Download Mesoderm progenitor cells of common origin contribute to the head

RESEARCH ARTICLE 1943
Development 133, 1943-1953 (2006) doi:10.1242/dev.02365
Mesoderm progenitor cells of common origin contribute to
the head musculature and the cardiac outflow tract
Libbat Tirosh-Finkel, Hadas Elhanany, Ariel Rinon and Eldad Tzahor*
During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm
(i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in
vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and
skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick
embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell
populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the
head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the
differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm
play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper
understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well
as pathological, aspects of heart and craniofacial development.
INTRODUCTION
A key event in the establishment of the body plan during vertebrate
embryogenesis is the regionalization of the mesoderm into paraxial
and lateral compartments. Paraxial mesoderm in the trunk forms the
somites, which give rise to the skeletal muscle lineage, as well as to
cartilage, endothelial and dermis precursors. During the past decade,
the tissues and signaling molecules that induce the formation of
skeletal muscle from somites have been intensively studied (Bailey
et al., 2001; Buckingham, 2001; Pourquie, 2001). Notably, Wnt
signaling molecules secreted from the dorsal neural tube, together
with Sonic hedgehog (Shh) from the notochord, induce myogenesis
in the trunk, whereas bone morphogenic protein (Bmp) signals from
the lateral plate mesoderm have been shown to block myogenesis in
the trunk (Borycki et al., 2000; Munsterberg et al., 1995; Pourquie
et al., 1996; Reshef et al., 1998; Stern et al., 1995; Tajbakhsh et al.,
1998).
In vertebrates, the head musculature is derived from the paraxial
mesoderm located anterior to the somites [termed cranial paraxial
mesoderm, CPM (Couly et al., 1992; Noden, 1983)]. Unlike the
paraxial mesoderm in the trunk, the CPM lacks any overt sign of
segmentation. Together with cranial neural crest cells, CPM cells fill
the branchial (pharyngeal) arches, paired thickenings around the
pharynx that will eventually give rise to the facial structures (Noden
and Trainor, 2005). It appears that different intrinsic and extrinsic
regulatory pathways control skeletal muscle formation in the trunk
and in the head, as indicated by the genetic loss of myogenic
transcription factors in mice (Kelly et al., 2004; Lu et al., 2002;
Rudnicki et al., 1993; Tajbakhsh et al., 1997), as well as by the
manipulation of tissues and signaling molecules in chick embryos
(Hacker and Guthrie, 1998; Mootoosamy and Dietrich, 2002; Noden
et al., 1999; Tzahor et al., 2003).
Department of Biological Regulation, Weizmann Institute of Science, Rehovot
76100, Israel.
*Author for correspondence (e-mail: [email protected])
Accepted 15 March 2006
Cardiac progenitor cells are derived from the lateral plate
mesoderm. This tissue splits into somatic and splanchnic layers;
cells within the splanchnic mesoderm form the cardiac crescent (also
called the primary heart field) that later forms the myocardium and
endocardium of the heart. In both Xenopus and chick embryos, Bmp
and fibroblast growth factor (Fgf) act as potent inducers of cardiac
differentiation during early stages of heart formation (Lough and
Sugi, 2000; Schlange et al., 2000; Schultheiss et al., 1997). By
contrast, members of the canonical Wnt signaling pathway can block
cardiac differentiation during these stages (Brott and Sokol, 2005;
Foley and Mercola, 2005; Marvin et al., 2001; Schneider and
Mercola, 2001; Tzahor and Lassar, 2001).
In the developing head, the lateral mesoderm (also termed
splanchnic mesoderm, SpM) is located on the ventral side of the
embryo, beneath the floor of the pharynx. It has been shown in both
chick (Mjaatvedt et al., 2001; Waldo et al., 2001) and mouse (Cai
et al., 2003; Dodou et al., 2004; Kelly et al., 2001; Zaffran et al.,
2004) embryos that cells in the head originating in various parts
(albeit obscure) of the mesoderm contribute to the formation of the
anterior pole of the heart following the formation of the linear heart
tube. It was originally thought that all cardiac myocytes arise from
the primary heart field; however, these studies suggest that the
arterial pole of the heart develops by the addition of cells derived
from mesoderm progenitors, now termed the second heart field
[SHF (Buckingham et al., 2005)]. In contrast to our relatively broad
understanding of the development of the primary heart field (Kirby,
2002; Olson and Schneider, 2003), the nature of the
secondary/anterior heart field and its exact anatomical location
remain elusive (Abu-Issa et al., 2004).
In a previous study in chick embryos, we demonstrated that
signals from the dorsal neural tube (e.g. Wnt1 and Wnt3a) block
cardiogenesis in the adjacent CPM (Tzahor and Lassar, 2001). The
identification of a secondary heart field in vertebrate embryos led us
to consider whether the CPM contributes in any way to this recently
discovered myocardial lineage. Accordingly, in the present study,
we established cellular and molecular models aimed at exploring,
both in vitro and in vivo, how head mesoderm cells are specified in
DEVELOPMENT
KEY WORDS: Myogenesis, Secondary heart field, Cranial paraxial mesoderm, Splanchnic mesoderm, Bmp4
the avian embryo. Our findings show that Bmp signals affect both
cardiac and skeletal muscle cell fates: Bmp4 promotes cardiogenesis
and, at the same time, blocks skeletal muscle differentiation in head
mesoderm progenitors. Furthermore, our fate-mapping studies
reveal, for the first time, that cells within the CPM contribute to the
myocardium and endocardium of the cardiac outflow tract (OFT).
This cell population may represent an additional source of cardiac
progenitors during vertebrate embryogenesis. We therefore propose
that the developmental programs of mesoderm progenitors that
contribute to the head musculature and the OFT are tightly linked,
and are controlled by Bmp signaling levels.
MATERIALS AND METHODS
Explant culture
In order to distinguish between the CPM and splanchnic mesoderm (SpM),
the tubular heart was first removed from stage 10 chick embryos. CPM
explants comprising surface ectoderm (along with some cranial neural crest
cells) plus endoderm from the roof of the pharynx, and SpM explants,
including the endoderm of the pharynx, were dissected separately. Explants
were cultured in a collagen drop covered with medium (10% FCS in ␣MEM
medium, chick embryo extract 2.5%, penicillin/streptomycin 0.5%) in fourwell plates. Control or BMP4-conditioned medium, harvested from HEK293 cells stably expressing human BMP4-HA, were added to the explants.
To block Bmp signaling, 500 ng/ml of recombinant Fc-Noggin protein
(R&D Systems) was added to the medium. RNA was harvested using a
Versagene Cell Kit (Gentra Systems). cDNA was synthesized from DNasetreated total RNA, using a M-MLV reverse transcriptase-mediated extension
of random primers, followed by a PCR amplification using different sets of
primers for cardiac and skeletal muscle markers (primer sequences are
available upon request).
In situ hybridization
Whole-mount in situ hybridization was performed using digoxigeninlabeled antisense riboprobes synthesized from the cDNA. A full list of the
in situ hybridization probes and a detailed protocol are available upon
request. Images were obtained using a Leica MZ16FA stereomicroscope
attached to a digital camera (DC300F, Leica Microsystems). For paraffin
sectioning, fixed embryos were dehydrated (ethanol/xylene), washed, and
transferred to paraffin. The embryos were sectioned at 10-15 ␮m, using a
Leica microtome.
Implantation of cell aggregates
HEK-293 or HEK-293-BMP4 cells were transferred to an agar plate for
24 hours to form aggregates, which were then selected for implantation
into stage 9 embryos (Tzahor et al., 2003). The eggs were sealed and
incubated for another 24 hours, embryos were fixed with 4% PFA, and in
situ hybridization analysis was performed.
In-ovo dye injection
Fate-mapping experiments were performed on stage 8-10 embryos.
Micropipettes attached to a micromanipulator were filled with DiI or CMDiI (D282, C7001, Molecular Probes) at 5 or 2.5 mg/ml, respectively, in
ethanol, followed by dilution in tetraglycol (1:2). The dye was pressureinjected into the right or left CPM. Following fixation, bright field and
fluorescent images were taken.
Quail-chick chimeras
Quail grafts of the CPM, at the level of rhombomere 1-3 (some of which
were labeled with DiI for visualization), were dissected out. A corresponding
piece of chick CPM was removed, and the quail graft was implanted at this
site. The eggs were incubated for 24 hours. Operated embryos were selected
for sectioning and QCPN detection.
Immunohistochemistry
Fixed embryos were embedded in paraffin and sectioned as described above.
Sections were blocked with 5% goat serum, 1% BSA in PBS, prior to
incubation with the primary antibody [MF20 (1:10), QCPN (DSHB)],
followed by Cy3-conjugated anti-mouse IgG (1:100).
Development 133 (10)
RESULTS
Characterizing the nature of cardiogenesis and
myogenesis in the head mesoderm, in vitro
We previously observed using explant culture assays that head
mesoderm cells undergo cardiac (Tzahor and Lassar, 2001), as well
as skeletal muscle, differentiation (Tzahor et al., 2003) in vitro. We
now applied a more defined explant culture system to study the
differentiation potentials of the cranial paraxial mesoderm (CPM) and
splanchnic mesoderm (SpM) populations (Fig. 1, see also Materials
and methods). At the time of dissection, stage 10 SpM explants
expressed the cardiac differentiation marker Nkx2.5, as well as
members of the Gata family of zinc finger transcription factors. The
expression of Gata4 and the structural myocardial marker ventricular
myosin heavy chain (vMHC) was augmented after three days in
culture (Fig. 1B). The SpM explants also expressed the recently
identified SHF marker Islet1 [Isl1 (Cai et al., 2003)], although its
levels gradually decreased in culture. Interestingly, Capsulin, a bHLH
transcription factor involved in head muscle formation in the mouse
(Lu et al., 2002), was expressed in SpM explants that were cultured
for three days (Fig. 1B). Thus, SpM explants are committed to the
cardiac lineage, and can undergo further cardiac differentiation in
vitro, a finding consistent with SHF cells.
In contrast to the SpM, CPM explants express the skeletal
muscle markers MyoD and Myogenin after three days in culture
(Fig. 1B). Notably, Myf5 expression was consistently detected in
both CPM and SpM explants. In addition, the expression of Gata5,
Gata6 and Isl1 was detected in both CPM and SpM explants,
suggesting a degree of plasticity in these mesodermal cells in vitro
(Fig. 1B).
In order to identify candidate signaling molecules that regulate
skeletal muscle and cardiac differentiation programs, we examined
the expression of members of the Bmp, Fgf (Fig. 1C) and Wnt (data
not shown) signaling pathways, which are known to be major
players during early cardiac and skeletal muscle development. Both
Fgf8 and Fgf10, as well as the receptors for Fgf and Bmp ligands,
were expressed in both CPM and SpM explants. Notably, both Bmp2
and Bmp4 were expressed at higher levels within cultured SpM
explants than within cultured CPM explants, a finding that strongly
correlates with cardiogenesis (Fig. 1C). Taken together, these
explant culture results demonstrate that myogenesis could be
observed in CPM explants, whereas cardiogenesis could be observed
in SpM explants. Moreover, Bmp signaling pathways may play a
crucial role in the specification of these lineages.
Candidate regulatory molecules for head
mesoderm specification in vivo
To gain in vivo information on the signaling molecules and tissuespecific transcription factors regulating the differentiation of
mesoderm precursors into cardiac and skeletal muscle lineages, we
initiated candidate gene expression analyses by in situ hybridization
in whole-mount and sectioned chick embryos (Fig. 2). Embryos
were sectioned at four different levels, the first branchial arch (BA),
the OFT/second BA, the heart/third BA and the inflow tract, to
demonstrate the expression of both myogenic and cardiogenic
markers (Fig. 2A). Because cells from the SHF migrate to the OFT
between embryonic stages 10-22 (Waldo et al., 2001), and because
myogenesis in the BAs can be observed at stages 14-15 (Hacker and
Guthrie, 1998; Noden et al., 1999), we focused our analyses on stage
15-17 embryos (Fig. 2).
Nkx2.5, an early cardiogenic marker, was broadly expressed in the
developing heart, as well as in surrounding tissues corresponding to
the SHF (Fig. 2A), whereas the cardiac myosin heavy chain (cMHC)
DEVELOPMENT
1944 RESEARCH ARTICLE
Cardiac and skeletal muscle cell fates
RESEARCH ARTICLE 1945
was expressed only in myocardial cells within the embryonic heart
(Fig. 2B). Nkx2.5 was expressed in the mesodermal core at the distal
region of the first BA, the ventral mesenchyme of the second BA
(which is directly connected to the OFT at stage 16), the ventral
pharyngeal endoderm (the floor of the pharynx), and in the OFT
(Fig. 2A1,A2). At the level of the third BA, the splanchnic
mesoderm was clearly seen as an epithelial layer underneath the
endoderm, both of which express Nkx2.5 (Fig. 2A3) but not cMHC
(Fig. 2B3). In the most posterior area of the embryonic heart,
Nkx2.5, but not cMHC, was expressed in the dorsal mesocardium,
whereas both genes were observed in the inflow tract (Fig. 2,
compare A4 with B4).
Nkx2.5 is known to associate with members of the Gata family.
Although we found that Gata4 was expressed throughout the
embryonic heart at stage 16 (Fig. 2C,C3) and at lower levels in
the OFT (Fig. 2C2), Gata5 and Gata6 were restricted more to
the OFT (Fig. 2D2,E2) and, to a lesser extent, to the tubular
heart (Fig. 2D,E,D3,E3). Unlike Gata4, the expression of Gata5
and Gata6 (as well as Nkx2.5) at the level of the second and third
BAs was detected in the splanchnic mesoderm beneath the
pharynx (Fig. 2C3,D3,E3). All three genes could be detected at
the inflow pole of the heart (Fig. 2C4,D4,E4). Thus, our gene
expression analyses suggest that in chick embryos, Gata5 and
Gata6, but not Gata4, may play important roles in the
specification of the SHF.
The LIM homeodomain transcription factor Isl1 is a key molecule
in the specification of the SHF lineage during mouse embryogenesis
(Cai et al., 2003). It was recently shown that this gene might confer
‘stem cell characteristics’ on cardiac myocytes after birth (Laugwitz
et al., 2005). The expression of Isl1 at stage 16 was not restricted to
the cardiogenic lineage; yet, similar to Nkx2.5, this gene was
expressed in the mesenchyme of the second BA, and in the pharyngeal
endoderm (Fig. 2F2), in the splanchnic mesoderm at the level of the
third BA (Fig. 2F3), and in the dorsal mesocardium (Fig. 2F4). Unlike
Nkx2.5, Gata5 and Gata6, Isl1 expression was not seen in the heart
myocardium (Fig. 2F2,F4) in a manner similar to the expression of
Isl1 in the mouse (Cai et al., 2003). Thus, Isl1 expression in stage 16
chick embryos represents a pool of undifferentiated cardiogenic cells
within the head mesoderm, consistent with previous reports (Cai et al.,
2003; Yuan and Schoenwolf, 2000).
The skeletal muscle marker Capsulin was expressed in the
mesodermal core of the first and second BAs (Fig. 2G1,G2), and in
the splanchnic mesoderm surrounding the gut (Fig. 2G4). In
addition, we observed the expression of Capsulin at the distal end of
the OFT (see Fig. S1 in the supplementary material). These findings,
together with our data from explant cultures demonstrating the
expression of Capsulin in differentiating SpM cells (Fig. 1), lead us
to propose that Capsulin, like Isl1, is specifically expressed in
paraxial and splanchnic mesoderm cells that contribute to both
myogenic and cardiogenic lineages.
DEVELOPMENT
Fig. 1. In vitro differentiation potential of cranial paraxial mesoderm and splanchnic mesoderm in chick embryos. (A) Dissected cranial
paraxial mesoderm (CPM, purple) and splanchnic mesoderm (SpM, orange) of a stage 10 chick embryo: (A1) Ventral view; (A2) following the
removal of the linear heart tube; (A3) transverse section of the embryo (dotted line in A2). (B,C) CPM and SpM explants were dissected from stage
10 embryos. RNA was harvested from the explants either immediately (0) or after 3 days in culture. RT-PCR analysis was performed for the indicated
cardiac markers and signaling molecules. Although cranial paraxial mesoderm explants underwent myogenesis in culture (n=17/20), splanchnic
mesoderm explants underwent cardiogenesis (n=20/20). T, total chick RNA; da, dorsal aorta; nc, notochord; nt, neural tube; oft, outflow tract; ph,
pharynx.
Development 133 (10)
Fig. 2. Gene expression analysis of candidate regulatory molecules that specify the head mesoderm. (A-J) Whole-mount in situ
hybridization for the indicated genes in stage 16 chick embryos. (A1-J4) Transverse sections at four levels (indicated by the dotted lines in A).
(A1-J1) First branchial arch. (A2-J2) Outflow tract/second branchial arch. (A3-J3) Epithelial splanchnic mesoderm/third branchial arch. (A4-J4) Inflow
tract. ba1, branchial arch 1; ba2, branchial arch 2; da, dorsal aorta; dm, dorsal mesocardium; end, endoderm; V, trigerminal ganglion; ift, inflow
tract; mn, motoneuron; nt, neural tube; oft, outflow tract; ov, otic vesicle; ph, pharynx; spm, splanchnic mesoderm.
DEVELOPMENT
1946 RESEARCH ARTICLE
Members of the T-box family of proteins were previously
shown to play a fundamental role in patterning the developing
vertebrate heart (Plageman, Jr and Yutzey, 2005). Tbx1, a T-box
transcription factor known to influence SHF formation (Xu et al.,
2004) and head muscle formation (Kelly et al., 2004), was
strongly expressed in the mesodermal core of the first and second
BAs, as well as in the pharyngeal endoderm and the otic vesicle
(Fig. 2H1,H2). Tbx1 expression at these axial levels resembled the
expression patterns of Isl1, Capsulin and Nkx2.5, although it was
not detected in the OFT. Tbx5, in contrast, was not expressed in
either the BA or OFT regions (see Fig. S2A in the supplementary
material).
Taken together, our gene expression analyses revealed a group of
transcription factors (i.e. Gata5, Gata6, Isl1, Tbx1 and Capsulin)
that, based on their spatiotemporal expression, are likely to play a
role in the development of the SHF lineage in the chick embryo.
Although both Tbx1 (Xu et al., 2004) and Isl1 (Cai et al., 2003) were
recently shown to be involved in this new myocardial lineage in
mice, Gata5, Gata6 and Capsulin represent new candidates for this
lineage in chick embryos.
Fgf10 and Fgf8 were also shown to play a crucial role in the
SHF lineage in mice (Brown et al., 2004; Kelly et al., 2001). Fgf8
expression was detected in both the endoderm and ectoderm of
stage 16 chick embryos, at the boundary between the splanchnic
and paraxial mesoderm (Fig. 2J2,J3; see also Fig. S2C in the
supplementary material). Furthermore, Mkp3, a MAP kinase
regulator that was shown to negatively affect the Fgf8
RESEARCH ARTICLE 1947
signaling pathway in the chick limb (Kawakami et al., 2003), is
expressed in a similar manner (see Fig. S2B in the supplementary
material).
We next looked at the expression of Bmp4 (as well as Bmp2 and
Bmp7; see Fig. S1D,E in the supplementary material) in the heart
and BA region (Fig. 2I). Bmp4 was expressed in the ectoderm of the
BAs, the pharyngeal endoderm, the splanchnic mesoderm, the aortic
sac and the dorsal mesocardium (Fig. 2I1-I4, Fig. 6). Notably, the
expression of Bmp4 overlapped with that of Isl1 in the splanchnic
mesoderm and in the dorsal mesocardium (Fig. 2, compare F3 and
F4 with I3 and I4), suggesting that Bmp4 may be involved in the
regulation of Isl1 expression.
Bmp4 induces cardiac gene expression, and blocks
skeletal muscle differentiation in the head, both
in vitro and in vivo
The expression of Bmp family members both in SpM explants and
in vivo (Figs 1, 2) suggests that this signaling pathway may play a
major role in the determination of cardiac and skeletal muscle cell
fates. To test this possibility, BMP4-conditioned medium was added
to both CPM and SpM explants that had been cultured for two or
three days (Fig. 3). BMP4 induced the expression of the cardiac
genes Nkx2.5, Gata4, Gata5, Gata6, vMHC and Capsulin in the
CPM explants. In parallel, Bmp4 blocked expression of the skeletal
muscle markers MyoD, Myf5 and Myogenin that appeared in these
same explants after three days in culture. Myf5 expression was also
abolished in SpM explants following BMP4 administration. TimeFig. 3. Bmp4 signaling promotes
cardiogenesis and blocks myogenesis in
vitro. (A) RT-PCR analysis of CPM and SpM
explants. Explants cultured in the absence (–)
or presence (+) of BMP4-conditioned medium.
RT-PCR analysis was performed after 3 days.
(B) RT-PCR analysis of explants cultured for 3
days in the absence (–) or presence (+) of
Noggin protein. BMP4 induced cardiogenesis
and blocked skeletal muscle differentiation in
CPM explants after 2 and 3 days in culture
(n=15/15), while Noggin induced myogenesis
in splanchnic mesoderm explants after 3 days
in culture (n=7/8).
DEVELOPMENT
Cardiac and skeletal muscle cell fates
course RT-PCR and real-time PCR analyses of CPM cells in culture
(data not shown) revealed the expression of cardiogenic markers
during the first day, followed by a downregulation of these markers
and an upregulation of myogenic differentiation at day three.
Although SpM explants expressed cardiac differentiation markers
in the absence of ectopic BMP4 (Fig. 1; these explants also express
endogenous Bmp2 and Bmp4), ectopic application of BMP4
increased the expression of Gata4 and Gata5 after two days in
culture (Fig. 3A). Importantly, in all cases we noted that
administration of BMP4 (or Bmp2, data not shown) induced beating
of the SpM-derived cardiomyocytes, suggesting that in vitro, BMP
levels control the differentiation and function of myocytes derived
from the splanchnic mesoderm.
In order to block the endogenous BMP signals, we applied the
Bmp antagonist Noggin to our explant culture system (Fig. 3B). In
SpM explants treated with Noggin, the expression level of vMHC
(but not other cardiac markers) was reduced, whereas the levels of
the skeletal muscle markers MyoD and Myogenin were slightly
elevated (Fig. 3B). Similar results were obtained using virally
expressed Noggin (data not shown). CPM explants treated with
Noggin were not significantly affected, in line with the significant
level of expression of endogenous Noggin in these cells (Fig. 1C).
These results, along with the expression of Myf5 and Capsulin in
SpM explants, suggest that SpM cells may contain a few myogenic
progenitors that can undergo myogenesis if BMP levels are
sufficiently low. Alternatively, SpM cells that are committed to the
cardiogenic lineage, but not fully differentiated, could transdifferentiate into myogenic progenitors if BMP levels remain low.
Together, our explant culture data demonstrate that, in vitro, BMP4
promotes a cardiac cell fate in both CPM and SpM explants, and
blocks skeletal muscle formation in CPM explants.
Because data from our in vitro assays demonstrated that
application of BMP4 to CPM cells promotes cardiogenesis, we next
tested whether ectopic Bmp4 could induce cardiogenesis in vivo
(Fig. 4). Pellets of cells overexpressing BMP4 were implanted into
the right side of the CPM of stage 9-10 chick embryos (Fig. 4A,B).
BMP4 strongly induced Nkx2.5 gene expression on the right side of
the head mesoderm and ectoderm, as evidenced in whole-mount
(Fig. 4C1) and sectioned embryos (Fig. 4C2,C3). We further tested
the impact of BMP4 on Gata gene expression (Fig. 4D). Although
we did not detect significant induction of Gata4 or Gata6 expression
(data not shown), Gata5 expression was induced by BMP4 in the BA
region (Fig. 4D1,D2,D3).
The expression patterns of Bmp4 and Isl1 genes (Fig. 2) indicated
that BMP4 might regulate Isl1 expression. Bmp4 cells that were
implanted in the right side of the CPM (Fig. 4E) blocked the
neuronal expression of Isl1 in the cranial nerve ganglia V (Fig.
4E1,E2). By contrast, BMP4 induced the expression of this gene in
the BA mesenchyme, as well as in the ectoderm and endoderm (Fig.
4E3). We therefore suggest that BMP4 acts upstream of Isl1 in vivo.
Capsulin is expressed in both paraxial and splanchnic mesoderm,
suggesting its possible role in both cardiac and skeletal muscle
lineage specification in the head (Figs 1, 2). BMP4 induced Capsulin
expression in the mesenchyme of the BA (Fig. 4F1,F2,F3), similar to
its effect on Isl1 and consistent with the effects of BMP4 in vitro (Fig.
3A). Considering that BMP signals are known to play an inhibitory
role in skeletal muscle differentiation, these results suggest that
Capsulin may act as a repressor of head muscle differentiation.
Furthermore, BMP4 efficiently blocked Myf5 expression (Fig.
4G1,G2,G3), and reduced Tbx1 expression (Fig. 4H1,H2,H3) in the
BA region. The expression of Myf5 at the site of lateral rectus
progenitors (Mootoosamy and Dietrich, 2002) was also inhibited in
Development 133 (10)
response to BMP4 application (Fig. 4G1,G2,G3). Thus, both in vitro
and in vivo experimental systems demonstrate that BMP4 plays a
crucial role in determining the fate of the head mesoderm: ectopic
application of BMP4 protein induced the expression of cardiac
marker genes, and blocked myogenic differentiation.
Cranial paraxial mesoderm cells contribute to
both the myocardial and endocardial layers of the
cardiac outflow tract
The fact that Bmp signaling induces cardiogenesis in cranial paraxial
mesoderm in vitro and in vivo, prompted us to investigate the
contribution of these cells to the developing heart at the looping
stages. To test, in vivo, our hypothesis that CPM cells are recruited
to the developing heart, we employed fate-mapping analyses in the
chick embryo (Figs 5, 6). DiI was injected into the left or right sides
of the CPM of stage 9-11 embryos in ovo at the axial level of
rhombomers 1-4 (Fig. 5A,E). The location of the incorporated DiI
was subsequently followed at stages 17-22. Most of the CPM cells
migrated into the first BA (Fig. 5B,F) in accordance with previous
studies (Hacker and Guthrie, 1998). Strikingly, in most embryos,
DiI-labeled cells from the CPM were detected in both the OFT and
the aortic sac (Fig. 5C,D,G,H). Although DiI-labeled CPM cells
from the left side of the embryos could be detected in the inner
curvature of the OFT (Fig. 5D), DiI injected into the right side was
detected in the outer curvature of the OFT (Fig. 5H; summarized in
Table S1 in the supplementary material). This new finding shows
that cells from the cranial paraxial mesoderm migrate to the OFT in
vivo.
Previous fate-mapping studies in the chick by Kirby and
colleagues demonstrated that neural crest cells originating from the
hindbrain corresponding to rhombomers 6-8 are required for normal
heart development [thus termed cardiac neural crest cells (Kirby et
al., 1983)]. In order to confirm that the DiI-labeled cells in the OFT
were derived from the CPM and not from cranial neural crest cells,
we injected the DiI at an earlier stage of development (stage 8), prior
to the onset of cranial neural crest migration (Fig. 5I-L). In these
embryos, DiI-labeled cells were detected in the OFT (Fig. 5K,L),
indicating that at the level of the first BA, and, to a lesser extent, the
second BA (see Fig. S3 in the supplementary material), CPM cells
migrate to the OFT. Furthermore, we observed DiI-labeled cells
from the CPM in the OFT in cranial neural crest-ablated embryos
(data not shown).
In order to confirm that DiI was injected into the CPM rather
than into the splanchnic mesoderm, some of the injected embryos
were immediately sectioned transversely to visualize the location
of the fluorescence signal. As expected, the injected DiI was
located in the CPM (Fig. 5N). Notably, DiI-labeled CPM cells
were found in both the myocardial and endocardial layers of the
OFT (Fig. 5O,P). Collectively, these DiI labeling experiments
demonstrate that cells from the CPM that migrate to the first and
second BAs contribute to both the endocardial and myocardial
lineages of the cardiac OFT.
To gain insights into the molecular make-up of the CPM cells en
route to the OFT, we compared our results from the fate mapping
and molecular analyses of sectioned chick embryos (Fig. 6). CMDiI was injected into the CPM of stage 10 embryos. Stage 16
embryos were fixed and sectioned transversely (Fig. 6A) at the level
of the first BA (Fig. 6C), the aortic sac/second BA (Fig. 6C1) and the
OFT (Fig. 6C2). The signal resulting from the labeled CPM cells
was compared with identical sections of embryos that were stained
for Nkx2.5, Gata5, Myf5, Bmp4 and the myocyte marker MF20. As
expected, labeled CPM cells were localized in the myogenic core of
DEVELOPMENT
1948 RESEARCH ARTICLE
RESEARCH ARTICLE 1949
Fig. 4. BMP4 induces cardiac gene expression and
blocks skeletal muscle differentiation in vivo.
(A) Dorsal view of a stage 9 embryo, implanted with cells
expressing HA-BMP4 (dashed circle). (B) Western blot
analysis of HA-tagged BMP4 in the conditioned medium of
the transfected cells. (C-H) In situ hybridization analyses for
the indicated genes. Whole-mount and transverse sections
of the embryos are shown (left panel, control; right panel,
Bmp4 treated). Induction (white arrowhead) or reduction
(open arrowhead) of the expression of cardiac and skeletal
muscle markers following BMP4 application is indicated.
Ectopically-applied Bmp4 induced expression of the cardiac
markers Nkx2.5 (n=7/7), Gata5 (n=7/14) and Capsulin
(n=5/6). By contrast, BMP4 cells blocked the expression of
the skeletal markers Myf5 (n=4/4) and Tbx1 (n=3/4). A dual
effect was observed for Isl1: although Bmp4 abolished the
expression of Isl1 in the trigeminal ganglion (V), an elevated
expression of this gene was found in the mesenchyme and
ectoderm of the first BA (n=7/7). as, aortic sac; ba1, ba2,
first, second branchial arches; lr, lateral rectus; nt, neural
tube.
DEVELOPMENT
Cardiac and skeletal muscle cell fates
1950 RESEARCH ARTICLE
Development 133 (10)
Fig. 6. Cellular and molecular analyses of cranial paraxial mesoderm en route to the heart. (A) Diagram of a stage 16 embryo (ventral
view), following removal of the heart. Dotted lines represent the three section levels displayed in the lower panels. (B) Diagram of quail-chick
chimera assay. (C-I) Transverse sections at the first branchial arch (ba) level (upper dotted line in A). (C1-I1) Transverse sections at the aortic sac level
(middle line). (C2-I2) Transverse sections at the outflow tract (OFT) level (lower line). (C-C2) Sections of CM-DiI-labeled cells at the aforementioned
levels. (D-D2) Transverse sections through the quail-chick chimera, followed by immunostaining with the quail-specific antibody (QCPN, ⫻10 bright
field and fluorescence images) (D⬘-D2⬘) ⫻20 fluorescence images of the marked area shown in the ⫻10 images (D-D2). (E-E2) Immunostaining for
Myosin heavy chain using MF20 antibody. (F-I2) Sections through embryos subjected to whole-mount in situ hybridization with the indicated
markers. aaa1, aortic arch artery 1; aaa2, aortic arch artery 2; as, aortic sac; ba1, ba2, first, second branchial arches; end, endocardium; myo,
myocardium; nt, neural tube; ov, otic vesicle; ph, pharynx.
DEVELOPMENT
Fig. 5. Cranial paraxial mesoderm
cells contribute to the myocardium
and endocardium of the cardiac
outflow tract in vivo. (A-P) Tracking
of CPM cells by Dil labelling. DiI
injections into the left (A) or right
(E,I,M) CPM of stage 10 (or stage 8, I)
chick embryos. Lateral (B,F,J) or
transverse (C,G,K) views of embryos at
stage 17-18 are shown as an overlay
of bright field and fluorescence
images. (D,H,L) Higher magnification
of dissected hearts. Cells labeled on
the left side of the CPM contribute to
the inner curvature of the cardiac OFT
(D), whereas cells from the right side
contribute to the outer curvature (H,L).
(O,P) The CPM cells contribute to both
the myocardium and endocardium:
cross section (O) and transverse
section (P) of the OFT. (M,N) Embryo
dissected shortly after the DiI injection
(M) to verify the location of the dye in
the paraxial mesoderm (N). ba1, first
branchial arch; ba2, second branchial
arch; cc, conus cordis; da, dorsal
aorta; end, endocardium; ic, inner
curvature; myo, myocardium; nc,
notochord; nt, neural tube; oc, outer
curvature; oft, outflow tract; ov, otic
vesicle; ph, pharynx; rv, right ventricle;
ta, truncus arteriosus.
the first BA, as indicated by Myf5 in situ hybridization (Fig. 6H). Of
the cardiogenic genes mentioned above, only Nkx2.5 could be
detected at the level of the first BA (Fig. 6F,G, Fig. 2).
During heart looping stages in chick embryos, the OFT shifts
caudally (Waldo et al., 2001), such that at stage 16-18 the aortic sac,
which connects the aortic arch arteries to the OFT, is connected
between the first and second BAs (Fig. 6A). CM-DiI-labeled cells at
the level of the aortic sac were detected in a region where Nkx2.5 and
Gata5, but not Myf5, were expressed (Fig. 6F1,G1,H1). These
results suggest that CPM cells that migrate into the BAs adopted
molecular characteristics of skeletal muscle cells (Myf5, compare
Fig. 6C with 6H). Cells that migrated through the aortic sac
presumably adopt a myocardial cell identity (Nkx2.5 and Gata5,
compare Fig. 6C1,F1 and G1). The lack of expression of Bmp4 at
the first BA level (Fig. 6I) and its expression in the aortic sac/second
BA (Fig. 6I.1) is correlated with the change in cell fate from
myogenic to cardiogenic progenitors. At the level of the OFT, CMDiI-labeled cells could be detected in the endocardium and
myocardium (Fig. 6C2,E2; myocardial cells are MF20 positive),
which also express Nkx2.5 and Gata5 (Fig. 6F2,G2).
We next used the quail-chick grafting technique to verify our dye
labeling results (Fig. 6B; see also Table S2 in the supplementary
material). CPM grafts (with the surface ectoderm) of stage 8 quail
embryos were transplanted into the CPM of stage-matched chick
embryos (Fig. 6B). Sections of the operated embryos revealed the
existence of quail-derived CPM cells (recognized by a QCPNspecific antibody) within the mesenchyme of the first BA (Fig.
6D,D⬘), the aortic sac/second BA (Fig. 6D1,D1⬘) and within the
endocardium and myocardium of the cardiac OFT (Fig. 6D2,D2⬘).
This experiment further indicates that CPM cells rather than cranial
neural crest contribute to the OFT during looping stages. Taken
together, our dye marking technique, quail chick grafting procedure
and retroviral infection (see Fig. S4 in the supplementary material)
confirm the observation that cranial paraxial mesoderm cells
contribute to the myocardial and endocardial components of the
cardiac OFT.
DISCUSSION
In the present study, we employed both in vitro and in vivo
experimental systems in the avian embryo to explore how mesoderm
progenitors in the head, differentiate into both heart and craniofacial
muscles. Our detailed gene expression analyses of candidate tissuespecific transcription factors and signaling molecules both in vitro
and in vivo (Figs 1, 2) revealed a group of transcription factors
(Gata5, Gata6, Isl1 and Capsulin) that, based on their
spatiotemporal expression, are likely to play a role in the
development of the secondary/anterior heart field (SHF) in the chick.
These analyses further revealed a considerable degree of plasticity
between cardiac and skeletal muscle precursors in the head
mesoderm. We also provide evidence that Bmp4 plays a key role in
specifying the cell fates of both myogenic and cardiogenic
progenitors in the cranial paraxial mesoderm (Figs 3, 4). Data from
both fate-mapping experiments and molecular analyses demonstrate
that cranial paraxial mesoderm cells represent a source of
cardiogenic progenitors that populate the cardiac outflow tract in
vivo (Figs 5, 6).
Bmp signaling induces cardiogenesis and represses
head myogenesis during heart looping stages
Bmp signaling molecules are required for early heart formation in
vertebrate embryos (Jiao et al., 2003; Liu et al., 2004; Schlange et
al., 2000; Schultheiss et al., 1997; Shi et al., 2000). Similarly, Dpp,
RESEARCH ARTICLE 1951
the Drosophila ortholog of Bmp, is required for the formation of the
dorsal vessel, the equivalent of the heart in flies (Frasch, 1995). It
was further suggested that Bmp2, which is expressed in the
SHF/OFT myocardium, affects the proliferation of these cells in
vitro (Waldo et al., 2001).
Here, we demonstrate that the expression of Bmp family members
during heart looping and the early stages of myogenesis is correlated
with cardiac gene expression (Figs 1, 2). Ectopic application of
BMP4 during these stages induced robust expression of Nkx2.5,
Gata5, Isl1 and Capsulin in the CPM in vitro (Fig. 3) and in vivo
(Fig. 4), whereas inhibition of the BMP signals by Noggin partially
blocked cardiogenesis, and induced myogenesis in SpM explants
(Fig. 3). Concomitant with the induction of cardiogenesis in the
CPM, BMP4 blocked skeletal myogenesis in this tissue, both in vitro
and in vivo (Figs 3, 4), consistent with previous studies (Pourquie
et al., 1996; Reshef et al., 1998; Tzahor et al., 2003). Ectopic
application of BMP2 and BMP4 to SpM cells induced a rhythmic
beating of cardiomyocytes in vitro, suggesting a possible role for
Bmp signaling in the terminal differentiation of cardiac progenitors.
Loss-of-function studies of Isl1 in mice had previously shown that
Bmp4 (as well as other Bmp and Fgf family members) is a target of
Isl1 in the SHF (Cai et al., 2003). We now demonstrate that BMP4
induces Isl1 expression in the SHF, while blocking its expression in
neuronal tissue. Thus, there appears to be a positive feedback loop
between Isl1 and Bmp4.
The involvement of cranial paraxial mesoderm in
cardiogenesis
Our fate-mapping experiments using vital dyes and our quail chick
transplantation experiments, demonstrate that paraxial mesoderm
cells that migrate primarily to the first BA contribute to both
myocardial and endocardial layers of the OFT (Figs 5, 6; see also
Figs S3, S4 in the supplementary material). DiI-labeled cells from
the left side of the CPM were found in the inner curvature of the
OFT, whereas those from the right side were detected at the outer
curvature of the OFT (Fig. 5). These findings suggest that the
differences in origin and in the migratory paths taken by CPM cells
as they move toward the OFT could affect the process of rightward
heart looping, a critical step during heart morphogenesis in
vertebrate embryos. Taken together, our data identify a novel
mechanism by which cranial paraxial mesoderm cells adopt a
myocardial cell fate. Although we confirmed that DiI-labeled cells
within the OFT were derived from the mesoderm and not from
cranial neural crest cells (Figs 5, 6), the influence of the cranial
neural crest on the migration of CPM cells into the OFT was not
addressed in this study.
It was previously shown that SHF cells in the chick embryo
contribute to the myocardium of the OFT (Waldo et al., 2001); later,
these mesodermal cells provide smooth muscle cells that form the
base of the great arteries (Waldo et al., 2005). Cardiac neural crest
cells provide yet another source for smooth muscle cells of the blood
vasculature (Hutson and Kirby, 2003). Studies in zebrafish embryos
have shown that cardiac neural crest cells contribute to the
developing myocardium (Li et al., 2003; Sato and Yost, 2003).
Results from quail-chick transplantation assays further suggest that
progenitor cells originating in the cephalic mesoderm can give rise
to angioblasts that populate the endocardium of the OFT (Noden,
1991). Our data demonstrate that CPM cells can be found in both
myocardial and endocardial cell populations. Collectively, these
studies suggest that endocardial and myocardial precursors within
the OFT derive from multiple origins within the embryo, rather than
a common progenitor population.
DEVELOPMENT
Cardiac and skeletal muscle cell fates
What, then, is the relationship between the cardiac progenitor
populations that constitute the SHF (Buckingham et al., 2005) and
those originating in the cranial paraxial mesoderm? In the chick, the
SHF includes the epithelial splanchnic mesoderm, beneath the floor
of the pharynx, that lies caudal to the OFT (Waldo et al., 2001),
whereas the anterior heart field is restricted to the cephalic
mesodermal cells surrounding the aortic sac (Mjaatvedt et al., 2001).
In mice, the anterior heart field includes the right ventricle and the
OFT, as well as the mesodermal core of the pharyngeal arches (Kelly
et al., 2001). Our results, which demonstrate that CPM cells at the
levels of the first and second BAs (which eventually form the
mesodermal core of the pharyngeal arches) contribute to the OFT,
are consistent with these descriptions of the anterior heart field. Our
results do not rule out a contribution of cardiac progenitors from the
splanchnic mesoderm into the OFT, as has been previously shown
(Waldo et al., 2005; Waldo et al., 2001). In view of the dynamic
nature of heart morphogenesis, our data is consistent with the
hypothesis that cardiac progenitor populations from multiple sources
(or heart fields) are recruited into the heart at different stages of
development, and from various locations (Abu-Issa et al., 2004).
A model for the specification of the cranial
paraxial mesoderm in response to Bmp signals
Our results are summarized in a schematic model (Fig. 7), which
demonstrates that in chick embryos, DiI-labeled cells from the CPM
can migrate along the myogenic core of the first BA, through the
aortic sac, and into the cardiac OFT. Initially, these cells represent
myoblast precursors, as evidenced by the expression of various
skeletal muscle markers (e.g. Myf5, Capsulin and Tbx1; highlighted
Fig. 7. A model for cranial paraxial mesoderm specification in the
chick embryo. Our model proposes that cells within the CPM that
migrate through the first branchial arch (marked by orange line), first
adopt a myogenic lineage (Myf5, Capsulin and Tbx1; highlighted in the
upper box). Those cells migrating further towards the aortic sac, which
connects the branchial arches to the OFT, initiate cardiogenesis (note
the expression of cardiac markers Gata5, Gata6, Capsulin, Isl1 and
Nkx2.5; middle box). A smaller portion of these cells may reach the
myocardium and endocardium of the OFT, where different cardiac
markers are expressed (e.g. Gata4 and cMHC; lower box). The gradual
shift from a skeletal muscle to a cardiac cell fate is correlated with the
spatiotemporal expression of Bmp4. Moreover, ectopic application of
Bmp4 both in vitro and in vivo promotes cardiogenesis in the cranial
paraxial mesoderm, and blocks the skeletal muscle differentiation
programs.
Development 133 (10)
in the upper box). CPM cells that surround the aortic sac presumably
adopt myocardial characteristics (Nkx2.5, Gata5, Isl1 and Capsulin;
middle box). CPM-derived cells within the OFT are co-localized
with cardiogenic cells expressing Nkx2.5, Gata4 and cMHC (lower
box). Our cellular and molecular analyses of the cranial paraxial
mesoderm cells suggest that these cells are gradually specified along
the dorsoventral axis, in correlation with Bmp signaling levels.
The expression of Bmp family members – in particular, Bmp4 –
in the head region is consistent with its positive role in cardiogenesis:
ectopic application of Bmp4 induces cardiac differentiation in CPM
cells, and reduces the differentiation of skeletal muscle precursors.
Thus, we propose that Bmp4 stands at the apex of a consortium of
signaling pathways that promote cardiogenesis and suppress
myogenesis in progenitor cell populations originating in the head
mesoderm (Fig. 7).
Although CPM cells can undergo myogenic, skeletogenic or
angiogenic differentiation (Couly et al., 1993; Noden, 1983; Noden,
1991), the present study demonstrates that these cells further adopt
unique cardiogenic properties. Based on our current results, as well
as on those of previous studies, we propose that paraxial mesoderm
cells in the head possess the ability to differentiate into a broader
range of lineages than does the trunk paraxial mesoderm (Tzahor
et al., 2003; Tzahor and Lassar, 2001). Although the initial
differentiation and patterning of the somites into both myogenic and
skeletogenic lineages take place at sites adjacent to the axial tissues,
the differentiation of the CPM cells is repressed by signals (e.g.
Wnt1 and Wnt3a) from the neural tube (Tzahor et al., 2003; Tzahor
and Lassar, 2001).
During early embryogenesis, heart and skeletal muscle progenitor
cells were originally thought to derive from distinct regions of the
mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm,
respectively). We propose that the developmental programs of
progenitor populations that contribute to both the head musculature
and the anterior pole of the heart are tightly linked, consistent with
the notion of a single cardiocraniofacial morphogenetic field
(Hutson and Kirby, 2003). Accordingly, insults to any component of
this field may lead to both cardiac and craniofacial abnormalities.
Whether there is a common progenitor cell in the cranial paraxial
mesoderm that differentiates into both cardiac and skeletal muscle
lineages, or whether this cell population is heterogeneous in nature
awaits detailed molecular and cellular analyses. In addition, the
impact and function of these cells within the OFT will constitute a
major focus of our future research efforts.
This work was supported by research grants to E.T. from the Estelle Funk
Foundation for Biomedical Research, and from Ruth & Allen Ziegler. E.T. is the
incumbent of the Gertrude and Philip Nollman Career Development Chair. We
thank Andrew Lassar, Elazar Zelzer, Herve Kempf and Stephane Zaffran for
critical reading of the manuscript, and members of our laboratory team for
their help and support.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/10/1943/DC1
References
Abu-Issa, R., Waldo, K. and Kirby, M. L. (2004). Heart fields: one, two or more?
Dev. Biol. 272, 281-285.
Bailey, P., Holowacz, T. and Lassar, A. B. (2001). The origin of skeletal muscle
stem cells in the embryo and the adult. Curr. Opin. Cell Biol. 13, 679-689.
Borycki, A., Brown, A. M. and Emerson, C. P., Jr (2000). Shh and Wnt signaling
pathways converge to control Gli gene activation in avian somites. Development
127, 2075-2087.
Brott, B. K. and Sokol, S. Y. (2005). A vertebrate homolog of the cell cycle
regulator Dbf4 is an inhibitor of Wnt signaling required for heart development.
Dev. Cell 8, 703-715.
Brown, C. B., Wenning, J. M., Lu, M. M., Epstein, D. J., Meyers, E. N. and
DEVELOPMENT
1952 RESEARCH ARTICLE
Epstein, J. A. (2004). Cre-mediated excision of Fgf8 in the Tbx1 expression
domain reveals a critical role for Fgf8 in cardiovascular development in the
mouse. Dev. Biol. 267, 190-202.
Buckingham, M. (2001). Skeletal muscle formation in vertebrates. Curr. Opin.
Genet. Dev. 11, 440-448.
Buckingham, M., Meilhac, S. and Zaffran, S. (2005). Building the mammalian
heart from two sources of myocardial cells. Nat. Rev. Genet. 6, 826-835.
Cai, C. L., Liang, X., Shi, Y., Chu, P. H., Pfaff, S. L., Chen, J. and Evans, S.
(2003). Isl1 identifies a cardiac progenitor population that proliferates prior to
differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877889.
Couly, G. F., Coltey, P. M. and Le Douarin, N. M. (1992). The developmental fate
of the cephalic mesoderm in quail-chick chimeras. Development 114, 1-15.
Couly, G. F., Coltey, P. M. and Le Douarin, N. M. (1993). The triple origin of skull
in higher vertebrates: a study in quail-chick chimeras. Development 117, 409429.
Dodou, E., Verzi, M. P., Anderson, J. P., Xu, S. M. and Black, B. L. (2004).
Mef2c is a direct transcriptional target of ISL1 and GATA factors in the anterior
heart field during mouse embryonic development. Development 131, 39313942.
Foley, A. C. and Mercola, M. (2005). Heart induction by Wnt antagonists
depends on the homeodomain transcription factor Hex. Genes Dev. 19, 387396.
Frasch, M. (1995). Induction of visceral and cardiac mesoderm by ectodermal Dpp
in the early Drosophila embryo. Nature 374, 464-467.
Hacker, A. and Guthrie, S. (1998). A distinct developmental programme for the
cranial paraxial mesoderm in the chick embryo. Development 125, 3461-3472.
Hutson, M. R. and Kirby, M. L. (2003). Neural crest and cardiovascular
development: a 20-year perspective. Birth Defects Res. C Embryo Today 69, 213.
Jiao, K., Kulessa, H., Tompkins, K., Zhou, Y., Batts, L., Baldwin, H. S. and
Hogan, B. L. (2003). An essential role of Bmp4 in the atrioventricular septation
of the mouse heart. Genes Dev. 17, 2362-2367.
Kawakami, Y., Rodriguez-Leon, J., Koth, C. M., Buscher, D., Itoh, T., Raya, A.,
Ng, J. K., Esteban, C. R., Takahashi, S., Henrique, D. et al. (2003). MKP3
mediates the cellular response to FGF8 signalling in the vertebrate limb. Nat. Cell
Biol. 5, 513-519.
Kelly, R. G., Brown, N. A. and Buckingham, M. E. (2001). The arterial pole of
the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm.
Dev. Cell 1, 435-440.
Kelly, R. G., Jerome-Majewska, L. A. and Papaioannou, V. E. (2004). The
del22q11.2 candidate gene Tbx1 regulates branchiomeric myogenesis. Hum.
Mol. Genet. 13, 2829-2840.
Kirby, M. L. (2002). Molecular embryogenesis of the heart. Pediatr. Dev. Pathol. 5,
516-543.
Kirby, M. L., Gale, T. F. and Stewart, D. E. (1983). Neural crest cells contribute to
normal aorticopulmonary septation. Science 220, 1059-1061.
Laugwitz, K. L., Moretti, A., Lam, J., Gruber, P., Chen, Y., Woodard, S., Lin, L.
Z., Cai, C. L., Lu, M. M., Reth, M. et al. (2005). Postnatal isl1+ cardioblasts
enter fully differentiated cardiomyocyte lineages. Nature 433, 647-653.
Li, Y. X., Zdanowicz, M., Young, L., Kumiski, D., Leatherbury, L. and Kirby,
M. L. (2003). Cardiac neural crest in zebrafish embryos contributes to
myocardial cell lineage and early heart function. Dev. Dyn. 226, 540-550.
Liu, W., Selever, J., Wang, D., Lu, M. F., Moses, K. A., Schwartz, R. J. and
Martin, J. F. (2004). Bmp4 signaling is required for outflow-tract septation
and branchial-arch artery remodeling. Proc. Natl. Acad. Sci. USA 101, 44894494.
Lough, J. and Sugi, Y. (2000). Endoderm and heart development. Dev. Dyn. 217,
327-342.
Lu, J. R., Bassel-Duby, R., Hawkins, A., Chang, P., Valdez, R., Wu, H., Gan, L.,
Shelton, J. M., Richardson, J. A. and Olson, E. N. (2002). Control of facial
muscle development by MyoR and capsulin. Science 298, 2378-2381.
Marvin, M. J., Di Rocco, G., Gardiner, A., Bush, S. M. and Lassar, A. B. (2001).
Inhibition of Wnt activity induces heart formation from posterior mesoderm.
Genes Dev. 15, 316-327.
Mjaatvedt, C. H., Nakaoka, T., Moreno-Rodriguez, R., Norris, R. A., Kern, M.
J., Eisenberg, C. A., Turner, D. and Markwald, R. R. (2001). The outflow
tract of the heart is recruited from a novel heart-forming field. Dev. Biol. 238,
97-109.
Mootoosamy, R. C. and Dietrich, S. (2002). Distinct regulatory cascades for head
and trunk myogenesis. Development 129, 573-583.
Munsterberg, A. E., Kitajewski, J., Bumcrot, D. A., McMahon, A. P. and
RESEARCH ARTICLE 1953
Lassar, A. B. (1995). Combinatorial signaling by Sonic hedgehog and Wnt
family members induces myogenic bHLH gene expression in the somite. Genes
Dev. 9, 2911-2922.
Noden, D. M. (1983). The embryonic origins of avian cephalic and cervical muscles
and associated connective tissues. Am. J. Anat. 168, 257-276.
Noden, D. M. (1991). Origins and patterning of avian outflow tract endocardium.
Development 111, 867-876.
Noden, D. M. and Trainor, P. A. (2005). Relations and interactions between
cranial mesoderm and neural crest populations. J. Anat. 207, 575-601.
Noden, D. M., Marcucio, R., Borycki, A. G. and Emerson, C. P., Jr (1999).
Differentiation of avian craniofacial muscles: I. Patterns of early regulatory gene
expression and myosin heavy chain synthesis. Dev. Dyn. 216, 96-112.
Olson, E. N. and Schneider, M. D. (2003). Sizing up the heart: development
redux in disease. Genes Dev. 17, 1937-1956.
Plageman, T. F., Jr and Yutzey, K. E. (2005). T-box genes and heart development:
putting the “T” in heart. Dev. Dyn. 232, 11-20.
Pourquie, O. (2001). Vertebrate somitogenesis. Annu. Rev. Cell Dev. Biol. 17, 311350.
Pourquie, O., Fan, C. M., Coltey, M., Hirsinger, E., Watanabe, Y., Breant, C.,
Francis-West, P., Brickell, P., Tessier-Lavigne, M. and Le Douarin, N. M.
(1996). Lateral and axial signals involved in avian somite patterning: a role for
BMP4. Cell 84, 461-471.
Reshef, R., Maroto, M. and Lassar, A. B. (1998). Regulation of dorsal somitic cell
fates: BMPs and Noggin control the timing and pattern of myogenic regulator
expression. Genes Dev. 12, 290-303.
Rudnicki, M. A., Schnegelsberg, P. N., Stead, R. H., Braun, T., Arnold, H. H.
and Jaenisch, R. (1993). MyoD or Myf-5 is required for the formation of
skeletal muscle. Cell 75, 1351-1359.
Sato, M. and Yost, H. J. (2003). Cardiac neural crest contributes to
cardiomyogenesis in zebrafish. Dev. Biol. 257, 127-139.
Schlange, T., Andree, B., Arnold, H. H. and Brand, T. (2000). BMP2 is required
for early heart development during a distinct time period. Mech. Dev. 91, 259270.
Schneider, V. A. and Mercola, M. (2001). Wnt antagonism initiates cardiogenesis
in Xenopus laevis. Genes Dev. 15, 304-315.
Schultheiss, T., Burch, J. and Lassar, A. (1997). A role for bone morphogenetic
proteins in the induction of cardiac myogenesis. Genes Dev. 11, 451-462.
Shi, Y., Katsev, S., Cai, C. and Evans, S. (2000). BMP signaling is required for
heart formation in vertebrates. Dev. Biol. 224, 226-237.
Stern, H. M., Brown, A. M. and Hauschka, S. D. (1995). Myogenesis in paraxial
mesoderm: preferential induction by dorsal neural tube and by cells expressing
Wnt-1. Development 121, 3675-3686.
Tajbakhsh, S., Rocancourt, D., Cossu, G. and Buckingham, M. (1997).
Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and
Myf-5 act upstream of MyoD. Cell 89, 127-138.
Tajbakhsh, S., Borello, U., Vivarelli, E., Kelly, R., Papkoff, J., Duprez, D.,
Buckingham, M. and Cossu, G. (1998). Differential activation of Myf5 and
MyoD by different Wnts in explants of mouse paraxial mesoderm and the later
activation of myogenesis in the absence of Myf5. Development 125, 41554162.
Tzahor, E. and Lassar, A. B. (2001). Wnt signals from the neural tube block
ectopic cardiogenesis. Genes Dev. 15, 255-260.
Tzahor, E., Kempf, H., Mootoosamy, R. C., Poon, A. C., Abzhanov, A., Tabin,
C. J., Dietrich, S. and Lassar, A. B. (2003). Antagonists of Wnt and BMP
signaling promote the formation of vertebrate head muscle. Genes Dev. 17,
3087-3099.
Waldo, K. L., Kumiski, D. H., Wallis, K. T., Stadt, H. A., Hutson, M. R., Platt,
D. H. and Kirby, M. L. (2001). Conotruncal myocardium arises from a
secondary heart field. Development 128, 3179-3188.
Waldo, K. L., Hutson, M. R., Ward, C. C., Zdanowicz, M., Stadt, H. A.,
Kumiski, D., Abu-Issa, R. and Kirby, M. L. (2005). Secondary heart field
contributes myocardium and smooth muscle to the arterial pole of the
developing heart. Dev. Biol. 281, 78-90.
Xu, H., Morishima, M., Wylie, J. N., Schwartz, R. J., Bruneau, B. G., Lindsay,
E. A. and Baldini, A. (2004). Tbx1 has a dual role in the morphogenesis of the
cardiac outflow tract. Development 131, 3217-3227.
Yuan, S. and Schoenwolf, G. C. (2000). Islet-1 marks the early heart rudiments
and is asymmetrically expressed during early rotation of the foregut in the chick
embryo. Anat. Rec. 260, 204-207.
Zaffran, S., Kelly, R. G., Meilhac, S. M., Buckingham, M. E. and Brown, N. A.
(2004). Right ventricular myocardium derives from the anterior heart field. Circ.
Res. 95, 261-268.
DEVELOPMENT
Cardiac and skeletal muscle cell fates