Craniofacial muscle, comprised of branchiomeric (branchial arch
muscle) and extraocular muscle, has distinct origins and
developmental regulatory mechanisms from that of the trunk
muscle. For example, Wnt signaling has been shown to promote
trunk skeletal muscle differentiation, while inhibiting craniofacial
muscle development (Tzahor et al., 2003). 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. MyoRand capsulin
probably function as survival factors in differentiating head muscle,
although there may also be a migration defect in MyoR; capsulin
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
Pitx2 promotes development of splanchnic mesoderm-
derived branchiomeric muscle
Feiyan Dong1, Xiaoxia Sun1, Wei Liu1, Di Ai1, Elizabetha Klysik1, Mei-Fang Lu1, Julia Hadley2, Laurent Antoni2,
Li Chen1,3, Antonio Baldini1, Pip Francis-West2and James F. Martin1,*
Recent experiments, showing that both cranial paraxial and splanchnic mesoderm contribute to branchiomeric muscle and cardiac
outflow tract (OFT) myocardium, revealed unexpected complexity in development of these muscle groups. The Pitx2 homeobox
gene functions in both cranial paraxial mesoderm, to regulate eye muscle, and in splanchnic mesoderm to regulate OFT
development. Here, we investigated Pitx2 in branchiomeric muscle. Pitx2 was expressed in branchial arch core mesoderm and both
Pitx2 null and Pitx2 hypomorphic embryos had defective branchiomeric muscle. Lineage tracing with a Pitx2creallele indicated that
Pitx2 mutant descendents moved into the first branchial arch. However, markers of both undifferentiated core mesoderm and
specified branchiomeric muscle were absent. Moreover, lineage tracing with a Myf5creallele indicated that branchiomeric muscle
specification and differentiation were defective in Pitx2 mutants. Conditional inactivation in mice and manipulation of Pitx2
expression in chick mandible cultures revealed an autonomous function in expansion and survival of branchial arch mesoderm.
KEY WORDS: Homeobox, Branchiomeric muscle, Mouse, Chick
Development 133, 4891-4899 (2006) doi:10.1242/dev.02693
1Institute of Biosciences and Technology, Texas A&M System Health Science Center,
2121 Holcombe Blvd, Houston, TX 77030, USA. 2Department of Craniofacial
Development, King’s College, London Guy’s Tower, London SE1 9RT, UK. 3Program
in Cardiovascular Sciences, Department of Medicine, Baylor College of Medicine,
One Baylor Plaza, Houston, TX 77030, USA.
*Author for correspondence (e-mail: email@example.com)
Accepted 12 October 2006
cardiac cells derived from the second cardiac lineage (Ai et al.,
2006). In this work, we investigated the role of Pitx2 in
Our data uncover an evolutionarily conserved role for Pitx2 in
growth and survival of branchiomeric muscle progenitors. Both
conditional ablation of Pitx2in mouse embryos and manipulation of
Pitx2 dose in chick embryo primary cultures reveal an autonomous
Pitx2function in branchiomeric muscle precursors. Our findings also
show that MyoR fails to be expressed in Pitx2 mutants, indicating a
defect in undifferentiated muscle progenitors. Expression of Tbx1 is
preserved in Pitx2 mutant embryos suggesting that the Pitx2 and
Tbx1-mediated genetic pathways in branchiomeric muscle are
distinct. Taken together, our data reveal a crucial role for Pitx2 in
branchiomeric muscle development and reveal a branching of genetic
pathways upstream of the MRFs in branchiomeric muscle.
MATERIALS AND METHODS
Mouse alleles used in this study
The Pitx2flox, Pitx2nulland Pitx2hypoalleles have been described. Briefly, the
Pitx2floxallele contains LoxP sites flanking Pitx2exon5 and has been shown
to be a true conditional null allele (Gage et al., 1999). The Pitx2nullallele is
a 4 kb deletion that removes Pitx2 exons 5 and 6 and the intervening intron
(Lu et al., 1999). The Pitx2hypoallele is a weak hypomorphic allele,
previously called Pitx2 ?ab, that contains a deletion of the Pitx2aand Pitx2b
isoforms and has reduced Pitx2c function (Liu et al., 2001). The ?-catenin
conditional null allele has been described (Brault et al., 2001).
Embryos were fixed, dehydrated and embedded in paraffin blocks and
sectioned at 5 ?m. 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% H2O2in 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
Na2HPO4pH 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 NaH2PO4pH 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? 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 ?m.
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 ?m wax sections as described (Francis-
West et al., 1994). The Pitx2probe is described by Yu et al. (Yu et al., 2001)
and the chick MyoD clone by Lin et al. (Lin et al., 1989).
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 dominant-
negative Pitx2 construct (Yu et al., 2001). Micromasses were cultured for 3
days, fixed briefly in ice-cold methanol and immunostained with the pan-
myosin 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 (?-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
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 ?m and stained with H&E. For TUNEL, embryos
Development 133 (24)
Fig. 1. Pitx2 is required for survival of branchiomeric muscle
precursors. (A,B) Right and left views of 9.5 dpc mouse embryos after
whole-mount in situ hybridization with a Pitx2 probe (arrows denote
hybridization signal). (C-H) Expression in chick embryos of Pitx2
(C,E,F,H) and MyoD (D,G) analysed by whole-mount in situ
hybridization (C,F) and radioactive in situ hybridization to tissue sections
(D,E,G,H) of stage 10 (C,F), 24 (D,E) and 26 (G,H) chick embryos. C is a
dorsal view and D,E,G,H are frontal sections through the facial
primordial. F is a transverse vibratome section through the head of the
embryo in C. Arrows in D,G indicate developing muscles, in E the
ectodermal and mesodermal expression of Pitx2, in H the
ectomesenchymal Pitx2 expression. do, dorsal oblique muscle; e, eye; f,
frontonasal mass; hy, hyoid arch; md, mandibular primordia.
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