Clonal analysis reveals common lineage relationships between … · RESEARCH ARTICLE 3269...

3269RESEARCH ARTICLE

INTRODUCTIONAll skeletal muscles are not identical. Notably, trunk and headmuscles differ in a number of important respects. They derive fromdifferent embryonic regions: trunk muscles form from paraxialmesoderm of the somites, whereas most head muscles are formedfrom unsegmented cranial paraxial mesoderm (Noden and Francis-West, 2006). Trunk and head muscles also have distinct generegulatory programmes such that Pax3, which is an importantupstream regulator of trunk myogenesis (Buckingham and Relaix,2007), is not expressed in head mesoderm, whereas other genes suchas Tbx1 or Pitx2 (Grifone and Kelly, 2007) are upstream regulatorsof skeletal myogenesis in the head, but not the trunk. Differences canalready be observed at the onset of gastrulation when cells that giverise to cranial paraxial mesoderm ingress through the streak beforecells that give rise to somitic paraxial mesoderm (Kinder et al., 1999;Parameswaran and Tam, 1995; Tam et al., 1997).

Craniofacial muscles can be classified into different groups:somite-derived neck and tongue muscles, branchiomeric musclesthat are involved in mastication, facial expression and function ofthe larynx and pharynx, and extraocular muscles that control eyemovement. Branchiomeric muscles are derived from pharyngealmesoderm, which includes both lateral splanchnic and paraxial

mesoderm. This forms the mesodermal core of the branchial archesand is then repositioned within the head during the morphogeneticmovements that accompany craniofacial development. Caudalbranchial arches give rise to laryngeal and pharyngeal muscles and,in mammals, the first and second arches give rise to progenitors ofjaw and facial expression muscles, respectively (Larsen et al.,2009). Extraocular muscles derive mainly from prechordalmesoderm, although it is not clear whether there is also acontribution from paraxial mesoderm (Noden and Francis-West,2006). However, extraocular muscles are clearly subject to differentgenetic regulation from branchiomeric muscles. Transcriptionfactors such as Tbx1, MyoR or capsulin, required forbranchiomeric myogenesis, do not play a role in their development(Kelly et al., 2004; Lu et al., 2002). Furthermore, the hierarchy ofmyogenic determination factors of the MyoD family differsbetween extraocular and branchiomeric muscles (Sambasivan et al.,2009). However, Pitx2 function is required for both extraocular andfirst-branchial-arch-derived muscles (Dong et al., 2006).

Classic fate mapping and lineage tracing experiments had indicateda close relationship between progenitors for cranial paraxial mesodermand mesoderm that will form the heart, which ingress through thestreak at the same stage (Kinder et al., 1999). In addition, graftingexperiments showed that cardiac and cranial paraxial mesodermprogenitors are present in the same region (Parameswaran and Tam,1995; Tam et al., 1997). In single-cell labelling experiments in theepiblast, 60% of clones contributed to more than one structure,including cranial paraxial mesoderm and lateral mesoderm from whichthe heart is derived (Buckingham et al., 1997; Lawson and Pedersen,1992). The first heart field arises from lateral splanchnic mesodermand forms the primitive heart tube. It is now established, fromexperiments in chick and mouse embryos, that pharyngeal splanchnic

Development 137, 3269-3279 (2010) doi:10.1242/dev.050674© 2010. Published by The Company of Biologists Ltd

1Institut Pasteur, Unité de Génétique Moléculaire du Développement, CNRS URA2578, 28 rue du Dr Roux, Paris 75015, France. 2Developmental Biology Institute ofMarseille-Luminy, UMR CNRS 6216 Université de la Méditerranée, Campus deLuminy, Institut PaseteurMarseille, France. 3Unité de Biologie Moléculaire duDéveloppement, CNRS URA 2578, 28 rue du Dr Roux, Paris 75015, France.

*Author for correspondence ([email protected])

Accepted 16 June 2010

SUMMARYHead muscle progenitors in pharyngeal mesoderm are present in close proximity to cells of the second heart field and showoverlapping patterns of gene expression. However, it is not clear whether a single progenitor cell gives rise to both heart andhead muscles. We now show that this is the case, using a retrospective clonal analysis in which an nlaacZ sequence, converted tofunctional nlacZ after a rare intragenic recombination event, is targeted to the c-actin gene, expressed in all developing skeletaland cardiac muscle. We distinguish two branchiomeric head muscle lineages, which segregate early, both of which also contributeto myocardium. The first gives rise to the temporalis and masseter muscles, which derive from the first branchial arch, and also tothe extraocular muscles, thus demonstrating a contribution from paraxial as well as prechordal mesoderm to this anterior musclegroup. Unexpectedly, this first lineage also contributes to myocardium of the right ventricle. The second lineage gives rise tomuscles of facial expression, which derive from mesoderm of the second branchial arch. It also contributes to outflow tractmyocardium at the base of the arteries. Further sublineages distinguish myocardium at the base of the aorta or pulmonary trunk,with a clonal relationship to right or left head muscles, respectively. We thus establish a lineage tree, which we correlate withgenetic regulation, and demonstrate a clonal relationship linking groups of head muscles to different parts of the heart,reflecting the posterior movement of the arterial pole during pharyngeal morphogenesis.

KEY WORDS: Retrospective clonal analysis, Head muscles, Second heart field, Mouse

Clonal analysis reveals common lineage relationshipsbetween head muscles and second heart field derivatives inthe mouse embryoFabienne Lescroart1, Robert G. Kelly2, Jean-François Le Garrec1, Jean-François Nicolas3, Sigolène M. Meilhac1

and Margaret Buckingham1,*

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mesoderm, which constitutes the second heart field (SHF), contributesto the growth of the developing cardiac tube (Buckingham et al.,2005). In the mouse embryo, cells from this field form the outflowtract myocardium and also contribute to the right ventricle, as well asto the venous pole of the heart. The SHF is regulated by a geneticnetwork that includes genes such as Islet1, the expression of whichmarks these cardiac progenitor cells (Cai et al., 2003). Pharyngealmesoderm, contributing to the outflow region of the heart, iscontiguous with that contributing to the branchiomeric muscles, asshown by dye labelling experiments of the mesodermal core of the firsttwo branchial arches in mouse and chick embryos (Kelly et al., 2001;Nathan et al., 2008). Furthermore, common gene expression profilesare observed in these cells (Bothe and Dietrich, 2006; Grifone andKelly, 2007; Tzahor, 2009), with a proximal-distal gradient of geneexpression within the mesodermal core, corresponding to markersassociated with branchiomeric rather than SHF progenitors (Nathan etal., 2008). Tbx1 provides an example of a regulatory gene that isimplicated in branchiomeric myogenesis (Kelly et al., 2004) and alsoplays an important role in the formation of the cardiac outflow tract(Xu and Baldini, 2007). Genetic tracing experiments with Islet1-Creor Mesp1-Cre activated in precardiac mesoderm, show expression ofthe conditional Rosa26 reporter in branchiomeric muscles as well asin the heart, indicating that these skeletal muscles also derive from cellsthat had expressed Islet1 or Mesp1. Dye labelling and manipulation ofsignalling pathways in explants of cranial mesoderm at earlier stagesin the chick embryo also show overlapping cardiac and skeletal musclepotential (Tirosh-Finkel et al., 2006).

Mesodermal cells in the pharyngeal region can thus contribute toheart and head muscle, and branchiomeric muscle progenitorsexpress genes that characterize the SHF, prior to entering themyogenic programme. However, it is not clear whether a singleprogenitor cell gives rise to descendants in both types of striatedmuscle or whether progenitors are initially intermingled. Dyelabelling of populations of cells and genetic tracing experiments donot distinguish between these possibilities. We have usedretrospective clonal analysis to investigate lineage relationshipsbetween head and heart muscle. We show that there are twobranchiomeric muscle lineages, the first of which also contributes toextraocular muscles. The first branchiomeric muscle lineage givesrise to the temporalis and masseter muscles, which are first-archderivatives (Larsen et al., 2009), and also unexpectedly contributesmyocardial cells to the right ventricle. The second branchiomericmuscle lineage gives rise to muscles of facial expression that aresecond-arch derivatives and also contributes to myocardium at thearterial pole of the heart. Within this second lineage, a furthersubdivision is observed between myocardium at the base of thepulmonary trunk and the aorta. These sublineages contribute to leftor right muscles of facial expression, respectively. There is thereforea clonal relationship between head and heart muscle progenitors,with further sublineages that form a lineage tree, as discussed here.

MATERIALS AND METHODSMiceThe Mlc1v-nlacZ-24 transgenic line (Kelly et al., 2001), the T4 transgenicline (Biben et al., 1996) and the c-actinnlaacZ1.1/+ mouse line (Meilhac etal., 2003) have been described previously. Mef2c-AHF-enhancer-Cre males(Verzi et al., 2005) were crossed to the Rosa26R-nlacZ reporter line(J.-F.N., E. Tzouanacou and V. Wilson, unpublished).

X-gal staining, immunochemistry and histologyDissected c-actinnlaacZ1.1/+ embryos were fixed in 4% paraformaldehyde(PFA) and X-gal staining was performed as previously described (Bajolleet al., 2006; Meilhac et al., 2003).

Mlc1v-nlacZ-24 and Mef2c-AHF-enhancer-Cre;Rosa26R-nlacZembryos were sectioned using a cryostat. Immunochemistry was performedwith MyoD (Dako) and -galactosidase (J.-F.N.) antibodies.

Retrospective clonal analysisA total of 627 embryos at embryonic day (E) 14.5 had been collected in aprevious study (Bajolle et al., 2008) and 1596 additional embryos werecollected here. Statistical analyses were carried out on the newly collectedembryos only, as some hearts of the first series had been sectioned for otherpurposes such that the collection was no longer complete and therefore nolonger fulfilled the random criterion required for statistical analysis. Mostof the E14.5 c-actinnlaacZ1.1/+ embryos present multiple clusters of -galactosidase-positive cells or fibres and it is therefore essential to establishby statistical analysis whether labelled cells derive from a singlerecombination event and are thus clonally related. Because recombinationis random, there is a low probability that such an event occurs in the samelocation a second time and, therefore, a cluster probably contains clonallyrelated cells (Meilhac et al., 2004; Meilhac et al., 2003). We distinguishedbetween large and small clusters of labelled cells and fibres as large clustersare derived from an earlier recombination event than small clusters and aretherefore more interesting for our study. Large clusters in skeletal musclesor in myocardium were defined as clusters with more than 10 fibres or 10cells labelled, respectively. Such large clusters in head muscles were seenin 1.8% of embryos, whereas small clusters occurred at a much higherfrequency (17.5%).

Statistical analysisWe estimated the expected frequency of double recombination events intwo different regions, which, according to the law of independentprobabilities, is equal to the product of the frequency of labelling in eachregion (Tables 1, 2). In order to decide whether the observed frequency ofcommon labelling in two distinct regions, for instance branchiomericmuscles and heart myocardium, was consistent with the expectedfrequency, we performed a statistical test. We have used the non-parametricFisher’s exact test that allows us to work with small numbers of labelledembryos. The null hypothesis is that the labelling in both regions resultsfrom two independent events. When the P-value is less than 0.05, the nullhypothesis can be confidently rejected, leading to the conclusion that thelabelling probably derives from a single recombination event.

RESULTSExpression of the Mlc1v-nlacZ-24 transgenereveals early continuity between splanchnicmesoderm and the mesodermal core of the firstand second branchial archesIn the Mlc1v-nlacZ-24 transgenic line, in which reporter geneexpression is driven by Fgf10 regulatory elements (Kelly et al.,2001), the outflow tract and part of the right ventricle of thedeveloping heart are -galactosidase-positive (Fig. 1A-C). At E8.5,X-gal staining was seen in the mesodermal core of the developingarches (Fig. 1A,B), where Fgf10 is also expressed (Kelly et al.,2001). This expression domain is contiguous with -galactosidase-positive cells in the splanchnic mesoderm of the SHF and itsmyocardial derivatives at the arterial pole of the heart. Thiscontinuity was maintained at E9.5 when transgene expression wasobserved in the mesodermal core of the arches where thebranchiomeric skeletal muscle programme initiates (Fig. 1C).Mlc1v-nlacZ-24 expression thus illustrates the continuity betweenmyocardial and skeletal muscle progenitor cells in its expressiondomain in pharyngeal mesoderm. As shown in Fig. 1D, the Mlc1v-nlacZ-24 transgene continued to be expressed in branchiomerichead muscles at later stages.

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A retrospective clonal analysis at E14.5In order to examine a possible lineage relationship betweenmyocardium at the arterial pole of the heart and branchiomeric headmuscles, we carried out a retrospective clonal analysis (Bonnerot andNicolas, 1993) using the c-actinnlaacZ1.1/+ line (Meilhac et al., 2003).In this line, the reporter has been introduced into an allele of the -cardiac actin gene, which is expressed throughout the myocardium(Fig. 1E) and also in all developing skeletal muscles (Fig. 1F) (Bibenet al., 1996; Sassoon et al., 1988). This gene therefore provides anappropriate endpoint for clonal analysis of these tissues. Theretrospective clonal approach avoids preconceived ideas aboutlineage relationships and is based on the analysis of a collection ofembryos rather than isolated examples. It employs an nlaacZ reporterin which a duplication introduces a stop codon into the -galactosidase coding sequence, rendering it non-functional. A rareintragenic recombination event results in random removal of theduplication, independently of gene expression, so that the reporternow makes functional -galactosidase when the gene into which itis integrated is expressed. Cells that are descended from a progenitorthat has undergone such a recombination event give rise to labelled

cells that are clonally related (see Fig. S1 in the supplementarymaterial). In the case of skeletal muscle, where cells fuse to formfibres, this is assessed as fibres with labelled nuclei.

At E14.5, head muscle primordia have formed and c-actin isstrongly expressed in these skeletal muscles, as well as in the heart(Fig. 1E,F). This timepoint has therefore been selected for theretrospective analysis using the c-actinnlaacZ1.1/+ line. At this stage,all embryos scored contained -galactosidase-positive cells. Thisfrequency means that multiple recombination events have probablytaken place per embryo to convert the nlaacZ sequence to afunctional nlacZ reporter. Although multiple clusters of -galactosidase-positive fibres were present in body muscles,labelling was much more rare in head muscles (19.3%), whichrepresent a small fraction of the total musculature. Most of theembryos with such labelling in head muscles (77.7%) only had oneor two -galactosidase-positive fibres. As small clusters of labelledfibres probably correspond to more recent recombination events,which are less informative about the progenitor cell pool, wesubsequently focused our analysis on clusters containing more than10 labelled fibres. Only 39 out of the 2223 embryos (1.8%) hadclusters of more than 10 -galactosidase-positive fibres in headmuscles (Table 1). Therefore, the probability that more than 10labelled fibres per embryo derive from multiple recombinationevents is very low (see Fig. S2 in the supplementary material). Thecalculation of probability is an essential feature of this clonalapproach; the statistical demonstration, based on observation ofmany embryos, that a double recombination event is veryimprobable leads to the conclusion that labelled cells descend froma single recombined progenitor and are therefore clonally related.

Two subpopulations of progenitor cells contributeto muscles derived from mesoderm of the firsttwo branchial archesExamples of c-actinnlaacZ1.1/+ embryos with -galactosidase activityin head muscles are shown in Fig. 2. Two distinct distributions oflabelled fibres were observed in subsets of branchiomeric muscles,as illustrated in Fig. 2A,B compared with Fig. 2C,D. These resultsare summarized in Fig. 2G,H. Fourteen embryos had -galactosidase-positive fibres that were restricted to the temporalis andmasseter muscles (category I). In 21 embryos, these muscles werenot labelled but -galactosidase-positive fibres were observed inmuscles of facial expression, as indicated (category II). Only fourembryos had -galactosidase-positive fibres in both categories ofmuscles. This labelling is highly unlikely to result from twoindependent recombination events as the expected frequency ofdouble recombination events in category I and category II musclesis very low (Table 2) and labelled cells are therefore clonally related.The number of muscles labelled increases with the number of -galactosidase-positive fibres, in particular for category II, whichincludes a greater number of distinct muscles and involveswidespread migration of branchial-arch-derived cells. In addition tothe number of muscles and their size, which affects the extent oflabelling, the number of labelled cells in the clone reflects thenumber of divisions and hence timing of the recombination event inthe progenitor cell. In embryo number 1304, for example,recombination probably occurred in a more recent progenitor thatgave rise only to the masseter muscle, whereas in most cases acommon progenitor gives rise to both category I type muscles(masseter and temporalis). In the case of category II muscles, thereis some indication that the zygomaticus, buccinator and auricularismuscles are more frequently labelled; however, these muscles arelarger than other category II muscles. In general, the distribution of

3271RESEARCH ARTICLEClonality between heart and head muscles

Fig. 1. Transgene expression patterns during heart and headmuscle development. (A-D)The Mlc1v-nlacZ-24 transgene expressionpattern. (A)Wholemount X-gal staining at E8.5 showing a lateral viewof transgene expression in myocardium of the outflow tract (OFT) andright ventricular regions of the heart and in the mesodermal core of thebranchial arches (1, 2 and 3). (B)Sagittal section at E8.5 showingexpression of the transgene in the mesoderm of the branchial arches(1, 2 and 3) and in myocardium of the OFT. Arrowheads indicate thecontinuity between these sites of expression. (C)Wholemount view ofthe pharyngeal region at E9.5 with the heart removed and stained withX-gal. Numbers indicate the branchial arches where the mesodermalcore is labelled. Arrowheads indicate the continuity between these sitesof expression. (D)Wholemount X-gal staining in the head at E14.5showing labelling of branchiomeric muscles. FL, forelimb.(E,F)Wholemount X-gal staining of the heart (E) and head (F) musclesof a T4 c-actin-nlacZ embryo at E14.5. ao, aorta; pt, pulmonary trunk;RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium.

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-galactosidase-positive fibres between muscles indicates adispersion of cells after the recombination event. The four embryosthat had labelling in both categories (Fig. 2E,F,H) had large numbers(>40) of -galactosidase-positive fibres, with two of the embryos(2880, 1779) also having a very large number of -galactosidase-positive fibres throughout the body, indicative of a very earlyrecombination event. The presence of two distinct labelling patternsand the fact that both were seen only in embryos with many labelledcells (+ or ++), indicates that category I and II derive from earlycommon progenitor cells that segregated into distinct lineages.

Most embryos showed labelling in muscles on either the left orright side of the head (Fig. 2H), with no particular bias. Someembryos had head muscle labelling on both sides. In four cases

(385, 2084, 284, 1436) this was restricted to category II muscles.In two other examples (2080, 2880), one side of the embryo hadextensive labelling in category II or both category I and II muscles,whereas only category I muscles were labelled on the other side(Fig. 2F). In these examples, a common progenitor for bothlineages must have given rise to asymmetrically distributeddescendants that were only of category I type on the right side. Thetwo very extensively labelled embryos (1779, 2688) had completelabelling on both sides (Fig. 2E). Bilateral labelling indicates thatthe recombination event preceded the onset of gastrulation,whereas monolateral labelling can arise before or after gastrulationand cannot be used as a criterion to date the clones (Lawson et al.,1991; Selleck and Stern, 1991).


Table 1. Frequency of labelling in head or somitic muscles and in heart myocardiumNumber of embryos with Number of embryos with clusters of

-gal+ cells and/or fibres (%) >10 -gal+ cells and/or fibres (%)

Category I muscles 2.6 0.9Category II muscles 18.2 1.1Somitic muscles 87.7 23.7AP myocardium 13.9 2.1RV myocardium 61.7 7.5LV myocardium 67 10.6

Number of embryos analysed: 1596 (see Materials and methods).AP, arterial pole; LV, left ventricle; RV, right ventricle; -gal+, -galactosidase-positive.

Fig. 2. Two distinct groups of head muscles derive from subpopulations of progenitor cells. (A-F�) Examples of c-actinnlaacZ1.1/+ embryos withX-gal stained fibres in head muscles. Two distinct patterns of labelling are observed either of temporalis (te) and masseter (ma) muscles (A,B), locateddeep in the head, or of facial expression muscles located closer to the surface. au, auricularis; bu, buccinator; fr, frontalis; oc, occipitalis; oo, orbitalisoculi; qua, quadratus labii; zy, zygomaticus (C,D). (E-F�) Some embryos have -galactosidase-positive cells in both categories of muscles. In the case of E,all muscles of the embryo are labelled, indicative of a very early recombination event. The identification number of the embryo is indicated in eachpanel. (G)Scheme of category I (blue) and category II (pink) muscles. (H)All embryos with labelling of >10 -galactosidase-positive (-gal +) fibres aresummarized in this table, which indicates category I and II type distributions. The presence of labelled fibres within a muscle is indicated by a blacksquare. The number of the embryo is indicated above each column. In some cases, -gal + fibres are present on both the left (L) and right (R) side of thesame embryo and this distribution is indicated. At the bottom, the number of fibres with -gal + nuclei is indicated. + indicates that more than 50 fibresare scored as positive; ++ indicates that all fibres in the body muscles appear positive. Embryos are ordered on this basis. D

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Extraocular muscles, which lie in close proximity to the eye,are thought to derive from both prechordal and paraxialmesoderm (Evans and Noden, 2006; Noden and Francis-West,2006). We have observed ten embryos (out of 2223), which had-galactosidase-positive fibres in extraocular muscles. Thisincludes three embryos that also show labelling in both categoryI and II muscles (2880, 1779, 2688). In three other cases (3243,394, 2901), there was labelling only in category I branchiomericmuscles (Fig. 3A-C). Given the high number of embryos scoredand the very low number of embryos with labelling in thesesmall muscles, it is very improbable that more than onerecombination event had occurred, leading to the conclusion thatthere is a clonal relationship between these muscle groups (Fig.3D; P0.0004). Embryos 3243 and 2880 showed labelling onlyin a subset of extraocular muscles (dorsal rectus muscles for3243 and dorsal rectus and lateral rectus muscles for 2880);however, the four other embryos with labelling in category Imuscles showed labelling in all six extraocular muscles as shownin Fig. 3B,C. No link was observed with category II muscles(P0.96).

Other branchiomeric muscles situated deep within the embryo,including those of the pharynx and larynx derived from posteriorbranchial arches, were not analysed in detail in our analysis,which concentrated on the muscles presented in Fig. 2H.

However, we also scored additional muscles located underneaththe mandible in the context of category I and category IIlabelling (see Fig. S3 in the supplementary material).

We also examined whether embryos with -galactosidase-positive fibres in branchiomeric head muscles also showedlabelling in muscles of the trunk and limbs that are derived fromsomites (Table 1). We conclude that there is an early segregationof branchiomeric and somitic muscle progenitor cells (Table 2; seeFig. S4 in the supplementary material).

Branchiomeric head muscles share commonprogenitors with right ventricular and arterialpole myocardiumWe next examined how many of the embryos with category I or IIlabelling in head muscles also showed labelling in myocardialderivatives of the anterior SHF, namely myocardium of the rightventricle and at the base of the pulmonary trunk and aorta, describedas the arterial pole of the heart. The frequency of embryos withlabelling in this myocardium is presented in Table 1. The leftventricle, which does not derive from the SHF (Meilhac et al., 2004),is presented as a negative control.

None of the embryos in category I had any -galactosidase-positive cells in the arterial pole of the heart; however, seven of themhad labelled clusters (>10 cells) in the right ventricle. A summary for


Table 2. Expected frequency of double recombination eventsExpected frequency of a Observed frequency

double recombination event of common labelling

Cat. I muscles + Cat. II muscles 1�10–4 3�10–3* (P1�10–5)Cat. I muscles + Somitic muscles 2�10–3 1�10–3 (P0.56)Cat. II muscles + Somitic muscles 3�10–3 2�10–3 (P0.37)Cat. I muscles + RV myocardium 7�10–4 4�10–3* (P3�10–5)Cat. II muscles + AP myocardium 2�10–4 3�10–3* (P9�10–5)Cat. I muscles + LV myocardium 1�10–3 0 (P0.32)Cat. II muscles + LV myocardium 1�10–3 0 (P0.23)

Number of embryos analysed: 1596 (see Materials and methods).The expected frequency of double recombination events is the product of the frequency of each single event (Table 1). The comparison between expected and observedfrequencies indicates clonality (see also Fig. S2 in the supplementary material).*Statistically significant according to the Fisher’s exact test (the P-value is indicated in brackets). AP, arterial pole; LV, left ventricle; RV, right ventricle.As left ventricular myocardium derive from the first myocardial cell lineage (Meilhac et al., 2004), we expect no clonal relationship between head muscles and left ventriclemyocardium. Statistical analysis has been carried out with this as a negative control; rare double labelling in head muscles and the left ventricle can be demonstrated to beindependent events.

Fig. 3. Head muscles share common progenitors with extraocular muscles. (A)Summary of labelling in head muscles with labelling inextraocular muscles (EOMs; >10 fibres labelled), represented as a black column (see Fig. 2H). (B,C)Examples of X-gal staining of c-actinnlaacZ1.1/+

embryos (with eyes removed) at E14.5 with labelled muscles and EOMs (indicated by arrowheads). Higher magnifications of the EOMs are shown inboxes. DR, dorsal rectus (also named superior rectus); DO, dorsal oblique (also named superior oblique); IO, ventral oblique (also named inferioroblique); IR, ventral rectus (also named inferior rectus); LR, lateral rectus; MR, medial rectus. (D)The percentage of total embryos (1596) that had -galactosidase-positive fibres in EOMs and category I head muscles. Statistical analysis indicates a clonal relationship. D

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all embryos with head muscle labelling is given in Fig. 4A and anexample is shown in Fig. 4B,B�. The low probability of doublerecombination events in the two regions reported in Table 2 suggeststhat labelling in category I muscles and right ventricular myocardiumprobably results from a single event. Furthermore, statistical analysis,using the Fisher’s exact test and based on the figures shown in Fig.4C, strongly supports the conclusion that the -galactosidase-positivecells in the right ventricle and fibres in category I skeletal musclesarise from a common progenitor (P3�10-5).

We then examined embryos with category II head muscle labellingand found in this case that many of them also had -galactosidase-positive cells in the arterial pole of the heart (12/21). The results aresummarized in Fig. 4A and an example is shown in Fig. 4E,E�.Statistical analysis, using the Fisher’s exact test and based on thenumbers shown in Fig. 4F, supports the conclusion that the -galactosidase-positive cells in arterial pole myocardium and fibres incategory II skeletal muscles arise from a common progenitor(P9�10-5). In some cases, labelling in the arterial pole extendedinto the right ventricle, but no labelling of only the right ventriclewas observed in category II embryos.

Category II labelling in left or right head musclescorrelates with labelling in myocardium at thebase of the pulmonary trunk or aortaBy E14.5, myocardium at the arterial pole of the heart was located atthe base of the pulmonary trunk or aorta. When we looked moreclosely at embryos with -galactosidase-positive cells in this region,

as well as labelled fibres in the head, we distinguished labelling in oneor both of these arteries (Fig. 5A�,B�,C�). Examination of skeletalmuscle labelling had indicated that most embryos had -galactosidase-positive fibres on either the left or right side of the head. We observeda striking correlation between left or right labelling of category IIskeletal muscles and labelling of myocardium at the base of thepulmonary trunk or aorta, respectively. In cases where both arteries had-galactosidase-positive cells, labelling was predominantly seen onboth sides of the head (Fig. 5A-D). This was observed in embryos withlarge numbers of labelled fibres in head muscles. The significance ofthis observation was examined and validated by phylogenetic tools togenerate the tree shown in Fig. S5 in the supplementary material. Thisresult indicates that there is a common progenitor for pulmonary trunkmyocardium and category II muscles on the left side of the head,whereas those on the right side of the head share a common progenitorwith myocardium at the base of the aorta.

Mef2c-AHF-Cre genetic tracing shows differencesin the two head muscle lineagesWe next examined the descendants of progenitors in which theMef2c-AHF-enhancer had been activated. This enhancer marksprogenitors of the outflow tract and right ventricular myocardium(Verzi et al., 2005), as well as some branchial-arch-derived muscles(Dong et al., 2006).

We crossed the Mef2c-AHF-enhancer-Cre line with a Rosa26R-nlacZ reporter line. At E10.5, labelled cells were found in themesodermal core of the first and second branchial arches, but also


Fig. 4. Category I embryos also have labelling in right ventricular myocardium, whereas category II embryos also have labelling inarterial pole myocardium. (A)Summary of head muscle labelling, with labelling in the right ventricle (RV; >10 cells labelled) or the arterial pole(AP) myocardium, represented as black boxes (see Fig. 2H). (B,B�) An c-actinnlaacZ1.1/+ embryo at E14.5 with extensive labelling in category I muscles(B) and with labelled cells in the right ventricle (B�). (C)The percentage of total embryos that had -galactosidase-positive cells in RV myocardiumand in fibres of category I head muscles. The four embryos with labelling in both categories I and II were not included in this test. Statistical analysisindicates a clonal relationship. (D)Scheme of the relationship between category I muscles and RV myocardium. (E,E�) Example of an embryo withextensive labelling in category II muscles (E) and AP myocardium (E�). (F)The percentage of total embryos that had -galactosidase-positive cells inAP myocardium or in fibres of category II head muscles. Statistical analysis indicates a clonal relationship. (G)Scheme of the relationship betweencategory II muscles and AP myocardium. Abbreviations for head muscles are as in Fig. 2.

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in the outflow tract, right ventricular myocardium and mesodermbehind the heart tube (Fig. 6A-B�). However, within the branchialarches, labelled cells were found only in the more distal part of thesecond branchial arch (Fig. 6A). Sections show that although in thefirst branchial arch there was overlapping expression of -galactosidase and MyoD, -galactosidase-positive cells weremainly negative for MyoD in the second branchial arch (Fig.6B,B�). This indicates that positive cells in the second branchialarch have not adopted a myogenic fate. In keeping with this, atE14.5, labelled cells were found only in category I head muscles(Fig. 6C), as well as in the right ventricular and outflow tractmyocardium as expected. We propose that first branchial arch headand heart derivatives are marked by the activation of this enhancer,whereas in the second arch, activation is restricted to cells givingrise to cardiac derivatives. This result reveals molecular differencesin the regulation of skeletal myogenic progenitor cells in the firstand second arch.

DISCUSSIONWe have shown the lineage relationships between head musclesand second heart field derivatives, as summarized in Fig. 6D.This retrospective clonal analysis demonstrates thatbranchiomeric skeletal muscle and SHF-derived myocardiumderive from common progenitors and that there are distinctlineage and sublineage relationships within this framework.Notably, we show clonality between skeletal muscles derivedfrom the first branchial arch and right ventricular myocardium.This unexpected finding, together with clonality betweenoutflow-tract-derived myocardium at the base of the great arteriesand second-branchial-arch-derived muscles, concords with theposterior movement of the arterial pole of the heart duringpharyngeal morphogenesis.

Two non-somitic head muscle lineagesSkeletal muscles of the head fall into two categories, both clonallydistinct from somitic muscles, indicating early segregation of thesedifferent myogenic lineages, as expected from fate mappingexperiments (Parameswaran and Tam, 1995; Tam et al., 1997).First, there are progenitors that contribute to temporalis andmasseter muscles, derived from the first branchial arch (Larsen etal., 2009). We also detected a significant clonal relationship withextraocular muscles. This comprised all the extraocular muscles,not just the dorsal oblique and lateral rectus, previously proposedto be derived from mesoderm at the level of the arches (Noden andFrancis-West, 2006). We observed two clones in which only asubset of extraocular muscles are labelled (dorsal rectus or dorsaland lateral rectus), suggesting differences in the timing ofprogenitor segregation. The clonality between extraocular and firstbranchial arch muscles indicates that paraxial mesoderm, as wellas prechordal mesoderm, contributes to all these muscles. This linkbetween first branchial arch muscle derivatives and extraocularmuscles is also observed at the level of Pitx2 function (Dong et al.,2006). The clonal relationship with right ventricular myocardialcells indicates a contribution from a progenitor for lateralsplanchnic, as well as paraxial pharyngeal, mesoderm. Pitx2 alsomarks cardiac progenitors; however, its dynamic expression withinthe myocardium complicates the interpretation of genetic tracingexperiments (Franco and Campione, 2003). We also identified asecond category of muscles, mainly involved in facial expression,that arise from the second branchial arch (Larsen et al., 2009). Firstand second branchial arch muscles are therefore derived fromdistinct lineages.

The progenitor cells that give rise to right or left head musclesreflect right-left segregation. Clones that contribute to both right andleft head muscles must derive from a progenitor that precedes


Fig. 5. Left-right labelling of category II musclescorrelates with myocardial labelling in thepulmonary trunk or aorta. (A-C�) X-gal staining(arrowheads) of c-actinnlaacZ1.1/+ embryos at E14.5with unilateral (A-B�) or bilateral (C-C�) labelling inhead muscles. 14K536 is an example of labelling inthe left facial expression muscles (A,A�) and thiscorrelates with a labelling at the base of thepulmonary trunk (pt; A�, ventral view). 14K1848 is anexample of right unilateral labelling (B,B�), withlabelling at the base of the aorta (ao; B�, dorsal view).14K1436 has bilateral labelling of head muscles(C,C�) and has labelling at the base of both arteries(C�, ventral view). (D)Summary of observations thatinclude all embryos with a labelling in category IImuscles (n12) or category I and II (n4, indicated byasterisks), which also show labelling in the arterialpole of the heart (see Fig. 2H). Correlations betweenthe left or right side of the head (cat II-L or cat II-R)and the pt or ao are indicated in purple (6/6) andgreen (4/4), respectively. Bilateral labelling correlateswith labelling of myocardium at the base of botharteries (in black, 5/6).

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bilateralisation of the mesoderm prior to gastrulation (Lawson et al.,1991). A spatial boundary between mesoderm of the first and secondbranchial arches has been demonstrated at E8.5 by orthotopic graftsof cranial paraxial mesoderm (Trainor et al., 1994). We now proposethat the cell lineage segregation between the mesoderm of the firsttwo arches has already taken place by the time of gastrulation.

Branchiomeric muscles share common progenitorswith right ventricular or arterial pole myocardiumOur data reveal a clonal relationship between branchiomericcraniofacial muscles and myocardial derivatives of pharyngealmesoderm in the SHF. From previous retrospective clonal

analyses, we know that the anterior SHF contributes to outflowtract and right ventricular myocardium (Meilhac et al., 2004).The Mlc1v-nlacZ-24 transgenic line shows early continuitybetween Fgf10-expressing cells in the SHF and the mesodermalcore of the first and second branchial arches. We now report thatcategory I, first-branchial-arch-derived head muscles show aclonal relationship with cells in the right ventricle. The anteriorboundary of the linear heart tube is positioned on the anterior-posterior axis at the level of the first branchial arch (Waldo et al.,2001). Mesoderm in the core of this arch may thus be continuouswith the arterial pole of the heart at the time cells migrate intothe early cardiac tube to contribute to the right ventricle. This


Fig. 6. A model for clonal relationships between head and heart muscle progenitors, including genetic tracing experiments.(A-C)Genetic tracing with Mef2c-AHF-enhancer-Cre:Rosa26R-nlacZ embryos. (A)Wholemount X-gal staining at E10.5 showing expression inmyocardium of the outflow tract (OFT) and right ventricular (RV) regions of the heart, and in the mesodermal core of the branchial arches (BA1,BA2; distal only). (B,B�) Immunofluorescence on DAPI-stained sections at E10.5 showing coexpression of -galactosidase (-gal) and MyoD in BA1but not BA2. (C)Wholemount X-gal staining at E14.5. Labelling is only detected in category I muscles (arrowheads). ma, masseter; te, temporalis.(D)A schematic cell lineage tree deduced from the results presented. Identification numbers of embryos are shown in each branch. Expression ofregulatory genes, deduced from genetic tracing experiments, are indicated in orange. Developmental time is indicated below in embryonic days.EOMs, extraocular muscles. (E,F)Schematic representations of cells in pharyngeal mesoderm that contribute to the core of the branchial arches, andto myocardium of the outflow tract and right ventricle. Cells migrate first (blue) from pharyngeal mesoderm at the level of the first branchial arch(BA) to the developing heart tube to contribute to right ventricular myocardium (E). Cells from the second branchial arch (pink) migrate later(around E9.5-E10) to contribute to outflow tract myocardium (F). (G)Schematic representation of the contribution of the different lineages to headmuscles and heart myocardium. A first lineage (blue) contributes to masticatory muscles (temporalis and masseter) and to right ventricular (RV)myocardium. A second lineage (pink) contributes to left or right facial expression muscles and myocardium at the base of the pulmonary trunk (pt)or aorta (ao), respectively.DEVELO

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result differs from the conclusions drawn for the chick embryo,where dye labelling experiments suggested that a population ofcells contributing to the mesoderm of the first branchial arch,and subsequently to the masseter muscle, also contribute to theoutflow tract of the heart (Tirosh-Finkel et al., 2006). This mayreflect a difference in developmental timing between birds andmammals, but may also be due to dye labelling of a populationof cells such that outflow tract progenitors are labelled at thesame time as the mesodermal core of both branchial arches.Category II, second-branchial-arch-derived head muscles sharea clonal relationship with myocardium at the base of the aortaand pulmonary trunk, which form by septation of the outflowtract. Consistent with these findings, as development proceeds,the heart tube moves posteriorly relative to the arches so that ata later stage its anterior boundary is aligned with the secondbranchial arch. Indeed, dye labelling of second branchial archmesoderm in the mouse embryo showed subsequent localisationof labelled cells in outflow tract myocardium (Kelly et al., 2001).

Different head muscles, derived from the first branchial arch, didnot show any distinction with respect to their clonal relationship tomyocardium and, similarly, no such clonal distinction was observedfor second branchial arch muscles.

Many large clones in the right ventricle and also in arterial polemyocardium do not show labelling in head muscles, suggesting thatonly a subset of myocardial progenitor cells share a clonalrelationship with head muscles. There is no evident regionalizationof such clones within the right ventricle. As expected, within thelarge clones that also contribute to head muscles, we see some thatcolonise both arterial pole and right ventricular myocardium,consistent with the contribution of the second myocardial celllineage (Meilhac et al., 2004).

Estimation of the date of lineage segregationThe retrospective clonal analysis gives information about the ageof a common progenitor cell, as well as the location of itsdescendants. To estimate the date of segregation of myocardiumand head muscles, we have compared E14.5 embryos showing thisdistribution with the pattern of labelling in the heart of E8.5 c-actinnlaacZ1.1/+ embryos (Meilhac et al., 2004). Most of these E14.5c-actinnlaacZ1.1/+ embryos have labelling that is restricted to theright ventricular (category I) or arterial pole (category II)myocardium. At E8.5, c-actinnlaacZ1.1/+ embryos, which also showlabelling that is restricted to the right ventricle or outflow region,have 3-10 -galactosidase-positive cells, indicative of a recentrecombination event. Therefore, we can propose that the lineagefor muscles and for myocardium segregates late in the context ofheart development. Embryos with labelling in head musclesderived from both first and second branchial arches showed a moreextensive pattern of labelling in the heart, with more chamberslabelled, including the left ventricle, suggesting that therecombination event predates the segregation of the first andsecond myocardial lineages (Meilhac et al., 2004).

Clonal distribution in the pulmonary trunk oraorta correlates with head muscle lateralityWe show that clones in head muscles derived from the secondbranchial arch have -galactosidase-positive cells inmyocardium of the pulmonary trunk or the aorta. Segregationbetween the base of the aorta and pulmonary trunk waspreviously reported for clones in the hearts of c-actinnlaacZ/+

embryos at E14.5, with regionalisation of clonal distributionnoted in outflow tract myocardium at E10.5 (Bajolle et al.,

2008). Gene and transgene expression patterns also show similarregionalisation, which extends to a subpopulation of cells in theSHF, indicating differences in transcriptional regulation in thesemyocardial subpopulations (Bajolle et al., 2008; Rochais et al.,2009; Theveniau-Ruissy et al., 2008). Strikingly, clones thatcolonised the pulmonary trunk contributed to second-branchial-arch-derived head muscles on the left side of the embryo,whereas clones in the aorta contributed to muscles on the rightside of the head. Dye injections in the chick embryo have shown,however, that when right pharyngeal mesoderm is marked(Hamburger and Hamilton stage 13), this subsequently labelspulmonary trunk myocardium and a timecourse on labelled cellsshows a spiralling movement of this mesoderm as it migratesinto the outflow tract of the heart (Ward et al., 2005). In mouseembryos, dye injection into the outflow tract shows that itundergoes rotation between E9.5 and E10.5 (Bajolle et al.,2006). These observations might lead one to expect that therewould be a correlation between clones in right head muscles andthe pulmonary trunk. The converse correlation that we observeleads to a re-evaluation of the timing of addition of cells to theoutflow tract. Previous experiments in the mouse embryo haveshown that cells from the second branchial arch, labelled at E9.5,are found within the outflow tract at E10.5 (Kelly et al., 2001),suggesting that cells from the mesodermal core of the secondarch are added to the arterial pole of the heart after E9.5 andtherefore probably after the initiation of rotation. This lateaddition is consistent with dye labelling and observations oncardiac gene expression within the core mesoderm of the archesin the chick embryo, indicating that cells located within the archcontribute to the outflow tract (Nathan et al., 2008; Tirosh-Finkelet al., 2006).

Correlation with genetic tracing experimentsusing Cre lines and the Rosa26 reporterMesp1 is expressed early in the common progenitor ofextraocular and branchiomeric muscles, as well as themyocardium (Harel et al., 2009; Saga et al., 2000). Islet1-Creand Nkx2.5-Cre lines with the Rosa26R reporter also showedlabelled head muscles and heart myocardium, but did not markextraocular muscles (Harel et al., 2009). The onset of Islet1 (andNkx2.5) expression is therefore too late or too limited to markcommon progenitors of extraocular and first arch muscles thatare revealed by retrospective clonal analysis. Differencesbetween first branchial arch muscle derivatives might have beenpredicted based on genetic tracing with the Islet1-Cre (Nathan etal., 2008); however, this was not observed in the clonal analysis.Tbx1 also plays a crucial role in the two first branchial archlineages. Tbx1 has been shown to be required for branchiomericmyogenesis (Grifone et al., 2008; Kelly et al., 2004) and isinvolved in the development of right ventricular and arterial polemyocardium (Xu and Baldini, 2007; Xu et al., 2005). Mutantsfor Tbx1 show defects in branchiomeric muscles and in arterialpole myocardium. We show here that the Mef2c-AHF-enhancer,activated in progenitors of the core mesoderm of the branchialarches and of the outflow tract and right ventricular myocardium(Dong et al., 2006; Verzi et al., 2005), distinguishes the first- andsecond-branchial-arch-derived muscle lineages. The Mef2c-AHF-enhancer-Cre is activated in the common progenitor ofright ventricular myocardium and first-branchial-arch-derivedhead muscles, but marks only arterial pole myocardium and nothead muscles derived from the second branchial arch,presumably reflecting a later activation of the enhancer in this


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lineage. This may correspond to the retinoic-acid-sensitivepopulation that contributes later to the outflow tract described byLi et al. (Li et al., 2010).

Correlation of lineage segregation obtained by clonal analysiswith the expression or role of transcriptional regulators (shownschematically in Fig. 6D) is delicate as genetic tracing experimentscan be misleading and may only give a partial picture. Signallingpathways also classically affect cell fate choices. This is clearlydemonstrated for the role of bone morphogenetic protein inpromoting myocardial versus myogenic differentiation (Tirosh-Finkel et al., 2006); however, the spatiotemporal complexity ofsignalling inputs makes it difficult to extend this analysis to lineagedomains.

In conclusion, this clonal analysis provides novel insights intothe lineage relationships between head muscles and the heart,both in terms of contributions to different muscle structures ormyocardial compartments and of the suggested timing of lineagesegregation during early development (summarised in Fig. 6).

Note added in proofA recent paper on the ascidian Ciona intestinals demonstrates a commonprecursor for head and heart muscles. (Stolfi et al., 2010).

AcknowledgementsWe thank C. Bodin for technical help. The work in M.B.’s laboratory wassupported by the Pasteur Institute and the CNRS, with grants from the E.U.Integrated Projects ‘Heart Repair’ [LH SM-CT2005-018630 (also to R.G.K.)] and‘CardioCell’ (LT2009-223372), which have funded J.-F.L.G. M.B. and R.G.K.also acknowledge the support of the Association Française contre lesMyopathies. S.M.M. and R.G.K. are INSERM research scientists. F.L. benefitsfrom a doctoral fellowship from the Ile de France region.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.050674/-/DC1

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