is preceded by several steps during which precursor cells are specified. Markers of myogenic specification are myf5, myoD, mrf4 and myogenin, which encode transcription factors of the basic helix-loop-helix family. These factors bind to promoters of many musclespecific genes and interact with MEF2 (myocyte enhancer binding factor-2) belonging to the MADS (MCM1, agamous, deficiens, serum response factor) box transcription factors. Signalling events leading to myogenic precursor cell specification and to the formation of muscle fibres are being elucidated. Inductive signals emanate from the neural tube, notochord and ectoderm. Controversial findings concerning the role of the notochord and neural tube in muscle development suggest that the epigenetic events leading to myogenesis are more complex than originally anticipated. Signals from the lateral plate counteract those from the axial organs and induce the locally restricted emigration of muscle precursor cells. Future investigations will have to show how signalling molecules and their receptors interact

Part of the work mentioned in this review article was generously sponsored by grants from the Deutsche Forschungsgemeinschaft to B.B.S. (Br 957/2–1; Br 957/5–1; Ch 44/12–2 and 3) and B.C. (Ch 44/12–1, 2 and 3). B. Brand-Saberi (✉) · B. Christ Anatomisches Institut II, Universität Freiburg, Albertstrasse 17, D-79104 Freiburg, Germany e-mail: [email protected]; Tel.: +49 761 203 5086; Fax: +49 761 203 5091

in the process of fine-tuning muscle formation in the embryo. Key words Myogenesis · Differentiation · bHLH · Transcription factors · MADS-box transcription factors · pax genes · Cell migration

Introduction Among the diversity of vertebrate cell types, skeletal muscle is in several respects remarkable. One reason is that its myogenic progenitor cells can originate in sites distant from where they differentiate, so that they have to undergo long migrations to reach their target sites. Muscle cells are incapable of proliferation, but they are usually associated with a pool of cells still capable of replicating and regenerating the muscle tissue. In adult muscle, these cells are termed satellite cells. Another reason is that once fully differentiated, skeletal muscle is no longer made up of individual cells, but consists of multinucleate muscle fibres. Finally, the specification of skeletal muscle precursors, as well as muscle differentiation and functional maintenance, are dependent on cell-cell interactions. In recent years, the molecular mechanisms underlying these processes have been unravelled. The steps leading to the formation of functional muscle fibres and the factors involved in the specification and the commitment of precursor cells have been the object of intense studies for decades. A landmark in our understanding of muscle development was the discovery of transcription factors capable of triggering muscle development even in non-myogenic cells such as fibroblasts (Choi et al. 1990; Cserjesi and Olson 1991; Weintraub et al. 1994). In this article, we focus not only on the inherent determination factors involved in muscle development, i.e. their genetic control, but also on the signals that have been found to induce their expression, i.e. the epigenetic events triggering this process. These mechanisms are interesting especially with respect to migrating myogenic cells, such as the muscle precursor cells for the limb buds, tongue and dia-

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Fig. 1 A A 21/2-day chick embryo (HH-stage 18). In situ hybridization for pax-3. Note the onset of expression in the segmental plate. Arrows point to strong expression in the lateral portions of the dermomyotomes. Migration to the limb buds (lb leg bud, sp segmental plate) has not started yet. B Cranial part of same embryo. Arrow indicates the pax-3-positive myogenic cells migrating from the first six somites to the tongue region, forming the hypoglossal cord. They will later give rise to the tongue muscles, which in the chick are extrinsic muscles. C Four-day chick embryo (HH-stage 24). pax-3 expression in the paraxial mesoderm is visible in the hypaxial muscle buds (arrow-

heads). It now reappears in the medial dermomyotome lip (arrows). pax-3-positive myogenic cells have now populated the limb buds (asterisks). D In situ hybridization for myoD in a 41/2-day chick embryo (HH-stage 25). myoD is expressed in the myotomes in a metameric pattern. The proximal myogenic zones of the limb buds, and myoblasts in the branchial arches and in the hypoglossal cord also express myoD. E Four-day chick embryo (HH-stage 23) stained with a monoclonal antibody against desmin. The myotomes are stained especially at the cranial and caudal edges; the limb buds are still devoid of staining. F Detail of myotomes shown in E

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phragm. Here, not only determination factors and their inducers are involved in muscle development, but also signals controlling their location within the body. Most of our information results from studies using the transgenic approach in the mouse and micromanipulations in the avian embryo. Due to highly conserved molecules and mechanisms, however, it is possible to formulate some universal rules that most likely also underlie early muscle development in the human and have a bearing on pathological situations. Apart from summarizing recent findings, it is our aim to pose questions where we see still unsolved problems in the fascinating process of muscle development.

The origin of skeletal muscle Skeletal muscle in the vertebrate embryo can be traced to three major embryonic groups: epaxial body muscle, hypaxial body muscle and head muscle. Body muscle (i.e. trunk and limb muscle) is entirely derived from the somites, whereas head muscle develops from three sources: somites, paraxial head mesoderm and prechordal mesoderm (Wachtler and Christ 1992; Christ and Ordahl 1995; Noden 1983). All muscle cells produced by somites have their origin in the dermomyotome, an epithelial structure located in the dorsal part of the somite. As the embryo develops, the dermomyotomes shift from the dorsal into an oblique position and come to lie more laterally. From each dermomyotome arises a second layer subjacent to and beneath it: the myotome. Myotome cells are longitudinally arranged postmitotic myocytes expressing muscle markers such as desmin and myosins (Holtzer 1957; Kaehn et al. 1988; Williams and Ordahl 1997; Fig. 1E,F). Myotome formation is still not entirely understood, but there is evidence that it develops from the craniomedial corner of the dermomyotome as well as its edges (Kaehn et al. 1988; Denetclaw et al. 1997). The medial myotome produces epaxial muscle which will yield the intrinsic back muscle (Christ and Ordahl 1995; Brand-Saberi et al. 1996a), whereas the lateral myotome and the lateral portion of the dermomyotome consisting of double-layered “muscle buds” produce the hypaxial muscle (Wachtler and Christ 1992; Spörle et al. 1996; Fig. 1C). The muscles of the ventral body wall such as the thoracic and abdominal muscles develop from these muscle buds. The attribute “hypaxial” is also applied to the limb muscle. Here, a special situation is found, because the limb musculature is derived from the lateral dermomyotomes only. The lateral dermomyotome disintegrates to release individually migrating, undifferentiated myogenic precursor cells capable of proliferation (Williams and Ordahl 1994). Individually migrating muscle precursor cells from the somites are also involved in muscle formation in the tongue and diaphragm (van Bemmelen 1889; Schemainda 1979; Wells 1954; Fig. 1B).

The genetic specification of muscle precursor cells Specification of the pool of cells that will give rise to muscle is a multistep process starting long before somitogenesis. The epiblast contains cells with the potential to form muscle after experimental dissociation (George-Weinstein et al. 1996). In vitro, the percentage of cells from the primitive streak that differentiate into muscle correlates with the number of N-cadherin-expressing cells. The switch from Eto N-cadherin takes place during gastrulation, because cultured epiblast cells predominantly express E-cadherin, whereas N-cadherin prevails in cells from the primitive streak (George-Weinstein et al. 1997). In vivo, the key events that stabilize myogenic competence and finally lead to myogenic commitment also appear to occur during gastrulation (Krenn et al. 1988; Kirschhofer et al. 1994; Christ and Ordahl 1995). Markers of myogenic specification belong to the family of basic helix-loop-helix (bHLH) transcription factors and to the MADS (MCM1, agamous, deficiens, serum response factor) box transcription factors. Proteins of the first group are Myf5, MyoD, MRF4 and Myogenin (Ott et al. 1991; Pownall and Emerson 1992; Sassoon 1993). Overexpression of the respective genes, especially myoD and myf5, can convert many different cell types to a myogenic fate (Choi et al. 1990; Weintraub 1993). Because of this, MyoD and Myf5 have been termed muscle determination factors, while myogenin and mrf4 are active genetically downstream of myf5 and myoD. myf5/myoD double knockout mice are unable to generate muscle precursor cells (Rudnicki et al. 1993). Since myf5 and myoD null mice both have muscle, it has been assumed that these two muscle determination factors act along separate pathways or can compensate for one another (Rudnicki et al. 1992; Weintraub 1993; Braun and Arnold 1996; Wang et al. 1996). Members of the bHLH family of myogenic transcription factors activate muscle-specific genes by binding to their promoters via their basic regions, while forming heterodimers with other HLH proteins via their HLH regions (Benezra et al. 1990). Myogenic bHLH factors are first expressed even before myotome formation in the dorsomedial area of the epithelial somite and subsequently in the myotome (Fig. 1D). In the mouse, myf5 is the first gene to be expressed in the myotomes (Ott et al. 1991), while myoD precedes myf5 in the avian somite (Pownall and Emerson 1992; Fig. 1D). Members of the bHLH-transcription factors also interact with the second type of myogenic regulators. MEF2 (myocyte specific enhancer factor-2) belongs to the MADS family of transcription factors (Cserjesi and Olson 1991; Lassar et al. 1991). mef2 is not restricted to skeletal muscle, but appears first in the differentiating myocard. However, for a stable commitment to the myogenic fate and for muscle differentiation to occur, mef2 has to be expressed in addition to members of the bHLH family, since it enhances and stabilizes their expression (Molkentin and Olson 1995). In addition to these synergistic mechanisms, myogenesis is controlled by antagonists. Myogenic inhibitors have been des-

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Fig. 2 A Operation scheme: the ventral half of an epithelial quail somite is extirpated and grafted into a dorsal position in a chick host embryo. B After 2 days of reincubation, the ventral half has differentiated according to its new surroundings: it yields desmin-positive myotome (my) and dermis of the back. Feulgen-staining reveals quail nuclei. C Barrier insertion (arrow) between axial organs and paraxial mesoderm. On the operated side, a desmin-positive myotome (my) is absent 2 days later (nt neural tube, no notochord, drg dorsal root ganglion, asterisk paraxial mesoderm). D mef2C in situ hybridization after removal of the neural tube in the caudal part of the embryo. Arrow-

head points to normal expression of mef2C in the myotomes. Arrows point to operation region where somites are fused and mef2C expression is absent. E Section through operated area shows fused dermomyotomes (arrows) in the dorsal position; neural tube is absent (no mef2C expression, no notochord). F Ectopic notochord graft (no) into the paraxial mesoderm prevents formation of dermomyotome (dm) and myotome (my) after a reincubation of 24 h. G Notochord removal results in ectopic muscle formation and fusion of myotomes (arrow) underneath the neural tube (nt). Desmin staining

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cribed, such as ID, Msx1, Twist and BMP-4 (Wang et al. 1992; Hebrok et al. 1994; Hogan 1996). Signals that tip the balance between induction and inhibition of myogenesis will be discussed below. In the embryo, the genes of the different transcription factors are expressed in temporally and spatially highly specific ways reflecting the origin of muscle precursor cells. It has recently been shown that myogenic bHLH-gene expression is preceded by the expression of a gene originally implicated in the function of myogenic cell migration: pax-3. The pax genes are paired domain and homeodomain homologues of Drosophila paired. pax-3 is first expressed throughout the segmental plate. As somitic compartments form, its expression becomes restricted to the dermomyotomes; later on it is highly expressed in the lateral portion of the dermomyotome and in migrating myogenic cells that are derived from them at limb bud and tongue level (Goulding et al. 1994; Williams and Ordahl 1994; Daston et al. 1996; Fig. 1A,B). Myogenesis was analysed in double mutant mice carrying a mutation of the pax-3 gene (splotch) in combination with a targeted deletion of myf5. In sp/myf5 double homozygous mutant embryos, no striated muscle forms in the body and myoD is not activated (Tajbakhsh et al. 1997). Moreover, forced expression of pax-3 using retroviral expression systems in chick tissue explants has revealed that pax-3 activates myoD, myf5 and myogenin (Maroto et al. 1997). These findings suggest that pax-3 acts genetically upstream of myogenic bHLH genes and therefore specifies cells with muscle-forming potential at a very early stage. Intriguingly, sp/myf5 double mutants do possess normally developed head muscle (Tajbakhsh et al. 1997). Since normal non-somitic muscle precursors do not express pax-3 or the highly related gene pax-7 (Mansouri et al. 1996), this finding does not contradict the notion that pax-3 is necessary for muscle development in the body. However, it raises the interesting question of how myogenesis is controlled in the head.

The epigenetic events: local signalling During normal development, the aforementioned genes underlying muscle specification and differentiation are only activated in particular locations of the embryo. By experimentally forced expression, they can be induced ectopically, resulting in a change of fate in the targeted cells (Choi et al. 1990; Weintraub et al. 1994; Maroto et al. 1997). What induces muscle regulatory genes in normal development? This key question can be answered by a classical experiment. An approach originally applied by Spemann and coworkers asks whether a grafted piece of tissue develops according to its programmed fate or whether this fate is established by local signalling in its new surroundings. When the ventral half of a newly formed somite is taken from a 2-day quail embryo and grafted to replace the dorsal half of a 2day chick embryo, it differentiates like a normal dorsal half, i.e. it gives rise to muscle and dermis (Fig. 2A,B).

This approach is characterized by the fact that it answers one question with yes or no, but it simultaneously opens the door to many new questions. What we have learned is that the paraxial mesoderm is not determined regarding its dorsoventral polarity up to somite III (the third somite from the segmental plate; Christ and Ordahl 1995). However, we do not know how this determination is achieved. The avian embryo provides suitable conditions to address this question by micromanipulations interfering with local tissue interactions that could be the basis of signalling events leading to determination of tissues. Observations of disturbed development following such manipulations date back to the middle of this century (Avery et al. 1956). If we remove one-half of the neural tube surgically, or insert a barrier between neural tube and paraxial mesoderm, muscle formation is inhibited on the operated side of the embryo (Fig. 2C). The absence of muscle in chick embryos is preceded by an absence of epithelial structures. pax-3 and the epithelialization gene paraxis are not expressed (Fig. 3A–C; Sosic et al. 1997; Schmidt et al. 1998). However, muscle regulatory factors of the bHLH-type are initially expressed in the absence of the neural tube, suggesting that they have already been activated or become activated by other signals (Bober et al. 1994a). After longer reincubation periods, however, myoD, myf5 and myogenin are downregulated and myogenesis does not continue. There may be several reasons for this, one being the fact that mef2A and C are not expressed in the absence of the neural tube (Sosic, Olson, Christ, Brand-Saberi, unpublished; Fig. 2D,E). Because expression of the myogenic bHLH factors is stabilized and enhanced by MEF2, which itself depends on signals from the neural tube, stable myogenic commitment and muscle differentiation are mediated by signals from the neural tube. Concerning the exact source of muscle-promoting signals within the neural tube, findings are controversial. While Buffinger and Stockdale (1995) have found that the ventral half of the neural tube supports myogenesis in vitro, several other studies attribute this activity to the dorsal part of the neural tube (Stern et al. 1995; Spence et al. 1996). The ventral neural tube and the notochord are sources of the signalling molecule Sonic Hedgehog (SHH), a key morphogen of vertebrate limb and somite patterning (Riddle et al. 1993; Johnson et al. 1994). Originally, the notochord was described to have a ventralizing influence on the somite-inducing ventral (sclerotome) markers such as pax1, when grafted ectopically, while the (dorsal) neural tube has a dorsalizing influence (Brand-Saberi et al. 1993a; Pourquié et al. 1993; Goulding et al. 1994; Ebensperger et al. 1995; Xue and Xue 1996; Fig. 2F). Conversely, the absence of the notochord can lead to an extension of the muscleforming domain ventrally, resulting in ectopic muscle underneath the neural tube (Fig. 2G). More recently, notochord signals have also been found to support or even induce myogenesis (Pownall et al. 1996; Buffinger and Stockdale 1994, 1995; Borycki et al. 1998). This apparent contradiction may be explained by different concentrations of the signalling molecule SHH (Dietrich et al. 1997) or by different stages of sensitivity and maturation of the paraxi-

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Fig. 3 A Operation scheme showing barrier insertion between axial organs and segmental plate alone and in combination with ectoderm removal. B In situ hybridization for pax-3 after barrier insertion. Arrows point to the region that had been separated from the axial organs: somites are absent and pax-3 expression is lacking. Barrier was removed during fixation. C In situ hybridization for paraxis after barrier insertion. Arrowheads point to operated area. Somites and paraxis expression are absent. Barrier removed during fixation. D In situ hybridization for bmp4 after combined operation (barrier insertion and ectoderm removal): bmp4 is induced in the paraxial mesoderm (ar-

rows). E Section through operated region. Arrow points to bmp4 expression in the paraxial mesoderm. Compare with contralateral side. F Anti-BrdU staining after administration of BrdU after combined operation. Fewer cells have taken up BrdU during S-phase on the operated side (arrow). G TUNEL (TdT-mediated dUTP-X nick end labeling) staining to reveal apoptotic cells: normal control side (dm dermomyotome). A few apoptotic cells are present only in the lateral and most medial portion of the dermomyotome. H TUNEL staining after combined operation: the paraxial mesoderm contains numerous apoptotic cells

al mesoderm (Xue and Xue 1996; Borycki et al. 1997). Especially regarding the latter, experimenters not only have to consider embryonic stages, but also the craniocaudal position of the somites and their particular stage (Christ and Ordahl 1995; Gossler and Hrabe de Angelis 1998). It has been shown in this context that pax-1 and myoD activation follow different developmental kinetics (Borycki et al. 1997).

On the other hand, SHH seems to function as a mitogen which expands the pool of muscle precursor cells under certain conditions (Duprez et al. 1998; Stockdale, personal communication). It has also been found that the avian notochord provides a survival factor for epaxial muscle precursors (Teillet et al. 1998). This would also account for the observation that the myoD expression domain is expanded, similarly to the pax-1 domain after retrovirally driven ectopic expression of shh (Johnson et al. 1994; Duprez et al.

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1998). In shh knockout mice, myf5 expression is strongly impeded while myoD is expressed normally (Chiang et al. 1996). This indicates that there is a difference in responsiveness of different subpopulations of the myogenic precursor pool. In the mouse embryo, myf5 first appears in the epaxial muscle precursors, whereas myoD marks the hypaxial muscle precursors (Kablar et al. 1997). It is also known that myf5 and myoD are probably (co-)induced by signals from different sources, i.e. the neural tube and ectoderm, respectively (Cossu et al. 1996). To what extent these findings also apply for the chick is not entirely clear, since myoD is expressed in the expaxial precursors before myf5 (Pownall and Emerson 1992). Another reason for differences in the results obtained from studies of notochord influence could be that in vitro experiments eliminate other signals that modify the action of the notochord in vivo. For example, it has been shown that lateral signals have to be taken into account as well when looking at somite patterning (Gamel et al. 1995; Pourquié et al. 1996; Dietrich et al. 1998; Capdevila et al. 1998; Capdevila and Johnson 1998). A member of the transforming growth factor beta (TGFβ) family of growth factors, BMP-4, diffuses from the lateral plate and specifies the lateral portion of the somite, thereby inducing lateral markers such as sim-1 (Pourquié et al. 1996). The BMPs (bone morphogenetic proteins) play important roles in embryonic patterning in a number of contexts (reviewed in Hogan 1996). In the 2-day chick embryo, administration of BMP-4 to the segmental plate inhibits the formation of an epithelial dermomyotome, decreases the number of mitoses and induces apoptosis (Schmidt et al. 1998, summarized in Fig. 3D–H). These BMP-mediated effects are concentration dependent and are counteracted in the paraxial mesoderm by several mechanisms: first, Noggin in the medial portion of the dermomyotome binds to BMPs, resulting in the inhibition of signalling through BMP receptors and counteracting their muscle-inhibiting influence (Re’em-Kalma et al. 1995; Zimmermann et al. 1996; Hirsinger et al. 1997; Capdevila and Johnson 1998; McMahon et al. 1998). Secondly, Follistatin in the medial and lateral dermomyotome and myotome also neutralizes BMP-4 (Amthor et al. 1996; Fainsod et al. 1997). Thirdly, Chordin may have a similar function (Sasai et al. 1995; Piccolo et al. 1996; Thomson 1997). The situation is further complicated by the fact that Noggin and Chordin are also synthesized by the notochord (Preobrazhenskii and Glinka 1985; Preobrazhensky et al. 1987; Holley et al. 1995; McMahon et al. 1998). This may also account for some of the observations following ectopic notochord grafting in the embryo. It is most likely that several signals are required in the control of muscle development: a mitotic or trophic signal is exerted by SHH, whereas an antagonistic signal against lateralization by BMP4 is exerted by Noggin and Chordin, resulting in the onset of muscle differentiation. Maybe at certain stages of development, the muscle-inducing signals (Noggin and Chordin) prevail over the mitotic (SHH) signals and vice versa. The dorsalizing signals acting on the paraxial mesoderm have been shown to emanate not only from the dorsal neu-

ral tube, but also from the ectoderm overlying the segmental plate (Gallera 1966; Fan and Tessier-Lavigne 1994; Spence et al. 1996; Sosic et al. 1997). Candidate molecules for dorsalization and subsequent myogenesis are the WNT family (Stern et al. 1995; Münsterberg et al. 1995; Fan et al. 1997; Capdevila and Johnson 1998). WNTs are secreted glycoproteins that are key modulators of embryonic cell fate and are highly conserved during evolution (reviewed by Moon et al. 1997). wnt1 (originally referred to as int-1) was first described in 1982 as a proto-oncogene involved in mammary cancer (Nusse and Varmus 1982). Subsequently, 16 related wnt genes have been described in the mouse (McMahon 1992; Moon et al. 1997). The wnt genes show sequence homology to Drosophila wingless, which induces nautilus, the fly homologue of genes coding myogenic bHLH proteins (Ranganayakulu et al. 1996). In the mouse embryo, wnt1 and wnt3a are expressed in the dorsal neural tube, while wnt4 and wnt6 are expressed in the dorsal ectoderm (Parr et al. 1993). The complexity of the in vivo situation is well documented by recent studies showing an interplay between BMP-4, Noggin and different WNTs to underlie myotome formation (Marcelle et al. 1997; Hirsinger et al. 1997).

Muscle growth and differentiation During the process of specification of muscle precursors, more cells have to be constantly generated to keep pace with embryonic growth and to provide material for the individual muscles. Since fully differentiated skeletal muscle fibres do not divide, proliferation normally has to precede differentiation. Thus, muscle growth results from a balance between proliferation of precursor cells and subsequent differentiation into myocytes and muscle fibres. A number of mitogens have been described for myogenic precursor cells, fibroblast growth factor-2 (FGF-2) being the most established one in vitro (Gospodarowicz et al. 1976; Clegg et al. 1987; Olwin et al. 1994a). Other FGFs such as FGF-4 and FGF-6 have been found to be expressed in skeletal muscle (Stark et al. 1991; de Lapeyriere et al. 1993). FGFs are capable of maintaining myoblasts derived from primary cultures in a proliferative and undifferentiated state in the presence of serum (Olwin et al. 1994b; Olson and Hauschka 1986). This is also true of TGFβs (Molkentin et al. 1986; Zappelli et al. 1996). Depletion of FGFs leads to withdrawal from the cell cycle, expression of muscle regulatory factors and terminal differentiation, at least in certain cell lines in vitro (Olson 1992). This process is accompanied by the downregulation of FGF receptors (Olwin and Hauschka 1988; Templeton and Hauschka 1992). In vivo, the expansion of the myogenic precursor cell pool is much more complicated due to the highly sensitive spatial and temporal interactions of signalling and responding tissues. In the developing myotome, the presence of FGFR1 is still controversial (Orr-Urtreger et al. 1991; Peters et al. 1992; Yamaguchi et al. 1992; Grothe et al. 1996), but it is unlikely that it mediates a mitogenic stimulus, since early myotome

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Fig. 4 A Transverse section through a 3-day chick embryo at limb bud level which received a graft of a quail dermomyotome. Antibody against quail nuclei reveals the grafted dermomyotome plus overlying ectoderm and emigrating myogenic cells in the limb bud mesenchyme (arrows). B In situ hybridization for sf/hgf in the wing bud of a 4-day chick embryo. The strongest expression domain is situated at the anterior margin and anterodistal portion of the wing bud. C Injection of SF/HGF into the flank region. pax-3-positive cells detach from the dermomyotomes (arrows) (wb wing bud, lb leg bud). D Chick embryo after grafting of a bead soaked in SF/HGF. pax-3-positive cells detach from the dermomyotomes (arrows). E Antibody staining of 3-day chick embryo with antibodies against N-cadherin. Myogenic cells in the dorsal and ventral myogenic zone are stained (dmz dorsal myogenic zone, vmz ventral myogenic zone, nt neural tube, my myotome, no notochord)

cells do not divide. Surprisingly, several signalling and receptor molecules involved in embryonic development seem to exert dual functions that are normally mutually exclusive: growth and differentiation. FGFR1 is one of them. While it is commonly accepted to mediate proliferation, it has a differentiation-promoting capacity in a particular skeletal muscle cell line (Templeton and Hauschka 1992). Two other FGF receptors have been described to be present in the myotome: FGFR4 in the mouse (Stark et al. 1991; Korkonen et al. 1992) and FREK in the chick (Marcelle et al. 1995). FREK-positive-replicating myoblasts populate the myotome some time after the appearance of myoD-positive muscle cells in the myotome. It is present in the trunk and limb muscle masses for days, reaching a maximum at E9 so that it appears to be expressed by embryonic and fetal (primary and secondary) myoblasts which represent subsequent myoblast types (Miller 1992; Miller and Stockdale 1986). Moreover, FREK is expressed in the satellite cells of the adult skeletal muscle (Mauro 1961; Bischoff 1990; Marcelle et al. 1994). In comparison with other myogenic markers, FREK follows pax-3 and precedes myoD. These findings imply that following precursor cell specification, replication has to take place which effectively expands the precursor cell population. The FGF-mediated control of proliferation and differentiation could be exerted by an autocrine mechanism, since several fgfs are expressed in the developing myotome (fgf-2: Joseph-Silverstein et al. 1989; Dono and Zeller 1994; fgf-4: Niswander and Martin 1992; fgf-6: de Lapeyriere et al. 1993; Han and Martin 1993; fgf-7: Mason et al. 1994). It has recently been shown that deletion of a gene controlling the growth of tissues and organs, the growth differentiation factor 8 (gdf8) or myostatin, results in the expansion of muscle tissue (McPherron and Lee 1997; McPherron et al. 1997; Szabo et al. 1998). This mutation in the mouse leads to a hypermuscular phenotype. The myostatin gene which encodes a growth factor of the TGFβ family has been found to be mutated in most double-muscled European cattle breeds (Grobet et al. 1998). Local signalling between neighbouring tissues is also involved in muscle growth and differentiation. One of the signalling molecules of the notochord introduced in the previous section, SHH, also appears to play a role in both processes, in addition to its function in somite and limb axis determination (Riddle et al. 1993; Johnson et al. 1994; Laufer et al. 1994). Duprez et al. (1998) found an extension of the pax-3 expression domain in the limb bud after retroviral overexpression of shh and an increase in bromodeoxyuridine (BrdU)-incorporating myoblasts after SHH administration in vitro. pax-3 being upstream of myoD and myf5 (Maroto et al. 1997; Tajbakhsh et al. 1997), this led to an increase in differentiated muscles in vivo and myotubes in vitro. It hence appears that the proliferative effect and the positive effect on differentiation are two aspects that are linked together and are only separable events when reincubation times are short. The expansion of the myoblast pool by SHH has also been observed in the zebrafish (Currie and Ingham 1996). However, in the avian embryo this effect has to be exerted well before epithelializa-

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tion of the paraxial mesoderm, since several reports show that the notochord has a de-epithelializing effect on the somite which can also convert the dorsal half into a mesenchyme and inhibits muscle formation (Brand-Saberi et al. 1993; Pourquié et al. 1993; Goulding et al. 1994; Xue and Xue 1996). Moreover, there is a difference between the response of epaxial and hypaxial muscle. The latter seems to be unaffected by notochord implantation (Rong et al. 1992; Pourquié et al. 1993; Brand-Saberi et al. 1993). This again shows that muscle formation is also controlled by mediolateral signalling and not only by dorsoventral signalling. BMP-4 from the lateral plate specifies the lateral myogenic lineage in the somites from which limb muscle and ventral body wall muscle develops (Ordahl and Le Douarin 1992; Pourquié et al. 1996). BMP-4 and its antagonist Noggin regulate the balance between proliferation and differentiation of muscles along the mediolateral axis (Hirsinger et al. 1997; Reshef et al. 1998; McMahon et al. 1998). In a complex way, BMP-4 is involved in the repression of muscle differentiation when emanating from the lateral plate, while it is also involved in myotome differentiation via the induction of wnt-1 in the dorsal neural tube (Marcelle et al. 1997). In this scenario, the same signalling molecule (BMP4) has an agonistic and antagonistic effect depending on its spatial context. Moreover, concentrations also play an important role in the quality of the response (Tonegawa et al. 1997). Not only proliferation is an inhibitor of muscle differentiation. Muscle precursor cells that have left the cell cycle in the G1 phase may be maintained in an undifferentiated state by intracellular suppressor molecules before undergoing terminal differentiation. Such inhibitors of muscle differentiation are the regulatory factors Id and Twist (Benezra et al. 1990; Sun et al. 1991; Jen et al. 1992; Hebrok et al. 1994; Peverali et al. 1994; Spicer and Lassar 1994; Hebrok et al. 1997). Id is an HLH protein that associates with ubiquitous bHLH proteins (E-proteins; Lassar et al. 1991; Davis and Weintraub 1992; Yutzey and Konieczny 1992). This association results in a competitive inhibition for the E-proteins that are needed by myogenic regulatory factors such as MyoD for obligate heterodimerization and subsequent binding to the CANNTG-consensus sequence of the DNA (E-box) in many muscle-specific genes (Wang et al. 1992). In contrast to Id, the bHLH protein Twist not only competes with myogenic bHLH proteins for E-proteins, but also acts through active repression of muscle-specific genes and by inhibiting the trans-activation by Mef2 (Spicer et al. 1996; Hebrok et al. 1997). In order to exert these functions in vivo, twist is expressed in the appropriate way temporally and spatially: It is expressed in the dermomyotomes and in the sclerotomes during somite development and also in the limb bud and head mesenchyme (Füchtbauer 1995; Stoetzel et al. 1995). Therefore it appears that Twist is an important regulator of growth and differentiation of muscle and other tissue in which rapid expansion requires cell populations to be maintained in an undifferentiated state. This is especially important where developing tissues move in relation to one another, which is the case for the neural-crest-derived head mesenchyme as well as for the hypaxial muscle domain, especially at limb bud levels.

Cell migration in muscle development There are two ways by which hypaxial muscle precursors invade new target sites: either as an epithelial sheet or by migration as individual mesenchymal cells. At cervical and flank levels, epithelial double-layered muscle buds comprising the lateral portions of the dermomyotome and the myotome move into the ventrolateral body wall mesenchyme (somatic mesoderm; Fischel 1895; Christ et al. 1983, 1986). During this process, the dermomyotomal portion is still replicating and pax-3-positive whereas the myotome is differentiated (Ordahl and Le Douarin 1992; Christ and Ordahl 1995). The invasion of the somatic mesoderm by somitic muscle buds has been shown to be mediated by fibronectin (Jaffredo et al. 1988), making it likely that migration is the underlying mechanism. However, since integrins as receptors for fibronectin and other extracellular matrix (ECM) components are involved in stimulating proliferation and differentiation (Adams and Watt 1989, 1993; Ingber 1991), the continuous elongation could also be the product of the mitotic activity of the lateral dermomyotome. During later development, the muscle buds disperse, with both layers intermingling to form the material for the layers of the ventrolateral body-wall muscle (Christ et al. 1983; Christ and Ordahl 1995). The migration of individual myogenic precursor cells occurs in the avian embryo in the occipital area, and at limb bud levels (reviewed by Christ and Ordahl 1995; Brand-Saberi et al. 1996a). In the mammalian embryo, myogenic cells additionally migrate from the cervical level to the anlage of the diaphragm (Wells 1954; Müntener 1968). The myogenic cells in the occipital area are derived from the first five to six somites in birds (Schemainda 1979; Huang et al. 1999) and the first five somites in murine embryos (Dietrich et al. 1998). They leave the lateral dermomyotomes of these somites and migrate in close association (“hypoglossal cord”), but still as individual cells towards the floor of the branchial arches. From here they move cranially to form the tongue muscles. During their migration, myogenic cells are pax-3 positive (Fig. 4B; Bladt et al. 1995; Huang et al. 1999; Dietrich et al. 1998). The entire skeletal muscle of the limbs is derived from the lateral dermomyotomes adjacent to the limb buds (Fischel 1895; Grim 1970; Christ et al. 1974, 1977; Chevallier et al. 1977). In the chick embryo, this is at somites 15–20/21 for the wing bud and at somites 26–32/33 for the leg bud. In the mouse embryo, somites 8–14 give rise to the forelimb muscles and somites 27–34 give rise to the hindlimb muscles (Theiler 1972; Lance-Jones 1988; Christ and Ordahl 1995; Zhi et al. 1996). The emigration of myogenic precursor cells for limbs, tongue and diaphragm is the result of a de-epithelialization occurring in these regions (Fig. 4A). It has been reported that de-epithelialization and subsequent emigration of somitic cells can be induced ectopically by grafting of proximal limb bud mesoderm to the flank level (Hayashi and Ozawa 1995). The underlying molecular mechanisms for this have also recently been unveiled. Knockout studies in

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the mouse have shown that myogenic cell emigration is achieved by an interaction between the transmembrane tyrosine kinase receptor c-Met and its ligand scatter factor/hepatocyte growth factor (SF/HGF; Bladt et al. 1995). Targeting of the genes for either the ligand or the receptor results in the absence of myogenic cells in the limb buds, tongue and diaphragm. Interestingly, the extrinsic tongue muscle is formed normally in the c-met mutant, whereas the intrinsic tongue muscle is absent (Bladt et al. 1995). The findings are supported by the fact that ectopic application of exogenous SF/HGF leads to de-epithelialization of the adjacent somites even at interlimb level (Fig. 4C,D; BrandSaberi et al. 1996b). The resulting phenotype of c-met and sf/hgf knockout mice resembles the phenotype of a naturally occurring mutation in the pax-3 gene called splotch (Franz et al. 1993; Bober et al. 1994b). Moreover, Pax-3 has been shown to regulate the expression of c-met (Epstein et al. 1996; Yang et al. 1996). sf/hgf is also expressed in the limb bud mesoderm during later stages of myogenic cell migration, suggesting a function in addition to myogenic cell recruitment by de-epithelialization. The question is whether SF/HGF might have a chemoattractive effect on myoblasts, thus guiding them towards the distal tip of the limb bud. Although the spatiotemporal expression of sf/hgf along the hypoglossal cord and in the diaphragm region suggests such a function (Dietrich et al. 1998; Birchmeier, personal communication), the pattern of expression in the limb buds is likely to be the product of a more complex interaction involving pattern formation, proliferation and differentiation and could have a bearing for all of these processes (Fig. 4B). The function of SF/HGF in the adult muscle may give us a clue concerning its possible role during development: upon injury, SF/HGF serves as an activator of satellite cells (Tatsumi et al. 1996; Cornelison and Wold 1997). It could therefore be reasoned that SF/HGF also controls the balance between migration and differentiation of myoblasts (Scaal et al. 1998). In this context, it is important to note that SF/HGF regulates the function of a molecule involved in myogenic cell migration and differentiation in the avian embryo: the cell adhesion molecule N-cadherin (Brand-Saberi et al. 1996c; George-Weinstein et al. 1997). The adhesiveness of cadherins can be modulated by phosphorylation of intracellular associated molecules (reviewed by Gumbiner 1995; Birchmeier et al. 1996; Huber et al. 1996). This mechanism is also believed to underlie the detachment of myogenic cells from the lateral dermomyotomes on stimulation of cMet by SF/HGF, since de-epithelializing cells continue to express N-cadherin (Brand-Saberi et al. 1996b). Another molecule has been suggested as a marker of myogenic cell migration: the homeobox gene lbx1, a homologue of Drosophila ladybird (Jagla et al. 1994, 1995; Dietrich et al. 1998). In birds and mammals, lbx-1 is expressed at the sites where hypaxial muscle de-epithelializes and in the entire occipital and cervical area. In this respect it differs from all other known markers of the lateral somite such as sim-1, pax-3 and c-met. It will be interesting to see how lbx-1 expression is controlled at the craniocaudal level.

As myogenic cells progress on their way through the limb bud mesoderm, there are several prerequisites. Fibronectin has to be available for migration through the intercellular spaces (Brand-Saberi et al. 1993b). Moreover, intercellular spaces have to be large enough, which in vivo is achieved by the distribution of hyaluronan (Krenn et al. 1991; Kosher et al. 1981). The invasion and homing to the dorsal and ventral myogenic zones where the premuscular masses are formed depends on homophilic interactions between the migrating cells and the stationary limb bud mesenchyme cells by means of N-cadherin (Fig. 4E; Brand-Saberi et al. 1996c). The myogenic cells invade the limb bud mesenchyme in a proximodistal direction, but never reach the most distal region of the growing limb bud, the socalled progress zone (Fig. 4A). Consequently, the distal mesenchyme is a muscle-free zone (Brand et al. 1985; Brand-Saberi et al. 1989). While proximally there is a predilection for the myogenic cells from the more cranially situated somites to occupy the anterior region of the limb bud and the ones from more caudally situated somites to occupy the posterior region of it, the distribution of myoblasts in the distal portion is more mixed (Zhi et al. 1996). hox genes seem to be involved in this process (Yamamoto et al. 1998). Likewise, early and late migrating cells do not have a preferential homing area with respect to the proximodistal axis (Zhi, unpublished). When the dorsal and ventral premuscle masses are formed, myogenic cells are still actively replicating and express pax-3 (Fig. 1C) (Goulding et al. 1994; Williams and Ordahl 1994; own observations). Small amounts of myf5 and myoD can be found in the segmental plate from HHstage 15 on using reverse transcriptase polymerase chain reaction (RT-PCR) detection (Lin-Jones and Hauschka 1996; Georg-Weinstein et al. 1996b). From HH-stage 24 on, the centrally located myoblasts in the limb buds start to express myoD (de la Brousse and Emerson Jr 1990; Williams and Ordahl). Also in the limb, the intermediate filament desmin can be detected at protein level in the postmitotic myocytes and fusing myotubes from HH-stage 24 on. Shortly after, myosins can be found immunohistochemically (Crow and Stockdale 1986). Individual muscles of the limb are formed by splitting of the dorsal and ventral muscle masses. The patterning of the definitive muscle in the limb bud is exerted by the stationary lateral-plate-derived limb mesoderm (Grim and Wachtler 1991; Brand-Saberi et al. 1996c; Yamamoto et al. 1998).

Concluding remarks Our understanding of the molecular events underlying muscle development has increased considerably during the last 10 years. Gene targeting in mice and microsurgical approaches in the chick embryo especially have revealed the function of genes expressed during myogenesis. Among the most recent achievements is the molecular analysis of signalling events leading to myogenic precursor cell specification, a process that is becoming increasingly more complex.

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We are now in a position of being able to study the fine-tuning of morphogenetic events in vivo. One of the most intriguing questions will be to fully understand the mechanisms that control the patterning of muscle precursor cells undergoing long-range migration. Another demanding task will be to study the signalling molecules and their receptors in more detail. Using molecular, biochemical and in vivo approaches, we may hope that present contradictions will not only be eliminated in the future, but at the same time open our eyes to unknown perspectives. Acknowledgements The authors thank Frank Stockdale for valuable discussions and comments on the manuscript. We thank Bruce Paterson for the donation of the avian myoD probe, Mike Stark and Christophe Marcelle for the pax3 probe, Claudio Stern for the avian sf/hgf probe, Drazen Sosic and Eric Olson for mef2 ISH and Ketan Patel for bmp4 ISH. We are grateful to Alexander Bonafede for probe preparation, Corina Schmidt, Verena Dathe and Martin Scaal for photographs of embryos and Ulrike Pein, Ellen Gimbel, Monika Schüttoff and Lidia Koschny for excellent technical assistance. We also thank Corina Schmidt for figure layout and Heike Bowe and Ulrike Uhl for manuscript preparation.

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