Download Clonal analysis reveals common lineage

RESEARCH ARTICLE 3269
Development 137, 3269-3279 (2010) doi:10.1242/dev.050674
© 2010. Published by The Company of Biologists Ltd
Clonal analysis reveals common lineage relationships
between head muscles and second heart field derivatives in
the mouse embryo
Fabienne Lescroart1, Robert G. Kelly2, Jean-François Le Garrec1, Jean-François Nicolas3, Sigolène M. Meilhac1
and Margaret Buckingham1,*
SUMMARY
Head muscle progenitors in pharyngeal mesoderm are present in close proximity to cells of the second heart field and show
overlapping patterns of gene expression. However, it is not clear whether a single progenitor cell gives rise to both heart and
head muscles. We now show that this is the case, using a retrospective clonal analysis in which an nlaacZ sequence, converted to
functional nlacZ after a rare intragenic recombination event, is targeted to the c-actin gene, expressed in all developing skeletal
and cardiac muscle. We distinguish two branchiomeric head muscle lineages, which segregate early, both of which also contribute
to myocardium. The first gives rise to the temporalis and masseter muscles, which derive from the first branchial arch, and also to
the extraocular muscles, thus demonstrating a contribution from paraxial as well as prechordal mesoderm to this anterior muscle
group. Unexpectedly, this first lineage also contributes to myocardium of the right ventricle. The second lineage gives rise to
muscles of facial expression, which derive from mesoderm of the second branchial arch. It also contributes to outflow tract
myocardium 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 with
genetic 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.
INTRODUCTION
All skeletal muscles are not identical. Notably, trunk and head
muscles differ in a number of important respects. They derive from
different embryonic regions: trunk muscles form from paraxial
mesoderm of the somites, whereas most head muscles are formed
from unsegmented cranial paraxial mesoderm (Noden and FrancisWest, 2006). Trunk and head muscles also have distinct gene
regulatory programmes such that Pax3, which is an important
upstream regulator of trunk myogenesis (Buckingham and Relaix,
2007), is not expressed in head mesoderm, whereas other genes such
as Tbx1 or Pitx2 (Grifone and Kelly, 2007) are upstream regulators
of skeletal myogenesis in the head, but not the trunk. Differences can
already be observed at the onset of gastrulation when cells that give
rise to cranial paraxial mesoderm ingress through the streak before
cells 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 muscles
that are involved in mastication, facial expression and function of
the larynx and pharynx, and extraocular muscles that control eye
movement. Branchiomeric muscles are derived from pharyngeal
mesoderm, which includes both lateral splanchnic and paraxial
1
Institut Pasteur, Unité de Génétique Moléculaire du Développement, CNRS URA
2578, 28 rue du Dr Roux, Paris 75015, France. 2Developmental Biology Institute of
Marseille-Luminy, UMR CNRS 6216 Université de la Méditerranée, Campus de
Luminy, Institut PaseteurMarseille, France. 3Unité de Biologie Moléculaire du
Développement, CNRS URA 2578, 28 rue du Dr Roux, Paris 75015, France.
*Author for correspondence ([email protected])
Accepted 16 June 2010
mesoderm. This forms the mesodermal core of the branchial arches
and is then repositioned within the head during the morphogenetic
movements that accompany craniofacial development. Caudal
branchial arches give rise to laryngeal and pharyngeal muscles and,
in mammals, the first and second arches give rise to progenitors of
jaw and facial expression muscles, respectively (Larsen et al.,
2009). Extraocular muscles derive mainly from prechordal
mesoderm, although it is not clear whether there is also a
contribution from paraxial mesoderm (Noden and Francis-West,
2006). However, extraocular muscles are clearly subject to different
genetic regulation from branchiomeric muscles. Transcription
factors such as Tbx1, MyoR or capsulin, required for
branchiomeric myogenesis, do not play a role in their development
(Kelly et al., 2004; Lu et al., 2002). Furthermore, the hierarchy of
myogenic determination factors of the MyoD family differs
between extraocular and branchiomeric muscles (Sambasivan et al.,
2009). However, Pitx2 function is required for both extraocular and
first-branchial-arch-derived muscles (Dong et al., 2006).
Classic fate mapping and lineage tracing experiments had indicated
a close relationship between progenitors for cranial paraxial mesoderm
and mesoderm that will form the heart, which ingress through the
streak at the same stage (Kinder et al., 1999). In addition, grafting
experiments showed that cardiac and cranial paraxial mesoderm
progenitors are present in the same region (Parameswaran and Tam,
1995; Tam et al., 1997). In single-cell labelling experiments in the
epiblast, 60% of clones contributed to more than one structure,
including cranial paraxial mesoderm and lateral mesoderm from which
the heart is derived (Buckingham et al., 1997; Lawson and Pedersen,
1992). The first heart field arises from lateral splanchnic mesoderm
and forms the primitive heart tube. It is now established, from
experiments in chick and mouse embryos, that pharyngeal splanchnic
DEVELOPMENT
KEY WORDS: Retrospective clonal analysis, Head muscles, Second heart field, Mouse
3270 RESEARCH ARTICLE
MATERIALS AND METHODS
Mice
The Mlc1v-nlacZ-24 transgenic line (Kelly et al., 2001), the T4 transgenic
line (Biben et al., 1996) and the c-actinnlaacZ1.1/+ mouse line (Meilhac et
al., 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 histology
Dissected c-actinnlaacZ1.1/+ embryos were fixed in 4% paraformaldehyde
(PFA) and X-gal staining was performed as previously described (Bajolle
et al., 2006; Meilhac et al., 2003).
Mlc1v-nlacZ-24 and Mef2c-AHF-enhancer-Cre;Rosa26R-nlacZ
embryos were sectioned using a cryostat. Immunochemistry was performed
with MyoD (Dako) and -galactosidase (J.-F.N.) antibodies.
Retrospective clonal analysis
A total of 627 embryos at embryonic day (E) 14.5 had been collected in a
previous study (Bajolle et al., 2008) and 1596 additional embryos were
collected here. Statistical analyses were carried out on the newly collected
embryos only, as some hearts of the first series had been sectioned for other
purposes such that the collection was no longer complete and therefore no
longer fulfilled the random criterion required for statistical analysis. Most
of the E14.5 c-actinnlaacZ1.1/+ embryos present multiple clusters of galactosidase-positive cells or fibres and it is therefore essential to establish
by statistical analysis whether labelled cells derive from a single
recombination event and are thus clonally related. Because recombination
is random, there is a low probability that such an event occurs in the same
location a second time and, therefore, a cluster probably contains clonally
related cells (Meilhac et al., 2004; Meilhac et al., 2003). We distinguished
between large and small clusters of labelled cells and fibres as large clusters
are derived from an earlier recombination event than small clusters and are
therefore more interesting for our study. Large clusters in skeletal muscles
or in myocardium were defined as clusters with more than 10 fibres or 10
cells labelled, respectively. Such large clusters in head muscles were seen
in 1.8% of embryos, whereas small clusters occurred at a much higher
frequency (17.5%).
Statistical analysis
We estimated the expected frequency of double recombination events in
two different regions, which, according to the law of independent
probabilities, is equal to the product of the frequency of labelling in each
region (Tables 1, 2). In order to decide whether the observed frequency of
common labelling in two distinct regions, for instance branchiomeric
muscles and heart myocardium, was consistent with the expected
frequency, we performed a statistical test. We have used the non-parametric
Fisher’s exact test that allows us to work with small numbers of labelled
embryos. The null hypothesis is that the labelling in both regions results
from two independent events. When the P-value is less than 0.05, the null
hypothesis can be confidently rejected, leading to the conclusion that the
labelling probably derives from a single recombination event.
RESULTS
Expression of the Mlc1v-nlacZ-24 transgene
reveals early continuity between splanchnic
mesoderm and the mesodermal core of the first
and second branchial arches
In the Mlc1v-nlacZ-24 transgenic line, in which reporter gene
expression is driven by Fgf10 regulatory elements (Kelly et al.,
2001), the outflow tract and part of the right ventricle of the
developing heart are -galactosidase-positive (Fig. 1A-C). At E8.5,
X-gal staining was seen in the mesodermal core of the developing
arches (Fig. 1A,B), where Fgf10 is also expressed (Kelly et al.,
2001). This expression domain is contiguous with -galactosidasepositive cells in the splanchnic mesoderm of the SHF and its
myocardial derivatives at the arterial pole of the heart. This
continuity was maintained at E9.5 when transgene expression was
observed in the mesodermal core of the arches where the
branchiomeric skeletal muscle programme initiates (Fig. 1C).
Mlc1v-nlacZ-24 expression thus illustrates the continuity between
myocardial and skeletal muscle progenitor cells in its expression
domain in pharyngeal mesoderm. As shown in Fig. 1D, the Mlc1vnlacZ-24 transgene continued to be expressed in branchiomeric
head muscles at later stages.
DEVELOPMENT
mesoderm, which constitutes the second heart field (SHF), contributes
to the growth of the developing cardiac tube (Buckingham et al.,
2005). In the mouse embryo, cells from this field form the outflow
tract myocardium and also contribute to the right ventricle, as well as
to the venous pole of the heart. The SHF is regulated by a genetic
network that includes genes such as Islet1, the expression of which
marks these cardiac progenitor cells (Cai et al., 2003). Pharyngeal
mesoderm, contributing to the outflow region of the heart, is
contiguous with that contributing to the branchiomeric muscles, as
shown by dye labelling experiments of the mesodermal core of the first
two branchial arches in mouse and chick embryos (Kelly et al., 2001;
Nathan et al., 2008). Furthermore, common gene expression profiles
are observed in these cells (Bothe and Dietrich, 2006; Grifone and
Kelly, 2007; Tzahor, 2009), with a proximal-distal gradient of gene
expression within the mesodermal core, corresponding to markers
associated with branchiomeric rather than SHF progenitors (Nathan et
al., 2008). Tbx1 provides an example of a regulatory gene that is
implicated in branchiomeric myogenesis (Kelly et al., 2004) and also
plays an important role in the formation of the cardiac outflow tract
(Xu and Baldini, 2007). Genetic tracing experiments with Islet1-Cre
or Mesp1-Cre activated in precardiac mesoderm, show expression of
the conditional Rosa26 reporter in branchiomeric muscles as well as
in the heart, indicating that these skeletal muscles also derive from cells
that had expressed Islet1 or Mesp1. Dye labelling and manipulation of
signalling pathways in explants of cranial mesoderm at earlier stages
in the chick embryo also show overlapping cardiac and skeletal muscle
potential (Tirosh-Finkel et al., 2006).
Mesodermal cells in the pharyngeal region can thus contribute to
heart and head muscle, and branchiomeric muscle progenitors
express genes that characterize the SHF, prior to entering the
myogenic programme. However, it is not clear whether a single
progenitor cell gives rise to descendants in both types of striated
muscle or whether progenitors are initially intermingled. Dye
labelling of populations of cells and genetic tracing experiments do
not distinguish between these possibilities. We have used
retrospective clonal analysis to investigate lineage relationships
between head and heart muscle. We show that there are two
branchiomeric muscle lineages, the first of which also contributes to
extraocular muscles. The first branchiomeric muscle lineage gives
rise to the temporalis and masseter muscles, which are first-arch
derivatives (Larsen et al., 2009), and also unexpectedly contributes
myocardial cells to the right ventricle. The second branchiomeric
muscle lineage gives rise to muscles of facial expression that are
second-arch derivatives and also contributes to myocardium at the
arterial pole of the heart. Within this second lineage, a further
subdivision is observed between myocardium at the base of the
pulmonary trunk and the aorta. These sublineages contribute to left
or right muscles of facial expression, respectively. There is therefore
a clonal relationship between head and heart muscle progenitors,
with further sublineages that form a lineage tree, as discussed here.
Development 137 (19)
Fig. 1. Transgene expression patterns during heart and head
muscle development. (A-D)The Mlc1v-nlacZ-24 transgene expression
pattern. (A)Wholemount X-gal staining at E8.5 showing a lateral view
of transgene expression in myocardium of the outflow tract (OFT) and
right ventricular regions of the heart and in the mesodermal core of the
branchial arches (1, 2 and 3). (B)Sagittal section at E8.5 showing
expression of the transgene in the mesoderm of the branchial arches
(1, 2 and 3) and in myocardium of the OFT. Arrowheads indicate the
continuity between these sites of expression. (C)Wholemount view of
the pharyngeal region at E9.5 with the heart removed and stained with
X-gal. Numbers indicate the branchial arches where the mesodermal
core is labelled. Arrowheads indicate the continuity between these sites
of expression. (D)Wholemount X-gal staining in the head at E14.5
showing labelling of branchiomeric muscles. FL, forelimb.
(E,F)Wholemount X-gal staining of the heart (E) and head (F) muscles
of 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.
A retrospective clonal analysis at E14.5
In order to examine a possible lineage relationship between
myocardium at the arterial pole of the heart and branchiomeric head
muscles, we carried out a retrospective clonal analysis (Bonnerot and
Nicolas, 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) (Biben
et al., 1996; Sassoon et al., 1988). This gene therefore provides an
appropriate endpoint for clonal analysis of these tissues. The
retrospective clonal approach avoids preconceived ideas about
lineage relationships and is based on the analysis of a collection of
embryos rather than isolated examples. It employs an nlaacZ reporter
in which a duplication introduces a stop codon into the galactosidase coding sequence, rendering it non-functional. A rare
intragenic recombination event results in random removal of the
duplication, independently of gene expression, so that the reporter
now makes functional -galactosidase when the gene into which it
is integrated is expressed. Cells that are descended from a progenitor
that has undergone such a recombination event give rise to labelled
RESEARCH ARTICLE 3271
cells that are clonally related (see Fig. S1 in the supplementary
material). In the case of skeletal muscle, where cells fuse to form
fibres, this is assessed as fibres with labelled nuclei.
At E14.5, head muscle primordia have formed and c-actin is
strongly expressed in these skeletal muscles, as well as in the heart
(Fig. 1E,F). This timepoint has therefore been selected for the
retrospective analysis using the c-actinnlaacZ1.1/+ line. At this stage,
all embryos scored contained -galactosidase-positive cells. This
frequency means that multiple recombination events have probably
taken place per embryo to convert the nlaacZ sequence to a
functional nlacZ reporter. Although multiple clusters of galactosidase-positive fibres were present in body muscles,
labelling was much more rare in head muscles (19.3%), which
represent a small fraction of the total musculature. Most of the
embryos with such labelling in head muscles (77.7%) only had one
or two -galactosidase-positive fibres. As small clusters of labelled
fibres probably correspond to more recent recombination events,
which are less informative about the progenitor cell pool, we
subsequently focused our analysis on clusters containing more than
10 labelled fibres. Only 39 out of the 2223 embryos (1.8%) had
clusters of more than 10 -galactosidase-positive fibres in head
muscles (Table 1). Therefore, the probability that more than 10
labelled fibres per embryo derive from multiple recombination
events is very low (see Fig. S2 in the supplementary material). The
calculation of probability is an essential feature of this clonal
approach; the statistical demonstration, based on observation of
many embryos, that a double recombination event is very
improbable leads to the conclusion that labelled cells descend from
a single recombined progenitor and are therefore clonally related.
Two subpopulations of progenitor cells contribute
to muscles derived from mesoderm of the first
two branchial arches
Examples of c-actinnlaacZ1.1/+ embryos with -galactosidase activity
in head muscles are shown in Fig. 2. Two distinct distributions of
labelled fibres were observed in subsets of branchiomeric muscles,
as illustrated in Fig. 2A,B compared with Fig. 2C,D. These results
are summarized in Fig. 2G,H. Fourteen embryos had galactosidase-positive fibres that were restricted to the temporalis and
masseter muscles (category I). In 21 embryos, these muscles were
not labelled but -galactosidase-positive fibres were observed in
muscles of facial expression, as indicated (category II). Only four
embryos had -galactosidase-positive fibres in both categories of
muscles. This labelling is highly unlikely to result from two
independent recombination events as the expected frequency of
double recombination events in category I and category II muscles
is 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, which
includes a greater number of distinct muscles and involves
widespread migration of branchial-arch-derived cells. In addition to
the number of muscles and their size, which affects the extent of
labelling, the number of labelled cells in the clone reflects the
number of divisions and hence timing of the recombination event in
the progenitor cell. In embryo number 1304, for example,
recombination probably occurred in a more recent progenitor that
gave rise only to the masseter muscle, whereas in most cases a
common progenitor gives rise to both category I type muscles
(masseter and temporalis). In the case of category II muscles, there
is some indication that the zygomaticus, buccinator and auricularis
muscles are more frequently labelled; however, these muscles are
larger than other category II muscles. In general, the distribution of
DEVELOPMENT
Clonality between heart and head muscles
3272 RESEARCH ARTICLE
Development 137 (19)
Table 1. Frequency of labelling in head or somitic muscles and in heart myocardium
Category I muscles
Category II muscles
Somitic muscles
AP myocardium
RV myocardium
LV myocardium
Number of embryos with
-gal+ cells and/or fibres (%)
Number of embryos with clusters of
>10 -gal+ cells and/or fibres (%)
2.6
18.2
87.7
13.9
61.7
67
0.9
1.1
23.7
2.1
7.5
10.6
Number of embryos analysed: 1596 (see Materials and methods).
AP, arterial pole; LV, left ventricle; RV, right ventricle; -gal+, -galactosidase-positive.
(385, 2084, 284, 1436) this was restricted to category II muscles.
In two other examples (2080, 2880), one side of the embryo had
extensive 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 both
lineages must have given rise to asymmetrically distributed
descendants that were only of category I type on the right side. The
two very extensively labelled embryos (1779, 2688) had complete
labelling on both sides (Fig. 2E). Bilateral labelling indicates that
the recombination event preceded the onset of gastrulation,
whereas monolateral labelling can arise before or after gastrulation
and cannot be used as a criterion to date the clones (Lawson et al.,
1991; Selleck and Stern, 1991).
Fig. 2. Two distinct groups of head muscles derive from subpopulations of progenitor cells. (A-F⬘) Examples of c-actinnlaacZ1.1/+ embryos with
X-gal stained fibres in head muscles. Two distinct patterns of labelling are observed either of temporalis (te) and masseter (ma) muscles (A,B), located
deep in the head, or of facial expression muscles located closer to the surface. au, auricularis; bu, buccinator; fr, frontalis; oc, occipitalis; oo, orbitalis
oculi; 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 each
panel. (G)Scheme of category I (blue) and category II (pink) muscles. (H)All embryos with labelling of >10 -galactosidase-positive (-gal +) fibres are
summarized in this table, which indicates category I and II type distributions. The presence of labelled fibres within a muscle is indicated by a black
square. 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 the
same embryo and this distribution is indicated. At the bottom, the number of fibres with -gal + nuclei is indicated. + indicates that more than 50 fibres
are scored as positive; ++ indicates that all fibres in the body muscles appear positive. Embryos are ordered on this basis.
DEVELOPMENT
-galactosidase-positive fibres between muscles indicates a
dispersion of cells after the recombination event. The four embryos
that 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 -galactosidasepositive fibres throughout the body, indicative of a very early
recombination event. The presence of two distinct labelling patterns
and the fact that both were seen only in embryos with many labelled
cells (+ or ++), indicates that category I and II derive from early
common progenitor cells that segregated into distinct lineages.
Most embryos showed labelling in muscles on either the left or
right side of the head (Fig. 2H), with no particular bias. Some
embryos had head muscle labelling on both sides. In four cases
Clonality between heart and head muscles
RESEARCH ARTICLE 3273
Table 2. Expected frequency of double recombination events
Cat. I muscles + Cat. II muscles
Cat. I muscles + Somitic muscles
Cat. II muscles + Somitic muscles
Cat. I muscles + RV myocardium
Cat. II muscles + AP myocardium
Cat. I muscles + LV myocardium
Cat. II muscles + LV myocardium
Expected frequency of a
double recombination event
Observed frequency
of common labelling
1⫻10–4
2⫻10–3
3⫻10–3
7⫻10–4
2⫻10–4
1⫻10–3
1⫻10–3
3⫻10–3* (P1⫻10–5)
1⫻10–3 (P0.56)
2⫻10–3 (P0.37)
4⫻10–3* (P3⫻10–5)
3⫻10–3* (P9⫻10–5)
0 (P0.32)
0 (P0.23)
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 observed
frequencies 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 ventricle
myocardium. 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 be
independent events.
However, we also scored additional muscles located underneath
the mandible in the context of category I and category II
labelling (see Fig. S3 in the supplementary material).
We also examined whether embryos with -galactosidasepositive fibres in branchiomeric head muscles also showed
labelling in muscles of the trunk and limbs that are derived from
somites (Table 1). We conclude that there is an early segregation
of branchiomeric and somitic muscle progenitor cells (Table 2; see
Fig. S4 in the supplementary material).
Branchiomeric head muscles share common
progenitors with right ventricular and arterial
pole myocardium
We next examined how many of the embryos with category I or II
labelling in head muscles also showed labelling in myocardial
derivatives of the anterior SHF, namely myocardium of the right
ventricle and at the base of the pulmonary trunk and aorta, described
as the arterial pole of the heart. The frequency of embryos with
labelling in this myocardium is presented in Table 1. The left
ventricle, 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 -galactosidasepositive cells in the arterial pole of the heart; however, seven of them
had labelled clusters (>10 cells) in the right ventricle. A summary for
Fig. 3. Head muscles share common progenitors with extraocular muscles. (A)Summary of labelling in head muscles with labelling in
extraocular 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 in
boxes. DR, dorsal rectus (also named superior rectus); DO, dorsal oblique (also named superior oblique); IO, ventral oblique (also named inferior
oblique); 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.
DEVELOPMENT
Extraocular muscles, which lie in close proximity to the eye,
are thought to derive from both prechordal and paraxial
mesoderm (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. This
includes three embryos that also show labelling in both category
I and II muscles (2880, 1779, 2688). In three other cases (3243,
394, 2901), there was labelling only in category I branchiomeric
muscles (Fig. 3A-C). Given the high number of embryos scored
and the very low number of embryos with labelling in these
small muscles, it is very improbable that more than one
recombination event had occurred, leading to the conclusion that
there is a clonal relationship between these muscle groups (Fig.
3D; P0.0004). Embryos 3243 and 2880 showed labelling only
in a subset of extraocular muscles (dorsal rectus muscles for
3243 and dorsal rectus and lateral rectus muscles for 2880);
however, the four other embryos with labelling in category I
muscles showed labelling in all six extraocular muscles as shown
in Fig. 3B,C. No link was observed with category II muscles
(P0.96).
Other branchiomeric muscles situated deep within the embryo,
including those of the pharynx and larynx derived from posterior
branchial arches, were not analysed in detail in our analysis,
which concentrated on the muscles presented in Fig. 2H.
3274 RESEARCH ARTICLE
Development 137 (19)
Fig. 4. Category I embryos also have labelling in right ventricular myocardium, whereas category II embryos also have labelling in
arterial 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 myocardium
and 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 analysis
indicates a clonal relationship. (D)Scheme of the relationship between category I muscles and RV myocardium. (E,E⬘) Example of an embryo with
extensive labelling in category II muscles (E) and AP myocardium (E⬘). (F)The percentage of total embryos that had -galactosidase-positive cells in
AP myocardium or in fibres of category II head muscles. Statistical analysis indicates a clonal relationship. (G)Scheme of the relationship between
category II muscles and AP myocardium. Abbreviations for head muscles are as in Fig. 2.
Category II labelling in left or right head muscles
correlates with labelling in myocardium at the
base of the pulmonary trunk or aorta
By E14.5, myocardium at the arterial pole of the heart was located at
the base of the pulmonary trunk or aorta. When we looked more
closely at embryos with -galactosidase-positive cells in this region,
as well as labelled fibres in the head, we distinguished labelling in one
or both of these arteries (Fig. 5A⬙,B⬙,C⬙). Examination of skeletal
muscle labelling had indicated that most embryos had -galactosidasepositive fibres on either the left or right side of the head. We observed
a striking correlation between left or right labelling of category II
skeletal muscles and labelling of myocardium at the base of the
pulmonary trunk or aorta, respectively. In cases where both arteries had
-galactosidase-positive cells, labelling was predominantly seen on
both sides of the head (Fig. 5A-D). This was observed in embryos with
large numbers of labelled fibres in head muscles. The significance of
this observation was examined and validated by phylogenetic tools to
generate the tree shown in Fig. S5 in the supplementary material. This
result indicates that there is a common progenitor for pulmonary trunk
myocardium and category II muscles on the left side of the head,
whereas those on the right side of the head share a common progenitor
with myocardium at the base of the aorta.
Mef2c-AHF-Cre genetic tracing shows differences
in the two head muscle lineages
We next examined the descendants of progenitors in which the
Mef2c-AHF-enhancer had been activated. This enhancer marks
progenitors 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 Rosa26RnlacZ reporter line. At E10.5, labelled cells were found in the
mesodermal core of the first and second branchial arches, but also
DEVELOPMENT
all embryos with head muscle labelling is given in Fig. 4A and an
example is shown in Fig. 4B,B⬘. The low probability of double
recombination events in the two regions reported in Table 2 suggests
that labelling in category I muscles and right ventricular myocardium
probably 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-positive
cells in the right ventricle and fibres in category I skeletal muscles
arise from a common progenitor (P3⫻10-5).
We then examined embryos with category II head muscle labelling
and found in this case that many of them also had -galactosidasepositive cells in the arterial pole of the heart (12/21). The results are
summarized in Fig. 4A and an example is shown in Fig. 4E,E⬘.
Statistical analysis, using the Fisher’s exact test and based on the
numbers shown in Fig. 4F, supports the conclusion that the galactosidase-positive cells in arterial pole myocardium and fibres in
category II skeletal muscles arise from a common progenitor
(P9⫻10-5). In some cases, labelling in the arterial pole extended
into the right ventricle, but no labelling of only the right ventricle
was observed in category II embryos.
Clonality between heart and head muscles
RESEARCH ARTICLE 3275
in the outflow tract, right ventricular myocardium and mesoderm
behind the heart tube (Fig. 6A-B⬘). However, within the branchial
arches, labelled cells were found only in the more distal part of the
second branchial arch (Fig. 6A). Sections show that although in the
first branchial arch there was overlapping expression of galactosidase and MyoD, -galactosidase-positive cells were
mainly negative for MyoD in the second branchial arch (Fig.
6B,B⬘). This indicates that positive cells in the second branchial
arch have not adopted a myogenic fate. In keeping with this, at
E14.5, labelled cells were found only in category I head muscles
(Fig. 6C), as well as in the right ventricular and outflow tract
myocardium as expected. We propose that first branchial arch head
and heart derivatives are marked by the activation of this enhancer,
whereas in the second arch, activation is restricted to cells giving
rise to cardiac derivatives. This result reveals molecular differences
in the regulation of skeletal myogenic progenitor cells in the first
and second arch.
DISCUSSION
We have shown the lineage relationships between head muscles
and second heart field derivatives, as summarized in Fig. 6D.
This retrospective clonal analysis demonstrates that
branchiomeric skeletal muscle and SHF-derived myocardium
derive from common progenitors and that there are distinct
lineage and sublineage relationships within this framework.
Notably, we show clonality between skeletal muscles derived
from the first branchial arch and right ventricular myocardium.
This unexpected finding, together with clonality between
outflow-tract-derived myocardium at the base of the great arteries
and second-branchial-arch-derived muscles, concords with the
posterior movement of the arterial pole of the heart during
pharyngeal morphogenesis.
Two non-somitic head muscle lineages
Skeletal muscles of the head fall into two categories, both clonally
distinct from somitic muscles, indicating early segregation of these
different myogenic lineages, as expected from fate mapping
experiments (Parameswaran and Tam, 1995; Tam et al., 1997).
First, there are progenitors that contribute to temporalis and
masseter muscles, derived from the first branchial arch (Larsen et
al., 2009). We also detected a significant clonal relationship with
extraocular muscles. This comprised all the extraocular muscles,
not just the dorsal oblique and lateral rectus, previously proposed
to be derived from mesoderm at the level of the arches (Noden and
Francis-West, 2006). We observed two clones in which only a
subset of extraocular muscles are labelled (dorsal rectus or dorsal
and lateral rectus), suggesting differences in the timing of
progenitor segregation. The clonality between extraocular and first
branchial arch muscles indicates that paraxial mesoderm, as well
as prechordal mesoderm, contributes to all these muscles. This link
between first branchial arch muscle derivatives and extraocular
muscles is also observed at the level of Pitx2 function (Dong et al.,
2006). The clonal relationship with right ventricular myocardial
cells indicates a contribution from a progenitor for lateral
splanchnic, as well as paraxial pharyngeal, mesoderm. Pitx2 also
marks cardiac progenitors; however, its dynamic expression within
the myocardium complicates the interpretation of genetic tracing
experiments (Franco and Campione, 2003). We also identified a
second category of muscles, mainly involved in facial expression,
that arise from the second branchial arch (Larsen et al., 2009). First
and second branchial arch muscles are therefore derived from
distinct lineages.
The progenitor cells that give rise to right or left head muscles
reflect right-left segregation. Clones that contribute to both right and
left head muscles must derive from a progenitor that precedes
DEVELOPMENT
Fig. 5. Left-right labelling of category II muscles
correlates with myocardial labelling in the
pulmonary trunk or aorta. (A-C⬙) X-gal staining
(arrowheads) of c-actinnlaacZ1.1/+ embryos at E14.5
with unilateral (A-B⬙) or bilateral (C-C⬙) labelling in
head muscles. 14K536 is an example of labelling in
the left facial expression muscles (A,A⬘) and this
correlates with a labelling at the base of the
pulmonary trunk (pt; A⬙, ventral view). 14K1848 is an
example of right unilateral labelling (B,B⬘), with
labelling 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 that
include all embryos with a labelling in category II
muscles (n12) or category I and II (n4, indicated by
asterisks), which also show labelling in the arterial
pole of the heart (see Fig. 2H). Correlations between
the 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) and
green (4/4), respectively. Bilateral labelling correlates
with labelling of myocardium at the base of both
arteries (in black, 5/6).
3276 RESEARCH ARTICLE
Development 137 (19)
bilateralisation of the mesoderm prior to gastrulation (Lawson et al.,
1991). A spatial boundary between mesoderm of the first and second
branchial arches has been demonstrated at E8.5 by orthotopic grafts
of cranial paraxial mesoderm (Trainor et al., 1994). We now propose
that the cell lineage segregation between the mesoderm of the first
two arches has already taken place by the time of gastrulation.
Branchiomeric muscles share common progenitors
with right ventricular or arterial pole myocardium
Our data reveal a clonal relationship between branchiomeric
craniofacial muscles and myocardial derivatives of pharyngeal
mesoderm in the SHF. From previous retrospective clonal
analyses, we know that the anterior SHF contributes to outflow
tract and right ventricular myocardium (Meilhac et al., 2004).
The Mlc1v-nlacZ-24 transgenic line shows early continuity
between Fgf10-expressing cells in the SHF and the mesodermal
core of the first and second branchial arches. We now report that
category I, first-branchial-arch-derived head muscles show a
clonal relationship with cells in the right ventricle. The anterior
boundary of the linear heart tube is positioned on the anteriorposterior axis at the level of the first branchial arch (Waldo et al.,
2001). Mesoderm in the core of this arch may thus be continuous
with the arterial pole of the heart at the time cells migrate into
the early cardiac tube to contribute to the right ventricle. This
DEVELOPMENT
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 in
myocardium 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 BA1
but 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 of
regulatory 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, and
to 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 head
muscles 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.
RESEARCH ARTICLE 3277
result differs from the conclusions drawn for the chick embryo,
where dye labelling experiments suggested that a population of
cells contributing to the mesoderm of the first branchial arch,
and subsequently to the masseter muscle, also contribute to the
outflow tract of the heart (Tirosh-Finkel et al., 2006). This may
reflect a difference in developmental timing between birds and
mammals, but may also be due to dye labelling of a population
of cells such that outflow tract progenitors are labelled at the
same time as the mesodermal core of both branchial arches.
Category II, second-branchial-arch-derived head muscles share
a clonal relationship with myocardium at the base of the aorta
and pulmonary trunk, which form by septation of the outflow
tract. Consistent with these findings, as development proceeds,
the heart tube moves posteriorly relative to the arches so that at
a later stage its anterior boundary is aligned with the second
branchial arch. Indeed, dye labelling of second branchial arch
mesoderm in the mouse embryo showed subsequent localisation
of labelled cells in outflow tract myocardium (Kelly et al., 2001).
Different head muscles, derived from the first branchial arch, did
not show any distinction with respect to their clonal relationship to
myocardium and, similarly, no such clonal distinction was observed
for second branchial arch muscles.
Many large clones in the right ventricle and also in arterial pole
myocardium do not show labelling in head muscles, suggesting that
only a subset of myocardial progenitor cells share a clonal
relationship with head muscles. There is no evident regionalization
of such clones within the right ventricle. As expected, within the
large clones that also contribute to head muscles, we see some that
colonise both arterial pole and right ventricular myocardium,
consistent with the contribution of the second myocardial cell
lineage (Meilhac et al., 2004).
2008). Gene and transgene expression patterns also show similar
regionalisation, which extends to a subpopulation of cells in the
SHF, indicating differences in transcriptional regulation in these
myocardial subpopulations (Bajolle et al., 2008; Rochais et al.,
2009; Theveniau-Ruissy et al., 2008). Strikingly, clones that
colonised the pulmonary trunk contributed to second-branchialarch-derived head muscles on the left side of the embryo,
whereas clones in the aorta contributed to muscles on the right
side 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 labels
pulmonary trunk myocardium and a timecourse on labelled cells
shows a spiralling movement of this mesoderm as it migrates
into the outflow tract of the heart (Ward et al., 2005). In mouse
embryos, dye injection into the outflow tract shows that it
undergoes rotation between E9.5 and E10.5 (Bajolle et al.,
2006). These observations might lead one to expect that there
would be a correlation between clones in right head muscles and
the pulmonary trunk. The converse correlation that we observe
leads to a re-evaluation of the timing of addition of cells to the
outflow tract. Previous experiments in the mouse embryo have
shown 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 second
arch are added to the arterial pole of the heart after E9.5 and
therefore probably after the initiation of rotation. This late
addition is consistent with dye labelling and observations on
cardiac gene expression within the core mesoderm of the arches
in the chick embryo, indicating that cells located within the arch
contribute to the outflow tract (Nathan et al., 2008; Tirosh-Finkel
et al., 2006).
Estimation of the date of lineage segregation
The retrospective clonal analysis gives information about the age
of a common progenitor cell, as well as the location of its
descendants. To estimate the date of segregation of myocardium
and head muscles, we have compared E14.5 embryos showing this
distribution with the pattern of labelling in the heart of E8.5 cactinnlaacZ1.1/+ embryos (Meilhac et al., 2004). Most of these E14.5
c-actinnlaacZ1.1/+ embryos have labelling that is restricted to the
right ventricular (category I) or arterial pole (category II)
myocardium. At E8.5, c-actinnlaacZ1.1/+ embryos, which also show
labelling that is restricted to the right ventricle or outflow region,
have 3-10 -galactosidase-positive cells, indicative of a recent
recombination event. Therefore, we can propose that the lineage
for muscles and for myocardium segregates late in the context of
heart development. Embryos with labelling in head muscles
derived from both first and second branchial arches showed a more
extensive pattern of labelling in the heart, with more chambers
labelled, including the left ventricle, suggesting that the
recombination event predates the segregation of the first and
second myocardial lineages (Meilhac et al., 2004).
Correlation with genetic tracing experiments
using Cre lines and the Rosa26 reporter
Mesp1 is expressed early in the common progenitor of
extraocular and branchiomeric muscles, as well as the
myocardium (Harel et al., 2009; Saga et al., 2000). Islet1-Cre
and Nkx2.5-Cre lines with the Rosa26R reporter also showed
labelled head muscles and heart myocardium, but did not mark
extraocular muscles (Harel et al., 2009). The onset of Islet1 (and
Nkx2.5) expression is therefore too late or too limited to mark
common progenitors of extraocular and first arch muscles that
are revealed by retrospective clonal analysis. Differences
between first branchial arch muscle derivatives might have been
predicted based on genetic tracing with the Islet1-Cre (Nathan et
al., 2008); however, this was not observed in the clonal analysis.
Tbx1 also plays a crucial role in the two first branchial arch
lineages. Tbx1 has been shown to be required for branchiomeric
myogenesis (Grifone et al., 2008; Kelly et al., 2004) and is
involved in the development of right ventricular and arterial pole
myocardium (Xu and Baldini, 2007; Xu et al., 2005). Mutants
for Tbx1 show defects in branchiomeric muscles and in arterial
pole myocardium. We show here that the Mef2c-AHF-enhancer,
activated in progenitors of the core mesoderm of the branchial
arches and of the outflow tract and right ventricular myocardium
(Dong et al., 2006; Verzi et al., 2005), distinguishes the first- and
second-branchial-arch-derived muscle lineages. The Mef2cAHF-enhancer-Cre is activated in the common progenitor of
right ventricular myocardium and first-branchial-arch-derived
head muscles, but marks only arterial pole myocardium and not
head muscles derived from the second branchial arch,
presumably reflecting a later activation of the enhancer in this
Clonal distribution in the pulmonary trunk or
aorta correlates with head muscle laterality
We show that clones in head muscles derived from the second
branchial arch have -galactosidase-positive cells in
myocardium of the pulmonary trunk or the aorta. Segregation
between the base of the aorta and pulmonary trunk was
previously reported for clones in the hearts of c-actinnlaacZ/+
embryos at E14.5, with regionalisation of clonal distribution
noted in outflow tract myocardium at E10.5 (Bajolle et al.,
DEVELOPMENT
Clonality between heart and head muscles
lineage. This may correspond to the retinoic-acid-sensitive
population that contributes later to the outflow tract described by
Li et al. (Li et al., 2010).
Correlation of lineage segregation obtained by clonal analysis
with the expression or role of transcriptional regulators (shown
schematically in Fig. 6D) is delicate as genetic tracing experiments
can be misleading and may only give a partial picture. Signalling
pathways also classically affect cell fate choices. This is clearly
demonstrated for the role of bone morphogenetic protein in
promoting myocardial versus myogenic differentiation (TiroshFinkel et al., 2006); however, the spatiotemporal complexity of
signalling inputs makes it difficult to extend this analysis to lineage
domains.
In conclusion, this clonal analysis provides novel insights into
the lineage relationships between head muscles and the heart,
both in terms of contributions to different muscle structures or
myocardial compartments and of the suggested timing of lineage
segregation during early development (summarised in Fig. 6).
Note added in proof
A recent paper on the ascidian Ciona intestinals demonstrates a common
precursor for head and heart muscles. (Stolfi et al., 2010).
Acknowledgements
We thank C. Bodin for technical help. The work in M.B.’s laboratory was
supported 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 les
Myopathies. S.M.M. and R.G.K. are INSERM research scientists. F.L. benefits
from a doctoral fellowship from the Ile de France region.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.050674/-/DC1
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DEVELOPMENT
Clonality between heart and head muscles
A
STOP
α-cardiac actin
nlaacZ
nlacZ
α-cardiac actin
βgal+
αc-actin expression domain
E14.5
skeletal muscles and heart myocardium
early recombination event
B
STOP
α-cardiac actin
nlaacZ
α-cardiac actin
nlacZ
βgal+
αc-actin expression domain
skeletal muscles and heart myocardium
late recombination event
E14.5
Supplementary Figure S2
A
number of labelled fibres in head
muscles
1
2
3
4
5
6
7
8
9
10
20
30
40
>50
total
frequency of labelling (%)
11
4
1
1
0.1
0.2
0
0.2
0.1
0.3
0.2
0.1
0.1
1
19.3
B
18
16
14
12
11
10
8
6
4
4
2
1
1
3
4
0
1
2
0.1
0.2
0
0.2
0.1
0.3
0.2
0.1
0.1
5
6
7
8
9
10
20
30
40
number of labelled fibres
C Does labelling in 30 fibres result from 3 independent clones of 10 labelled fibres:
expected= 3x10 -8
observed= 1x10 -3
Does labelling in 10 fibres result from 10 independent clones of 1 labelled fibre:
expected= 1x10 -10
observed= 3x10 -3
1
>50
Supplementary Figure S3
A
labelling in
sub-mandibular muscles
category I only
+
+
+
+
+
+
+
+
10 10 11 15 15 16 22 24 24 28 35 35 40 +
C
B
+
+
+
+
+
+
+
+
LR
D
fac exp
ant dig
+
++
10 10 13 20 20 +
++
number of β−gal fibres labelled in
head muscles
categories
I+II
category II only
ant dig
my
fac exp
my
14K2216
category I
14K1117
category II
14K2880
category I+II
Supplementary Figure S4
A.
labelling in somitic muscles
*
category I only
+
C.
14K2216
G.
+
10/11
14K2136
+
+
+
+
+
D.
1/11
14K2084
11/19
H.
Do `-gal + fibres in category I and in somitic muscles
result from two independent recombination events?
labelling in cat I (%)
no labelling in cat I (%)
total
Fisher's test: P-value=0.56
+
E.
category I
labelling in
somitic (%)
0.1
23.6
18.7
10 10 11 15 15 16 22 24 24 28 35 35 40 +
no labelling in
somitic (%)
total
0.5
75.8
81.3
0.5
99.5
100
14K1848
+
+
+
+
+
+
+
+
++
B.
+
category II only
F.
8/19
14K2607
category II
Do `-gal + fibres in category II and in somitic muscles
result from two independent recombination events?
labelling in cat II (%)
no labelling in cat II (%)
total
Fisher's test: P-value=0.37
labelling in
somitic (%)
0.3
13.5
18.7
no labelling in
somitic (%)
0.6
75.7
81.3
total
0.8
99.5
100
The figures indicate the % of total embryos (1596) that had `-galactosidase positive fibres in somitic or category I (G) or category II (H) head muscles.
++
number of `<gal fibres labelled in
10 10 13 20 20 +
head muscles
*
categories
I+II
Supplementary Figure S5
A
Cat I-left
Cat I-right
Cat II-left
Cat II-right
B.
Pulmonary
trunk
Aorta
011000010000000001111 11111 00000000
111000 0110000000000000000010000000
011111 11111 11000000000000000010000
011111 1000000111 1000000000011011 11
011111 11111 11000000000000000000000
011111 0000000111 100000000000000000
B
99
left category II muscles
pulmonary trunk myocardium
93
right category II muscles
myocardium of the aorta
83
left category I muscles
right category I muscles