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First published online 15 November 2006
doi: 10.1242/dev.02693
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1 Institute of Biosciences and Technology, Texas A&M System Health Science
Center, 2121 Holcombe Blvd, Houston, TX 77030, USA.
2 Department of Craniofacial Development, King's College, London Guy's Tower,
London SE1 9RT, UK.
3 Program in Cardiovascular Sciences, Department of Medicine, Baylor College of
Medicine, One Baylor Plaza, Houston, TX 77030, USA.
* Author for correspondence (e-mail: jmartin{at}ibt.tamhsc.edu)
Accepted 12 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Homeobox, Branchiomeric muscle, Mouse, Chick
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Moreover, investigation of transcriptional regulation
of Myf5, one of the four muscle regulatory factors (MRFs), revealed
separable elements controlling Myf5 expression in trunk and
craniofacial muscle (Carvajal et al.,
2001; Hadchouel et al.,
2003). Mrf4 (Myf6 - Mouse Genome Informatics),
another MRF, appears to be dispensable for head muscle development but is
crucial in trunk muscle (Kassar-Duchossoy
et al., 2004). The paired domain factors, Pax3 and Pax7, have
important functions in trunk muscle development but are not expressed in head
muscle (Tajbakhsh et al.,
1997).
Classically, two sources have been shown to contribute to branchiomeric and
ocular muscle, cranial paraxial mesoderm (CPM) and the prechordal plate
mesoderm (Noden and Francis-West,
2006; Chai and Maxson,
2006). Recent work has revealed overlap in the progenitors that
contribute to branchiomeric and cardiac muscle. For example, lineage tracing
in mouse embryos revealed the existence of the second cardiac lineage, derived
from splanchnic mesoderm, that contributes to both cardiac and branchiomeric
muscle (Buckingham et al.,
2005). Fate-mapping studies in mouse and chick embryos revealed
that CPM, in addition to branchiomeric muscle, also contributes to the cardiac
outflow tract (OFT) (Tirosh-Finkel et al.,
2006; Trainor et al.,
1994). The significance of separate precursor populations, with
distinct developmental histories, in branchiomeric muscle development is
unknown.
Heterotopic grafting experiments in mouse embryos revealed substantial
plasticity in the CPM, as transplanted CPM was competent to assume the
characteristics of the recipient site
(Trainor et al., 1994;
von Scheven et al., 2006a).
This observation is consistent with the demonstration that environmental cues
are crucial for the normal diversification of CPM. Bmp4 was shown to
promote cardiac differentiation and inhibit skeletal muscle differentiation
(Tirosh-Finkel et al., 2006).
Similarly, Fgf8 was shown to promote branchiomeric muscle development while
inhibiting extraocular muscle (EOM) development
(von Scheven et al., 2006a).
These findings indicate that signaling from surrounding tissues determines the
fate of progenitor cells within the CPM.
Less is known about the cell-autonomous mechanisms regulating branchiomeric
muscle development. Tbx1 has been shown to be required for
branchiomeric muscle and cardiac OFT development. In the OFT, Tbx1
regulates proliferation of progenitor cells by regulating expression of Fgf
ligands (Vitelli et al., 2002;
Xu et al., 2004). A similar
mechanism may underlie Tbx1-mediated regulation of branchiomeric muscle
development (Kelly et al.,
2004). Capsulin and MyoR (Tcf21 and Msc, respectively - Mouse
Genome Informatics), two basic helix-loop-helix (bHLH) transcription factors
that mark undifferentiated progenitor cells, are necessary for branchiomeric
muscle development (Lu et al.,
2002; von Scheven et al.,
2006b). Mice that are double mutant for MyoR and capsulin
lack a subset of first branchial arch-derived muscles, such as the temporalis,
masseter and pterygoids. MyoR and capsulin probably function as
survival factors in differentiating head muscle, although there may also be a
migration defect in MyoR; capsulin mutants.
Pitx2 is a paired-related homeobox gene mutated in Rieger syndrome
type I, an autosomal dominant, haploinsufficient disorder that includes tooth
anomalies, anterior segment eye defects and facial dysmorphologies as cardinal
features (Diehl et al., 2006;
Gage et al., 1999;
Kitamura et al., 1999;
Lin et al., 1999;
Lu et al., 1999;
Semina et al., 1996).
Pitx2 also plays an essential role in the late aspects of left right
asymmetry (LRA) and cardiac OFT development
(Ai et al., 2006;
Kioussi et al., 2002). Recent
work has shown that the Pitx2 OFT phenotype can be traced to a defect
in cardiac cells derived from the second cardiac lineage
(Ai et al., 2006). In this
work, we investigated the role of Pitx2 in branchiomeric muscle.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
Pitx2null allele is a 4 kb deletion that removes
Pitx2 exons 5 and 6 and the intervening intron
(Lu et al., 1999). The
Pitx2hypo allele is a weak hypomorphic allele, previously
called Pitx2 
ab, that contains a deletion of the
Pitx2a and Pitx2b isoforms and has reduced Pitx2c
function (Liu et al., 2001).
The ß-catenin conditional null allele has been described
(Brault et al., 2001).
Immunohistochemistry
Embryos were fixed, dehydrated and embedded in paraffin blocks and
sectioned at 5 µm. The slides were deparaffinized and rehydrated according
to standard protocols. Antigen retrieval was performed by heating the slides
in a 95°C water bath for 30 minutes in 0.01 mol/l sodium citrate (pH 6.0)
followed by slowly cooling down to room temperature. Sections were blocked in
3% H2O2 in methanol for 10 minutes at room temperature.
The primary antibody used was mouse anti-chicken polyclonal antibody (from
Developmental Studies Hybridoma Bank, The University of Iowa) diluted in 1:100
and incubated overnight. The Zymed Histostain-Plus kit was used according to
the manufacturer's protocol.
Whole-mount LacZ staining and section
After dissection, the embryos were fixed in the fresh-made fixing buffer
(0.2 glutaraldehyde, 2% formalin, 5 mmol/l EGTA, 2 mmol/l MgCl2, in
0.1 mol/l Na2HPO4 pH 7.3) for 20-30 minutes. Following
three washes with the rinse buffer (0.1% sodium deoxycholate, 0.2% NP40, 2
mmol/l MgCl2, in 0.1 mol/l NaH2PO4 pH 7.3),
the samples were stained with the staining buffer (1 mg/ml X-gal, 5 mmol/l
potassium ferricyanide, 5 mmol/l potassium ferrocyanide, in rinse buffer)
until the optimized results appeared. After removing the staining, the embryos
were then rinsed with 1 x PBS for 5 minutes. All the above procedures
were performed at room temperature. The embryos were finally post-fixed with
10% formalin and could be stored in this buffer at 4°C. The
LacZ-stained embryos were dehydrated in ethanol and isopropanol,
embedded in paraffin blocks and sectioned at 10 µm.
Whole-mount and section in situ hybridization
Whole-mount in situ hybridization was performed as previously described
(Lu et al., 1999). The mouse
Pitx2 probe was an exon6 fragment that hybridizes to all
Pitx2 isoforms. The myogenin, MyoD, Tbx1 and MyoR
probes have been previously described
(Kelly et al., 2004). In situ
hybridization to whole chick embryos was carried out as described by
Francis-West et al. (Francis-West et al.,
1995). 35S-in situ hybridization to tissue sections was
performed on 7 µm wax sections as described
(Francis-West et al., 1994).
The Pitx2 probe is described by Yu et al.
(Yu et al., 2001) and the
chick MyoD clone by Lin et al. (Lin et
al., 1989).
Chick embryology
Fertilized Ross White chicken eggs were supplied by Henry Steward & Co.
Ltd (Lincolnshire, UK) and were incubated at 37±1°C. Embryos were
staged according to Hamburger and Hamilton
(Hamburger and Hamilton,
1951). Stage 20/21 mandibular primordia micromass cultures were
prepared as described (Anakwe et al.,
2003) and were plated in the presence of high titer RCASBP viruses
encoding an activated version of Pitx2 or a dominantnegative Pitx2 construct
(Yu et al., 2001). Micromasses
were cultured for 3 days, fixed briefly in ice-cold methanol and immunostained
with the panmyosin antibody, A4.1025 (1 in 100), and A4.840 (1 in 50), which
recognizes cells expressing the slow MyHC isoforms SM3 and SM1 (from the
Developmental Studies Hybridoma Bank). This was followed by incubation with
horse anti-mouse IgG (
-specific) conjugated to FITC (Vector; 1:400) and
donkey anti-mouse IgM (µ-specific) conjugated to Cy3 (Jackson; 1:800) for
at least 1 hour at room temperature. Following three PBS washes for 5 minutes,
cultures were mounted under coverslips with PBS:glycerol (1:9) with 0.1%
phenylenediamine as an antifade reagent. Values shown are the mean and
standard error of the mean of at least nine cultures from three independent
experiments. The data was analysed using Student's t-test.
Histology and apoptosis
For histology, embryos were fixed overnight in Bouin's fixative or buffered
formalin, dehydrated through graded ethanol and embedded in paraffin. Sections
were cut at 7-10 µm and stained with H&E. For TUNEL, embryos were first
stained for LacZ using the whole-mount protocol, then embedded in
paraffin and sectioned. TUNEL staining was performed according to the
manufacturer's protocol (Serologicals Corporation).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mucchielli et al.,
1997). At 9.5 dpc, Pitx2 was expressed in
mesoderm-derived, core cells of the first branchial arch
(Fig. 1A,B)
(Mucchielli et al., 1997). In
chick embryos, Pitx2 was expressed in head mesoderm
(Fig. 1C,F) and in the core
mesoderm of the branchial arch (Fig.
1D,E). More dorsal sections indicated that Pitx2 was also
expressed in periocular mesenchyme and extraocular muscles
(Fig. 1G,H). As in the mouse,
Pitx2 was also expressed in oral ectoderm
(Fig. 1E). Pitx2
expression colocalized with the muscle marker MyoD in branchial arch
mesoderm and extraocular muscle (Fig.
1D,E,G,H). Taken together, these findings suggest that the
function of Pitx2 in branchiomeric muscle is probably conserved between mouse
and chick.
Pitx2 function is required for MyoR but not Tbx1 expression in first branchial arch muscle precursors
We next looked at markers of muscle development in
Pitx2null and Pitx2 hypomorphic mutant embryos.
myogenin encodes one of the four bHLH MRFs and is required for muscle
development (Hasty et al.,
1993). Moreover, myogenin is a late muscle marker that is
important for muscle differentiation. In Pitx2 control embryos,
myogenin was highly expressed in the branchial arch core cells, while in
Pitx2 hypomorphic embryos, myogenin was greatly reduced
(Fig. 2A,B). Importantly, in
Pitx2null-/- embryos myogenin was absent,
indicating that muscle precursors in the branchial arch core fail to activate
myogenin, an essential regulator of muscle differentiation
(Fig. 2C). MyoD is another MRF
and is a marker of committed myoblasts. Similar to myogenin, we found that
MyoD expression was absent in the Pitx2null
mutant embryos, indicating that myoblasts failed to be specified in the first
branchial arch of Pitx2null mutants
(Fig. 2D,E). It is also
possible that defects in myogenin and MyoD expression result from a
loss of progenitor cells (see below).
Previous studies have identified a requirement for the bHLH transcription
factors MyoR and capsulin in development of first branchial arch muscle
(Lu et al., 2002). In
addition, MyoR marks undifferentiated muscle precursor cells
(von Scheven et al., 2006b).
Moreover, in the MyoR; capsulin double-mutant embryos, elevated
apoptosis was detected in the core branchial arch cells. In
Pitx2null mutant embryos, MyoR failed to be
expressed in the core cells of the first branchial arch
(Fig. 2F,G). Previous work has
also established that the T-box transcription factor Tbx1 is
expressed in core cells of the branchial arches and is required for first
branchial arch muscle development, although MyoR continues to be
expressed in the Tbx1 mutants
(Kelly et al., 2004). In
Pitx2null mutants, Tbx1 was still expressed in
the first branchial arch, although in a reduced expression domain
(Fig. 2H,I). In addition,
expression of Pitx2 in Tbx1 null mutants was also unaffected
(Fig. 2J,K). Taken together,
these findings indicate a defect in undifferentiated muscle progenitors in
Pitx2 mutant embryos.
Committed myoblasts are absent in the Pitx2 null mutant first branchial arch
The expression analysis suggested that specification of myoblasts was
defective in Pitx2null mutant embryos. To study this
question in more detail, we performed lineage tracing with a
Myf5cre allele that marks cells that have activated
expression of Myf5 in committed myoblasts
(Tallquist and Soriano, 2003).
This is a very sensitive method for following the developmental progression of
Myf5-expressing descendents. Induction of recombination at the
Rosa 26 reporter locus by cre recombinase is heritable and
irreversible and so is a reliable method for performing lineage tracing in
mouse embryos (Soriano, 1999).
Myf5 expression is activated in the branchial arches at approximately
9.25 dpc. In Pitx2null mutants, the number of
LacZ-marked Myf5 descendents was drastically reduced in the
first branchial arch, consistent with the expression data indicating that
myoblast specification in core cells of the first branchial arch was defective
(Fig. 3A,B).
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;
Trainor et al., 1994). In
Pitx2null mutants, Myf5cre-marked
cells were excluded from the first branchial arch but were still present in
the dorsally located migrating myoblasts
(Fig. 3C,D). By 11.5 dpc,
Myf5cre-marked cells had started to form the masseter
muscle mass spanning the maxilla and mandible. In the
Pitx2null embryos, a small group of
Myf5cre-marked cells was apparent, indicating that a
reduced number of cells in the mandibular process activated Myf5
expression (Fig. 3E,F). It is
also notable that Myf5cre-marked cells, contributing to
extraocular muscles, were present but failed to localize properly around the
developing eye (Fig. 3D,F).
Taken together, these findings indicate that specified branchiomeric myoblasts
are drastically reduced in Pitx2null mutant embryos. We
interpret these data to indicate a defect in muscle specification, although it
should be noted that defective migration or a developmental delay may also
contribute to the phenotype.
Pitx2 descendents move into the first branchial arch but fail to form mature muscle
We performed a lineage-tracing experiment with the
Wnt1cre transgenic line that directs cre activity in the
neural crest that surrounds, and therefore outlines, the mesoderm in the
branchial arch (Chai et al.,
2000). In control embryos, LacZ-negative mesoderm-derived
cells were outlined by blue, neural crest derivatives
(Fig. 4A). In the
Pitx2null mutant embryo, there was a reduction in the
number of mesoderm cells; however, core mesoderm was present in the
Pitx2null mutant (Fig.
4A,B).
To establish more firmly that Pitx2 descendents were present in
the first branchial arch of Pitx2 mutant embryos, we used the
Pitx2cre allele to mark Pitx2 descendents
(Liu et al., 2002). This
strategy also marks cells that are fated to express Pitx2. In control
Pitx2creneo;R26R embryos, we found that
Pitx2 descendents contributed to the core mesoderm of the first
branchial arch (Fig. 4C). In
Pitx2null embryos, Pitx2 descendents were still
present in the first branchial arch core, although in reduced numbers
(Fig. 4D).
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To investigate the possibility that in Pitx2 mutants, the core mesoderm cells of the branchial arch underwent apoptosis, we performed TUNEL analysis on embryos in which the Pitx2 lineage was LacZ-marked.
In agreement with previous observations, control core mesoderm had little
cell death (Fig. 4G)
(Lu et al., 2002). By
contrast, in Pitx2null embryos, LacZ-marked
Pitx2 mutant descendents were TUNEL-positive, indicating that
Pitx2null cells were undergoing cell death
(Fig. 4G,H). Together these
data indicate that Pitx2 is required for development of the first
branchial arch muscle. Moreover, Pitx2 mutant descendents are present
in the branchial arch at 11.5 dpc, but they undergo apoptosis and are gone by
16.5 dpc.
Pitx2 is required for splanchnic mesoderm to contribute to branchiomeric muscle
Previous work revealed that a Mef2c enhancer element specifically
directed LacZ expression in splanchnic mesoderm that contributed to
the cardiac OFT but was not expressed in branchiomeric muscle
(Dodou et al., 2004).
Subsequent experiments using this Mef2c enhancer to direct cre
activity indicated that descendents of the Mef2c-expressing
splanchnic mesoderm contributed to branchiomeric muscle
(Fig. 5A,B)
(Verzi et al., 2005). Thus,
the Mef2c AHF cre provides a valuable reagent to dissect the role of
splanchnic mesoderm in branchiomeric muscle.
We used the Mef2c AHF cre to trace the splanchnic mesoderm lineage in Pitx2null mutant embryos. At 9.5 dpc, Mef2c AHF descendents were drastically reduced in the Pitx2null mutant embryos (Fig. 5C,D). One day later, at 10.5 dpc, a few LacZ-positive cells were visible in the first branchial arch of Pitx2null mutant embryos (Fig. 5E,F). At 11.5 dpc, Mef2c AHF descendents were no longer detectable in the Pitx2null mutant embryos. At this late stage, we noted an abnormal dispersion of Mef2c AHF cre descendents in the caudal branchial arches (Fig. 5G,H). The significance of this is unclear and is currently under investigation. Together, these data reveal a defect in development of the splanchnic mesoderm component of branchiomeric muscle in Pitx2null mutant embryos.
Pitx2 autonomously promotes muscle expansion
Because Pitx2 regulates Fgf8 and Bmp4-signaling
pathways in branchial arch morphogenesis, we wanted to investigate the
cellautonomous role of Pitx2 in branchiomeric muscle
(Lu et al., 1999). We turned
to the chick embryo system because of its utility as an experimental system.
Furthermore, Pitx2 expression is highly conserved between mice and
chicks (Fig. 1) and
Pitx2 is expressed in the early chick cranial mesoderm before any
onset of myogenesis (Fig.
1C,F). Then at later stages of development, Pitx2
transcripts are found in all the cranial muscles derived from the unsegmented
cranial mesoderm - i.e. those found in the mandibular and hyoid arch and the
extraocular muscles (Fig.
1D,E,G,H).
Muscle precursor cells, including the surrounding ectomesenchyme but not
the ectoderm, were isolated from the developing mandible and infected with a
retrovirus expressing Pitx2a. This resulted in a statistically
significant elevation in the number of myosin-positive cells when compared
with control cells (Fig. 6A,B).
We next used a retrovirus expressing a dominantnegative form of
Pitx2a to decrease Pitx2 activity in muscle precursors. Primary
cultures with reduced Pitx2a activity had lower numbers of myosin-positive
cells, which was a statistically significant difference from the control
(Fig. 6A,B). We also assessed
whether Pitx2a had a differential effect on slow versus fast myocyte
differentiation by immunostaining with a slow MyHC antibody
(Hughes and Blau, 1992). This
showed that loss and gain of Pitx2a function affected the development
of both slow and fast myocytes (Fig.
6B). These data indicate that Pitx2a is necessary and
sufficient for myocyte development in the context of craniofacial
mesenchyme.
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; Zhang et al.,
2006). Lineage tracing with the Mesp1cre and R26R alleles revealed that the Mesp1-expressing lineage contributed efficiently to masseter muscle in the control (Fig. 7A). By contrast, in Pitx2 conditional mutants (Mesp1cre; Pitx2flox/null (f/n)) there was a deficiency in masseter development (Fig. 7B). Investigation of myogenin expression in the Mesp1cre; Pitx2f/n embryos also indicated that branchiomeric muscle was defective in Pitx2 conditional mutants (Fig. 7C,D). Sections with H&E staining (Fig. 7E-H) and immunohistochemistry with a muscle-specific myosin antibody of control and Mesp1cre; Pitx2f/n embryos also indicated that branchiomeric muscle was severely defective in Pitx2 mutants (Fig. 7I-L).
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; Verzi et al.,
2005), to conditionally ablate Pitx2 in the splanchnic
mesoderm component of branchiomeric muscle. We have recently shown that
conditional deletion of Pitx2 with Mef2c AHFcre resulted in
severe cardiac defects (Ai et al.,
2006). We used lineage tracing with R26R to follow the progression
of branchiomeric muscle progenitors in the control and Pitx2
conditional mutant embryos. At 9.5 dpc, the Mef2c AHF lineage was
clearly marked in the core cells of the first branchial arch, while in the
Pitx2 conditional mutant there was a deficiency in
LacZ-marked cells (Fig.
8A,B). Similarly, for 11.5 and 12.0 dpc embryos, we consistently
found a deficiency in the LacZ-marked Mef2c AHF lineage in
the Pitx2 conditional mutants
(Fig. 8C-F). It is notable that
in all embryos examined, we found a small contribution of LacZ-marked
cells in the Pitx2 conditional mutant embryos. This observation
suggests that the Mef2c AHF cre failed to completely inactivate
Pitx2 in branchiomeric muscle progenitors. It is also possible that
there is a subpopulation of muscle precursors that are Pitx2
independent. Nonetheless, taken together with the Mesp1cre
data, our findings indicate that Pitx2 has an autonomous function in
the development of branchiomeric muscle progenitors. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pitx2 function in muscle
Pitx2 is expressed in multiple muscle types, including extraocular
muscle, branchiomeric muscle, cardiac muscle and trunk skeletal muscle
(Ai et al., 2006;
Kitamura et al., 1999). The in
vivo function of Pitx2 in trunk skeletal muscle is poorly understood.
Previous experiments investigating Pitx2 in the C2C12 myoblast cell
line, derived from satellite cells of the adult leg, uncovered a direct role
for Pitx2 in regulating myoblast proliferation through a mechanism
mediated by the N-terminus of Pitx2a
(Kioussi et al., 2002). In the
heart, Pitx2 regulates proliferation of cardiomyocytes of the OFT
(Ai et al., 2006). In
extraocular muscle, it has been suggested that Pitx2 may directly regulate MRF
transcription (Diehl et al.,
2006).
Our data indicate that, in branchiomeric muscle, Pitx2 regulates
undifferentiated precursor cells and probably controls expression of genes
that are involved in muscle expansion and survival. Recent experiments
revealed a role for MyoR and capsulin in the survival of a subset of
first branchial muscle precursors (Lu et
al., 2002). The Pitx2null mutant branchiomeric
muscle precursors fail to express MyoR and undergo apoptosis. It is
notable that there is evidence in the pituitary that Pitx2 and the related
factor Pitx1 promote cell survival by regulating expression of Lhx3
(Charles et al., 2005;
Zhao et al., 2006). In
addition, the third member of the Pitx family, Pitx3, is
required for postnatal survival of midbrain dopaminergic neurons
(van den Munckhof et al.,
2003). The requirement for Pitx2 in undifferentiated
precursor cells contrasts with the function of Pitx2 in asymmetric
organ morphogenesis. In left right organ morphogenesis, Pitx2 activity is
needed in the organ primordium rather than in undifferentiated precursors
(Ai et al., 2006;
Shiratori et al., 2006). This
may reflect a difference in tissues that only express Pitx2c.
Pitx2 and Tbx1 in craniofacial muscle development
Tbx1 mutants have sporadic failure of craniofacial muscle
development with loss of Tlx1 and Fgf10 expression
(Kelly et al., 2004).
Moreover, Tbx1 has been suggested to directly activate Pitx2
in the second cardiac lineage by binding to an element upstream of exon 6
(Nowotschin et al., 2006).
Tbx1 was still expressed in undifferentiated cells of the
Pitx2null mutant branchial arch core mesoderm consistent
with the notion that Tbx1 is an upstream regulator of Pitx2.
However, Pitx2 was still expressed in Tbx1null
mutants, indicating that a simple epistatic relationship is unlikely. In
addition, by contrast to Pitx2null mutant embryos,
Tbx1 mutants continue to express MyoR in the branchiomeric
progenitors, further arguing against a linear, epistatic relationship
(Kelly et al., 2004).
Alternatively, it may be that Pitx2 and Tbx1 regulate
parallel pathways that may converge on common target genes. Pitx and
Tbx genes have been shown to coordinately regulate gene expression in
the pituitary. Pitx1 and Tpit (Tbx19) bind to
proximate but distinct recognition elements in the POMC promoter
(Lamolet et al., 2001). In
this system, Pitx1 synergized with Tbx19 but failed to
transcriptionally synergize with Tbx1, suggesting that
cell-type-specific co-factors may be required for any potential synergism
between Pitx2 and Tbx1 in branchiomeric muscle progenitors
(Lamolet et al., 2001). It is
notable that in the zebrafish mutant van gogh, which carries a mutant
allele of Tbx1, muscle expression of endothelin 1 (edn1) is
reduced (Piotrowski et al.,
2003). In Pitx2null mutants, edn1
expression is reduced in the oral ectoderm, suggesting the possibility that
the Tbx1 and Pitx2-mediated pathway may converge on edn1
(Liu et al., 2003). Further
experiments will be required to investigate this idea.
Splanchnic mesoderm contribution to branchiomeric muscle
Similar to that described for the cardiac OFT, multiple lineages with
distinct developmental histories contribute to branchiomeric muscle. In the
heart, the primary heart field contributes to the linear heart tube, while the
second lineage is sequestered and moves into the OFT at a later stage. The
later addition of the secondary heart field is required for proper OFT
lengthening and morphogenesis (Buckingham
et al., 2005). In branchiomeric muscle, the addition of cells from
multiple lineages may play a similar role in controlling the size and pattern
of craniofacial muscle.
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ACKNOWLEDGMENTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pitx2a RCASBP retrovirus
showing myocytes that have been visualized with the pan MyHc antibody A4.1025.
(B) Bar chart quantifying these effects and also showing the effect on
slow (red bar) and fast (yellow bar) myocyte development.
*P<0.05.
