Genetic regulation of skeletal muscle development

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1 EXPERIMENTAL CELL RESEARCH 316 (2010) available at Review Genetic regulation of skeletal muscle development Keren Bismuth, Frédéric Relaix INSERM UMR S 787-Myology Group, Avenir Team Mouse Molecular Genetics, UPMC- Faculté de Médecine Pitié-Salpêtrière, Institut de Myologie, 105 Bd de l'hôpital Paris, France ARTICLE INFORMATION ABSTRACT Article Chronology: Received 1 March 2010 Revised version received 30 August 2010 Accepted 31 August 2010 Available online 7 September 2010 Keywords: Muscle development Transcription factor Myogenic Regulatory Factors During development, skeletal muscles are established in a highly organized manner, which persists throughout life. Molecular and genetic experiments over the last decades have identified many developmental control genes critical for skeletal muscle formation. Developmental studies have shown that skeletal muscles of the body, limb and head have distinct embryonic and cellular origin, and the genetic regulation at work in these domains and during adult myogenesis are starting to be identified. In this review we will summarize the current knowledge on the regulatory circuits that lead to the establishment of skeletal muscle in these different anatomical regions Elsevier Inc. All rights reserved. Contents Introduction Genetic control of trunk musculature Genetic control of limb muscle development Genetic control of facial muscles development From the embryo to the adult Concluding remarks Acknowledgments References Introduction Skeletal muscle cells, or myotubes, are contractile, multinucleated cells, which constitute the minimal functional unit of all muscles in the body. Undifferentiated muscle cells are called myoblasts; they are mononucleated and are characterized by the expression of members of the myogenic regulatory factors (MRFs). Classic experiments in embryology have clearly established that all body muscles, but not head, are derived from the condensation of paraxial mesoderm into epithelial structures called somites. Somites are formed along the rostro-caudal axis of the embryo and are organized in dorso-ventral compartments. The most ventral part is the sclerotome (that will give rise to the axial skeleton); most dorsally, the dermomyotome is comprised of cells that will give rise to dermis and muscle progenitors cells (MPC). The borders of the dermomyotome will undergo an epithelial to Corresponding author. Fax: address: frelaix@gmail.com (F. Relaix) /$ see front matter 2010 Elsevier Inc. All rights reserved. doi: /j.yexcr

2 3082 EXPERIMENTAL CELL RESEARCH 316 (2010) mesenchyme transition and form the third somitic compartment: the myotome, which contains the first differentiated myofibers. The epaxial (dorso-medial) part of the dermomyotome and myotome will give rise to the back muscles while the hypaxial (ventro-lateral) somite will generate the rest of the trunk and limb muscles [1]. Genetic in vivo invalidation of genes in the mouse, as well as studies in the avian model and in vitro experiments made it possible to identify genetic regulatory networks that are involved in specifying muscle progenitors and precursors cells (see Fig. 1). Genetic control of trunk musculature The dermomyotome contains the reservoir of proliferative skeletal muscle progenitors cells [1]. Key markers of these cells are the Paired-Homeobox transcription factors Pax3 and Pax7. Pax3 expression is found during organogenesis in various tissues among which the dorsal neural tube, neural crest cells, and body muscle cells. In addition to neural crest cell loss and dorsal neural tube closure defects, the absence of PAX3 causes impaired muscle development. Mice homozygous for the Pax3 Splotch allele, which have a spontaneous mutation in Pax3, have a complete loss of the hypaxial domain of the somite and as a result a loss of limb and some of the trunk muscles, while epaxial-derived muscles are less affected, revealing that hypaxial and epaxial muscles do not have the same requirement for PAX3 (Fig. 1), which also acts as a survival factor in these domains [2]. Pax7, however, is expressed later in PAX3-expressing MPCs and is not mandatory for muscle development; the consequence of Pax7 absence is only revealed later on, at the post-natal stage [3]. The combined lack of Pax3 and Pax7 however, is deleterious for trunk muscles formation since Pax3/Pax7-deficient MPCs are unable to enter the myogenic program, underlining the importance and overlapping functions of these two factors in building the muscle lineage [4]. What, then, induces these factors? The genetic network Six-Eya- Dach is a major regulator of Pax3 expression in the dermomyotome. Notably, the overexpression of both Dach2/Eya2 or Six1/Eya2 in chick somite explants lead to an up-regulation of Pax3 [5]. Six1 and Six4 control the hypaxial expression of Pax3 [6], and in their absence, limb muscles, which originate from the migrating hypaxial cells, are absent. Six genes also control early MRFs expression in the myotome [7] (Fig. 1) and misexpression of both Six1 and Eya2 is sufficient to trigger MRFs expression in avian somite [5]. SIX proteins bind MEF3 sites, which have been found in promoter and many others myogenic differentiation genes [8 10]. On the next level down in the myogenic genetic hierarchy, in the myotome, myogenesis is driven by the four MRFs (Myf5,, and ). These are basic helix loop helix (bhlh) transcription factors, which play somewhat redundant role to ensure proper muscle differentiation, in combination with less specific factors such as members of the MEF2 family. The first MRF expressed in the myogenic differentiation network is Myf5, it is first transiently expressed in the paraxial mesoderm before the onset of myogenesis [11], and is then expressed in the lips of the dermomyotome, inducing migration within the myotome and initiation of the myogenic differentiation pathway. The regulation of Myf5 expression has been thoroughly investigated leading to the identification of a large number of discrete enhancers [12]. Notochord and floor plate produce Sonic Hedgehog, regulating epaxial expression of Myf5. Mice mutant for Shh have a reduced Myf5 expression and lack epaxial musculature [13]. This regulation signals through GLI1, which directly binds the somite enhancer of Myf5 [14]. Furthermore compound Gli mutant display somite defects [15]. In mouse, PAX3 acts upstream of Myf5, in the hypaxial somite, by directly binding to a separate 145 bp regulatory element [2]. Strikingly, mice lacking Pax3 in addition to Myf5 and will lack all body muscles, demonstrating that these factors also act genetically upstream of [16] (Fig. 1). In the absence of, or, Myf5 alone is not able to drive muscle differentiation [17]. In the myotome, expression closely follows that of Myf5 [18]. However, is not necessary for muscle differentiation and its absence does not lead to muscle defect [19]. Nevertheless, the importance of MRF4 function as a determination and differentiation factor is revealed GENETIC HIERARCHY IN MYOGENESIS Hypaxial somite Epaxial somite Limb Branchiomeric muscles Adult Paraxis Paraxis Meox2 Pitx2 Pax3/7 Pax3 Pax3 Pax3 Tbx1 MyoR, Capsulin Myf5 My5 My5 Myf5 Myf5 >E10 >E10 Fig. 1 The molecular genetic pathways engaged in hypaxial and epaxial somite, limb, branchiomeric and adult myogenesis, are schematically represented. We distinguished two modes of interaction: known direct interaction at the transcriptional level (full line), from genetic interaction (dashed line). In both hypaxial and epaxial somites, starts to play its role in the genetic hierarchy at E10 (red line).

3 EXPERIMENTAL CELL RESEARCH 316 (2010) in a / ; Myf5 loxp/loxp context, indeed, when and Myf5 are lacking, MRF4 alone is able to sustain muscle development [20]. Interestingly, if both and Myf5 are missing, there is a delayed formation of the myotome accompanied by a delayed expression of [16]. Paraxis, a bhlh transcription factor is also involved in this early control of myogenic specification. In Paraxis null embryo, the paraxial mesoderm does not form epithelial somites, but disrupted skeletal muscles develop [21], despite the loss of Pax3 expression. Analysis of Paraxis: Myf5 mutant mice demonstrated that Paraxis, acting upstream of Pax3 in the epaxial somite also controls expression [22]. MYOD is considered as a major transcription factor for muscle lineage formation, on the basis of its ability (in fact shared with other MRFs) to induce myogenic fate in fibroblasts and other cell types [1]. Moreover, double and triple compound mutant mice have shown that, in combination with other MRFs, is involved both in myogenic determination and differentiation of skeletal muscle cells, underlining MYOD pivotal role in myogenesis (Fig. 1). During development, is first detectable at E10 in hypaxial somite, its expression is under the control of the transcription factors PAX3, MRF4, MYF5 and SIX1/SIX4. Indeed, overexpression of Pax3 in chick somite explants is sufficient to trigger expression [23]. Extra-cellular signals coming from the dorsal ectoderm (such as WNTs) and the lateral plate mesoderm (including BMPs and Notch signaling) regulate positively and negatively, respectively, the expression of [1]. The absence of is not deleterious for embryonic development, still its importance is revealed during adult muscle regeneration. / embryos show no obvious phenotype in the trunk or in head muscles, likely due to functional compensation by other MRFs. In the limb, however, myogenesis is paused between E11.5 and E13.5. MYOD triggers myoblasts differentiation by activating the expression of the fourth and last MRF implicated in myogenesis:. In somites expression is first detectable at E9.25 in the myotome, at that stage is genetically downstream of Myf5 and (Fig. 1), and indeed the promoter regions contain two E-Boxes directly bound by MYF5, MRF4 or MYOD, in addition to MEF2 and MEF3 sites. / pups die at birth due to respiratory failure and the histology analysis revealed that limb muscles area are populated with mononucleated cells and rare myofibers, revealing that in vivo, is necessary for mononucleated myoblasts fusion to form myotubes [1]. Interestingly, -derived myoblasts fuse normally in vitro, suggesting that in vivo, in the absence of, the environment negatively regulates myoblasts fusion, or that other MRFs are deregulated in this context and can compensate for the requirement of. In vivo, however, other MRFs, whose expression is apparently not altered in /, could not compensate for the loss of. Strikingly, : double mutant mice also display similar differentiation defects, yet, is still expressed in these double mutant mice, suggesting that MYF5 may be sufficient to activate, but the overall level of MRF (MYOGENIN and MYF5) in this context does not reach a threshold required for myogenic differentiation. Genetic control of limb muscle development Limb musculature develops from a few thousands of muscle progenitors cells that have delaminated from the hypaxial part of the dermomyotome and migrated into the opposite limb bud. This process starts around E9.25 in the forelimb and ends around E11.0 in the hindlimb. During their migration, progenitor cells proliferate and do not express any MRF. Once they reach their final destination in the limb, they will quickly start to differentiate (first expressing Myf5, then after a few hours, and ) and form the ventral and dorsal muscle masses of the fore- and hindlimbs. Pax3 but not Pax7 is expressed in migrating progenitor cells. Pax3 mutant embryos are devoid of limb muscles, due to apoptosis in the hypaxial somites, and display a loss of the tyrosine kinase receptor c-met expression. c-met and its ligand, Hepatocyte Growth Factor (HGF) or Scatter factor (SF), are essential for the delamination and migration of progenitors cells. While c-met is expressed in muscle progenitors cells, its ligand is secreted by surrounding mesenchymal cells [24]. The absence of c-met or HGF/SF prevents the progenitor cells from delaminating and migrating from the dermomyotome and no muscles are subsequently found in the limbs [25]. Lbx1 is another homeobox gene co-expressed with Pax3 in migrating MPCs. Dorsal muscles from forelimb and all muscles from hindlimb are missing in Lbx1 mutants. While cells do migrate out from the dermomyotome they cannot find their way to the limb bud [25]. This observation also revealed that the cell autonomous effect of Lbx1 affects differently forelimb from hindlimb and ventral from dorsal muscle masses. Mutations in the homeobox Meox2 gene also lead to specific loss of a subset of limb muscles [26]. Meox2 is expressed in the paraxial mesoderm and in the migrating MPCs into the limb bud. Interestingly, in the limb, Pax3 and Myf5 expression, but not that of, is downregulated in Meox2 mutant embryos, indicating that in the limb, unlike in the trunk, regulation is not under the control PAX3 and MYF5. In any case, in the context of a MEOX2 depleted environment, MYOD is not sufficient to sustain myogenesis [26]. The role and regulations of the MRFs are distinct in the trunk and limb (Fig. 1) : while SIX1/4 does not control Myf5 expression in the dermomyotome, it does so in the limb, via the very same 145 bp enhancer that PAX3 uses to control Myf5 expression in the hypaxial somite [27]. Moreover, myogenesis is delayed in somites from Myf5 nlacz/nlacz [Myf5: double mutant], it proceeds normally in the limb, revealing that neither MYF5 nor MRF4 are necessary for limb muscle development. While is dispensable for trunk myogenesis, where is expressed and can functionally replace, in the limb, myogenesis is stalled for 2 embryonic days between E11.5 and E13.5 in mutant embryos, time at which expression is induced in the limb, rescuing the phenotype [28]. Since Myf5 and have two distinct roles, the question whether all trunk and limb muscles are derived from a single lineage, has also been addressed. The specific ablation of Myf5 expressing cells, using Myf5 Cre ; R26R DTA/+, revealed the presence of a distinct myogenic +lineage [29,30]. Genetic control of facial muscles development Head muscles find their singularity in their developmental origin and in the unique genetic network of transcriptions factors involved in the establishment of this musculature. While all trunk and limb muscles originate from paraxial mesoderm, head

4 3084 EXPERIMENTAL CELL RESEARCH 316 (2010) muscles are derived from pre-chordal and pharyngeal head mesoderm. Branchiomeric muscle progenitor cells migrate and differentiate within the mesodermal core of the different head muscles [31], and are surrounded by endodermal and migrating neural crest cells. Although the same MRFs are involved in the myoblasts formation in the head, trunk and limb, the upstream factors and regulation of the MRFs differ. The first evidence of this difference came from the analysis of Pax3 Sp/Sp ; Myf5 nlacz/nlacz embryos [Pax3:Myf5: mutants], in which all trunk and limb muscles are missing but head muscles are unaffected [16]. In the head, Pax3 is expressed in cranial neural crest-derived cells, but not in mesodermal derivatives. Pax7, however, is expressed in branchiomeric muscles and other head muscles, but its role as a master regulator of head musculature has been excluded since Pax7 / mice do not display head muscle phenotype [32]. Instead, four transcription factors have been identified to control the induction of MRFs expression: MyoR, Capsulin, Tbx1 and Pitx2. MyoR and Capsulin belong to the bhlh family of transcription factors, they play a redundant function in the specification of the future masticatory muscles. Single MyoR / or Capsulin / mutants do not display head muscle defects, however the compound homozygote MyoR / ; Capsulin / lack 1st branchial arch (BA)-derived muscles, whereas other head muscles were not affected. This defect was the result of an absence of MRFs expression in the first BA and an increased apoptosis in the cells that failed to enter the myogenic program [33]. This finding reveals that distinct genetic programs regulate different groups of muscles within the head [31]. Tbx1 belongs to the family of the T-boxcontaining genes, and is expressed in the branchial arches before the activation of the MRFs. Tbx1 mutant mice die around birth and have defects in craniofacial and cardiovascular structures [31], in addition to impaired branchiomeric muscles formation. Tbx1 / muscle precursor cells correctly migrate into the 1st BA as indicated by the presence of MyoR and Capsulin mrna, however they fail to robustly activate Myf5 or, leaving only a small population of cells to enter the myogenic program. At later stages, branchiomeric muscles are lost except for sparse unilateral muscle masses retaining MRFs expression. Tbx1 ensures the strong bilateral triggering of branchiomeric myogenesis [34]. Pitx2 is another factor part of the core regulatory network of transcription factor engaged in head musculature development. Pitx2 is a bicoidrelated homeobox transcription factor; its expression precedes that of the myogenic factors and remained in all MRFs positive cells during myogenesis. In the head, Pitx2 mutation results in the loss of 1st BA-derived muscles including absence of peri-ocular and jaw muscles [35,36]. Lineage tracing studies established that at E10.5 Pitx2 Cre -derived cells are found in the mesodermal core of the 1st BA albeit in small number [36]. These cells failed to activate Myf5 and a strong reduction of expression was also observed. Myogenic cells specification failure was accompanied by an increased level of apoptosis and by E13.5, the 1st BA was lost. The expression of Tbx1, Capsulin and MyoR was also compromised in the myogenic field of the 1st BA, suggesting that PITX2 acts genetically upstream of these myogenic progenitor cells regulators in the mandibular arch. Analyses of a combination of MRFs mutants shed light on head muscle-specific requirement for Myf5 and. Myf5 nlacz/nlacz embryos lack extra ocular muscles (EOM) while mandibular muscles developed normally. In Myf5 nlacz/nlacz EOM, expression is not initiated despite the presence of Pitx2, revealing that in normal condition it is MYF5 or MRF4 that trigger expression and EOM accurate differentiation. In the BA, the absence of Myf5 and is not deleterious, and like in the somite, is able to drive myogenesis. While in the somite, PAX3 is responsible for the rescue of activation; analysis of the compound Tbx1 / ; Myf5 loxp/loxp revealed the sequence of events during BA development. Strikingly, expression is lost in these embryos. This has several implication, first, unlike in the EOM, MRF4 cannot rescue expression, nor can PITX2, second it suggests that Tbx1 is genetically equivalent to Pax3 during BA development [37]. Hence, it appears that the core network of genes engaged in head myogenesis not only differ from that of the body (Fig. 1), but also differ among the different groups of head muscles. From the embryo to the adult Muscle masses are composed of a pool of skeletal muscle progenitors cells, which continue to proliferate and at same time provide differentiated cells, building the embryonic and fetal muscle masses. The differential origin of the embryonic and fetal muscle cells has been addressed in a recent study. Using a Pax3 Cre/+ ; R26R DTA/+, in which cells that express Pax3 are specifically deleted, it was shown that PAX3+ progenitor cells give rise to all PAX7+ cells and are necessary for embryonic (limb in this case) myogenesis. Limb fetal myogenesis, however, is established from Pax7+ progenitors cells [38], that will later give rise to the adult muscle stem cells, called satellite cells (SC). SC have first been recognized by Alexander Mauro in 1961, and have been defined by their anatomic location beneath the basal lamina of the myofiber [39]. Since then, other cells present in the muscle, non-somitically derived, have been proposed to serve too, as resident muscle stem cells [40] or, to the least, to participate in muscle regeneration after injury [41]. Lineage tracing experiments by way of GFP labeling as well as classic quail/ chick grafting rendered possible to track the origin of satellite cells to the Pax3/Pax7 muscle progenitor cells in the dermomyotome, embryonic and fetal muscle masses [42,4]. However, studies conducted both in murine and avian models have established that head muscle stem cells, unlike trunk and limbs, do not derive from Pax3 expressing cells, but from cells which have expressed Isl1, a marker of the pharyngeal mesoderm [43], therefore the origin of SC is closely dependent on the origin of the muscle itself. Despite their different developmental origin, SC from branchiomeric, or extra-ocular, muscles are able to regenerate muscle when transplanted into a damaged limb [43,37]. Interestingly,atbirth there is a switch of myogenesis mode, as first suggested by work on single fibers that showed that quiescent muscle satellite cells can be derived from committed + cells [39,44]. This was recently confirmed genetically by analysis of icre/+ mice, demonstrating that all satellite cells have expressed at some point in their history [45]. A classic question in stem cell biology regards the mechanisms and factors that ensure stem cells maintenance: while it remains unclear whether embryonic muscle progenitor cells self-renew efficiently, SC are able to accurately replenish their pool. The set of transcription factors, which activate myogenesis during embryonic development, is redeployed in the adult (Fig. 1) to control muscle stem cells activation during post-natal growth, tissue injury and the replenishment of the stem cell niche. SC express

5 EXPERIMENTAL CELL RESEARCH 316 (2010) Pax7 and, in a subset of muscles, Pax3 [40]. Pax7-deficient mice present a rapid loss of SC after birth, suggesting that PAX3 cannot compensate for the loss of Pax7 and a role of Pax7 in SC post-natal survival and self-renewal [3,46,47]. The absolute requirement for Pax7 and Pax3 has recently been challenged. Generation of conditional Pax7, Pax3 as well as Pax7 cre-ert2 mouse line that allow the suppression of either genes expression in a time and tissuespecific manner, revealed that the requirement of PAX3 and PAX7 exists up to three weeks after birth but is strikingly lost thereafter [48]. This observation suggests that Pax3 and Pax7 are needed for early post-natal growth but not after, although they remain express in adult SC. The need for Pax3 and Pax7 is associated with embryonic, fetal muscle progenitors and young SC, whether this independency towards Pax3 and Pax7 marks the decline of SC stemness, or a switch of myogenic specification mechanisms remain to be elucidated. The role of Myf5 during adult muscle regeneration can be brought to light in a specific Myf5 null allele [49]. After muscle injury, these mice show an increased number of hypertrophic fibers, delayed differentiation and less efficient muscle regeneration. After injury, the quiescent SC will quit the G0 cell cycle state, reenter the myogenic program and quickly express, they are then called activated SC. Numerous studies have taken advantage of the isolated single fibers model to dissect adult myogenesis [44]. After activation, PAX7+ MYOD+ SC actively proliferate, most of the cells will then activate the myogenin differentiating gene and fuse with existing myofibers, while a small proportion of the cells will go back to an undifferentiated PAX7+ only stage and repopulate the SC niche. The role of MYOD in adult myogenesis is revealed by the phenotype of / mice, these one have a reduced body size and have an impair muscle regeneration due to the failure of SC to differentiate into mature myoblasts, as a consequence more SC accumulate in / muscles [50]. Interestingly, while is very important during embryonic myogenesis, adult mouse deficient for did not show any muscle defects [51], suggesting that MYOGENIN have different function during development in adult myogenesis. SC have to maintain their stemness but also their lineage memory. Hence, it is the equilibrium between genes that will act to prevent differentiation and promote self-renewal with genes that will allow the dormant SC to efficiently re-enter and progress through the myogenic lineage, that will make SC to function as bona fide healing cells for the damaged muscle. Concluding remarks In the embryos and adult, it appears that a core set of transcription factors, namely, the MRFs, are engaged in the development of all muscle cells in the body; the expression of their upstream regulators, however, is tissue and time specific. While our understanding of the genetic control of muscle formation has increased, many questions are left unanswered: clearly, this battery of muscle genes may not only be regulated by transcription factors. For instance, to what extent microrna [12] and epigenetic modifications control myogenesis? How do all of these pathways interact with one another to ensure proper muscle development? How do they respond to the neighboring environment in normal and pathological situation? Can we mimick in non-muscle cells the genetic signature of muscle stem cells for regenerative medicine? It makes no doubt that in the next decade, more and more layers will be added to the complexity of the genetic control of muscle lineage. Acknowledgments Our Research is funded by grants from INSERM Avenir program, Association Francaise contre les Myopathies, Association Institut de Myologie, La Ligue contre le Cancer, European consortium FP7 HEALTH-2009 Endostem, Decrypthon program grant, INCa network grant. REFERENCES [1] M.H. Parker, P. Seale, M.A. Rudnicki, Looking back to the embryo: defining transcriptional networks in adult myogenesis, Nat Rev Genet 4 (2003) [2] L. Bajard, F. Relaix, M. Lagha, D. Rocancourt, P. Daubas, M.E. 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