regulatory networks and cell lineages that underlie the formation of

Margaret Buckinghama,1

aDepartment of Developmental and Stem Cell Biology, CNRS UMR 3738, Institut Pasteur, 75015 , France

Edited by Ellen V. Rothenberg, California Institute of Technology, Pasadena, CA, and accepted by Editorial Board Member Neil H. Shubin January 18, 2017 (received for review August 27, 2016) Skeletal muscle in vertebrates is formed by two major routes, as with emphasis on the formation of head muscles and lineage rela- illustrated by the mouse embryo. Somites give rise to myogenic tionships with the . The focus will be on amniote , progenitors that form all of the muscles of the trunk and limbs. based on the mouse as a genetic model, with embryological back- The behavior of these cells and their entry into the myogenic ground provided by experimental manipulations in avians. program is controlled by gene regulatory networks, where paired box gene 3 (Pax3) plays a predominant role. Head and some neck Skeletal Muscles of the Trunk and Limbs muscles do not derive from somites, but mainly form from meso- All of the skeletal muscles present in the trunk and limbs are derm in the pharyngeal region. Entry into the myogenic program derived from somites, segments of paraxial mesoderm that form also depends on the myogenic determination factor (MyoD) family progressively on either side of the body axis from the anterior to of , but Pax3 is not expressed in these myogenic progenitors, the posterior of the developing embryo (1). Muscles form after where different gene regulatory networks function, with T-box delamination of cells from the dorsal somite, the dermomyo- factor 1 (Tbx1) and paired-like homeodomain factor 2 (Pitx2)as tome, which maintains an epithelial structure after cells in the key upstream genes. The regulatory genes that underlie the for- ventral part of the somite have acquired a mesenchymal phe- mation of these muscles are also important players in cardiogen- notype, forming the sclerotome, which subsequently gives rise to esis, expressed in the second heart field, which is a major source of bone and cartilage of the vertebral column and ribs. At different myocardium and of the pharyngeal arch mesoderm that gives rise axial levels, myogenic progenitors from the part of the somite to skeletal muscles. The demonstration that both types of striated adjacent to the axial structures of the neural tube and notochord muscle derive from common progenitors comes from clonal anal- will form the epaxial myotome, which contributes to back mus- yses that have established a lineage tree for parts of the myocardium cles, while the hypaxial myotome gives rise to body wall muscles. and different head and neck muscles. Evolutionary conservation of At limb level, myogenic progenitors migrate out into the limb the two routes to skeletal muscle in vertebrates extends to chordates, bud and subsequently differentiate to form the muscles of the to trunk muscles in the cephlochordate Amphioxus and to muscles limb. Cell migration from cervical somites is also important for derived from cardiopharyngeal mesoderm in the urochordate Ciona, the formation of the diaphragm muscle more anteriorly. As de- where a related gene regulatory network determines cardiac or skel- velopment proceeds, the initial muscle primordia, under the ’ etal muscle cell fates. In conclusion, Eric Davidson s visionary contri- action of surrounding connective tissue, become subdivided into bution to our understanding of gene regulatory networks and their distinct muscle masses; innervation takes place; and tendinous evolution is acknowledged. junctions with bones are established. In the initial waves of epaxial and hypaxial myogenesis, myogenic progenitors enter the skeletal myogenesis | muscle origins | second heart field | muscle program and differentiate into muscle. It is only later, gene regulatory networks | cell lineages when the epithelial structure of the central dermomyotome breaks down and cells enter the underlying muscle masses, that ovement is a fundamental requirement for animal survival, some of these cells do not differentiate, but constitute a reserve Mboth for finding food/feeding and for avoiding predators/ of myogenic progenitors for subsequent muscle growth and later locating to a propitious environment, and depends on cells with muscle regeneration. In the limbs, part of the population of contractile properties, mainly based on actomyosin motor activ- myogenic cells is also retained as a progenitor pool. ity. In more sophisticated organisms, specialized contractile pro- The gene regulatory network (2) that governs muscle pro- teins are present in muscle cells. In vertebrates, striated muscle genitor cell behavior (Fig. 1A) and controls activation of the permits movement and also underlies the pumping activity of the myogenic determination genes (Fig. 1B) is dominated by paired heart, which, like the simpler peristaltic pumps present in the box factor 3 gene (Pax3) (3), which is first expressed in pre- vascular system of many animals, performs a vital function in somitic mesoderm immediately anterior to the first somite and ensuring the circulation of nutrients within the body. Striated then throughout the early epithelial somite, before becoming muscles contain a range of distinct and overlapping contractile restricted to myogenic progenitors of the dermomyotome. protein isoforms, adapted to functional requirements of the motor Forkhead box protein factor c2 (Foxc2) is coexpressed with Pax3 activity of different skeletal muscles or of contractility in different in the early somite and then is expressed, together with Foxc1,at cardiac compartments of the heart. Although the contractile ap- paratus is similar at the protein level, the upstream regulation of cardiac or skeletal muscle genes is different. Skeletal muscle for- This paper results from the Arthur M. Sackler Colloquium of the National Academy of mation depends on myogenic regulatory factors of the myogenic Sciences, “Gene Regulatory Networks and Network Models in Development and Evolu- tion,” held April 12–14, 2016, at the Arnold and Mabel Beckman Center of the National determination factor (MyoD) family, whereas in cardiac muscle, Academies of Sciences and Engineering in Irvine, CA. The complete program and video these basic helix–loop–helix factors do not play a role, and other recordings of most presentations are available on the NAS website at www.nasonline.org/ families of transcription factors lead to the activation of striated Gene_Regulatory_Networks. muscle genes. The mesodermal progenitor cells that contribute to Author contributions: M.B. wrote the paper. cardiac and skeletal muscle also have distinct embryological origins, The author declares no conflict of interest. although it has now emerged that the skeletal muscles of the head This article is a PNAS Direct Submission. E.V.R. is a guest editor invited by the Editorial share common ancestry with the myocardium of the heart. In this Board. review, the formation of vertebrate skeletal muscle will be discussed, 1Email: [email protected].

5830–5837 | PNAS | June 6, 2017 | vol. 114 | no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1610605114 Downloaded by guest on September 24, 2021 PAPER A myogenic progenitor cell B Sim2 COLLOQUIUM survival and proliferation Pitx2 MyoD Msx1 myogenic limb Lbxl CXCR4 progenitor Pax3 Myf5 Meox2 Pax3 c-Met cell migration

Dmrt2 Myf5 epaxial somite FgfR4 myogenesis versus Six1/4 MyoD hypaxial Foxc2 Sproutys cell renewal (Eya1/2) Myf5 somite/limb derm

brown vascular endothelial and fat smooth muscle

bone/cartilage

Fig. 1. Gene regulatory networks at the onset of myogenesis in the trunk and limbs, where Pax3 plays a central role in controlling many aspects of myogenic progenitor cell behavior (A) including choice of the myogenic cell fate, survival, proliferation, migration, and entry into the differentiation program, which depends on activation of the myogenic determination genes at different sites where skeletal muscle formation is initiated in the mouse embryo (B). derm refers to the dorsal dermis.

a high level in the sclerotome, where these Foxc transcription directly regulates enhancer sequences that govern most of the factors are required for the formation of bone and cartilage. spatiotemporal expression of this early myogenic determination Foxc2 remains expressed at a lower level in the dermomyotome, gene. The early epaxial enhancer that controls Myf5 expression in notably in the hypaxial domain. Genetic manipulations in the the epaxial somite does not depend directly on Pax3, but is acti- mouse embryo have shown that Pax3 and Foxc2 reciprocally vated by wingless-related integration site (WNT) signaling mole- inhibit each other (4). When the dose of Foxc2 is diminished, cules signaling from the neural tube and by doublesex- and mab- Pax3-dependent myogenic progenitors predominate, whereas related transcription factor 2 (Dmrt2), which is also implicated in diminution of the level of Pax3 favors nonmuscle cell fates, such maintaining somite integrity. Dmrt2 is a direct Pax3 target. Later as vascular smooth muscle or endothelial cells. Somitic cells are activation of MyoD is not directly dependent on Pax3, but depends maintained in a multipotent stem cell state, until the balance is on other factors acting at different sites of myogenesis, including tipped toward Pax3 or Foxc2. In this model, signaling from ad- the transcription factor encoded by the paired-like homeodomain jacent cells can shift the equilibrium, as illustrated for Notch factor 2 (Pitx2), which lies genetically downstream of Pax3, acting signaling in limb-level somites (5). In addition to its role in cell in part of the hypaxial somite and the limb. During the migration fate determination, Pax3 is required for cell survival in the so- of myogenic progenitors to the limb, it is important to prevent the mite, illustrating the point that, in the absence of a key upstream premature onset of myogenesis. Msx1 directly represses the limb regulator, potentially dangerous unguided cells tend to die. Pax3 enhancer of Myf5 and also delays the activation of MyoD.Sim2 also contributes to control of the important choice between also performs this function in the limb. In this context, Meox2 myogenic progenitor cell self-renewal vs. entry into the muscle plays a role in the onset of activation of the Myf5 limb enhancer

differentiation program by regulating fibroblast growth factor (6). Signaling pathways also control the spatiotemporal activation BIOLOGY

(FGF) signaling, via the genes for Sprouty inhibitors and fibro- of the myogenic determination genes. Shh signaling from the DEVELOPMENTAL blast growth factor receptor4 (FgfR), which is a direct Pax3 tar- notchord and floor plate, as well as from the zone of polarizing get. Myogenic progenitor cell migration also depends on Pax3, activity in the limb, directly acts on Myf5 enhancers to up-regulate which directly activates the c-met gene. In the absence of c-Met them in the epaxial somite and limb bud, respectively. WNT sig- receptor tyrosine kinase signaling, myogenic progenitors do not naling is also promyogenic, whereas bone morphogenetic protein leave the somite, and muscles such as those in the limb are ab- (BMP) signaling prevents the expression of Myf5 and MyoD.Inthe sent. The chemokine receptor type 4 (CXCR4) is also required somite, BMP activity from the dorsal neural tube or lateral me- for migration of a subset of myogenic progenitors into the limb. soderm is buffered by inhibitors such as noggin. In general, the Activation of this gene depends on a transcription factor encoded effects of signaling pathways on the onset of myogenesis are fine- by ladybird homeobox 1 (Lbx1), which lies genetically down- tuned by a complex combination of components that modulate the stream of Pax3. Thus, Pax3 orchestrates key steps—cell fate signal at precise sites where muscle will form as development choice, survival, self-renewal, and migration—in the progression proceeds (1). of progenitor cells toward myogenesis. Sine oculis homeobox transcription factors 1/4 (Six1/4), with Pax3 also controls the onset of myogenesis (Fig. 1B). Entry their coactivators Eya1/2, are also important players in myo- into the myogenic program (2) depends on the myogenic de- genesis (2). Together with Pax3, they control the enhancers re- termination factors MyoD and myogenic factor 5 (Myf5), with sponsible for hypaxial somite and limb expression of Myf5. They myogenic regulatory factor 4 (Mrf4) performing this function also directly activate enhancer elements on the MyoD gene. In with Myf5 when muscle formation is initiated in the somite, Six1/4/Myf5(Mrf4) compound mutants, MyoD is not activated, before expression of MyoD. Muscle-cell differentiation depends and skeletal muscles do not form in the body and limbs. Pre- on subsequent expression of the myogenic differentiation factor, mature activation of the myogenic determination genes in the Myogenin, which, together with other transcription factors, such as limb is prevented by Dach, which is a corepressor of Six. Unlike myocyte enhancer factor 2 (Mef2) family members, plays an es- Pax3—which, if maintained in muscle cells, inhibits differentia- sential role in activating downstream muscle genes. The myogenic tion—Six factors activate Myogenin and, together with Myogenin, determination factors, notably Mrf4 and MyoD, can also perform regulate downstream muscle genes. In the Six1/4 double mutant, this function. Activation of Myf5 is controlled by Pax3, which Pax3 expression is compromised, suggesting that these factors

Buckingham PNAS | June 6, 2017 | vol. 114 | no. 23 | 5831 Downloaded by guest on September 24, 2021 also play an upstream role. Reciprocally, in the Pax3/Myf5(Mrf4) In general, the same downstream muscle genes are expressed compound mutant, which has a similar phenotype to the Six1/4/ in skeletal muscles of the head and body, although head muscles Myf5/(Mrf4) mutant, Six1/4 do not rescue MyoD activation, in- may express specific isoforms (11), exemplified by extraocular dicating that Pax3 lies genetically upstream of Six1/4. In this myosin heavy chain. Formation of muscles in the head, like those discussion, the focus is on Pax3; however, the sister gene, Pax7,is in the body, depends on the myogenic regulatory genes of the coexpressed in the central domain of the dermomyotome and MyoD family. Different combinations of myogenic determination becomes the predominant Pax factor in myogenic progenitors, genes are expressed in cells at different sites of myogenesis (12). because Pax3 is down-regulated during fetal development. In the Activation of the extraocular muscle program depends on Mrf4 perinatal/postnatal period and in satellite cells, the myogenic and Myf5, whereas MyoD, together with Myf5, is a driver of progenitors that permit adult muscle regeneration, Pax7 pre- myogenesis in cells that will form branchiomeric muscles. How- dominates (3). ever, the upstream gene regulatory network (2) that leads to activation of myogenic determination genes is different. Pax3 is Skeletal Muscles of the Head and Neck not a player. Pax7 is only activated later during fetal develop- In mesoderm anterior to the first cervical somite, the distinction ment, when it marks the reserve cell population of myogenic between paraxial and lateral splanchnic mesoderm is not clear- progenitors and is also a regulator of adult satellite cells, as in cut. During development of this region, protrusions known as the trunk and limbs. Six genes are expressed in nonsomitic branchial or pharyngeal arches progressively form in the pha- muscle progenitors. However, the Six1/4 mutant does not have a ryngeal region on either side of the embryo. Mesoderm pro- phenotype in these muscles, possibly because of compensation by trudes into these pouches, overlain by surface ectoderm with Six5, coexpressed with Six1 and Six4 in head muscle progenitors underlying pharyngeal endoderm. Many head muscles (known as (13). Pitx2 and Tbx1 (T-box factor 1 gene) are major upstream branchiomeric muscles) derive from this pharyngeal mesoderm regulators of head myogenesis (Fig. 2). In extraocular muscle (7). The first branchial arches give rise to muscles, such as the progenitors, Pitx2 is required both for cell survival and for acti- masseter and temporalis that control jaw movements, whereas vation of the myogenic determination genes Myf5 and Mrf4.In the second arches are the source of myogenic progenitors of the the Pitx2 mutant, MyoD is not activated, and eye muscles do not facial expression muscles. The mesodermal core of the more form. Similarly, in the first branchial arch, where a mesodermal posterior arches (numbered 3, 4, and 6) contributes pharyngeal deletion of Pitx2 has been examined in detail, myogenic cell and laryngeal muscles and also some neck muscles, which are not survival and growth are compromised, with failure to activate somite-derived. The skeletal muscle of the mammalian esopha- myogenic determination genes, leading to defective masticatory gus is also of nonsomitic origin. Myogenic progenitors from the muscle formation. The role of Pitx2 therefore resembles that of pharyngeal region colonize this structure, moving posteriorly Pax3 at sites of myogenesis in the body. The upstream regulation along the smooth muscle layer into the body from the early fetal of Pitx2 in the head is not well understood, but it is expressed at stage (8). Somitic cells that have moved anteriorly after de- higher levels in extraocular compared with limb muscle pro- lamination contribute to some muscles positioned in the head genitors. In Tbx1 mutants, branchial arches posterior to the first region, as seen for intrinsic laryngeal and tongue muscles that fail to develop, and masticatory muscles derived from the first derive from myogenic cells in the hypoglossal chord, a structure arch are hypoplastic, with sporadic unilateral formation. Tbx1, formed from the hypaxial region of occipital somites. These cells like Pitx2, is not only expressed in mesoderm; however, a me- move anteriorly as a coherent mass and begin to activate myo- sodermal specific deletion demonstrates the direct function of genic determination genes en route; however, as for myogenic Tbx1 in this tissue. In Tbx1/Myf5 double mutants, muscles de- progenitors that migrate to the diaphragm or limb, formation of rived from the first branchial arch are absent, indicating that the hypoglossal chord depends on Pax3 activation of c-met (3). Tbx1 lies genetically upstream of MyoD in this context where The six extraocular muscles (7), which constitute the most an- MyoD would normally determine myogenic cell fate in the ab- terior muscles in the embryo, are thought to be mainly derived sence of Myf5. In the gene regulatory network that leads to from prechordal mesoderm, which, as its name implies, lies an- myogenesis in the arches (Fig. 2), genes encoding mesenchymal terior to the notochord and is contiguous laterally with paraxial mesoderm. A distinguishing feature of head vs. body myogenesis is the major role played by neural crest in the development of head musculature (7). In the trunk, neural crest cells that mi- Pitx2 Tbx1 grate out from the dorsal neural tube transitorily affect early epaxial myogenesis (9). However, anteriorly, neural crest cells invade the branchial arches and occupy the head region, giving rise to the connective tissue, as well as to the craniofacial skel- eton and smooth muscle cells of the vasculature. Connective Tcf21 Msc tissue, which signals to myogenic progenitors and models mat- uration of the muscle masses, is therefore of neurectodermal origin in the head, whereas it is mesodermal in the body. Myo- Myf5 Myf4 Lhx2 genic cells from the branchial arches or from prechordal meso- derm undergo extensive cell displacement to form the developing muscle masses, moving as coherent groups of cells that have often MyoD already activated the myogenic determination genes. This move- MyoD Myf5 ment is in contrast to the migration of isolated progenitor cells from trunk somites to the diaphragm or limbs. Nonsomitic mus- Extraocular muscles Masticatory muscles cles differentiate during fetal stages, so that most head muscles are clearly distinguishable at embryonic day 14.5 (E14.5) in the mouse (1st branchial arch derivatives) embryo (10), and the esophagus skeletal muscle is only laid down Fig. 2. Gene regulatory networks that control the formation of head at E15.5 (8). This process differs from the first skeletal muscles of muscles, highlighting the role of Tbx1 and Pitx2 in extraocular and first the trunk, the myotomes, which form immediately adjacent to branchial arch-derived myogenic progenitors, for which most functional the somites from E8.5 (1). information is available in the mouse embryo.

5832 | www.pnas.org/cgi/doi/10.1073/pnas.1610605114 Buckingham Downloaded by guest on September 24, 2021 PAPER

stem cell transcription factor (Msc or MyoR) and transcription considered to be derivatives of the first heart field. At the arterial COLLOQUIUM factor 21 (Tcf21), basic helix–loop–helix transcription factors pole, the SHF is the major source of the right ventricle and also expressed in myogenic progenitors, also play a role. In the ab- of all of the myocardium of the outflow tract, which will con- sence of Msc, Tbx1 is up-regulated with reduction of Myf5 and stitute the base of the pulmonary trunk and aorta. The SHF MyoD expression, consistent with maintenance of a progenitor contributes the myocardium of the venous pole at the base of the cell pool. In the double Msc/Tcf21 mutant, major masticatory pulmonary vein and superior caval veins, as well as the dorsal muscles are lost, and Myf5 expression is down-regulated. These part of the right and left atria (21). Proliferating cardiac pro- factors bind regulatory sequences of both Myf5 and MyoD genes genitor cells lying posterior to the heart feed into the tube over a with functional consequences shown for Myf5 transcription. The period of several days, such that venous pole myocardium is still LIM homeobox 2 (Lhx2) transcription factor is also an in- forming at E12.5. At the arterial pole, as the branchial arches termediate in branchiomeric myogenesis. Lhx2 acts genetically develop, mesoderm in the core of the arches is recruited into the upstream of Myf5, and its mutation leads to defects in the heart tube, exemplified by labeling of cells in the second bran- specification of myogenic cells and abnormal head muscle pat- chial arch that contribute to the distal outflow tract at E9.5 (22). terning. It lies genetically downstream of Tbx1, Pitx2, and Tcf21, The heart tube is initially positioned adjacent to the first bran- which bind to putative regulatory elements of Lhx2.InTbx1/ chial arches as they form, subsequently moving caudally as a Lhx2 double mutants, branchiomeric muscles are absent. This result of morphogenetic movements, so that it becomes aligned finding is consistent with Lhx2 and Tbx1 activation of Myf5 and with the more posterior arches. The tube undergoes looping, MyoD, respectively. resulting in the anterior positioning of both poles, and expansion Not only are the upstream transcriptional regulators distinct, of the cardiac chambers with their subsequent septation. This but the impact of signaling pathways on activation of the myo- complex morphogenesis, which occurs as the heart beats, also genic program is different from that documented for the somites. involves neural crest cells, which invade the anterior SHF and Thus, WNT signaling is inhibitory, and sonic hedgehog signaling play a major role in septation of the outflow region and valve (Shh) appears to have little effect (7). Signals are released by formation. surrounding tissues, and notably by neural crest, which is a major The myogenic regulatory factors of the MyoD family are component of the anterior region of the embryo where muscles specific to skeletal muscle. The formation of cardiac muscle (23) form. Neural crest cells release the WNT antagonist Frzb and depends on a combination of more widely expressed transcrip- also noggin and gremlin to counteract BMP signaling, which, as tion factors—such as NK homeobox protein 2-5 (Nkx2-5), in the trunk, inhibits myogenesis. In the pharyngeal arches, GATA DNA binding factor 4 (Gata4), and members of the Mef2 FGF signaling plays a major role in regulating progenitor cell family—that activate downstream striated muscle genes, some of proliferation vs. differentiation (14). which encode proteins also present in skeletal muscle. In this Genetic tracing also underlines the differences between head case, regulation often depends on skeletal or cardiac-specific and body muscles. By using a Pax3Cre allele as a driver for re- enhancers, as for the α-cardiac actin gene (24). Expression of porter gene expression from a floxed Rosa26 allele, all skeletal cardiac or skeletal muscle-specific isoforms of the same gene muscles in the trunk and limbs are marked by the reporter, family also distinguish the two striated muscles (11). In general, whereas this labeling is not seen for non-somite-derived muscles head vs. body muscles do not preferentially express cardiac iso- (3). Lbx1Cre is also a useful tool (15) because it marks myogenic forms. An exception is the expression of an α-myosin heavy progenitors that migrate from the somite, without the con- chain, otherwise specific to the heart, found in tongue and some founding effect of Pax3 expression in cranial neural crest. mammalian jaw muscles, where the presence of the cardiac Comparison with a mesodermal posterior 1 Cre (Mesp1Cre) transcription factor Nkx2-5 has also been reported. These mus- driver, expressed in head muscle progenitors, distinguishes these cles are labeled after genetic tracing with an Nkx2.5Cre (25). muscles. Many non-somite-derived muscles are also marked when Nkx2-5 is expressed in cardiac progenitors of the SHF, as well as the Cre is under insulin gene enhancer protein (Isl1) control in the myocardium, and plays an important role in cardiogenesis, (14). This labeling is seen for muscles in the neck region, as well as evidenced by the severe developmental defects in Nkx2-5 BIOLOGY

as facial expression muscles, whereas masticatory muscles de- mutant (26). Other regulatory genes, discussed in the DEVELOPMENTAL rived from the first branchial arch are partially labeled. Isl1 context of nonsomitic skeletal muscle progenitors, are also es- mutants die before these muscles form, but overexpression of sential for the formation of the heart (18, 27). Genetic tracing this transcription factor represses myogenesis, consistent with with Mesp1Cre, expressed in precardiac mesoderm in the primi- the expression of Islet1 in myogenic progenitor cells. An en- tive streak, marks myocardium in all compartments of the heart hancer of the Mef2C gene also drives reporter gene expression in and its progenitors. Mesp1 mutants have cardiac defects. Islet1 some branchiomeric muscles, including those in the neck (16), has been considered as a marker of the SHF, although it is whereas somite-derived muscles are not labeled. The neck con- expressed at a low level in the cardiac crescent. The Isl1 mutant stitutes a transition zone where some muscles, like those in the has a phenotype typical of SHF function with major defects af- body, are somite-derived, whereas others arise from pharyngeal fecting the right ventricle and the atria, leading to embryonic mesoderm similar to head muscles (17). death (28). The SHF is a heterogeneous population of cardiac progeni- Cardiac Muscle and the Second Heart Field tors, with regional patterns of gene expression (27). Pitx2 is ini- Many of the genes that mark and regulate nonsomitic skeletal tially expressed in the left SHF, reflecting its response to Nodal muscle formation are also major players in cardiogenesis (18, signaling in left/right patterning. Pitx2 mutants have outflow tract 19). The early heart tube forms from ∼E7.5 in the mouse embryo defects, and left vs. right atrial specification is lost, with mutant by fusion at the midline of a bilateral cardiac crescent containing hearts showing right atrial isomerism, due to the loss of Pitx2 differentiating cells. These cells derive from lateral splanchnic repression of right atrial identity in progenitors of the left SHF. mesoderm. Subsequently, the tube grows by the addition of Pitx2 also represses progenitor cell proliferation in the sinus cardiac progenitor cells that initially lie medial and caudal to the venosus. This repressive effect is in contrast to the proliferative crescent and then behind the developing heart tube and in ad- defects in skeletal muscle progenitors reported for the Pitx2 jacent pharyngeal mesoderm. These cells constitute the second mutant. However, the function of Pitx2, its cofactors, and targets heart field (SHF) (20). The initial tube will mainly contribute the are poorly understood in these contexts. Tbx1 expression is re- myocardium of the left ventricle. This early cardiac tube and the stricted to an anterior domain of the SHF. In its absence, outflow differentiated cells of the cardiac crescent that precedes it are tract formation is defective, and pulmonary trunk myocardium is

Buckingham PNAS | June 6, 2017 | vol. 114 | no. 23 | 5833 Downloaded by guest on September 24, 2021 reduced, leading to a common arterial trunk at the arterial pole sequence containing such a duplication (laacZ) will only encode of the heart. TBX1 is a major candidate gene for Di-George functional β-galactosidase if the duplication is recombined so syndrome, which is characterized by congenital heart defects, that it reverts to lacZ. To look at skeletal and cardiac muscle, a notably the arterial pole malformation known as Tetralogy of laacZ sequence was introduced into an allele of the α-cardiac Fallot, thought to be due to reduction of subpulmonary myo- actin gene, expressed in developing skeletal muscle, as well as in cardium. In addition to the cardiac phenotype, absence of Tbx1 the myocardium. Collections of several thousand embryos were results in craniofacial defects, including defects in branchial examined for β-galactosidase staining in both types of muscle. arch-derived skeletal muscles (29). Although its expression is Statistical analysis of the frequency of labeling is essential to limited to the anterior part of the SHF, Tbx1 is also implicated in establish that this labeling results from a single recombination the addition of cells to the venous pole of the heart (30), asso- event in a progenitor cell and thus establish clonality. This ap- ciated with an arterial pole contribution from the posterior SHF proach had demonstrated that two cell lineages form the myo- (Cell Lineage Analyses). Another marker of cells in the anterior cardium of the heart (33), with the contribution of the second SHF is provided by a transgene under the control of an SHF lineage corresponding to that of the SHF. Analysis of head and enhancer of the Mef2c gene. In this case, the complete outflow neck muscles led to the lineage tree shown in Fig. 3. Somite- tact and right ventricular myocardium are labeled, suggesting derived muscles are not clonally related to myocardium, whereas that this element is active in a larger progenitor cell population nonsomitic muscles of the head (10) and neck (16) derive from than Tbx1. An extensive gene regulatory network is active in the common progenitors of the second myocardial cell lineage. They SHF and its subdomains (27, 19); however, cells expressing many have no clonal relationship with muscles of the trunk and limbs. of these genes have not been genetically traced in head muscles, This finding demonstrates that these two skeletal muscle groups and mutant phenotypes have not been documented in this con- not only differ in terms of the regulatory gene network that text, apart from the examples discussed here. In many cases, controls their entry into myogenesis, but also derive from dif- conditional mutants would be required because genes that are ferent embryological cell lineages. A first sublineage that forms essential for heart development result in death of the embryo nonsomitic muscles is constituted by progenitors for the tem- before head muscle formation, as seen for Foxc1/2 genes, which poralis and masseter muscles derived from the first branchial are expressed in the SHF and are required for arterial pole de- arches. Extraocular muscles are related to this group, suggesting velopment. These transcription factors regulate Isl1 and are that paraxial mesoderm as well as prechordal mesoderm con- potential regulators of Tbx1 (18); however, any role in cardiac/ tribute to eye muscles. The first branchial arch-derived skeletal skeletal muscle cell fate specification equivalent to their role in muscles show clonality with right ventricular myocardium. Fa- the somite context remains to be explored. cial-expression muscles, derived from the second branchial Within the mesodermal core of the branchial arches, where arches, are clonally related to the outflow region of the heart, to progenitor cells for cardiac and skeletal muscle are present, myocardium at the base of the pulmonary trunk and the aorta. A distal and proximal domains can be distinguished, in which MyoD third sublineage forms the nonsomitic muscles of the neck, no- and Myf5 have been activated or where expression of Isl1 and tably the trapezius and sternocleidomastoid muscles, and in- other SHF markers are maintained (14). Signaling pathways cluding muscles of the larynx and the nonsomitic component of functioning in pharyngeal mesoderm affect the behavior of these the splenius muscle (16). This finding is in keeping with obser- progenitor cells. FGF signaling from surrounding ectoderm, vations of Pax3 independence and Tbx1 dependence of the tra- pharyngeal endoderm, and neural crest, as well as from the SHF pezius and sternocleidomastoid muscles (34). The trapezius cells themselves, promotes proliferation and prevents premature group of muscles is clonally related to the venous pole of the differentiation in both skeletal and cardiac progenitors. BMP heart, including the right and left atria, as well as the myocar- signaling, conversely, has opposite effects, inhibiting differenti- dium at the base of the pulmonary vein and superior caval veins. ation of skeletal muscle progenitors and promoting cardiac dif- This sublineage is clonally distinct from the second branchial ferentiation as progenitors are recruited into the cardiac tube. arch sublineage that contributes to the pulmonary trunk and BMP and WNT signaling will tend to inhibit myogenesis of head aorta at the arterial pole of the heart. However, the third sub- muscles (7), which may be why myogenic regulatory genes are lineage also contributes to the pulmonary trunk, indicating that only activated in the distal domain of the arches. this myocardium has two origins. This dual origin is of major The overlap between paraxial and lateral splanchnic mesoderm clinical importance because many heart malformations seen at and the expression of SHF genes in skeletal muscle progenitors in birth involve this component of the arterial pole (18, 19). The pharyngeal mesoderm raise the question of a common progenitor function of Tbx1 at both poles of the heart probably reflects its for cardiac and skeletal muscle. However, these observations do implication in this sublineage (30). Dye-I labeling of cells in not distinguish between the presence of a common progenitor or cultured mouse embryos shows that some cells from the poste- the coexistence of distinct progenitors. rior part of the SHF, which is the source of myocardial cells at the venous pole of the heart, migrate anteriorly, acquire gene Cell Lineage Analyses expression patterns typical of the anterior SHF, and contribute Gene-expression profiles and functional analyses cannot resolve to outflow tract myocardium that will form the base of the this issue, which can be addressed by cell lineage studies (31). In pulmonary trunk (35). the avian embryo and, to a limited extent, in cultured mouse The three sublineages for nonsomitic skeletal muscles corre- embryos, grafting experiments or introduction of dyes or other spond to derivatives of the first, second, and more posterior markers have made it possible to follow the fate of progenitor branchial arches. Their contribution to different regions of the cells. However, these procedures usually involve labeling groups myocardium reflects the progressive posterior movement of the of cells and have temporal limitations. Genetic analyses in the heart in relation to the arches during pharyngeal morphogenesis. mouse embryo, adapted to clonal conditions, provide an alter- The skeletal muscles that derive from the arches show left/right native approach. Retrospective clonal analysis (32) is a powerful segregation of clones. In the case of second arch derivatives, this method that does not involve any preconceived idea about the segregation correlates with left-hand clones that also colonize location of progenitors. This method depends on the use of a the base of the pulmonary trunk, whereas clones on the right- reporter sequence, which contains a duplication that introduces hand side of the face colonize the base of the aorta (10). Simi- a stop codon, so that the reporter protein is not made. A rare larly for trapezius and sternocleidomastoid neck muscles, left/ intragenic recombination event resulting in removal of the du- right labeling correlates with clones in the left part of the venous plication leads to a functional coding sequence. Thus, a lacZ pole—the left atrium, left superior caval vein, and pulmonary

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early mouse embryo COLLOQUIUM (epiblast) Extraocular muscles (Right Ventricle) Masticatory muscles (1st arch) 1st lineage Left E6.5 aSHF Right Ventricle Ventricle FHF Facial Expression muscles (right) Atria (2nd arch)

Aorta

aSHF Facial Expression muscles (left) (2nd arch) 2nd lineage E7 SHF Pulmonary Trunk

Left Atrium (dorsal)

Left Superior Caval Vein

Pulmonary Vein

Neck muscles (left) (posterior arches) pSHF

Right Atrium (dorsal)

Right Superior Caval Vein

Neck muscles (right) (posterior arches)

Fig. 3. A lineage tree for the myocardium of the heart and for nonsomitic head and neck muscles (in bold), based on retrospective clonal analyses in the mouse embryo. In the case of nonsomitic neck muscles, clonal analysis focused on the trapezius and sternocleidomastoid muscles. The two myocardial lineages can be compared with the two heart fields that constitute sources of equivalent parts of the myocardium. a, anterior; FHF, first heart field; p, posterior.

vein, as well as the pulmonary trunk at the venous pole—vs. give rise to the different nonsomitic muscles lineages during or right-hand labeling in the right atrium and right superior caval after gastrulation, after the segregation of the second myocardial BIOLOGY vein (16). This left/right segregation takes place at the onset of cell lineage (Fig. 3). The lineage segregation for head or neck DEVELOPMENTAL gastrulation (36). Deducing the timing of events in retrospective muscles from myocardium is a late event in the context of heart clonal analysis (37) depends on how widespread the phenome- development. A late-segregation event is consistent with the non is across sublineages and also on the size of clones, which shared branchial arch origin of many skeletal and cardiac reflects how early the recombination event took place. Rarely, derivatives and the expression of SHF marker genes in some very large clones label most skeletal muscles and all compart- of these skeletal muscle progenitors. ments of the heart, reflecting a recombination event in the very Sophisticated genetic tracing (31) has the potential for more early embryo. In most cases, it is possible to estimate an ap- precise estimates of the time of lineage segregation. By using proximate timing of sublineage segregation based on clone size inducible conditional reporter genes, clonal analysis can be and therefore an estimated number of progenitor cell divisions. achieved with limiting levels of inducer given over a narrow time The segregation of somitic and nonsomitic muscles lineages is window. This approach is exemplified by induction by doxycy- probably a very early event, as also suggested by fate-mapping cline of a Mesp1–rtTA transgene with a responder Tet0–Cre experiments (38). In the case of extraocular muscle clones, some transgene on a genetic background with a conditional Rosa– showed labeling in head muscles derived from the second as well Confetti reporter. These experiments confirmed the presence as the first branchial arches, and in these cases, also had exten- of two myocardial cell lineages and showed that the second sive labeling in the heart, including the left ventricle. This finding lineage also contributes to head muscles (39). The timing of suggests that recombination took place in a progenitor that doxycycline administration indicated that the first myocardial predates the segregation of the two myocardial cell lineages lineage is established at ∼E6.5 (E6.25–6.75), whereas the during gastrulation (10), perhaps reflecting the prechordal me- second emerges later, at ∼E7 (E6.75–7.25). By using the soderm contribution. However, other clones extended only to Confetti reporter, all cell types are visualized, with the dem- labeling in muscles derived from the first branchial arch, in- onstration that the first lineage gives rise to monopotent en- dicating a later common progenitor. The common progenitors dothelial or cardiac muscle cells, whereas the second lineage that contribute to each of the branchiomeric muscle groups and generates bipotent cardiac muscle/endothelial, cardiac muscle/ to the corresponding parts of the heart probably segregate to smooth muscle, or cardiac muscle/skeletal muscle cell types.

Buckingham PNAS | June 6, 2017 | vol. 114 | no. 23 | 5835 Downloaded by guest on September 24, 2021 Alternative genetic approaches (40) to clonal analysis of cardiac circuitry in the cardiopharyngeal mesoderm that will form both progenitors have used a Mesp1Cre–MADM (mosaic analysis with cardiac and skeletal muscle (17). The heart derives from two double markers) method or the conditional Rosa–Confetti reporter Mesp1-positive cells that go on to generate bipotent cells in with a tamoxifen-inducible Smarcd3–CreERT2 transgene. The trunk ventral mesoderm that give rise to both the heart and atrial transgene is driven by an enhancer of Smard3,encodinga siphon muscles. These cardiopharyngeal progenitors express chromatin-remodeling factor expressed early in a subset of homologs of genes such as Nkx2-5 and Tbx1, and functional Mesp1-positive cardiac progenitor cells. In these experiments, studies have shown a remarkable degree of conservation with the two myocardial cell lineages with the properties described vertebrate gene regulatory network, reminiscent of cells in the above were identified; however, no labeling of head muscles SHF (49). Isl1 is also expressed in these progenitors, although it was reported. The presence of a fluorescent reporter in such is maintained in the muscle derivatives, whereas in vertebrates, inducible Cre genetic approaches also opens the possibility of Islet1 is thought to repress skeletal myogenesis (14). Another cell separation by flow cytometry and single-cell transcriptome apparent difference is the upstream role of Collier/Olf1/EBF in analyses of different cardiac progenitor types (39), including specifying muscle identity in atrial siphon muscles at the expense cardiac/skeletal muscle progenitor cells from the developing of heart myocardium. In Drosophila, the homolog of Collier embryo. confers identity to specific muscles, as indeed does the Isl1 ho- molog Tup (50). A myogenic function for Collier may also exist in Evolutionary Considerations vertebrates where the homologous genes Ebf2 and Ebf3 have All vertebrates have somites as a source of trunk and limb/wing/ been reported to be potential upstream regulators at sites of fin muscles, with a conserved gene regulatory network underlying muscle formation, including jaw muscles, in Xenopus (51). An- myogenesis. There is a great diversity in the deployment of head other intriguing characteristic of the Ciona cardiopharyngeal muscles between vertebrates, according to their feeding re- gene regulatory network is antagonism between the Nkx2-5 ho- quirements and predatory behavior (7); however, the origin and molog NK4 and Tbx1/10, such that NK4 promotes activation of genetic regulation of these muscles is conserved. Many bran- GATAa (Gata 4/5/6) and heart formation, whereas Tbx1/10 in- chiomeric muscles seen in mammals and birds have their ho- hibits GATAa and activates Collier, leading to skeletal muscle mologs in gnathostome (jawed) fish, such as the shark, and formation (52), a situation reminiscent of the Pax3:Foxc2 an- cyclostomes (jawless) fish, such as the hagfish or lamprey, also tagonism that controls cell fates in the mouse somite (4). In have some of these muscles, together with extraocular muscles general, conserved fragments of gene regulatory networks, even and an additional somite-derived group of epibranchial muscles. in remote species, are instructive, a classic example being the In the lamprey, homologous genes to Isl1, Nkx2-5, and Tbx1 are Paired (Pax)/Sine oculis (Six)/Eyes absent (Eya) network first expressed in myogenic progenitors of branchiomeric muscles. identified for the Drosophila eye (53), which is echoed in the The link between head and heart is therefore present, with the upstream regulation of trunk and limb myogenesis. concept of a cardiopharyngeal field as a mesodermal source of common progenitors (17). In this context, it is intriguing that the Concluding Remarks emergence of a heart with cardiac chambers (41), with its impli- Our understanding of the evolution of gene regulatory network cations in terms of two myocardial cell lineages, occurred in par- circuitry owes much to Eric Davidson’s conceptual insight (54). allel with head muscle diversity at the base of vertebrate radiation. His and his colleagues’ dissection at a single-cell level of tran- Chordates, which are closely related to vertebrates, show in- scriptional regulation in the early sea urchin embryo was a tour teresting analogies with vertebrate muscle formation. The de force that led to the precise delineation of gene regulatory cephalochordate Amphioxus has somites, where a Pax3/7 gene networks, such as that governing the specification of the endo- homolog (42) is expressed, and forms myotomal muscles with derm (55). The gene regulatory networks discussed here for expression of two MyoD family genes (43). It has branchiomeric skeletal muscle formation in the mouse embryo are very crude by head muscles analogous to those of jawless fish (44), which are comparison. They are averaged over populations of cells and lost at metamorphosis and then reformed in the adult. However, lack precise spatiotemporal resolution. As discussed in the con- their formation appears to be independent of myogenic regula- text of current studies on myocardial cell lineages, the use of tory genes, expressed exclusively in somites (43). Nevertheless, inducible genetic tools and fluorescent markers opens the way to homologs of the same upstream regulatory genes, such as Tbx1/ single-cell analysis, but reconstructing lineages and networks at 10 (45) or Isl1 (46), are expressed in this pharyngeal region in the embryo. Amphioxus does not have a well-defined heart, although this level in the mouse embryo, or other vertebrates, remains a the contractile branchial artery expresses some cardiac markers, challenge. In this context, the ascidian Ciona, with a small number such as the homolog of Nkx2-5 (47), and the analogy with car- of well-defined embryonic progenitor cells, is much more ame- diopharyngeal mesoderm, which gives rise to heart and head nable. However, research on vertebrate gene regulatory networks and the nature of cell heterogeneity in progenitor cell populations muscles is less evident. ’ Urochordates, such as the ascidian Ciona, do not form somites continues to advance. Eric Davidson s pioneering work provides a and lack the Pax3 gene regulatory network. As in vertebrates, conceptual framework for current and future understanding of skeletal muscle formation depends on myogenic regulatory how gene regulatory networks function to determine cell fate genes, represented by a homolog of the MyoD gene family, which during development. is activated at a high level in the few cells in the embryo that are ACKNOWLEDGMENTS. M.B. thanks all her previous laboratory members and destined to form muscles, exemplified by the tail muscle cell colleagues who have contributed to the work on skeletal myogenesis and lineage (48). Ciona has a clearly defined beating heart and cardiogenesis in her laboratory. The and CNRS UMR 3738 provides a striking example of the conservation of genetic have provided ongoing support.

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