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How is myogenesis initiated in the embryo?
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How is myogenesis initiated in the embryo?
Skeletal muscle of the vertebrate body is derived from the somites, segmental blocks of paraxial mesoderm, which form on either side of the neural robe (reviewed in Refs 1, 2). Anterior head muscles are derived from ~ prechordal and paraxial head mesoderm and their GIULIO COSSO, ~ G I M formation will not be discussed here. Initially, somites I~,RGARI~ NtJCglgfdl/~ are epithelial spheres surrounding a central cavity of mesenchymal calls. They are delimited by a basement $ ~ n a / m y a g ~ s m'e a ~ , d f r m u p a n ~ a r a / ~ s o d e n ~ membrane of condensed extracellular matrix, which but how m2/oblasts acquire their idatity is stiH a matter o f separates them from adjacent structures, such as the specalat~ The ¢~rracterO~ou of molecalar markers neural tube, notochord, dorsal ectoderm and lateral and, in sonw caseg the a a ~ s i s o f mutattotls im tbe mesoderm. In the mouse (reviewed in Ref. 3), somito- correslamdtag geme~ lms m~w made # posstlae to ask genesis begins from embryonic day eight and proceeds specific qaesUoas a ~ a tMs ln~ces~ speciftca~m o f fador~ Adjaceat in a rostml-caudal gradient for several days. The newly somite ce~fate depemds om ~ formed somites rapidly differentiate dorsally into the tissue~ such as the ~ r a l tube, amtochord dorsal columnar epithelial cells of the dermomyotome, and eao#erm mul lateral ~soCerK aa eitber postttvely or pre~rsor ventrally into the sclerotome. While the sclerotome negativety o~ t~e different ~ gives rise to the cartilage of the vertebral colunm and polmlattoas is the s o m ~ ~__#~J_e moleculesf o r this comp~x stg~a~g aat~ay i~c~ie soatc hedgehog aad the ribs, the dermomyotome is the source of muscle precursor cells and cells that contribute to other tissues. Cells w~tprotetws as posatve stgmu~ ald BMP4as a poss~e iahibitor. Althtmgb it is ge~wa~ assumed that ttlduclioB from the dorsomedial part of the somite adjacent to the neural tube migrate under the dermomyotome to form is reqwtre~ some observmio~ suggest that to umlergo ~ s i s as a the myotome. These cells rapidly become post-mitotic cells might have a ~ and constitute the first differentiated skeletal muscle in "default'pathway. By amalogy with Drosophil~ where the the embryo. The dorsal myotome later contributes to aeurogentc genes affect myogemesig the vertebrate the back muscles, which are referred to as epaxial. Cells homok~q~es o f aotch and its llgaads could be caa~lidate derived from the lateral part of the dermomyotome will • ~geclaesfor a repressio~ or derepressio~ raecha~is~ migrate to the limb, or form hypaxial muscles, such as Similar studies with cuRured muscle c e ~ also implicate those of the ventral body wall. The connective tissue of other HLHfactors as potewlial iahibitors of the MyoD the back muscles and the dermis that covers them are fami~ a ~ hence, of iuappropriate myogetwsi~ also formed by cells from the dermomyotome. The somite also gives rise to some endothelial precursors. In receptor, cells do not migrate correctly and Pax3addition to the different cell types contributed by the expressing cells are not detectable in the mutant limb ~0. somite, neural cress cells, which originate from the Expression of Sire I, which encodes a basic helix-loop-dorsal neural tube, migrate through this region. helix (bHLH) factor, has also been described in the lateral part of the dermomyotome~ L Specific markers of Molecular markers in the somite dermal or endothelial cell precursors in the somite are Somites, therefore, contain a number of different still lacking, although expression of Quekl, a vascular progenitor cell types, some of which can nov,, be identi- EGF-type receptor, has been described in the dorsofied by molecular markers. Paxl and Pax9 mark lateral quadrant of early somites1-'. sclerotomal cells, and the undulated mutations in Paxl Of the myogenic bHLH regulatory factors (MRFs) in affect the axial skeletonL The basic helix-loop-helix mammals, only mff5 is expressed in the dermomyo(bHLH) protein, sclemxis, is also an early marker of tome, in the medial pa.q~3. When the myotome subsesclerotome as well as connective tissues. Pax3 and quently forms, early myotomal cells continue to express Pax7 (Ref. 6) mark cells in the dermomyotome and in re)f5. In birds, M)~D is expressed first in this part of newly forming muscle masses, such as the myotome, the somite~. A simplified scheme, showing somitic exwhen the somite matures. The expression of Pax3 in pression of those genes for which a function has been the dermomyotome later becomes more concentrated documented, is shown in Fig. 1. In cells derived from in the lateral half, where it plays a role in progenitor the lateral part of the dermomyotome of mice ~s, MyoD muscle cell formation"r-9. The mouse mutant, splotcb, is activated early in the thoracic region. The apparent which has mutations in the Pax3 gene, lacks limb heterogeneity of MRF 'gene expression in the myomusculature and other muscle masses in the body that tome 16 is probably partly due to this hypaxial comare formed by migrating cells. Pax3 is, therefore, egsential ponent. At the level of the limb, Pax3- and c-metfor the definition and/or migration of these cells. expressing precursor cells, which migrate away from although it might also mark non-muscle precursor cells the lateral dermomyotome, do not initially express any in the dermomyotome. Another marker of muscle pre- member of the MyoD family17; differentiation in the cursor cells migrating from the lateral dermomyotome is limb is initiated by myj"5 expressior~, followcd rapidly the proto-oncogene, c-met, encoding a tyrosine kinase by expression of MyoD and the gene encoding myoreceptor. Mice carrying modified alleles of this gene genin t3. The observation that myf5 and/or MyoD are show deficiencies in muscle formation (C. Ponzetto, expressed early is consistent with the results of gene pets. commun.), for example in the limb, where it has knockout experiments with the MyoD family, which been shown that the c-met ligand, hepatocyte growth points to a role for MyoD and myf5 in the eady stages factor/scatter factor, is present. In the absence of the of myogenesis. Although mice that lack one or the TIG JUNE 1996 VOL. 12 NO. 6 PII:SO1f~8-9525(96111gJ25-1
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other MRF have skeletal muscle, in the absence of both, the double mutant not only lacks skeletal muscle, but the precursor myoblast population is absent TM.
Medial
Myotom¢formation The early expression of myf5 (or MyoD in birds) throughout the dorsomedial part of the dermomyotome, adjacent to the neural tube, would suggest that these are myotomal precursor cells, which will migrate under the dorsomedial edge of the dermomyotome (Fig. 2a). Markers of differentiating muscle cells in the early myotome often show a triangular-like panem, consistent with this model. However, ultrastructural studies on mature myotomal cells, which remain mononucleated initially, indicate that myofibrillar components are aligned along the cranial-caudal axis 1. To achieve this orientation, if cells migrate laterally from the dorsomedial edge, they would have to realign along the A-P axis once they enter the myotome, indeed observations in birds suggest that myotomal precursor cells migrate from the cranial edge of the dermomyotome (Fig. 2b), projecting apical extensions parallel to the axis of the embryo in order to contact the caudal margin of the dermomyotome I. An unresolved problem with the latter model is that only those MRF-positive cells in the medial-cranial comer of the dermomyotome would contribute directly to myotome formation; the nature of the other MRF-positive cells along the medial edge 17 remains an enigma. In addition, influences of the neural tube (see later) on myotome formation are more difficult to explain, without invoking cell-cell inductive signalling or the formation of complex gradients specifically affecting cells more-laterally located along the cranial edge. More detailed analysis of cell movements should help to clarify this issue. In insects, a class of migratory, muscle precursor cells, termed 'muscle pioneers', has been identified, which subsequendy recruit other cells to myogenesis19. In lower vertebrates, such as the zebrafish, where epaxial muscles are a major component, muscle pioneer cells have been described, but it is not clear that they perform a similar function2°. In birds and mammals it is unknown whether such a process occurs. However, recent observations on the FREK (fibroblast growth factor) receptor in i~irds might point to an analogous situationZL This receptor is expressed in replicating muscle precursor cells after the activation of Pax3, but before expression of the myogenic factors, in the muscles of the limb, for example. However, early myotomal cells do not express FREK and it is only later that FREKpositive myoblasts migrate into this region. It might be that these cells are derived from the lateral dermomyotome only; alternatively they might represent a second wave of medial epaxial-type muscle precursors, recruited from the dermomyotome by the early myotomezi. The induction of myog~atsis With identifiable progenitor cell populations in the somite, it becomes possible to address tile question of how these distinct differentiation pathways are induced. Classical embryological experiments point to the flexibility of the early somite. Rotation of the epithelial somite in a dorsal-ventral or medial-lateral direction does not prevent subsequent formation of, for example,
NT
0
lateral
myf5 I~1
paxs
~
P6ocl
Fic,t ~ 1. Representationof a somite at the limb level (transverse section), at the time when epaxial muscleis beginningto form in the myotome,in the medialpart of the somite.Migratorymuscle precursorcells are shown leavingthe lateral part of the dermomyotome(DM). Someof these will differentiaterapidlyto formhypaxialmuscle,while others will migrateinto the limb field. Markersof cells in the somirefor which a functionhas been demonstratedare indicated.Abbreviations:NC, notochord; NT, neural tube; SC, sclerotome. sclerotome and muscle, which takes place according to the final orientation of the somitek The plasticity of the early somite suggests strongly that signals from the local environment (epigenetic) determine the identity of cells within it. It is now generally accepted 22-26that the axial structures (neural tube and notochord) play a positive role in the process leading to differentiation of epaxial skeletal muscle27. A simple model would predict that the dorsal portion of the neural tube dorsalizes the somite and, thus, induces the dermomyotome, while the ventral part of the neural tube and the notochord ventralize the somite and induce the sclerotome. While this model can be applied to the sclerotome28, the picture appears to be more complicated in the case of somite dorsalization. Experiments testing ventral versus dorsal portions of the neural tube and the notochord give conflicting results, and appear to be influenced to a certain extent by the experimental conditions employed, such as in vi~, transplantation or ablation versus in vitro reconstitution of different embryonic rudiments29"33. These controversial results might also be due to the existence of more than one signal activating myogenesis. A ventralizing signal emanating from the notochord, and subsequently from the floor plate of the neural tube, is required to activate Paxl and sclerotome differentiation. It has been shown that sonic hedgehog, which is normally produced by the notochord (and floor plate), can mimic its action34. The salne or another ventral signal is also necessary for the initiation of myogenesis, acting via polarization of the neural tube, and also more directly on the somite, probably to promote the competence of cells to respond to signals from the dorsal neural tube3t,3z. Members of the WNT family (Wntl, -3 and --4) might constitute a
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the somites are dissected free of the neural tube and of any other adjacent tissue, no differentiation is observed in culture 15,23-~6. It was recently shown that dorsal ectoderm will induce cells in the paraxial mesoderm to undergo myogenesis in vitro15, but only if a close contact between mesoderm and ectoderm is preserved. This is in agreement with the observation that maintenance of Pax3 expression, which temporally and spatially precedes expression of myogenic markers, takes place iu explants of somites in close contact with dorsal Cranial ectodermM. Thus, the precursors of (d) dm (e) dm hypaxial muscle, located in the lateral portion of the unsegmented paraxial mesoderm and newly formed somites, probably receive an unidentified myogenic signal from the R dorsal ectoderm, but, in vivo, do m~t :~ express any member of the MyoD :~ family initially• It is, therefore, probable that their differentiation is revl pressed by signals derived from adjacent tissues. Mechanical sepaFmttlm2. Two models for myotomeformation,based on migrationof muscleprecursors ration of the lateral plate from the from the dorsomedial(a) or cranial (b) edge of the dermomyotome.The shaded area paraxial mesoderm in the chick represents rayf5(or M)~Din birds) expression in the dermomyotome,adiacentto the embryo35 induced expression of neural tube. (c) Showsthe developing somites from the thoracic region of an E9.5targeted MyoD in the lateral half of somites heterozygotemyJ5/nlacZ(wheren is the nuclearlocalizationsignal)mouse embryo,stained where it is not normally observed. for I~-galacto~idase.The arrowhead indicatesthe initialtriangularshape of the myotome as it begins to form. (d,e) Show a single somite viewed along the lateral face and the Similarly, in organ cultures of mouse caudal edge, respectively.[Eyeindicatesdirection of view of (d).] Abbreviations:C, caudal: paraxial mesoderm, terminal differdin, dorsomedial;E9.5,embryonicday 9.5; R, rostralor cranial;vl, ventrolateral. entiation of lateral myogenic precursor cells is delayed by the lateral mesoderm tS. Furthermore it was second signal produced by the dorsal neural tube and demonstrated that this inhibitory activity produced by components of the lateral plate mesoderm in birds can required to activate fully and stabilize the myogenic programme in the dorsal part of the somite3L3z. A word be replaced by cells expressing BMP4 (Ref. 11). Again, it might be simplistic to assume that BMP4 is the only of caution should be added to conclusions drawn from implanting cells overexpressing a given molecule. Even potential inhibitor because other molecules, such as ff the natural molecule is expressed at the right time and FGF5 (which might stimulate proliferation and inhibit in the right place to perform the postulated action, other differentiation), are expressed in son~atic lateral mesomolecules might have the same pattern of expression derm at the time myotome forms36. in conclusion, a preliminary picture begins to and be the natural inducer. Furthermore, overexpressing cells could activate, through an autocrine loop, the syn- appear, where positive and negative signals both reach the dorsal portion of somites from axial structures, dorthesis of other molecules not expressed by the parental sal ectoderm and lateral mesoderm. Sonic hedgehog, cells used as a control. In contrast to the above results, all the existing evi- various Wnt proteins and BMP4 seem likely candidates dence suggests that axial structures are not necessary for this regulatory signalling, and a possible model is for myogenic determination of the precursors of hypax- outlined in Fig. 3. It is clear that this picture is still ial muscles, located in the lateral half of the unseg- incomplete and the list of molecules will no doubt grow • m e n t e d p~,raxial mesoderm and somites1.27. Here the rapidly in the future. In keeping with the in vivo observations on the situation appears to be more complex, because the differentiation of muscle precursor cells coming from the mouse, cells from the medial half of the segmental plate, lateral region of the dermomyotome is delayed in order when cultured in the presence of axial structures, actito allow migration to the limb and body wall, before vate myf5, whereas cells from the lateral half, cultured they form muscle. In experiments where the neural tube with their own dorsal ectoderm, will activate MyoD is removed, and the lateral portion of the somite remains (which, in vivo, is probably initially repressed by the in contact with the lateral structures of the embryo, cells lateral mesoderm)15. This suggests that in mammals axial located there migrate nonnally and then undergo mus- struc~res activate myogenesis through a Myf5-dependent cle differentiation1. In contrast, in experiments where pathway while dorsal ectoderm can act through a
(a)
~
omyotome
(b)
~-..-.,
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MyoD-dependent pathway. Subsequendy, the great majority of myogenic cells express MyoD and myf5, although with variable intensity. This correlates with observations on mice where the myf5 gene has been targeted with lacZ, In older heterozygote myf5/nlacZ embryos, different types of muscle fibres express lacZ, as well as MyoD (Ref. 37). Furthermore, it correlates with observations on MyoD knockout mice where myf5 continues to be expressed in adult muscle fibres38. It is tempting to speculate that myf5 and MyoD might have duplicated from a common ancestor with diversification of medially and laterally derived muscles, possibly with the appearance of primitive vertebrates, because urochordates possess only one MRF (Ref. 39). Whether the Myf5 and MyoD proteins have intrinsically different properties remains to be established. However, it is clear that during the radiation of this gene family, the two genes acquired different regulatory sequences, which diversify their response to signals from axial and dorsolateral structures.
Is myoge~, is induced~ The discussion of environmental influences on the onset of myogenesis has tended to assume that induction is required. However, a number of observations suggest that this might not be the complete story. Cells from the chick epiblast layer (pregastrulation), cultured in vitro, will undergo myogenesis't0, even when grown at low clonal density in protein-free medium4t. Furthennore, although the epiblast normally gives rise to all types of embryonic tissue, the great majority of the clones differentiate into skeletal muscle. Co-culture of the epiblast cells with adjacent tissue will inhibit myogenesis41. One interpretation of these experiments, therefore, is that epiblast cells, even before their entry into the primitive streak and conversion to mesoderm, are already programmed for myogenesis and that in the absence of repression exerted by the in vwo context, they will differentiate into muscle. If this is the case, then the influence of structures surrounding the early somites is not to induce myogenesis but to relieve its repression. Even within the somites and unsegmented paraxial mesodenri there could be repressive mechanisms operating, because the requirement for induction by axial structures is no longer absolute when the medial and lateral halves are separated 4z. In addition to suggesting that derepression, not induction, is the issue, these observations are also in apparent contradiction with the need for a 'community effect' required for amphibian43 and mammalianz4 myogenesis, because induction implies that surrounding (similar) cells are necessary to support differentiation of committed cells, rather than a situation where surrounding cells are inhibiting differentiation. Beside species differences, which might be relevant, it is possible that a community of cells is required only after gastrulation when it has probably more to do with defining territories and borders, rather than directly specifying fate. Indeed, it has been reported that the Xenopus blastula will differentiate in culture into different cell types, including skeletal muscle44. At the molecular level there are some indications of how repression might function. By PCR analysis, MyoD-encoding messenger RNA is detectable in the
Fmt~ 3. How myogenesismight be induced in cells fromthe dorsal part of the somite, under the influenceof factorsfrom the surroundingtissuesacting positively(arrows) or negatively(lines ending with bar) on myf5andMyoDgenes.In the ventralpart of the somite, sonic hedgehog (SHH),which is first produced in the notochord (NC)and then the floor plate of the neural tube, induces ~lerotome formation. chick epiblasfl t and in the mouse embryo before somitogenesis45. In myf5/nlacZ heterozygote embryos, [3-galactosidase-positive cells are detectable in the presomitic mesoderm (Fig. 4) (S. Tajbakhsh and M. Buckingham, unpublished). It remains to be shown whether they are the direct precursors of myf5-expressing cells in the somite; however, it is clear that they do not immediately undergo myogenesis. Some repression mechanism must be operating in vivo, as in the case of myfS-expressing
Fmtm~4).Wholemount stainingof the caudal part of a targeted myf5/nlacZ(nis the nuclear localizationsignal)hetemZygote embryonicday 9 embryo showing myf5expressionin presomitic mesoderm.The four last-fom'~edsomites with somite I indicated by the arrow are seen on the right-handside with increasing numbers of ~-galactosidasestained (therefore myf5-expressing) cells in the more mature rostral (R) somites.Morecaudally(C). ~-galactosidasestained cells are seen in the unsegmented paraxial mesoderm(*).
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cells in the central nervous system46, to prevent myogenesis. Notch has been proposed as a possible mediator of such a mechanism45. In Drosophila, the neurogenic genes play a role in myogenesis'i7; in the absence of notch enlarged clusters of cells in the mesoderm behave as muscle founder cells, while Notch activation leads to downregulation of the expression of the gene encoding Drosophila MyoD (Ref. 48). In vertebrates, there are several Notch homologues. Notchl is expressed in presomitic mesoderm and is particularly high in the region immediately adjacent to the newly forming somites"i9, where Myf5 is not detectable (Fig. 4). Notchl is downregulated in the somites whereas Notch2 and Notch3 are expressed initially in newly formed somites 50. In ~ b t c h l mutant mice somitogenesis is disorganized, although some muscle markers are expressed49. Notch is a tmnsmembrane receptor and homologues of its ligands - delta and serrate in Drosophila - have been cloned in mammals (delta and jagged). Binding of ligand activates notch. Co-culture of jagged-expressing fibroblasts with Notchl-expressing C2 muscle cells inhibits differentiation of the latter St. In keeping with this, in C2 cells, notch, which has been activated by removal of the extmcellular domain, inhibits myogenesis and downregulates expression of the myogenic factor genes, notably that of ruff5 and M.t~oD(Ref. 45). The ligand jagged is not expressed in mesoderm, but deltal expression is high in presomitic mesoderm5z. As suggested by Kopan el alJ 5, notch might play a direct role in regulating myogenesis. This is far from proven in vi~9, and aspects of the distribution of the different notch and delta proteins remain puzzling in this context (e.g. Notch2 and deltal transcripts in the myotome'SZ), however, it is tempting to speculate that if it is necessary to prevent myogenesis taking place before somitogenesis this might be effected by activating notch. The recent suggestion that, in Drosophila, wingless could be another ligand of notchs3, potentially competing with delta, raises the possibility that in vertebrates the Wnt proteins (homologues of wingless) produced by the neural tube or dorsal ectoderm, relieve repression of myogenesis by blocking the notch receptor. Concluding comments Muscle as a "default" pathway remains a somewhat heretical view at present. However, myogenesis already stands out from other differentiation progmmmes in that the MyoD transcription factors wiii convert other cell types to activate muscle gene# 4. The interest in notch, in the context of repressing myogenesis, lies in its potential as a mediator of external signals. However, other intracellular factors, which will form heterodimers with the MyoD family or its partners, have also been shown to inhibit myogenesis when expressed in cultured muscle cells. These include twist (Ref. 55) and Id (Ref. 56), notably absem from differentiated muscle masses such as the myotome57. In Drosophila, twist is essential for mesoderm formation, thus acting as an upstream positive regulator of myogenesis, although the quiescent precursor cells of adult muscle are characterized by persistent twist expression58. It remains to be seen what tile role, if any, of these molecules is in vivo in preventing inappropriate myogenesis. Indeed, the role of many of the 'marker' molecules of the somite,
such as Siml, remains to be established. In addition, man)" transcription factors, such as MyoD and its partners, are subject to modification, via phosphorylation, for example. The effect of growth factor# in inhibiting or even stimulating myogenesis, via signal transduction pathways, is another important area that remains to be explored in rqvo. We are just beginning to understand how different progenitor cell populations are established and then induced in the somite by local environmental signals. Such external signals are also required for the survival and proliferation of somitic cells and their subsequent migration. The c-met receptor and its ligand, scatter factor, provide a new insight into the latter process. In the future we can hope to learn more, not only about the molecules that govern the movement of muscle precursor cells from the dermomyotome, but also the way in which muscle masses are patterned by the surrounding connective tissue. Key questions in myogenesis still require clarification: how are myogenic progenitors determined in the embryo, and how ~re they subsequently programmed in space and time to form differem types of skeletal muscle? The molecular answers to these questions are now beginning to emerge, making this an exciting period for muscle research. Acknowledgements Research in M.B.'s laboratory in Paris is supported by the Pasteur Institute, the CNRS, the AFM and the MERS(GREG), and in G.C.'slaboratoryin Rome by grants from the Telethon, the Fondazione Cenci Bolognetti and the MURST. Both laboratories are supported by an HCMgram from the EC. G.C. held an EC senior visiting fellowship at the Pasteur Institute. We are grateful to Didier Rocancourt for assistance with the diagrams. We thank the many colleagues who communicated results before publication.
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Poster on Gene Therapy GENE THERAPY ¢==:....--m
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The poster included in this issue of Trends in Genetics p r o v i d e s an o v e r v i e w o, t,,e c u r r e n t strategies b e i n g u s e d for t h e g e n e t h e r a p y o f h u m a n disease, T h e p o s t e r w a s a u t h o r e d b y K e n n e t h Culver, a n d p r o v i d e s t h e b a c k g r o u n d f o r a s h o r t series o f articles to b e p u b l i s h e d later this y e a r o n technical i n n o v a t i o n in g e n e therapy.
The articles will discuss the use of ribozymes for gene
therapy, targeted gene transfer and extrachromosomal replicating vectors as vehidcs for gene therapy.
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How is myogenesis initiated in the embryo?
Skeletal muscle of the vertebrate body is derived from the somites, segmental blocks of paraxial mesoderm, which form on either side of the neural robe (reviewed in Refs 1, 2). Anterior head muscles are derived from ~ prechordal and paraxial head mesoderm and their GIULIO COSSO, ~ G I M formation will not be discussed here. Initially, somites I~,RGARI~ NtJCglgfdl/~ are epithelial spheres surrounding a central cavity of mesenchymal calls. They are delimited by a basement $ ~ n a / m y a g ~ s m'e a ~ , d f r m u p a n ~ a r a / ~ s o d e n ~ membrane of condensed extracellular matrix, which but how m2/oblasts acquire their idatity is stiH a matter o f separates them from adjacent structures, such as the specalat~ The ¢~rracterO~ou of molecalar markers neural tube, notochord, dorsal ectoderm and lateral and, in sonw caseg the a a ~ s i s o f mutattotls im tbe mesoderm. In the mouse (reviewed in Ref. 3), somito- correslamdtag geme~ lms m~w made # posstlae to ask genesis begins from embryonic day eight and proceeds specific qaesUoas a ~ a tMs ln~ces~ speciftca~m o f fador~ Adjaceat in a rostml-caudal gradient for several days. The newly somite ce~fate depemds om ~ formed somites rapidly differentiate dorsally into the tissue~ such as the ~ r a l tube, amtochord dorsal columnar epithelial cells of the dermomyotome, and eao#erm mul lateral ~soCerK aa eitber postttvely or pre~rsor ventrally into the sclerotome. While the sclerotome negativety o~ t~e different ~ gives rise to the cartilage of the vertebral colunm and polmlattoas is the s o m ~ ~__#~J_e moleculesf o r this comp~x stg~a~g aat~ay i~c~ie soatc hedgehog aad the ribs, the dermomyotome is the source of muscle precursor cells and cells that contribute to other tissues. Cells w~tprotetws as posatve stgmu~ ald BMP4as a poss~e iahibitor. Althtmgb it is ge~wa~ assumed that ttlduclioB from the dorsomedial part of the somite adjacent to the neural tube migrate under the dermomyotome to form is reqwtre~ some observmio~ suggest that to umlergo ~ s i s as a the myotome. These cells rapidly become post-mitotic cells might have a ~ and constitute the first differentiated skeletal muscle in "default'pathway. By amalogy with Drosophil~ where the the embryo. The dorsal myotome later contributes to aeurogentc genes affect myogemesig the vertebrate the back muscles, which are referred to as epaxial. Cells homok~q~es o f aotch and its llgaads could be caa~lidate derived from the lateral part of the dermomyotome will • ~geclaesfor a repressio~ or derepressio~ raecha~is~ migrate to the limb, or form hypaxial muscles, such as Similar studies with cuRured muscle c e ~ also implicate those of the ventral body wall. The connective tissue of other HLHfactors as potewlial iahibitors of the MyoD the back muscles and the dermis that covers them are fami~ a ~ hence, of iuappropriate myogetwsi~ also formed by cells from the dermomyotome. The somite also gives rise to some endothelial precursors. In receptor, cells do not migrate correctly and Pax3addition to the different cell types contributed by the expressing cells are not detectable in the mutant limb ~0. somite, neural cress cells, which originate from the Expression of Sire I, which encodes a basic helix-loop-dorsal neural tube, migrate through this region. helix (bHLH) factor, has also been described in the lateral part of the dermomyotome~ L Specific markers of Molecular markers in the somite dermal or endothelial cell precursors in the somite are Somites, therefore, contain a number of different still lacking, although expression of Quekl, a vascular progenitor cell types, some of which can nov,, be identi- EGF-type receptor, has been described in the dorsofied by molecular markers. Paxl and Pax9 mark lateral quadrant of early somites1-'. sclerotomal cells, and the undulated mutations in Paxl Of the myogenic bHLH regulatory factors (MRFs) in affect the axial skeletonL The basic helix-loop-helix mammals, only mff5 is expressed in the dermomyo(bHLH) protein, sclemxis, is also an early marker of tome, in the medial pa.q~3. When the myotome subsesclerotome as well as connective tissues. Pax3 and quently forms, early myotomal cells continue to express Pax7 (Ref. 6) mark cells in the dermomyotome and in re)f5. In birds, M)~D is expressed first in this part of newly forming muscle masses, such as the myotome, the somite~. A simplified scheme, showing somitic exwhen the somite matures. The expression of Pax3 in pression of those genes for which a function has been the dermomyotome later becomes more concentrated documented, is shown in Fig. 1. In cells derived from in the lateral half, where it plays a role in progenitor the lateral part of the dermomyotome of mice ~s, MyoD muscle cell formation"r-9. The mouse mutant, splotcb, is activated early in the thoracic region. The apparent which has mutations in the Pax3 gene, lacks limb heterogeneity of MRF 'gene expression in the myomusculature and other muscle masses in the body that tome 16 is probably partly due to this hypaxial comare formed by migrating cells. Pax3 is, therefore, egsential ponent. At the level of the limb, Pax3- and c-metfor the definition and/or migration of these cells. expressing precursor cells, which migrate away from although it might also mark non-muscle precursor cells the lateral dermomyotome, do not initially express any in the dermomyotome. Another marker of muscle pre- member of the MyoD family17; differentiation in the cursor cells migrating from the lateral dermomyotome is limb is initiated by myj"5 expressior~, followcd rapidly the proto-oncogene, c-met, encoding a tyrosine kinase by expression of MyoD and the gene encoding myoreceptor. Mice carrying modified alleles of this gene genin t3. The observation that myf5 and/or MyoD are show deficiencies in muscle formation (C. Ponzetto, expressed early is consistent with the results of gene pets. commun.), for example in the limb, where it has knockout experiments with the MyoD family, which been shown that the c-met ligand, hepatocyte growth points to a role for MyoD and myf5 in the eady stages factor/scatter factor, is present. In the absence of the of myogenesis. Although mice that lack one or the TIG JUNE 1996 VOL. 12 NO. 6 PII:SO1f~8-9525(96111gJ25-1
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other MRF have skeletal muscle, in the absence of both, the double mutant not only lacks skeletal muscle, but the precursor myoblast population is absent TM.
Medial
Myotom¢formation The early expression of myf5 (or MyoD in birds) throughout the dorsomedial part of the dermomyotome, adjacent to the neural tube, would suggest that these are myotomal precursor cells, which will migrate under the dorsomedial edge of the dermomyotome (Fig. 2a). Markers of differentiating muscle cells in the early myotome often show a triangular-like panem, consistent with this model. However, ultrastructural studies on mature myotomal cells, which remain mononucleated initially, indicate that myofibrillar components are aligned along the cranial-caudal axis 1. To achieve this orientation, if cells migrate laterally from the dorsomedial edge, they would have to realign along the A-P axis once they enter the myotome, indeed observations in birds suggest that myotomal precursor cells migrate from the cranial edge of the dermomyotome (Fig. 2b), projecting apical extensions parallel to the axis of the embryo in order to contact the caudal margin of the dermomyotome I. An unresolved problem with the latter model is that only those MRF-positive cells in the medial-cranial comer of the dermomyotome would contribute directly to myotome formation; the nature of the other MRF-positive cells along the medial edge 17 remains an enigma. In addition, influences of the neural tube (see later) on myotome formation are more difficult to explain, without invoking cell-cell inductive signalling or the formation of complex gradients specifically affecting cells more-laterally located along the cranial edge. More detailed analysis of cell movements should help to clarify this issue. In insects, a class of migratory, muscle precursor cells, termed 'muscle pioneers', has been identified, which subsequendy recruit other cells to myogenesis19. In lower vertebrates, such as the zebrafish, where epaxial muscles are a major component, muscle pioneer cells have been described, but it is not clear that they perform a similar function2°. In birds and mammals it is unknown whether such a process occurs. However, recent observations on the FREK (fibroblast growth factor) receptor in i~irds might point to an analogous situationZL This receptor is expressed in replicating muscle precursor cells after the activation of Pax3, but before expression of the myogenic factors, in the muscles of the limb, for example. However, early myotomal cells do not express FREK and it is only later that FREKpositive myoblasts migrate into this region. It might be that these cells are derived from the lateral dermomyotome only; alternatively they might represent a second wave of medial epaxial-type muscle precursors, recruited from the dermomyotome by the early myotomezi. The induction of myog~atsis With identifiable progenitor cell populations in the somite, it becomes possible to address tile question of how these distinct differentiation pathways are induced. Classical embryological experiments point to the flexibility of the early somite. Rotation of the epithelial somite in a dorsal-ventral or medial-lateral direction does not prevent subsequent formation of, for example,
NT
0
lateral
myf5 I~1
paxs
~
P6ocl
Fic,t ~ 1. Representationof a somite at the limb level (transverse section), at the time when epaxial muscleis beginningto form in the myotome,in the medialpart of the somite.Migratorymuscle precursorcells are shown leavingthe lateral part of the dermomyotome(DM). Someof these will differentiaterapidlyto formhypaxialmuscle,while others will migrateinto the limb field. Markersof cells in the somirefor which a functionhas been demonstratedare indicated.Abbreviations:NC, notochord; NT, neural tube; SC, sclerotome. sclerotome and muscle, which takes place according to the final orientation of the somitek The plasticity of the early somite suggests strongly that signals from the local environment (epigenetic) determine the identity of cells within it. It is now generally accepted 22-26that the axial structures (neural tube and notochord) play a positive role in the process leading to differentiation of epaxial skeletal muscle27. A simple model would predict that the dorsal portion of the neural tube dorsalizes the somite and, thus, induces the dermomyotome, while the ventral part of the neural tube and the notochord ventralize the somite and induce the sclerotome. While this model can be applied to the sclerotome28, the picture appears to be more complicated in the case of somite dorsalization. Experiments testing ventral versus dorsal portions of the neural tube and the notochord give conflicting results, and appear to be influenced to a certain extent by the experimental conditions employed, such as in vi~, transplantation or ablation versus in vitro reconstitution of different embryonic rudiments29"33. These controversial results might also be due to the existence of more than one signal activating myogenesis. A ventralizing signal emanating from the notochord, and subsequently from the floor plate of the neural tube, is required to activate Paxl and sclerotome differentiation. It has been shown that sonic hedgehog, which is normally produced by the notochord (and floor plate), can mimic its action34. The salne or another ventral signal is also necessary for the initiation of myogenesis, acting via polarization of the neural tube, and also more directly on the somite, probably to promote the competence of cells to respond to signals from the dorsal neural tube3t,3z. Members of the WNT family (Wntl, -3 and --4) might constitute a
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the somites are dissected free of the neural tube and of any other adjacent tissue, no differentiation is observed in culture 15,23-~6. It was recently shown that dorsal ectoderm will induce cells in the paraxial mesoderm to undergo myogenesis in vitro15, but only if a close contact between mesoderm and ectoderm is preserved. This is in agreement with the observation that maintenance of Pax3 expression, which temporally and spatially precedes expression of myogenic markers, takes place iu explants of somites in close contact with dorsal Cranial ectodermM. Thus, the precursors of (d) dm (e) dm hypaxial muscle, located in the lateral portion of the unsegmented paraxial mesoderm and newly formed somites, probably receive an unidentified myogenic signal from the R dorsal ectoderm, but, in vivo, do m~t :~ express any member of the MyoD :~ family initially• It is, therefore, probable that their differentiation is revl pressed by signals derived from adjacent tissues. Mechanical sepaFmttlm2. Two models for myotomeformation,based on migrationof muscleprecursors ration of the lateral plate from the from the dorsomedial(a) or cranial (b) edge of the dermomyotome.The shaded area paraxial mesoderm in the chick represents rayf5(or M)~Din birds) expression in the dermomyotome,adiacentto the embryo35 induced expression of neural tube. (c) Showsthe developing somites from the thoracic region of an E9.5targeted MyoD in the lateral half of somites heterozygotemyJ5/nlacZ(wheren is the nuclearlocalizationsignal)mouse embryo,stained where it is not normally observed. for I~-galacto~idase.The arrowhead indicatesthe initialtriangularshape of the myotome as it begins to form. (d,e) Show a single somite viewed along the lateral face and the Similarly, in organ cultures of mouse caudal edge, respectively.[Eyeindicatesdirection of view of (d).] Abbreviations:C, caudal: paraxial mesoderm, terminal differdin, dorsomedial;E9.5,embryonicday 9.5; R, rostralor cranial;vl, ventrolateral. entiation of lateral myogenic precursor cells is delayed by the lateral mesoderm tS. Furthermore it was second signal produced by the dorsal neural tube and demonstrated that this inhibitory activity produced by components of the lateral plate mesoderm in birds can required to activate fully and stabilize the myogenic programme in the dorsal part of the somite3L3z. A word be replaced by cells expressing BMP4 (Ref. 11). Again, it might be simplistic to assume that BMP4 is the only of caution should be added to conclusions drawn from implanting cells overexpressing a given molecule. Even potential inhibitor because other molecules, such as ff the natural molecule is expressed at the right time and FGF5 (which might stimulate proliferation and inhibit in the right place to perform the postulated action, other differentiation), are expressed in son~atic lateral mesomolecules might have the same pattern of expression derm at the time myotome forms36. in conclusion, a preliminary picture begins to and be the natural inducer. Furthermore, overexpressing cells could activate, through an autocrine loop, the syn- appear, where positive and negative signals both reach the dorsal portion of somites from axial structures, dorthesis of other molecules not expressed by the parental sal ectoderm and lateral mesoderm. Sonic hedgehog, cells used as a control. In contrast to the above results, all the existing evi- various Wnt proteins and BMP4 seem likely candidates dence suggests that axial structures are not necessary for this regulatory signalling, and a possible model is for myogenic determination of the precursors of hypax- outlined in Fig. 3. It is clear that this picture is still ial muscles, located in the lateral half of the unseg- incomplete and the list of molecules will no doubt grow • m e n t e d p~,raxial mesoderm and somites1.27. Here the rapidly in the future. In keeping with the in vivo observations on the situation appears to be more complex, because the differentiation of muscle precursor cells coming from the mouse, cells from the medial half of the segmental plate, lateral region of the dermomyotome is delayed in order when cultured in the presence of axial structures, actito allow migration to the limb and body wall, before vate myf5, whereas cells from the lateral half, cultured they form muscle. In experiments where the neural tube with their own dorsal ectoderm, will activate MyoD is removed, and the lateral portion of the somite remains (which, in vivo, is probably initially repressed by the in contact with the lateral structures of the embryo, cells lateral mesoderm)15. This suggests that in mammals axial located there migrate nonnally and then undergo mus- struc~res activate myogenesis through a Myf5-dependent cle differentiation1. In contrast, in experiments where pathway while dorsal ectoderm can act through a
(a)
~
omyotome
(b)
~-..-.,
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MyoD-dependent pathway. Subsequendy, the great majority of myogenic cells express MyoD and myf5, although with variable intensity. This correlates with observations on mice where the myf5 gene has been targeted with lacZ, In older heterozygote myf5/nlacZ embryos, different types of muscle fibres express lacZ, as well as MyoD (Ref. 37). Furthermore, it correlates with observations on MyoD knockout mice where myf5 continues to be expressed in adult muscle fibres38. It is tempting to speculate that myf5 and MyoD might have duplicated from a common ancestor with diversification of medially and laterally derived muscles, possibly with the appearance of primitive vertebrates, because urochordates possess only one MRF (Ref. 39). Whether the Myf5 and MyoD proteins have intrinsically different properties remains to be established. However, it is clear that during the radiation of this gene family, the two genes acquired different regulatory sequences, which diversify their response to signals from axial and dorsolateral structures.
Is myoge~, is induced~ The discussion of environmental influences on the onset of myogenesis has tended to assume that induction is required. However, a number of observations suggest that this might not be the complete story. Cells from the chick epiblast layer (pregastrulation), cultured in vitro, will undergo myogenesis't0, even when grown at low clonal density in protein-free medium4t. Furthennore, although the epiblast normally gives rise to all types of embryonic tissue, the great majority of the clones differentiate into skeletal muscle. Co-culture of the epiblast cells with adjacent tissue will inhibit myogenesis41. One interpretation of these experiments, therefore, is that epiblast cells, even before their entry into the primitive streak and conversion to mesoderm, are already programmed for myogenesis and that in the absence of repression exerted by the in vwo context, they will differentiate into muscle. If this is the case, then the influence of structures surrounding the early somites is not to induce myogenesis but to relieve its repression. Even within the somites and unsegmented paraxial mesodenri there could be repressive mechanisms operating, because the requirement for induction by axial structures is no longer absolute when the medial and lateral halves are separated 4z. In addition to suggesting that derepression, not induction, is the issue, these observations are also in apparent contradiction with the need for a 'community effect' required for amphibian43 and mammalianz4 myogenesis, because induction implies that surrounding (similar) cells are necessary to support differentiation of committed cells, rather than a situation where surrounding cells are inhibiting differentiation. Beside species differences, which might be relevant, it is possible that a community of cells is required only after gastrulation when it has probably more to do with defining territories and borders, rather than directly specifying fate. Indeed, it has been reported that the Xenopus blastula will differentiate in culture into different cell types, including skeletal muscle44. At the molecular level there are some indications of how repression might function. By PCR analysis, MyoD-encoding messenger RNA is detectable in the
Fmt~ 3. How myogenesismight be induced in cells fromthe dorsal part of the somite, under the influenceof factorsfrom the surroundingtissuesacting positively(arrows) or negatively(lines ending with bar) on myf5andMyoDgenes.In the ventralpart of the somite, sonic hedgehog (SHH),which is first produced in the notochord (NC)and then the floor plate of the neural tube, induces ~lerotome formation. chick epiblasfl t and in the mouse embryo before somitogenesis45. In myf5/nlacZ heterozygote embryos, [3-galactosidase-positive cells are detectable in the presomitic mesoderm (Fig. 4) (S. Tajbakhsh and M. Buckingham, unpublished). It remains to be shown whether they are the direct precursors of myf5-expressing cells in the somite; however, it is clear that they do not immediately undergo myogenesis. Some repression mechanism must be operating in vivo, as in the case of myfS-expressing
Fmtm~4).Wholemount stainingof the caudal part of a targeted myf5/nlacZ(nis the nuclear localizationsignal)hetemZygote embryonicday 9 embryo showing myf5expressionin presomitic mesoderm.The four last-fom'~edsomites with somite I indicated by the arrow are seen on the right-handside with increasing numbers of ~-galactosidasestained (therefore myf5-expressing) cells in the more mature rostral (R) somites.Morecaudally(C). ~-galactosidasestained cells are seen in the unsegmented paraxial mesoderm(*).
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cells in the central nervous system46, to prevent myogenesis. Notch has been proposed as a possible mediator of such a mechanism45. In Drosophila, the neurogenic genes play a role in myogenesis'i7; in the absence of notch enlarged clusters of cells in the mesoderm behave as muscle founder cells, while Notch activation leads to downregulation of the expression of the gene encoding Drosophila MyoD (Ref. 48). In vertebrates, there are several Notch homologues. Notchl is expressed in presomitic mesoderm and is particularly high in the region immediately adjacent to the newly forming somites"i9, where Myf5 is not detectable (Fig. 4). Notchl is downregulated in the somites whereas Notch2 and Notch3 are expressed initially in newly formed somites 50. In ~ b t c h l mutant mice somitogenesis is disorganized, although some muscle markers are expressed49. Notch is a tmnsmembrane receptor and homologues of its ligands - delta and serrate in Drosophila - have been cloned in mammals (delta and jagged). Binding of ligand activates notch. Co-culture of jagged-expressing fibroblasts with Notchl-expressing C2 muscle cells inhibits differentiation of the latter St. In keeping with this, in C2 cells, notch, which has been activated by removal of the extmcellular domain, inhibits myogenesis and downregulates expression of the myogenic factor genes, notably that of ruff5 and M.t~oD(Ref. 45). The ligand jagged is not expressed in mesoderm, but deltal expression is high in presomitic mesoderm5z. As suggested by Kopan el alJ 5, notch might play a direct role in regulating myogenesis. This is far from proven in vi~9, and aspects of the distribution of the different notch and delta proteins remain puzzling in this context (e.g. Notch2 and deltal transcripts in the myotome'SZ), however, it is tempting to speculate that if it is necessary to prevent myogenesis taking place before somitogenesis this might be effected by activating notch. The recent suggestion that, in Drosophila, wingless could be another ligand of notchs3, potentially competing with delta, raises the possibility that in vertebrates the Wnt proteins (homologues of wingless) produced by the neural tube or dorsal ectoderm, relieve repression of myogenesis by blocking the notch receptor. Concluding comments Muscle as a "default" pathway remains a somewhat heretical view at present. However, myogenesis already stands out from other differentiation progmmmes in that the MyoD transcription factors wiii convert other cell types to activate muscle gene# 4. The interest in notch, in the context of repressing myogenesis, lies in its potential as a mediator of external signals. However, other intracellular factors, which will form heterodimers with the MyoD family or its partners, have also been shown to inhibit myogenesis when expressed in cultured muscle cells. These include twist (Ref. 55) and Id (Ref. 56), notably absem from differentiated muscle masses such as the myotome57. In Drosophila, twist is essential for mesoderm formation, thus acting as an upstream positive regulator of myogenesis, although the quiescent precursor cells of adult muscle are characterized by persistent twist expression58. It remains to be seen what tile role, if any, of these molecules is in vivo in preventing inappropriate myogenesis. Indeed, the role of many of the 'marker' molecules of the somite,
such as Siml, remains to be established. In addition, man)" transcription factors, such as MyoD and its partners, are subject to modification, via phosphorylation, for example. The effect of growth factor# in inhibiting or even stimulating myogenesis, via signal transduction pathways, is another important area that remains to be explored in rqvo. We are just beginning to understand how different progenitor cell populations are established and then induced in the somite by local environmental signals. Such external signals are also required for the survival and proliferation of somitic cells and their subsequent migration. The c-met receptor and its ligand, scatter factor, provide a new insight into the latter process. In the future we can hope to learn more, not only about the molecules that govern the movement of muscle precursor cells from the dermomyotome, but also the way in which muscle masses are patterned by the surrounding connective tissue. Key questions in myogenesis still require clarification: how are myogenic progenitors determined in the embryo, and how ~re they subsequently programmed in space and time to form differem types of skeletal muscle? The molecular answers to these questions are now beginning to emerge, making this an exciting period for muscle research. Acknowledgements Research in M.B.'s laboratory in Paris is supported by the Pasteur Institute, the CNRS, the AFM and the MERS(GREG), and in G.C.'slaboratoryin Rome by grants from the Telethon, the Fondazione Cenci Bolognetti and the MURST. Both laboratories are supported by an HCMgram from the EC. G.C. held an EC senior visiting fellowship at the Pasteur Institute. We are grateful to Didier Rocancourt for assistance with the diagrams. We thank the many colleagues who communicated results before publication.
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G. Cossu Is IN THE UNIVERSnY OF ROME "LA SAPIENZA~, INS'I'ilZrFEOF HISTOLOGYAND GENERAL EMBRYOLOGI6, VIA ~L SCaWA 14, 0 0 1 6 1 ROM~ ITALW ~ T.4JR~agHSM AND M. BUCKINGHAM ARE IN THE DEPARTMENT OF MOLECULAR BIOLOGI', URA C N R S 1947, PASTEURINSrHtW~ 2 5 RUE mY DR Roo'x, 75724 PAPds CEDEX 15, FRa~Cll
Poster on Gene Therapy GENE THERAPY ¢==:....--m
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The poster included in this issue of Trends in Genetics p r o v i d e s an o v e r v i e w o, t,,e c u r r e n t strategies b e i n g u s e d for t h e g e n e t h e r a p y o f h u m a n disease, T h e p o s t e r w a s a u t h o r e d b y K e n n e t h Culver, a n d p r o v i d e s t h e b a c k g r o u n d f o r a s h o r t series o f articles to b e p u b l i s h e d later this y e a r o n technical i n n o v a t i o n in g e n e therapy.
The articles will discuss the use of ribozymes for gene
therapy, targeted gene transfer and extrachromosomal replicating vectors as vehidcs for gene therapy.
TIC Jb~E 1996 VOL. 12 NO. 6
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