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First published online 25 July 2007
doi: 10.1242/dev.002501
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1 Department of Biological Regulation, Weizmann Institute of Science, Rehovot
76100, Israel.
2 Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
3 Swiss Federal Institute of Technology, ETH-Hoenggerberg HPM E38, CH-8093
Zürich, Switzerland.
4 Department of Pharmacology, Graduate School of Medicine, Kyoto University,
Kyoto 606-8501, Japan.
* Author for correspondence (e-mail: eldad.tzahor{at}weizmann.ac.il)
Accepted 27 June 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Skeletal myogenesis, Cranial neural crest, Cranial paraxial mesoderm, Mouse, Chick
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Trainor and Krumlauf, 2001).
CNC differentiates into a wide variety of cell types, including neurons, glia,
and pigment cells. These cells differ from trunk neural crest cells in various
respects; most importantly, in their ability to give rise to the skeletal
elements of the head (Le Douarin and
Kalcheim, 1999). CPM located anterior to the somites provides the
precursors for cranial skeletal muscles.
Both CPM and CNC cells stream into the neighboring branchial arches (BAs,
also known as pharyngeal arches), which form the templates of adult
craniofacial structures (Noden and
Trainor, 2005). Within the BAs, CNC cells surround the muscle
anlagen in a highly organized fashion, thereby separating the myoblasts from
the overlying surface ectoderm (Hacker and
Guthrie, 1998; Noden,
1983b; Trainor and Tam,
1995; Trainor et al.,
1994). Mesoderm-derived myoblast cells subsequently fuse together
to form a myofiber, which is attached to a specific CNC-derived skeletal
element, through CNC-derived connective tissue, in a precisely coordinated
manner. However, the molecular mechanisms underlying head muscle patterning -
myoblast guidance, positioning and connection to specific attachment sites -
remain poorly understood. Furthermore, the degree to which skeletal muscle
specification, differentiation and patterning is intrinsic to muscle
(mesoderm) progenitors or controlled by extrinsic environmental signals (e.g.
CNC cells) is a fundamental embryological question.
Craniofacial shapes are amazingly diverse in vertebrates but also within
species [e.g. dogs, birds (Helms et al.,
2005)]. This diversity apparently reflects a tight linkage between
the skeletal elements (CNC), connective tissue (CNC) and skeletal muscle
(mesoderm). Indeed, it has long been suggested that in addition to
contributing to the formation of skeletal elements and connective tissue in
the head, CNC cells may also be involved in the patterning of the head
musculature (Couly et al.,
1992; Ericsson et al.,
2004; Grammatopoulos et al.,
2000; Kontges and Lumsden,
1996; Noden,
1983a; Noden,
1983b; Olsson et al.,
2001; Schilling and Kimmel,
1997).
Because skeletal muscles in the head still form (albeit in a distorted
fashion) following in vivo ablation of the CNC cells in amphibian and chick
embryos (Ericsson et al., 2004;
Olsson et al., 2001;
Tzahor et al., 2003;
von Scheven et al., 2006)
(reviewed in Noden and Trainor,
2005), the precise impact of CNC cells on head muscle formation
remains unclear. Several genetic knockout models in mice have provided
insights into CNC development, however; the link between these genetic
perturbations and cranial muscle formation has not been explored. Thus,
although it is generally accepted that CNC influences cranial muscle
formation, exactly how CNC cells participate in this process remains to be
elucidated.
Previously, we identified signals that regulate head muscle differentiation
(Tzahor et al., 2003). In the
head, both bone morphogenetic protein (BMP) and the canonical Wnt signaling
molecules secreted by the dorsal neural tube act to repress skeletal muscle
formation. This may occur via inhibition of the myogenic differentiation of
the CPM in the vicinity of the neural tube. By contrast, these same Wnt
ligands are required to stimulate myogenesis in the trunk. Moreover, CNC cells
secrete both BMP inhibitors (Noggin, Gremlin) and Wnt inhibitors (Frzb), which
together induce myogenic differentiation of the CPM in vitro
(Tzahor et al., 2003).
Therefore, head muscle differentiation is subject to a complex balance between
neural tube-derived inhibitors and CNC-derived activators.
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; Gavalas et al.,
2001; Lee et al.,
2004) and cellular perturbations of CNC development in avian
embryos. Our results indicate that although CNC cells are not necessary for
the early specification of skeletal muscle progenitors, they play a crucial
role in the migration, positioning and differentiation of cranial muscle
precursors in vertebrate embryos. Our findings also demonstrate that in the
absence of CNC cells, other tissues and signals are capable of promoting
skeletal muscle differentiation in the head. |
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chick and quail embryos
Fertilized chicken and quail eggs were incubated for 1-5 days at 38.5°C
in a humidified incubator up to Hamburger-Hamilton stages 8-26.
CNC ablation
Dorsal neural tube ablation was performed at around stage 8, as previously
described (Tzahor et al.,
2003).
In situ hybridization and histological analyses
A full list of the in situ hybridization probes and detailed protocols are
available upon request (see also
Tirosh-Finkel et al.,
2006).
Cell proliferation assay combined with in situ hybridization
Stage 8 chick embryos were incubated for 
45 hours, followed by the
addition of 200 µl of 10 mM 5'-bromo-2'-deoxyuridine (BrdU) for
1 hour in ovo. Thereafter, embryos were fixed and processed for in situ
hybridization and sectioning. Selected sections were subjected to an
immunostaining protocol (Tirosh-Finkel et
al., 2006). BrdU-positive cells in the myogenic core, demarcated
by Myf5 staining, were counted and divided by the total number of
DAPI-positive nuclei in the same region.
In-ovo dye injection
DiI, CM-DiI or DiO labeling experiments were performed on stage 8 chick
embryos (Tirosh-Finkel et al.,
2006).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); Dlx5 (in the mouse) was used to label the CNC. We
began by investigating whether CNC cells are a prerequisite to the
specification of the skeletal muscle lineage. Studies in amphibians
(Ericsson et al., 2004;
Olsson et al., 2001) as well
as our previous work with chick embryos
(Tzahor et al., 2003) suggest
that early steps of muscle development in the vertebrate head are
CNC-independent. However, this interpretation is complicated by the fact that
neural crest cells in chick embryos are known to regenerate following
extirpation (Saldivar et al.,
1997; Scherson et al.,
1993; Vaglia and Hall,
1999).
Therefore, we used a mouse model involving a genetic loss in a specific
subset of CNC cells. In the mouse, previous studies of combined
Hoxa1/Hoxb1 mutants revealed extensive synergy between these
two genes. The combination of a homozygous null Hoxa1 allele
(Lufkin et al., 1991) and a
homozygous Hoxb1-3'RARE allele, a mutant of
the retinoic acid enhancer required for Hoxb1 expression in the
neural tube (Marshall et al.,
1994), resulted in the specific failure of CNC cells to form and
migrate into the second branchial arch (BA2), whereas CNC cells in the other
BAs remained unaffected (Gavalas et al.,
2001). This genetic ablation of CNC cells in BA2 did not
significantly affect the early patterning of endoderm and surface ectoderm in
the arch; however, its effect on mesodermal cells was not examined.
The Hoxa1/Hoxb1-3'RARE double-knockout
mouse model therefore provides means to determine whether CNC cells are
required for early myogenesis. In order to confirm the mutant BA2 phenotype,
we performed in situ hybridization for Hoxa2
(Fig. 1A; note the specific
loss of Hoxa2 expression in BA2 of the double-mutant embryo in
A'). Next, control (double heterozygote) or mutant (double homozygote)
E9.5 mouse embryos were subjected to in situ hybridization for the early
skeletal muscle markers capsulin and Tbx1
[Fig. 1B,C
(Kelly et al., 2004;
Lu et al., 2002)]. Because
these early skeletal muscle markers were detected in BA2 of mutant embryos, we
propose that CNC cells are not necessary for the early stages of head muscle
specification.
Muscle patterning defects could be observed in the Hoxa1/Hoxb1-3'RARE mutants: first, the expression patterns of capsulin and Tbx1 in BA2 were slightly expanded in the mutant embryos, as compared with controls (Fig. 1B',C'), and second, capsulin expression in the myogenic core seemed to be more condensed, presumably because of the absence of infiltrating CNC cells within the core of BA2 (inset in Fig. 1B'). These results are consistent with a possible role for CNC during skeletal muscle patterning at later stages of muscle development.
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). Of particular interest to us is the fact that this molecule
is intricately involved in craniofacial development. The loss-of-function of
Twist in mouse embryos has revealed its essential role in CNC
migration and differentiation, although mutant embryos do not survive beyond
E10.5 (Chen and Behringer,
1995; Soo et al.,
2002). Importantly, in Twist mutant embryos, CNC cells
are formed at all levels of the brain (Fig.
2K'), despite the failure of the neural tube to close, but
their patterns of migration had changed. In addition, it has been suggested
that CNC development was arrested at an early phase of skeletogenic
differentiation in Twist mutants
(Soo et al., 2002). In the mouse, at E8.5, Twist expression is detected in the head mesenchyme (Fig. 2A,A'), although its expression differed from that of the early myogenic marker capsulin (Fig. 2B,B'D,D'). From E9 onwards, Twist is expressed exclusively in CNC cells as shown by its overlapped expression pattern with the CNC marker Dlx5 (Fig. 2E,E'F,F', and data not shown). Therefore, we used the Twist mouse model to determine its indirect impact on skeletal muscle formation. In situ hybridization of control and Twist mutants at E9.5-10.5 indicated a profound Twist-dependent alteration in the expression of the skeletal muscle markers capsulin, Tbx1, Myf5 and MyoD (Fig. 2G-J'). In control embryos, muscle markers are typically expressed in the core of the BAs (Fig. 2G,H,I,J); this pattern of expression was altered in Twist mutants (Fig. 2G',H',I',J'). These analyses indicate that patterning and differentiation of CNC cells are tightly linked to those of the skeletal muscle precursors, but they are dispensable for initial myogenic specification.
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), we analyzed the distribution of the cadherin mRNAs in the
CPM and CNC (Fig. 2L,M). The
expression patterns of both cadherin 6
(Fig. 2L') and cadherin
11 (Fig. 2M') (see also
Soo et al., 2002) were
severely altered in Twist mutants compared with the controls. Taken
together, these findings suggest that head muscle patterning is regulated
non-cell-autonomously by Twist, expressed by CNC cells. This regulation may
also involve cadherin molecules expressed by both CNC and mesoderm cells.
Wnt signaling has previously been implicated in the early stages of neural
crest development [e.g. neural crest induction and lineage specification
(Garcia-Castro et al., 2002;
Ikeya et al., 1997)].
Activation of the Wnt signaling pathway in mice by expressing a stabilized
ß-catenin in neural crest cells (using the Wnt1-Cre transgene)
promoted the development of sensory neurons, at the expense of other neural
crest derivatives (Lee et al.,
2004). Dlx5 is thought to be expressed in CNC-derived
skeletogenic progenitors (Holleville et
al., 2003); its expression was markedly reduced in BA1 and was
undetected in BA2 of E10.5 mutant [constitutively active
(CA)-ß-catenin/Wnt1-Cre] embryos compared with controls
(Fig. 3A,A'), supporting
the findings of Lee et al. (Lee et al.,
2004). Accordingly, we used this mouse model to determine how the
forced differentiation of CNC cells into the neuronal lineage affects skeletal
muscle formation in mouse embryos. In contrast to the typical expression of
capsulin in the core of the BAs in control embryos at E9.5
(Fig. 3B), capsulin expression
was severely mispatterned and upregulated in the mutant
(Fig. 3B'). Likewise, the
expression of Tbx1 was upregulated in the core of the mutants' BA1,
as compared with that of control embryos
(Fig. 3C,C').
We next explored myogenic determination and differentiation at later developmental stages in both control and CA-ß-catenin/Wnt1-Cre embryos. At E10.5, we detected traces of Myf5 expression in the mutants' BAs compared with the controls (Fig. 3D,D'); MyoD expression was undetectable in those mutants (Fig. 3E,E'). Consistent with the loss of MyoD in the head musculature, expression of Mgn in cranial muscles was also undetectable at E11.5 (Fig. 3F,F'). These findings indicate that abnormal CNC fate determination can lead to defects in patterning and differentiation of muscle precursors in the head. Taken together, our analyses in mouse models suggest that CNC cells are not necessary for the initial specification of the head muscle progenitors; however, they play key roles in regulating the patterning and differentiation of the cranial skeletal muscles during later stages of myogenesis.
Ablation of the CNC cells in chick embryos alters myogenic gene expression
To complement our mouse genetic studies, we extended this analysis to avian
embryos. Our previous finding in chick embryos that Myf5 was
expressed following CNC ablation (Tzahor
et al., 2003) led us to consider that CNC ablation in the chick
may impact the patterning and/or the kinetics of myogenesis (presumably
downstream of Myf5). We employed the CNC-ablation model in stage 8
chick embryos (Tzahor et al.,
2003) (Fig.
4A,A'). After 36-48 hours, embryos were subjected to in situ
hybridization for the muscle markers capsulin, Tbx1, Myf5 and
MyoD (Fig. 4).
Expression of Tbx1 and Myf5 in the BAs was upregulated and
expanded to fill the entire arch mesenchyme
(Fig. 4B,D). Capsulin
expression was detected between the BAs in the operated embryos
(Fig. 4C'). MyoD
was slightly upregulated in the proximal region of BA1 (maxilla) in these
embryos, whereas its expression in the distal arch (mandible) and in BA2 was
diminished (Fig. 4E').
Our findings indicate that the expression of the skeletal muscle markers was
maintained following CNC ablation in chick embryos. However, removal of the
CNC in chick embryos severely distorted the expression patterns of myogenic
genes. These results corroborate our findings in the mouse mutant embryos, and
suggest that the nature of these interactions is conserved in vertebrates.
CNC cells influence mesoderm migration and axial registration
Because both CNC and CPM migrate en route to the BAs via overlapping
migratory pathways (Hacker and Guthrie,
1998; Trainor and Tam,
1995), we explored the idea that CNC cells might influence the
migration of mesodermal cells. In order to gain insights into these migratory
events, DiI was used as a lineage tracer to unilaterally label the CPM at
stage 8 in both control and CNC-ablated embryos
(Fig. 5A,B). In control
embryos, DiI-labeled mesodermal cells migrated in a typical crescent-shaped
pattern into BA1, whereas in CNC-ablated embryos some mesoderm cells failed to
enter BA1 (Fig. 5B').
Cell death was not observed in the CNC-ablated embryos, indicating that these
cells were not lost because of increased apoptosis (data not shown). In view
of the robust expression of mesodermal markers in BA1 of CNC-ablated embryos
(see Tbx1 and Myf5 expression data,
Fig. 4), and the reduced
migration of mesodermal cells into BA1
(Fig. 5), we speculated that in
the absence of CNC cells, other mesodermal cells are able to enter BA1.
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To confirm these findings by another approach, we employed quail-chick
transplantations in conjunction with fate mapping labeling
(Fig. 5E,F,G,H). Quail CPM
cells at the level of rhombomere 4, which normally migrate to BA2
(Hacker and Guthrie, 1998;
Trainor and Tam, 1995), were
labeled with DiI, transplanted into a stage-matched chick embryo, and left to
develop for an additional 24 hours. Cells from the control graft migrated into
BA2, as shown in both whole-mount embryos and sections stained with
quail-specific QCPN antibodies (Fig.
5F). In the absence of the CNC, CPM cells failed to enter BA2
(Fig. 5H). Instead, some
grafted cells shifted anteriorly toward BA1
(Fig. 5H'). In addition,
QCPN-labeled cells in the ablated embryo were located more dorsally, compared
with quail-derived mesoderm cells in control embryos
(Fig. 5, compare panels
F'' with H'). Taken together, these different approaches
demonstrate that CNC cells regulate the migration and axial registration of
CPM cells en route to the BAs.
An additional mechanism that could account for the upregulation of Myf5 in the BAs of CNC-ablated embryos (Fig. 4) involves increased myoblast proliferation. To explore how ablation of the CNC affects mesoderm proliferation, we performed in situ hybridization for Myf5 followed by BrdU immunostaining on transverse sections (Fig. 6A-D). In the trunk, Myf5-expressing cells in the myotome were mostly BrdU-negative (Fig. 6A''), indicating that these cells underwent myogenic differentiation. In a similar manner, myogenic cells in the core of the BAs seemed to be BrdU-negative in control embryos (Fig. 6B'', quantified in D). In sharp contrast, BrdU staining in the myogenic cores of CNC-ablated BAs was significantly increased (Fig. 6C'',D). Similar results were obtained using immunofluorescence analysis for Myf5 and BrdU in the myotome (Fig. 6E-G''''), in BA2 of control (Fig. 6F-F''''), or in CNC-ablated embryos (Fig. 6G-G''''). These analyses revealed that there are more Myf5+/BrdU+ cells in BA2 of CNC-ablated embryos than in the control (note the yellow spots in Fig. 6G''''). We propose that in the absence of CNC cells, proliferating myoblasts (Myf5+/BrdU+) accumulate in the BAs. These cells apparently fail to exit the cell cycle and thus skeletal muscle differentiation may be reduced or delayed.
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; Waldo et al.,
2005). Along these same lines, significant upregulation of
Fgf8 could be observed in the ventral (distal) ectoderm of BA1 (in
both maxilla and mandible) and in the BA2 of the CNC-ablated embryos
(Fig. 6H,H',I,I').
Furthermore, application of Fgf8 protein to CPM explants in vitro reduced
myogenic differentiation (Fig.
6J). It appears that other signaling pathways were deregulated as
a result of CNC ablation, as indicated by the moderate upregulation of both
Frzb and Bmp4 in the BA ectoderm (data not shown). We
propose that these changes in signaling molecules in the BA ectoderm following
CNC ablation in chick embryos can increase myogenic cell proliferation,
resulting in delayed or reduced differentiation of the branchiomeric
musculature. To gain a deeper understanding of the effect(s) of CNC cells on mesodermal cell proliferation/differentiation, we analyzed control and CNC-ablated chick embryos, using a combined in vitro-in vivo approach (Fig. 6K). Ablation of the CNC was performed at stage 8 and the embryos were left to develop in ovo until stage 10. Explants of the CPM (including the ectoderm and endoderm) were then dissected from these embryos and assayed by RT-PCR after 4 days. The reduced levels of the CNC markers Noelin and Frzb indicated that the ablation was successful. MyoD, Mgn and MHC were reduced in the CNC-ablated embryos, compared with their levels in the controls, whereas Myf5 was slightly upregulated in the ablated embryos (Fig. 6K), in line with the upregulation of Myf5 after CNC ablation in vivo (Fig. 4). These results further suggest that CNC cells exert their effect on myogenic differentiation downstream of Myf5.
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)].
To study the impact of CNC cells on head muscle differentiation in vivo, we
followed this timetable of differentiation by performing immunofluorescence
staining for desmin, MHC and F-actin on sections from both control and
CNC-ablated chick embryos at E4.5-5. In control embryos, desmin and MHC were
detected in BA1-derived jaw muscle (e.g. intermandibular and mandibular
adductor muscles, Fig. 7B,D,F).
Higher magnifications of these sections revealed a scaffold of skeletal muscle
progenitors, with their subsequent organization into myofibers in the control
embryos (Fig.
7B'',D'',F'',H'',J''). However, in the
CNC-ablated embryos, we observed a dramatic reduction in myogenic
differentiation and overall myofiber organization in the BA1-derived jaw
muscles was severely disrupted (Fig.
7C'',E'',I'',K''). Furthermore, in some of
these CNC-ablated embryos, the BA musculature was missing
(Fig. 7G'). Thus, normal
cranial skeletal muscle differentiation and myofiber architecture are
regulated by the CNC. Taken together, our findings demonstrate that in vertebrates, although early myogenic specification is CNC-independent, the patterning, migration, proliferation and differentiation of skeletal muscle progenitors are all influenced by CNC cells (Fig. 7L). Furthermore, the early effects of CNC cells on myoblast migration, proliferation and the onset of differentiation could impact upon muscle fiber morphogenesis at later developmental stages. In summary, our results demonstrate that during vertebrate embryogenesis, CNC cells play varying roles in the regulation of skeletal muscle precursors during craniofacial development.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kelly et al., 2004;
Lu et al., 2002;
Mootoosamy and Dietrich, 2002;
Noden et al., 1999;
Rudnicki et al., 1993;
Tajbakhsh et al., 1997;
Tzahor et al., 2003;
von Scheven et al., 2006). In
the present study, our results have provided insights into the extrinsic
regulatory mechanisms that affect head muscle formation, by focusing on the
crosstalk between CNC and CPM during myoblast specification, migration,
patterning and differentiation.
In vertebrates, CNC cells contribute to the majority of the skeletal and
connective tissue within the head but not the muscle fibers, which originate
from the mesoderm. However, the tight anatomical proximity between CNC and
skeletal muscle precursors, as well as experimental evidence
(Couly et al., 1992;
Ericsson et al., 2004;
Kontges and Lumsden, 1996;
Noden, 1983a;
Noden, 1983b;
Olsson et al., 2001;
Schilling and Kimmel, 1997),
have led to suggestions that CNC cells play an indirect role during head
muscle formation. Explicitly how CNC cells control head muscle patterning, and
whether CNC cells promote myogenic differentiation, are issues that remain
unresolved. Although our previous study provided evidence that in the chick,
CNC cells promote myogenic differentiation in vitro
(Tzahor et al., 2003), a
recent report by von Scheven et al. suggested that CNC cells are dispensable
for early cranial muscle differentiation
(von Scheven et al.,
2006).
Our latest findings concerning head skeletal muscle specification, patterning and differentiation in three mouse genetic models: complete loss of a specific population of CNC cells (Hoxa1/Hoxb1-3'RARE), along with defects in CNC cell differentiation and migration (CA-ß-catenin/Wnt1-Cre and Twist), in combination with loss-of-function experiments in the chick, demonstrate that CNC cells regulate skeletal muscle patterning and differentiation in vivo. These results highlight the multiple and dynamic interactions between mesoderm and neural crest cells, crucial to our understanding of head muscle development as well as craniofacial evolution, diversity and pathogenesis.
Myogenic specification
In this study, we show that both capsulin and Tbx1 were expressed
in BA2 of Hoxa1/Hoxb1-3'RARE double
mutants, despite the lack of CNC cells. We further demonstrate that early
myogenic markers are expressed (although mispatterned) in CNC-ablated chick
embryos. These findings, in combination with other studies in amphibians
(Ericsson et al., 2004;
Olsson et al., 2001) and chick
(Tzahor et al., 2003;
von Scheven et al., 2006),
strongly support the idea that CNC cells are not necessary for the early
specification of the skeletal muscle lineage in vertebrates. However, our data
clearly demonstrate that CNC cells are involved in diverse aspects of cranial
muscle patterning following specification of the myogenic cells.
Head muscle patterning
Our analyses of the skeletal muscle markers in Twist mutants
demonstrated pronounced defects in the expression of myogenic genes in the
head region. These results imply that the location and/or the differentiation
of the CNC affect the patterning of the adjacent skeletal muscle markers in a
non-cell-autonomous manner. We demonstrated that Twist is expressed
in CNC cells between E9-E10.5; however, we cannot rule out the possibility
that there is a transient expression of Twist in the head mesoderm
prior to CNC delamination. Tissue-specific knockout of Twist in CNC
cells should clarify its exact, direct or indirect, impact on myogenesis.
How does Twist, a bHLH transcription factor expressed in the CNC cells,
affect skeletal muscle formation in the adjacent mesodermal cells? One
possibility is that Twist might regulate the cell adhesion properties of the
CNC cells, and these cells, in turn, could influence skeletal muscle
patterning. Indeed, it was recently shown that Twist directly regulates the
expression of members of the cadherin family of adhesion molecules during
tumor development and metastasis (Yang et
al., 2004). The observation that cadherin molecules, normally
expressed by both mesoderm and CNC cells, were altered in Twist
mutants may provide a clue as to the nature of the molecular mechanisms
underlying the crosstalk between the CNC and skeletal muscle precursors.
Interestingly, there is some evidence that myogenesis can be regulated by
cell-cell contact mediated by the cell surface receptors CDO and BOC, both of
which are related to the cadherin family
(Cole et al., 2004).
Using a Cre/loxP system in which a constitutively active form of
ß-catenin (Harada et al.,
1999) was specifically expressed in neural crest cells, it was
shown that the Wnt/ß-catenin signaling pathway induced sensory
neurogenesis by acting instructively on neural crest progenitors while, at the
same time, blocking other CNC-derived cell types
(Lee et al., 2004). We show
that capsulin, Tbx1 were aberrantly expressed in the BAs of
CA-ß-catenin/Wnt1-Cre mutants, whereas the myogenic
markers, MyoD and Mgn, were not detected in these mutant
embryos. These findings demonstrate that fate specification of CNC progenitors
is tightly coupled to the patterning and differentiation of the skeletal
muscle progenitors.
CNC ablation experiments in the chick corroborated our genetic studies in the mouse, by showing that in the absence of CNC cells severe muscle patterning defects were seen. In addition, mesoderm cells migrated in an abnormal manner in the CNC-ablated embryos. Based on these results, we propose that in the absence of the CNC, the axial registration of the CPM is disrupted in either an active or a passive manner.
It is well-documented that the axial registration between the CNC and the
CPM is maintained as both cell populations remain coherent throughout their
migration and subsequent musculoskeletal morphogenesis
(Evans and Noden, 2006;
Grammatopoulos et al., 2000;
Hacker and Guthrie, 1998;
Kontges and Lumsden, 1996;
Trainor and Tam, 1995). The
CNC cells, which anatomically envelop the mesodermal core within the BAs,
create barriers to mesodermal cell movement, thus preventing the mixing of
mesoderm cells from different axial levels
(Noden and Trainor, 2005;
Trainor and Tam, 1995). In the
absence of these CNC barriers, it is conceivable that abnormal migration of
mesoderm cells could occur, resulting in mixing of the normally separate BA
streams, and a corresponding disruption of the axial registry. The abnormal
migratory behaviors of CPM cells in the chick model could be attributed to the
lack of a steric hindrance by the CNC cells, or because of their active
(anterior) migration in response to signals from BA1.
Analogous to the head muscles, limb muscle patterning is dependent upon
signals from the surrounding skeletogenic mesenchyme derived from the lateral
plate mesoderm (Kardon et al.,
2003), although in the head, most of the skeletogenic mesenchymal
cells are of CNC origin. Ectopic activation of the Wnt/ß-catenin pathway
in limb mesoderm induced ectopic limb muscles in regions where myotubes do not
normally differentiate (Kardon et al.,
2003). Conversely, in the head we showed that Frzb, a Wnt
antagonist, promoted MyoD expression in vitro and in vivo
(Tzahor et al., 2003). Thus,
muscle patterning is extrinsically controlled by the surrounding mesenchymal
cells in both the head and the limb, although these signals seem to play
distinct roles in each compartment.
Skeletal muscle proliferation and differentiation
We previously demonstrated that CNC induced myogenic differentiation in CPM
explants, although in vivo ablation of CNC cells did not significantly affect
the expression of Myf5 (Tzahor et
al., 2003; von Scheven et al.,
2006). It remains possible that CNC cells could affect myogenic
differentiation downstream of Myf5. We now show that Myf5 expression
(RNA and protein) is upregulated following CNC ablation in chick embryos.
Furthermore, in the CA- ß-catenin/Wnt1-Cre model,
myogenesis seems to be initiated in BA1-2 (low levels of Myf5 were
detected in these areas); however, MyoD and Mgn, which are
downstream genes, failed to be activated in these mutants. These findings are
consistent with a previous study demonstrating that Myf5 expression
during limb myogenesis correlates with myoblast proliferation, whereas MyoD
acts at a later developmental stage, during post-mitotic differentiation
(Delfini et al., 2000).
Along these same lines, we further demonstrate that the upregulation of Myf5 could be linked to increased cell proliferation (observed by Myf5+/BrdU+ co-staining) in the BAs of the CNC-ablated embryos. This finding suggests that CNC cells regulate cranial myogenesis by specifically influencing the rate of cell proliferation/differentiation within the myogenic core. Thus, the progression of myoblasts through differentiation appears to be controlled by the CNC to ensure myogenesis at an appropriate place and time during craniofacial development. In the absence of CNC, some muscle precursor cells presumably fail to exit the cell cycle and to undergo terminal myogenic differentiation.
Interestingly, ablation of the cardiac neural crest cells, a distinct
population of neural crest cells originating from the caudal hindbrain
(Kirby et al., 1983), resulted
in a similar increase in cell proliferation
(Waldo et al., 2005), which
was attributed to increased Fgf8 signaling in the ventral pharynx
(Hutson et al., 2006). We show
that Fgf8 is upregulated in the ectoderm of the BAs in the
CNC-ablated embryos. This observation is in line with our in vitro results and
in vivo Fgf8 bead application (von Scheven
et al., 2006), which demonstrate the reduced myogenic
differentiation capacity of this signaling pathway. We propose that CNC
ablation induces Fgf8 upregulation in the BA ectoderm. This, in turn,
increases cell proliferation and delays differentiation. Furthermore, in a
striking similarity to the cranial mesoderm, mesoderm cells from the secondary
heart field failed to migrate into the outflow tract after cardiac neural
crest ablation in chick embryos (Waldo et
al., 2005). Thus, failure of mesoderm precursors to migrate
ventrally at the appropriate time resulted in ectopic sites of cardiac
(Waldo et al., 2005) and
skeletal muscle differentiation (this study). We suggest that CNC-dependent
regulation of mesoderm proliferation and migration (presumably mediated by
Fgf8 signaling) constitutes a general regulatory mechanism during vertebrate
development.
The increased Myf5+/BrdU+ co-staining in the head
mesoderm compared with the trunk is consistent with the delayed
differentiation of the head versus the trunk musculature
(Noden et al., 1999).
Likewise, it has been suggested that the head mesoderm expresses high levels
of putative negative regulators for myogenic differentiation
(Bothe and Dietrich, 2006).
Indeed, we observed increased expression of capsulin [and MyoR (also
known as Msc - MGI), data not shown] in the CNC-ablated chick and
mouse embryos. The pronounced increase in cell proliferation following CNC
ablation could explain the significant reduction in late muscle
differentiation markers. Immunofluorescence analyses of desmin and MHC in
CNC-ablated chick embryos as well as MyoD and Mgn expression
in CA-ß-catenin/Wnt1-Cre mouse mutants indicate that
myogenic differentiation, as well as myofiber architecture and positioning, is
regulated by CNC cells.
In summary, our study on craniofacial muscle development in mouse and chick embryonic models has clarified the extent to which the myogenic program is controlled by extrinsic environmental signals. We provide direct evidence that CNC cells play diverse and crucial roles during skeletal muscle formation in vertebrates (Fig. 7L). The appearance of early myogenic markers following surgical ablation of the CNC in chick embryos, or genetic ablation of CNC cells in mouse embryos, shows that early specification of the skeletal muscle lineage is not dependent upon the presence of CNC cells. However, the subsequent migration of skeletal muscle progenitors, along with their patterning, proliferation and differentiation, are tightly controlled by CNC cells. Our findings also demonstrate that other tissues and signals are capable of promoting skeletal muscle differentiation in the head, in the absence of CNC cells. We therefore propose that CNC cells provide guidance cues that enable muscle precursor cells to migrate to the correct positions in the head, and to resume myogenic differentiation in a coordinated manner.
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ACKNOWLEDGMENTS |
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REFERENCES |
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