Clonal Analysis Reveals a Common Origin Between Nonsomite-Derived Neck Muscles and Heart Myocardium

Clonal analysis reveals a common origin between nonsomite-derived neck muscles and heart myocardium

Fabienne Lescroarta,1, Wissam Hamoua, Alexandre Francoub, Magali Théveniau-Ruissyb, Robert G. Kellyb, and Margaret Buckinghama,2

aUnité de Génétique Moléculaire du Développement, CNRS Unité de Recherche Associée 2578, Institut Pasteur, 75015 Paris, France; and bAix-Marseille Université, Institut de Biologie du Développement de Marseille, CNRS Unité Mixte de Recherche 7288, 13288 Marseille, France

Contributed by Margaret Buckingham, December 23, 2014 (sent for review November 10, 2014; reviewed by Michael Levine and Drew Noden) Neck muscles constitute a transition zone between somite-derived Masticatory and facial expression muscles derive from the skeletal muscles of the trunk and limbs, and muscles of the head, mesodermal core that extends into the first and second branchial which derive from cranial mesoderm. The trapezius and sterno- (or pharyngeal) arches, respectively (11). These arches also con- cleidomastoid neck muscles are formed from progenitor cells that tain SHF cardiac progenitors (12). Cells with divergent cardiac have expressed markers of cranial pharyngeal mesoderm, whereas and skeletal muscle fates in this cardiopharyngeal mesoderm are other muscles in the neck arise from Pax3-expressing cells in the similarly labeled by genetic tracing (13). Furthermore, retrospec- somites. Mef2c-AHF-Cre genetic tracing experiments and Tbx1 mu- tive clonal analysis in the mouse embryo has shown that masti- tant analysis show that nonsomitic neck muscles share a gene catory muscles of the head and right ventricular myocardium regulatory network with cardiac progenitor cells in pharyngeal me- originate from common progenitor cells, whereas facial expres- soderm of the second heart field (SHF) and branchial arch-derived sion muscles share a common clonal origin with the arterial pole head muscles. Retrospective clonal analysis shows that this group of the heart, such that myocardium at the base of the pulmonary of neck muscles includes laryngeal muscles and a component of the trunk or the aorta is clonally related to left or right facial ex- splenius muscle, of mixed somitic and nonsomitic origin. We dem- pression muscles, respectively (14). Cardiopharyngeal mesoderm onstrate that the trapezius muscle group is clonally related to myo- cells that give rise to both cardiac and skeletal muscle derivatives cardium at the venous pole of the heart, which derives from the are already present in urochordates, such as the ascidian Ciona posterior SHF. The left clonal sublineage includes myocardium of intestinalis, where the equivalent of an SHF can be distinguished the pulmonary trunk at the arterial pole of the heart. Although (15). A number of genes, including homologs of vertebrate Islet1 muscles derived from the first and second branchial arches also and Tbx1, are expressed in ascidian cardiopharyngeal mesoderm share a clonal relationship with different SHF-derived parts of the and form a gene regulatory network that governs both heart and heart, neck muscles are clonally distinct from these muscles and pharyngeal muscle formation (16, 17). define a third clonal population of common skeletal and cardiac Muscles of the neck play an essential role in coordination of muscle progenitor cells within cardiopharyngeal mesoderm. By link- movement between the head and trunk. In this transition zone, ing neck muscle and heart development, our findings highlight the muscles of both branchial and somitic origin are found. Myo- importance of cardiopharyngeal mesoderm in the evolution of the blasts in the more caudal branchial arches (3rd, 4th, and 6th) are vertebrate heart and neck and in the pathophysiology of human thought to give rise to neck muscles such as the cucullaris muscle, congenital disease.

neck muscles | myocardium | retrospective clonal analysis | mouse embryo | Tbx1 Significance

n mammals, all skeletal muscles are not equivalent. Entry into Head muscles, derived from the first and second pharyngeal Ithe skeletal muscle program and subsequent differentiation arches, share common progenitors with myocardial cells of the depend on transcription factors of the MyoD (myogenic differ- heart. This is in contrast to somite-derived skeletal muscles of the trunk and limbs. Neck muscles, located in the transition zone entiation 1) family. However, upstream regulators of the myo- between head and trunk, have both a somitic and nonsomitic genic program differ in different parts of the body. Skeletal origin. We now demonstrate a clonal relationship between muscles of the trunk and limbs derive from the somites and thus nonsomitic neck muscles and myocardial cells located in the are descendants of progenitors expressing Pax3, a paired box atria, inflow and outflow regions of the heart. This is distinct transcription factor that plays a major role in the control of −/− −/− from that of the two head muscle lineages. Formation of these myogenesis (1, 2). In Pax3 ;Myf5 double mutants most neck muscles, like those in the head, depends on a gene regu- skeletal muscles are lost. However, muscles in the cranial part of latory network shared with myocardial progenitors. We thus the embryo are not affected in these mutant mice (3), showing identify a third clonal group within cardiopharyngeal meso- that myogenesis in head skeletal muscles is controlled by a dif- derm, with implications for human malformations. ferent upstream genetic network (2). Indeed, head muscles are − + formed from cranial mesoderm, derived from Pax3 ;Mesp1 Author contributions: F.L. and M.B. designed research; F.L., W.H., A.F., and M.T.-R. per- (mesoderm posterior homolog transcription factor 1) cells (4, 5). formed research; F.L., R.G.K., and M.B. analyzed data; and F.L., R.G.K., and M.B. wrote the paper. Many of these myogenic progenitors express genes, including Islet1 (Isl1 transcription factor), Nkx2-5,orTbx1 (T-box tran- Reviewers: M.L., University of California Berkeley; and D.N., Cornell University. scription factor 1), that are also expressed in cardiac progenitor The authors declare no conflict of interest. 1 cells of the second heart field (SHF) within pharyngeal meso- Present address: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, B61070, Belgium. derm populations that form myocardium at the poles of the heart 2To whom correspondence should be addressed. Email: margaret.buckingham@ (6, 7). Among these regulators, Tbx1 is required for development pasteur.fr. of both the arterial pole of the heart and head muscle specifi- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cation (8–10). 1073/pnas.1424538112/-/DCSupplemental.

1446–1451 | PNAS | February 3, 2015 | vol. 112 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1424538112 Downloaded by guest on September 27, 2021 or its mammalian derivatives the trapezius and sternocleido- some neck muscles, including the trapezius and sternocleido- mastoidius, which, in keeping with a branchial origin, are in- mastoid muscles, are labeled (Fig. 1 C and D). The Mef2c nervated by a cranial nerve (18). Recent analysis of gene (myocyte enhancer factor 2c) enhancer is activated in the ante- expression and genetic tracing supports this view. The cucullaris rior SHF (21), and when Mef2c-Cre transgenic mice are crossed R-nlacZ/+ muscle in birds and turtles derives from cells in adjacent lateral with the Rosa26 reporter line, labeling is seen in head mesoderm that do not express Pax3 but express Tbx1 (19). De- muscles, notably those derived from the first branchial arch (14), spite evolutionary changes in cranial somites, this is also the case as well as in myocardium of the pulmonary trunk, aorta, and right for the progenitors of the trapezius and sternocleidomastoid ventricle. The trapezius and sternocleidomastoid muscles are also muscles in the mouse (19), where progenitors were also shown to labeled using this Cre line, as shown for the trapezius muscles on have expressed Islet1. Furthermore the trapezius muscle is missing whole-mount X-gal–stained embryos at embryonic day (E)14.5 in Tbx1 mutants (10, 19), consistent with an origin in cranial me- and E12.5 (Fig. 1 E and F). At E10.5 (Fig. 1G), labeling can be soderm rather than somites, in the mouse embryo. observed in the posterior pharyngeal region, where trapezius Here we address the question of a link between nonsomite- progenitors are potentially present. In keeping with this, these derived neck muscles and the myocardium. Using genetic tracing cells also express MyoD, indicating commitment to the skeletal in WT and Tbx1 mutant embryos and retrospective clonal anal- muscle lineage (1) (Fig. 1H). These results show that progenitors ysis, we characterize such muscles and show that neck muscle of nonsomitic neck muscles express common markers with, and progenitor cells share a common clonal origin with the venous originate in close proximity to, cardiac progenitors. pole of the heart. Our results identify a third common skeletal and cardiac progenitor population in cardiopharyngeal meso- No Clonal Relation with Head or Somite-Derived Muscles. To inves- derm, and we discuss the importance of this tissue in the evo- tigate a potential clonal relationship between these neck muscles, lution of the vertebrate heart and head/neck and the implications muscles derived from the first and second branchial arches, and for human pathology. somite-derived muscles of the neck and body, we performed a retrospective clonal analysis. This approach (Fig. S1A) depends Results on a rare intragenic recombination event in an nlaacZ sequence nlacZ Regulatory Gene Expression. We first verified regulatory gene ex- that converts it to a functional reporter (22). In this case pression in the progenitors of neck muscles, by using specific Cre the nlaacZ sequence is targeted to an allele of the α-cardiac actin drivers with a conditional Rosa26R-nlacZ reporter. Pax3 is ex- (αc-actin) gene (23), so that after recombination β-galactosidase β pressed in myogenic progenitor cells in all somites (1), and as ex- ( -gal) positive cells will be generated in the myocardium and in R-nlacZ/+ pected, many neck muscles were labeled when the Rosa26 developing skeletal muscles where the gene is expressed. Statis- Cre/+ reporter line was crossed with a Pax3 line (Fig. 1 A and B), tical analysis on the collection of embryos determines whether the indicating that they originate from somites. However, the tra- frequency of labeling corresponds to one or more recombination B β pezius and sternocleidomastoid muscles are negative with only events (Fig. S1 ). In the case of the former, -gal positive cells residual labeling, which is due to connective tissue, as previously are clonally related and derive from a common progenitor. Cre/+ α -actinnlaacZ reported (19). With the Mesp1 line, which is activated in In a collection of 2,018 embryos with the c allele, cardiac progenitors in the primitive streak (20), the heart and at E14.5, we found 30 embryos (1.5%) with labeling in the neck, BIOLOGY

limited to the trapezius and/or sternocleidomastoid muscles (Fig. DEVELOPMENTAL 2A). These muscles (Fig. 2B), referred to as the trapezius group, are often labeled together, and statistical analysis of the fre- AB EF quency of this labeling shows that they are a clonal unit (Table a-trap 1stBA 1stBA S1). When these embryos were sectioned, we observed that la- trap β s-trap ryngeal muscles are also -gal positive, suggesting a lineage re- s-trap lationship between these muscle groups (Fig. 3 B–E). These sterno R-nlacZ/+ Cre/+ heart muscles are not labeled in Rosa26 ;Pax 3 embryos (Fig. 3A), in keeping with a nonsomitic origin (11). No clonal Pax3-Cre; R26R E14.5 E14.5 relationship was observed between the trapezius group of mus- E14.5 E12.5 A MyoD cles and masticatory, or facial expression, head muscles (Fig. 2 CD G H and Table 1), derived from the first and second branchial arches, sterno 1stBA trap trap 1stBA respectively (14). Somitic neck muscles, marked by genetic Cre s-trap prog prog tracing with Pax3 , but not by the Mef2c-AHF-Cre transgene, and muscles in the trunk or limbs also showed no clonality with

Mef2c-AHF-enhancer-Cre; R26R Mef2c-AHF-enhancer-Cre; heart heart the trapezius group (Fig. 2A and Table 1). This was also the case for the tongue muscle, which is clonally related to somite-derived Mesp1-Cre; R26R E14.5 E14.5 E10.5 E10.5 neck muscles. The nonclonal nature of the labeling observed in these muscles is also indicated by random left/right distribution Fig. 1. Genetic tracing of the trapezius and sternocleidomastoid neck + + A muscles. (A and B) Transverse sections of an E14.5 Pax3Cre/ ;Rosa26R-nlacZ/ compared with labeling in the trapezius group (Fig. 2 ). We (Pax3-Cre;Rosa26R) embryo, stained with X-gal and eosin. The level of the therefore conclude that the trapezius and sternocleidomastoid sections is indicated at the bottom right. The sternocleidomastoid (sterno) muscles derive from common progenitors that are distinct from and spino-trapezius (s-trap) muscles are mostly negative for β-galactosidase those that give rise to first and second arch-derived head mus- (β-gal) (arrowheads). (C and D) Transverse sections of an E14.5 Mesp1Cre/+; cles, as well as to somite-derived muscles, and thus constitute an + Rosa26R-nlacZ/ (Mesp1-Cre;Rosa26R) embryo, stained with X-gal and eosin. independent muscle lineage. The level of the sections is indicated at the bottom right. Arrowheads in- β dicate muscles that are positive for -gal, including the trapezius (a-trap and Clonal Relation with Myocardium. The Mef2c-AHF-Cre genetic s-trap) and sternocleidomastoid (sterno) muscles (outlined with a discontin- – tracing of the trapezius group suggests a potential common ori- uous line). (E G) Whole-mount X-gal staining of Mef2c-AHF-enhancer-Cre; gin with a subset of heart progenitors. These are different from R26R embryos at E14.5 (E), E12.5 (F), and E10.5 (G), showing β-gal positive cells (arrowheads) in the first branchial arch-derived head muscles (1stBA), the common cardiac and skeletal myogenic progenitor cells acromio-trapezius (a-trap), and spino-trapezius (s-trap) muscles (E, F), or giving rise to head muscles that, as we show, are clonally distinct their progenitors (trap prog) (G) and in the heart. (H) Whole-mount in situ from neck muscles. Six of 30 embryos (20%) with labeling in hybridization with a MyoD riboprobe on an E10.5 embryo. the trapezius and/or sternocleidomastoid muscles also showed

Lescroart et al. PNAS | February 3, 2015 | vol. 112 | no. 5 | 1447 Downloaded by guest on September 27, 2021 A B 1317 3198 1553 1996 1331 2383 2010 2810 2940 2469 2185 2128 2353 2436 3057 1471 2036 2634 1342 1677 2703 2724 1445 1804 1631 1942 1374 2573 2215 2188 Sterno RL L L LR R LR LRRLL+RL+R a-trap RL RL RLRLLRRL LRLRLLRRLRRL+RL+RL+R L LLRRLRLLRR RLRLRLLRRLRRL+RL+RR sterno s-trap acromio Mast R Fac R -trap Tongue RL FL spino AP LL LL L L V L L L+R R R L L+R L+R L -trap A LR L L+RRLLRL+RLLRL L+R SCV L L L+R R L R L+R L L+R PV LLLLLLL nb of -gal positive fibers10 10 10 14 12 19 20 26 27 30 30 31 40 + + + + + + + + + + + + + + + + + Left trapezius muscles Right trapezius muscles C ratio ra(observed/expected)tio (observ 0 5 10 15 20 25 D D’ LA -3 EE’ AP * p=3x10 AP left trapezius a-trap a-trap RA * p=7x10-3 right trapezius * p=2x10-5 LA s-trap 14K1804-ventral s-trap 14K2215-ventral PV D” E” RA RSCV * p=2x10-4 LA * p=1x10-3 RSCV LSCV -2 * p=1x10 * p=2x10-4 PV * p=4x10-3 14K1804-left 14K1804-dorsal 14K2215-right 14K2215-dorsal

nlaacZ/+ Fig. 2. Trapezius neck muscles share common progenitors with myocardium. (A)Allαc-actin embryos at E14.5 with labeling of >10 β-galactosidase positive fibers (β-gal+) in the trapezius or sternocleidomastoid muscles of the neck are shown, with labeling indicated by a box. L, left side; R, right side; nb, + number of fibers with β-gal nuclei in the trapezius muscles; +, >50 positive fibers; Sterno, sternocleidomastoid muscle; a-trap, acromio-trapezius muscle; s-trap; spino-trapezius muscle; Mast, masticatory muscles; Fac, facial expression muscles; Tongue, tongue muscles; FL, forelimb muscles; AP, arterial pole; V, ventricles; A, atria; SCV, superior caval veins; PV, pulmonary vein. (B) Schematic representation of an E14.5 embryo showing superficial labeling of the two trapezius (trap) and sternocleidomastoid (sterno) muscles. (C) Significant clonal relationships estimated on the basis of the ratio between the observed and nlaacZ/+ expected numbers of double labeling events in trapezius muscles on the left or right side and myocardium in the collection of αc-actin embryos. The black vertical line indicates a ratio of observed labeling compared with labeling expected from more than one recombination event of 1. *P values showing nlaacZ/+ + a statistically significant relationship are indicated. (D–D″) Example of an αc-actin embryo (reference number 1804) with β-gal cells (arrowheads) in the left a-trap and s-trap muscles (D) and in the arterial pole (AP), left atrium (LA), and pulmonary vein (PV) (D′ ventral, D″ dorsal views of the heart of the same nlaacZ/+ + embryo.). (E–E″) Example of an αc-actin embryo (reference number 2215) with β-gal cells (arrowheads) in the right a-trap and s-trap muscles (E) and in the right atrium (RA) and right superior caval vein (RSCV) (E′ ventral, E″ dorsal views of the heart of the same embryo).

labeling in the arterial pole of the heart, specifically in pulmo- Furthermore, these muscles are absent in Tbx1 null embryos at nary trunk myocardial cells. Unexpectedly, labeling was also fetal stages (10, 19). We confirm this result at E14.5 in a cross observed in myocardial cells at the venous pole of the heart in 16 with the Mlc3f-nlacZ-2E transgene, expressed in differentiated of 30 embryos (53.3%), 5 of which also had β-gal positive cells at skeletal muscle cells, (Fig. 4 A and B), showing the loss of the the arterial pole (Fig. 2A). Venous pole labeling was seen in trapezius muscles as well as first and second arch-derived head myocardial cells of the atria and in the superior caval vein (or vena cava) and pulmonary veins, which in the mouse have a myocardial sleeve labeled by cardiac actin (24, 25). Statistical trap muscle group trap muscle group analysis shows that this colabeling is very unlikely to be due to + splenius muscle independent recombination events (example for the left atrium, − AA’B sterno D P = 6 × 10 7) (Table 1). We thus conclude that the trapezius a-trap group of muscles is clonally related to the myocardium of both a-trap sterno the arterial pole, and of the atria and veins at the venous pole of 14K1317 14K2929 the heart. In addition, we found a left/right regionalization, such C E that labeling in the trapezius muscle group on the left side of the a-trap neck correlates with labeling in the left side of the heart (pul- sterno monary trunk, left atrium, left superior caval vein, and pulmo- sterno 14K2703 14K2260 a-trap nary vein) (Fig. 2 C, D, D′, and D″), and labeling in the trapezius Pax3-Cre; R26R -actinnlaacZ/+ embryos group on the right side correlates with labeling in the right ve- c C nous pole (right atrium and right superior caval vein) (Fig. 2 , Fig. 3. Genetic tracing of laryngeal muscles and representative examples of E E″ C nlaacZ/+ Cre/+ , and ). This regionalization is statistically significant (Fig. 2 αc-actin embryos. (A and A′) Transverse sections of an E14.5 Pax3 ; + and Table 1). This is in keeping with our previous results showing Rosa26R-nlacZ/ (Pax3-Cre;R26R) embryo, stained with X-gal and eosin. The clonal relationships with venous pole myocardium of the right level of the section is indicated at the bottom left. β-galactosidase (β-gal) negative muscle masses in the region of the larynx are surrounded by a atrium and right superior caval vein or of the left atrium, left + – α nlaacZ/ superior caval vein, and pulmonary vein and of the pulmonary dotted line. (B E) Transverse sections of c-actin embryos (numbers at the bottom left refer to the specific embryo) in which β-gal positive trunk at the arterial pole of the heart (25). muscle masses are indicated (blue arrowheads). a-trap, acromio-trapezius; β Mef2c- sterno; sternocleidomastoid; black arrowheads indicate -gal positive cells Tbx1 Dependence. In addition to genetic labeling with the close to the larynx. Of 6 embryos with nonsomitic neck muscle labeling that AHF-Cre (Fig. 1 C–G), the trapezius and sternocleidomastoid were sectioned, all had labeling both in the trapezius group and in laryn- muscles are also genetically labeled using a Tbx1-Cre allele (26). geal muscles.

1448 | www.pnas.org/cgi/doi/10.1073/pnas.1424538112 Lescroart et al. Downloaded by guest on September 27, 2021 Table 1. Statistical analysis of the probability of independent the 22 embryos in the E14.5 collection that had labeling in this recombination events muscle, 9 also had labeling in somitic neck muscles (40.9%), and Muscle/myocardium Trapezius m Splenius m Somitic neck m 5 showed labeling in the trapezius muscle group (22.7%) (Fig. 5 A–E). In both cases this is statistically significant, indicating Cre Skeletal muscles clonality (Table 1 and Table S1). Genetic tracing with a Pax3 − Trapezius m — 3 × 10 5* 0.51 allele (Fig. 5F) confirms that some progenitor cells for this −5 −11 Splenius m 3 × 10 * — 8 × 10 * muscle had expressed Pax3 and are therefore somite-derived. −11 Somitic neck m 0.51 8 × 10 * — We conclude that this muscle, which is located in a transition Masticatory m 0.24 1 0.27 zone, is of mixed origin, formed by progenitors derived from the Facial expression m 0.27 0.18 0.30 somites that are clonally related to other neck muscles and also × −2 Tongue m 0.09 0.15 2 10 * by cardiopharyngeal progenitor cells that contribute to the tra- × −8 Forelimb m 0.13 0.04* 6 10 * pezius group of skeletal muscles and to myocardium. Myocardium − AP 4 × 10 5* 1 0.51 Discussion RV 0.18 1 0.19 In Fig. 6, we integrate our results on the trapezius group of LV 0.64 0.22 0.18 nonsomitic neck muscles with a lineage tree for the second × −3 RA 3 10 * 1 0.27 myocardial lineage contribution to the heart (27). We identify × −7 LA 6 10 *1 1 three distinct sublineages that contribute to first or second RSCV 9 × 10−6*1 1 − branchial arch-derived head muscles or to neck muscles that LSCV 2 × 10 7* 0.13 0.38 − derive from the caudal branchial arches. These sublineages also PV 1 × 10 9*0.071 contribute to myocardium on the anterior/posterior axis, with We have used the nonparametric Fisher’s exact test to assess whether dou- right ventricular, arterial pole, and venous pole contributions α nlaacZ/+ ble labeling results from two independent events in the 30 c-actin that follow the progressive posterior positioning of the heart in embryos with labeling in the trapezius group of muscles at E14.5. Each cate- the pharyngeal region as the branchial arches form. Common gory of neck muscles was tested for independence with other skeletal muscles progenitors for both myocardial cells and skeletal muscles are < × −2 or myocardium. *P 5 10 , indicating that the two regions are clonally probably a small proportion of the overall cardiac progenitor related. m, muscle; AP, arterial pole; RV, right ventricle; LV, left ventricle; RA, Mesp1 right atrium; LA, left atrium; RSCV, right superior caval vein; LSCV, left superior population marked by expression because they represent caval vein; PV, pulmonary vein. only 10% (out of 161 embryos studied) of the clones in Mesp1- inducible clonal analysis (28) and were not reported in the 38 embryos studied by clonal analysis using the MADM system muscles, also marked by Mef2c-AHF-Cre genetic tracing (Fig. 4 E driven by Mesp1-Cre (29). The common progenitors that con- and F). In addition, we observe that other neck muscles such as tribute to each of these three groups of skeletal and cardiac those of the larynx (Fig. 4 C and D), that are clonally related muscle derivatives probably segregate early. Inducible genetic Tbx1−/− to the trapezius group, are also absent in embryos. We tracing with clonal resolution of the kind performed to determine BIOLOGY Mef2c-AHF-Cre

further show, using genetic tracing and expres- the timing of first and second myocardial lineage segregation (28) DEVELOPMENTAL sion of the myogenic determination gene, MyoD, that trapezius will be required to investigate when these sublineages arise. It is muscle progenitor cells fail to emerge from the posterior pharyn- striking that neural crest cells that migrate through the pharyngeal region from E10.5, in the absence of Tbx1 (Fig. 4 G–R). geal region also do so in three distinct evolutionarily conserved streams, through the first, the second, and the more caudal arches The Splenius Muscle, of Somitic and Nonsomitic Origin. The splenius (30), thus paralleling the patterning of cardiopharyngeal meso- muscle, lying in an epaxial location caudal to the ear, provides an derm revealed by our clonal analysis. The development of cranial example of a muscle of mixed somitic and nonsomitic origin. Of neural crest and mesoderm are closely connected, crest being

Fig. 4. The trapezius muscle group and la- WT Tbx1-/- WT Tbx1-/- − − ryngeal muscles are affected in Tbx1 / em- AB bryos. (A–D) The comparison between Mlc3f- G H I J 2E transgene expression, that marks all skeletal muscle, in WT (A and C)orTbx1−/− E10.5 mutants (B and D) stained with X-gal shows the loss of the trapezius and sternocleidomastoid muscles (blue arrowheads in A) and the laryngeal muscles, both intrinsic and ex- E14.5 E14.5 K L M N trinsic (black arrowheads in C), in the mutant. Mlc3f-2E The tongue muscles (black arrow in C and D) CD E11.5 are still present in the absence of Tbx1. (E and F) Whole-mount X-gal staining of Mef2c-AHF- + enhancer-Cre (Mef2c-Cre);Rosa26R-nlacZ/ (R26R) − − E14.5 E14.5 embryos on a WT (E)orTbx1 / mutant (F) O P Q R genetic background at E14.5, showing that EF * * the trapezius muscle (blue arrowheads in E) E12.5 as well as the branchial arch-derived head muscles (white arrowhead in E) are severely affected in the mutant. (G–R) Whole-mount E14.5 E14.5 MyoD ISH Mef2c-Cre; R26R MyoD ISH Mef2c-Cre; R26R in situ hybridization with a MyoD riboprobe, Mef2c-Cre; R26R − − that marks skeletal muscle, on WT (G, K, and O) and Tbx1 / mutant (I, M, and Q) embryos at E10.5 (G and I), E11.5 (K and M) and E12.5 (O and Q). X-gal − − staining of Mef2c-Cre;R26R embryos in WT (H, L, and P)orinTbx1 / mutant (J, N, and R) embryos at E10.5 (H and J), E11.5 (L and N) or E12.5 (P and R). The forming trapezius (blue arrowheads) and head muscles (white arrowheads) fail to develop in the absence of Tbx1. The asterisk in F and R indicates asymmetric residual first arch-derived muscles that form stochastically in the absence of Tbx1.

Lescroart et al. PNAS | February 3, 2015 | vol. 112 | no. 5 | 1449 Downloaded by guest on September 27, 2021 A B splenius EOMs 2260 2484 2126 3004 3037 3058 1793 1560 2026 2286 1384 2282 2483 2165 2760 2929 1340 1830 2717 2421 3288 splenius LLRLLRLRR LRRR LRLRLLLL/R Trap LRRL/R 1st BA-derived muscles other neck L/RL/RL L L LL/R Mast right ventricle Fac RR L FL nb of -gal+ 10101015203030 + + 15 + + + 2020 + + + + + + left left 2nd BA-derived fibres muscles splenius splenius splenius only + Trap + other neck muscles pulmonary trunk C DE F right right 2nd BA-derived splenius splenius som muscles aorta LescroartLescroart et al. 20102010 s-trap splenius splenius left neck non somitic muscles a-trap left -gal pulmonary trunk 14K1560-left 14K2929-right 14K2421-left MF20 left atrium splenius + splenius + somitic splenius muscle Pax3-Cre; R26R Trap muscles neck muscles left superior caval vein pulmonary vein Fig. 5. The splenius muscle has a dual clonal origin. (A) All embryos with > β β + right neck non labeling of 10 -galactosidase positive ( -gal ) fibers in the splenius muscle somitic muscles are shown, with labeling indicated by a box. L, left side; R, right side; nb, + number of fibers with β-gal nuclei; +, >50 positive fibers; Trap, the trape- right atrium zius group of muscles; Other neck, somite-derived neck muscles; Mast, right right superior caval vein masticatory muscles; Fac, facial expression muscles; FL, forelimb muscles. (B) Schematic representation of an E14.5 embryo showing the labeled splenius splenius muscles nlaacZ/+ muscle, situated behind the ear. (C–E) Examples of αc-actin embryos with β-gal+ cells (arrowheads) in the left splenius muscle (C), in the right splenius muscles splenius and spino-trapezius (s-trap) muscles (D), and in the left splenius and somitic neck muscles (som) (E). Figures at bottom right indicate the refer- somitic-derived ence numbers of individual embryos. (F) Transverse section of an E14.5 + + neck muscles Pax3Cre/ ;Rosa26R-nlacZ/ (Pax3-Cre; R26R) embryo stained for X-gal (blue). Skeletal muscles are marked by immunohistochemistry with a myosin anti- Fig. 6. Schema showing the lineage relationship between nonsomitic neck body (MF20) (brown). The splenius muscle has β-gal positive cells, whereas muscles of the trapezius group and myocardium. The first two lineages (in the trapezius (a-trap) is negative. The level of the section is indicated at the blue) contribute to the nonsomitic head muscles, derived from the first (1st) bottom left. and second (2nd) branchial arches (BA), and to arterial pole myocardium (14). A third lineage (in mauve) contributes to the trapezius muscle group as well as to venous pole myocardium, with an additional contribution to the required for patterning of branchiomeric muscles as well as for pulmonary trunk, and a contribution to the splenius muscle (orange). An second heart field progenitor cell addition to the arterial pole of independent lineage (green) contributes to the somite-derived neck muscles, the heart (30–32). including part of the splenius muscle. We had previously analyzed clonal relationships between myocardium at the venous pole of the heart, which derives from the posterior SHF (33), and were surprised to find clonality between muscle first evolved in gnathostomes (bony fish) (36). The pres- the left venous pole and pulmonary trunk myocardium at the ar- ence of common lineages giving rise to specific compartments of terial pole (25). This sublineage is distinct from the sublineage that the heart and specific head and neck muscle groups suggests that contributes to pulmonary trunk myocardium and to left head modulation of the developmental potential of cardiopharyngeal muscles derived from the second branchial arch, and, as we show mesoderm has played an important role in the coevolution of the here, also gives rise to left nonsomitic neck muscles. Pulmonary neck and heart during vertebrate evolution. Indeed, the existence trunk myocardium thus has two different origins. Dye labeling of of multiple common myogenic progenitor cell populations in the cells in different regions of the SHF, followed by mouse embryo mammalian pharyngeal region may reflect reiteration of a devel- culture, showed that some cardiac progenitor cells move from opmental motif regulating the segregation of skeletal and cardiac a caudal to a more rostral location to contribute to outflow tract myogenic fate from common progenitor cells in cardiopharyngeal myocardium (34). We therefore propose that some of the pro- mesoderm. The transition zone, constituted by the neck, between genitors that are clonally related to neck muscles move rostrally head and trunk, shows no clear boundary between somitic and before they enter the arterial pole of the heart. craniopharyngeal muscle derivatives; and indeed our analysis of Tbx1 is required to regulate proliferation and delay differen- the splenius muscle, which is located relatively anteriorly, shows tiation in the SHF and is essential for outflow tract development that there is also no obligatory segregation between muscles, so −/− (9). However, Tbx1 embryos have recently been shown to have that a muscle like the splenius, with cervical innervation, can be of inflow as well as outflow tract defects, with abnormal development mixed origin. In the future it will be important to define when the of the dorsal mesenchymal protrusion and impaired addition of three sublineages that we describe segregate and how the ap- cells to the venous pole of the heart, resulting in atrioventricular pearance of different muscle and cardiac derivatives is regulated septal defects (35). Tbx1 thus regulates the behavior of cardiac by cardiopharyngeal patterning in the mouse. Our findings also progenitors that contribute to both poles of the heart tube as well have relevance for human pathology. TBX1 is the major gene as being required for the development of nonsomitic neck muscles, involved in del22q11.2 or DiGeorge syndrome, characterized by regulating all of the myogenic derivatives of the common lineage a range of cardiovascular and craniofacial anomalies. Under- defined here. standing of the etiology of these defects will depend on identifying The role of Tbx1 in the deployment of cardiopharyngeal me- the mechanisms by which Tbx1 regulates cardiac and skeletal soderm is already presaged by its expression in ascidians (16). myogenic fates within the three clonally related populations of The sublineage that we characterize here probably originated common cardiac and skeletal muscle progenitor cells in car- later during radiation of the vertebrates, because the cucullaris diopharyngeal mesoderm.

1450 | www.pnas.org/cgi/doi/10.1073/pnas.1424538112 Lescroart et al. Downloaded by guest on September 27, 2021 Materials and Methods were found with labeling in neck skeletal muscles. Because recombination is nlaacZ1.1/+ Cre/+ Cre/+ random, there is a low statistical probability that such an event occurs in the Mice. The αc-actin (23), Pax3 (37), Mesp1 (38), conditional Rosa26R-nlacZ/+ (14), Gtrosa26tm1Sor (39), Tbx1+/− (40), and Mlc3f-nlacZ-2E (41) same location a second time, and therefore a cluster of labeled cells in the mouse lines and the Mef2c-AHF-enhancer-Cre transgenic line (21) have been neck probably contains clonally related cells (23, 43). described previously. Animal care was in accordance with national and To establish clonal relationships between two distinct regions, we esti- institutional guidelines. mated the expected frequency of double recombination events in two different regions, which, according to the law of independent probabilities, is Xgal Staining, Immunochemistry, in Situ Hybridization, and Histology. E14.5 + equal to the product of the frequency of labeling in each region (Fig. S1). We α -actinnlaacZ1.1/ embryos were fixed in 4% paraformaldehyde, and whole- c then assessed with the Fisher’s exact test whether this number differed from mount X-Gal staining was performed as previously described (23, 42). Sec- the observed frequency of colabeling in the two regions. The null hypothesis tions, obtained using a cryostat, were X-gal stained, and immunochemistry was performed with MyoD (Dako) or MF20 (DSHB) antibodies with the is that the labeling in both regions results from two independent events. rabbit vectastain ABC kit (Vector Laboratories). Peroxidase activity was re- When the P value is lower than 0.05, the null hypothesis may be confidently vealed with SIGMAFAST DAB tablets. In situ hybridization using an antisense rejected, leading to the conclusion that the two regions are clonally related. MyoD riboprobe was carried out as previously described (10). ACKNOWLEDGMENTS. We thank C. Bodin for technical help. The work in ’ Retrospective Clonal Analysis and Statistical Analysis. A total of 2,018 embryos M.B. s laboratory was supported by the Pasteur Institute and the CNRS, with + “ ” at E14.5 were collected (14). Most of the E14.5 α -actinnlaacZ1.1/ embryos grants from the European Union Integrated Projects Heart Repair [LH SM- c CT2005-018630 (also to R.G.K.)] and “CardioCell” (LT2009-223372). M.B. and present multiple clusters of β-gal positive cells/fibers in skeletal muscles. R.G.K. also acknowledge the support of the Association Française contre Because small clusters are derived from a late recombination event and are les Myopathies (AFM). R.G.K. is an INSERM research scientist and acknowl- not relevant for investigating clonal relationships between skeletal muscles edges the support of the Fondation pour la Recherche Médicale (Equipe FRM and heart myocardium, we scored in our analysis only embryos with labeling DEQ20110421300). F.L. has benefitted from a doctoral fellowship from the of more than 10 β-gal positive cells/fibers. A total of 83 embryos (4.11%) Ile de France region and was supported by the AFM.

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