Development 140, 1034-1044 (2013) doi:10.1242/dev.085225
© 2013. Published by The Company of Biologists Ltd
Foxc1 controls the growth of the murine frontal bone
rudiment by direct regulation of a Bmp response threshold
Knowing how signaling molecules elicit transcriptional responses of
target genes at specific thresholds is crucial for understanding cell
specification during embryonic patterning. A key element of such
mechanisms is the complement of transcription factors that respond
to the signal and influence the expression of downstream genes
(Gurdon et al., 1995; Gurdon and Bourillot, 2001). Although a
number of these factors have been identified, how their transcription
regulatory activity is translated into events at the level of cell
specification and patterned growth is still incompletely understood.
Here, we investigate the role of Foxc1 in modulating the influence
of Bmp signaling on the expression of the Bmp effector Msx2, and
the specification of osteogenic precursor cells in the developing
The mammalian skull vault consists of a group of intricately
patterned bones that develop in close coordination (Morriss-Kay
and Wilkie, 2005; Chai and Maxson, 2006). These include the
paired frontal and parietal bones. The frontal bones are derived from
cranial neural crest and the parietal bones from head mesoderm. In
the mouse embryo, skull vault precursor cell populations migrate
into positions above the eye by E12.5. There they condense and
begin to differentiate into the calvarial rudiments. From E13.5 into
postnatal development, the paired frontal and parietal bone
rudiments grow apically, coming into apposition at the midline at
the frontal and sagittal sutures, and laterally at the coronal suture.
The morphogenetic mechanisms underlying the patterned growth
of the calvarial bones are under active investigation. Proliferation of
osteogenic cells within the rudiments contributes to growth (Ishii
et al., 2003), as does the apical migration of osteogenic precursor
cells. DiI labeling experiments show that a population of such
migratory osteogenic precursor (MOP) cells is located outside
(ectocranial to) the rudiments; cells from this population migrate
apically, insert into the leading edge of the growing rudiment and
differentiate into osteoblasts (Yoshida et al., 2008; Ting et al., 2009;
Roybal et al., 2010).
Msx genes, effectors of Bmp signaling, are crucial for calvarial
development (Satokata et al., 2000; Wilkie et al., 2000; Chai and
Maxson, 2006). Msx genes are an ancient and highly conserved
homeobox gene family with a variety of functions in vertebrate
organogenesis (Satokata and Maas, 1994; Phippard et al., 1996;
Bach et al., 2003; Chen et al., 2007). Msx1and Msx2are well known
as downstream targets of the Bmp signaling pathway, and at the
same time can regulate the expression of Bmp ligands (Bei and
Maas, 1998). In Msx2−/−embryos, the growth of the calvarial
rudiments is deficient, resulting in a large ossification defect in the
frontal bone (Ishii et al., 2003). Humans with heterozygous loss of
MSX2 function are similarly affected (Wilkie et al., 2000).
Combination Msx1/2 mutants exhibit agenesis of the frontal and
parietal bones (Han et al., 2007).
Msx2is regulated by the Bmp pathway through an upstream Bmp
responsive enhancer element (Brugger et al., 2004). This element,
~560 bp in length, contains a 52 bp fragment necessary for the Bmp
responsiveness of an Msx2 transgene in embryos and in cultured
cells. This fragment contains an AT-rich central sequence flanked by
Smad-binding sites. Both the AT-rich site and the Smad sites are
required for Bmp responsiveness. The AT-rich region contains a
partial Fox consensus site (Gao et al., 2003; Brugger et al., 2004;
Benayoun et al., 2011), leading us to propose that Fox proteins
function together with Smads to regulate the ability of Msx2 to
respond to Bmp signaling (Brugger et al., 2004). Here, we present
genetic and molecular evidence that the Fox protein Foxc1 controls
the Bmp responsiveness of Msx2.
Fox genes are involved in a variety of developmental processes
during organogenesis, as well as in aspects of energy homeostasis
and cancer (Hannenhalli and Kaestner, 2009; Benayoun et al.,
2011). Both targeted (Foxc1lacZ) and spontaneous (Foxc1ch) Foxc1-
null mice die perinatally with identical skeletal, ocular,
genitourinary, cardiovascular and somitic defects, as well as
hydrocephalus and cerebellar defects (Kume et al., 1998; Kume et
Department of Biochemistry and Molecular Biology, Norris Cancer Hospital,
University of Southern California Keck School of Medicine, 1441 Eastlake Avenue,
Los Angeles, CA 90089-9176, USA.
*Author for correspondence (email@example.com)
Accepted 18 December 2012
The mammalian skull vault consists of several intricately patterned bones that grow in close coordination. The growth of these bones
depends on the precise regulation of the migration and differentiation of osteogenic cells from undifferentiated precursor cells
located above the eye. Here, we demonstrate a role for Foxc1 in modulating the influence of Bmp signaling on the expression of Msx2
and the specification of these cells. Inactivation of Foxc1 results in a dramatic reduction in skull vault growth and causes an expansion
of Msx2 expression and Bmp signaling into the area occupied by undifferentiated precursor cells. Foxc1 interacts directly with a Bmp
responsive element in an enhancer upstream of Msx2, and acts to reduce the occupancy of P-Smad1/5/8. We propose that Foxc1 sets
a threshold for the Bmp-dependent activation of Msx2, thus controlling the differentiation of osteogenic precursor cells and the rate
and pattern of calvarial bone development.
KEY WORDS: Skull vault, Foxc1, Msx2, Bmp responsive element, Mouse
Jingjing Sun, Mamoru Ishii, Man-Chun Ting and Robert Maxson*
Foxc1-mediated regulation of Msx2
al., 2000; Kume et al., 2001; Seo and Kume, 2006). The most
striking skeletal phenotype in Foxc1 mutant mice is the lack of
calvarial bones. This defect is not secondary to hydrocephalus (Rice
et al., 2003). Recently, a new mouse model with a hypomorphic
allele of Foxc1 (Foxc1hith) was found to have meningeal defects in
association with frontal foramina and cortical dysplasia (Zarbalis et
al., 2007). In humans, mutations in FOXC1 are associated with
Axenfeld-Rieger and Dandy Walker syndromes, both inherited as
autosomal dominant traits. Individuals affected with Axenfeld-
Rieger syndrome exhibit a spectrum of malformations, including
dysgenesis of the anterior segment of the eye, tooth abnormalities,
maxillary hypoplasia, hypertelorism and cardiac outflow tract
defects (Tümer and Bach-Holm, 2009). Dandy-Walker syndrome,
the most common human cerebellar malformation, is associated
with deletions or duplications spanning the FOXC1 gene (Aldinger
et al., 2009).
We report here that Foxc1 controls the differentiation of
osteogenic precursor cells that contribute to the frontal bone, and
we present evidence that it does so by inhibiting the Bmp
responsiveness of Msx2and the activity of the Bmp pathway in such
cells. We show further that Foxc1 interacts directly with a Bmp-
responsive element in the Msx2 560 bp upstream enhancer, and we
confirm, by means of gain- and loss-of-function experiments in a
newly developed cranial neural crest cell line (Ishii et al., 2012),
that Foxc1 acts as a negative regulator of the Bmp responsiveness
of Msx2. We show finally that Foxc1 acts to restrict the occupancy
of P-Smad1/5/8 on the Msx2 Bmp responsive element. We propose
that the normal function of Foxc1 is to set a transcriptional threshold
for the Bmp-dependent activation of Msx2, thus controlling the
differentiation of osteogenic precursor cells and the initial phases
of calvarial bone development.
MATERIALS AND METHODS
Msx2 enhancer constructs pBSK-560bpMsx2-hsplacZ, pBSK-52bpMsx2-
hsplacZ, pBSK-52bpMsx2-mut-hsplacZand pGL2-560bpMsx2-tk-luciferase
have been described previously (Brugger et al., 2004). The Foxc1
expression vector pcDNA3-mFoxc1was a kind gift from Dr Tsutomu Kume
(Northwestern University, Chicago, IL, USA).
Cell culture, co-transfections and luciferase assay
10T1/2 cells were propagated in DMEM with 10% FBS. O9-1 cells were
maintained and differentiated into osteoblast as recently reported (Ishii et al.,
2012). Lipofectamine 2000 (Life Technologies) was used to introduce
pcDNA3-mFoxc1or siFoxc1 with pGL2-560bpMsx2-tk-lucinto 10T1/2 and
O9-1 cells. BMP2 (100 ng/ml) treatment and luciferase assay was
performed as described previously (Brugger et al., 2004).
Mouse strains and genotyping
Heterozygous Foxc1ch/+(ch: congenital hydrocephalus) mice were
purchased from Jackson Laboratory on the CHMU/Le background. Msx2
mutant mice, originally obtained from Dr Richard Maas (Harvard
University, Boston, MA, USA), were crossed into a C57BL/6 background
(Ishii et al., 2003). 560bpMsx2-hsplacZ transgenic mice have been
described previously (Kwang et al., 2002). The ch allele of Foxc1 was
identified by PCR amplification followed by Cac8I digestion (Kume et al.,
2000). The Msx2 mutant allele and the lacZ transgene were identified by
PCR (Satokata et al., 2000; Kwang et al., 2002).
Preparation of the agarose beads and calvarial explant culture were
performed as previously described (Rice et al., 2003; Brugger et al., 2004).
Histochemistry, immunostaining and in situ hybridization
lacZ, ALP histochemistry and immunofluorescent detection for P-Smad1/5/8
(Cell Signaling) have been described previously (Ishii et al., 2003; Ting et al.,
2009). Msx2 full-length cDNA was cloned into pBSKII(+) (Kwang et al.,
2002). A cloned Foxc1cDNA was a kind gift from Dr David Rice (University
of Helsinki, Finland). Bmp2and Bmp4full-length cDNAs were obtained from
Dr Malcolm Snead (USC, Los Angeles, CA, USA). NoggincDNA was a gift
from Dr Richard Harland (UCLA, Berkeley, CA, USA). Runx2 cDNA was
obtained from Dr Gérard Karsenty (Columbia University Medical Center,
NY, USA). Ribonucleotide probes were generated as described previously
(Kwang et al., 2002). Fluorescent in situ hybridization followed by tyramide
signal amplification (TSAPLUS, Perkin Elmer) was carried out as reported
previously (Paratore et al., 1999; Ting et al., 2009).
To quantify ALP, Msx2, Bmp4and Bmp2expression, continuous sections
from at least two wild-type and two mutant embryos were photographed
and analyzed with Adobe Photoshop. We used the magic wand tool to select
the region of interest and calculate the pixel numbers of selected area. Data
show the average pixel count of ~10-15 pictures from one representative
pair. For Foxc1 and Msx2 colocalization (Fig. 2G), yellow pixels were
selected by color and counted from a total 16 sections of three pairs of
control and mutant embryos. At least 10 pictures from three individuals of
each genotype were analyzed to quantify the influence of Msx2 dose on
ectopic ALP (Fig. 4I) and P-Smad1/5/8 (Fig. 4N) expression.
RNAi and qRT-PCR
siRNAs targeting Foxc1 (Ambion, SMARTpool Dharmacon) were
introduced into O9-1 or C3H10T1/2 cells by Lipofectamine 2000 (Life
Technologies) according to the manufacturer’s protocol. siGFP was used as
control (supplementary material Table S1). Cells were collected ~24-
48 hours after transfection and subjected to total RNA extraction with the
RNeasy Mini Kit (Qiagen). First-strand cDNA was synthesized with the
SuperScript III system (Life Technologies) for RT-PCR analysis. Relative
expression levels were normalized to Gapdh mRNA. Primer sequences are
given in supplementary material Table S1.
Chromatin immunoprecipitation (ChIP) assays
ChIP was performed as described previously (Ma et al., 2003; Brugger et al.,
2004) with minor modifications. Cells were treated with 60 ng/ml BMP2 for
30 minutes prior to fixation. Anti-Foxc1 pull-down was achieved by two
sessions of incubation: first with a goat anti-Foxc1 antibody (Abcam), then
with a rabbit anti-goat IgG (Sigma-Aldrich). Rabbit IgG was used for mock
immunoprecipitation (Sigma-Aldrich). qPCR was performed to amplify a
350 bp region of the endogenous Msx2enhancer. For transfected cells, DNA
constructs or siRNA were introduced into cells 24 hours prior to BMP2
treatment. A 200 bp region of the hsp68 mini-promoter immediately
downstream of the 52 bp Msx2 Bmpre in pBSK was amplified by qPCR.
Each result is controlled by IgG, and is from a single experiment
representative of three independent experiments. Primer sequences are
given in supplementary material Table S1.
Foxc1 regulates the size of the frontal bone
rudiment and the osteogenic differentiation of
cultured cranial neural crest cells
We first examined the expression of an early osteoblast marker,
alkaline phosphatase (ALP), in Foxc1mutant and control embryos at
E12.5 (Fig. 1B-C?), E13.5 (Fig. 3D,D?,F,F?) and E14 (supplementary
material Fig. S1A-B?). Consistent with previous findings (Ishii et al.,
2003; Rice et al., 2003), ALP was expressed in control embryos in a
group of cells that make up the frontal and parietal bone rudiments
(Fig. 1B,C; Fig. 3D,F; supplementary material Fig. S1A,B). In
Foxc1ch/chmutants, apical growth did not progress beyond the
primordium stage, and the rudiments mineralized by E15 with little
or no further growth (supplementary material Fig. S1A?,B?) (Rice et
al., 2003). These results are consistent with those of Rice and
colleagues (Rice et al., 2003). In addition to this previously reported
lack of apical growth, we found that the ALP-positive area expanded
laterally toward the epidermis and ventrally towards the midline of the
cranial base (arrows, Fig. 1C?). Quantitation of the ALP signal in
serial sections through the supraorbital ridge confirmed this expansion
(P<5.00?10–5, Fig. 1F). Runx2, also a pre-osteoblast marker,
exhibited a similar expansion in Foxc1 mutant embryos
(supplementary material compare Fig. S1C with S1C?). Foxc1
mutants have hydrocephalus, precluding analysis of skull vault
growth and patterning in embryos after E15.
Lineage tracing with Wnt1-Cre/R26R (neural crest) and Mesp1-
Cre/R26R (mesoderm) showed no major differences between Foxc1
mutant and control embryos in the distribution of neural crest and
mesoderm in the head region of E9.5 or E12.5 embryos
(supplementary material Fig. S2). Thus, the lack of apical extension
of the frontal and parietal bones is not likely to be caused by a failure
of osteogenic precursor cells to migrate into position in the
In situ hybridization revealed that Msx2, which is normally
expressed in the osteogenic cells of the frontal and parietal
rudiments (Fig. 1E), expanded laterally in Foxc1ch/chmutants,
coincident with the expansion in the ALP domain (arrows, Fig. 1C?;
P<1.00×10–16, Fig. 1F). We used Foxc1 and Msx2 probes
simultaneously to determine the relationship between the Foxc1
expression domain and the expansion of Msx2 expression (Fig. 2).
Because the Foxc1 ch mutation is a single base change, the Foxc1
transcript is detectible in sections of mutant embryos. At E11.5,
Msx2 was expressed broadly in cranial mesenchyme. Foxc1
transcripts were detected mainly in the periocular mesenchyme and
not in the mesenchyme where the frontal bone rudiment forms (data
not shown). By E12.5, in control embryos, Foxc1 was expressed in
the meninges and in the cranial mesenchyme, overlapping partially
with Msx2 in the lateral region of the Foxc1 domain (Fig. 2A,B).
The number of cells expressing both Foxc1 and Msx2 transcripts
(yellow pixels, Fig. 2G) increased in Foxc1mutants (P<5.00×10–4),
Development 140 (5)
consistent with an increase in Msx2 transcripts in cells expressing
the mutant Foxc1 mRNA. Msx2 expression also increased laterally
outside the Foxc1 domain (Fig. 2B?), suggesting that loss of Foxc1
results in non-cell autonomous effects on Msx2 expression. Similar
changes in Msx2expression in Foxc1mutants were evident at E13.5
(Fig. 2E?,F?). These results suggest that Foxc1 acts to restrict the
osteogenic domain and the Msx2 expression domain within the
supraorbital ridge. We note that Msx2 expression does not expand
into the innermost (endocranial) region of the Foxc1 domain,
suggesting that additional factors may be required for Msx2
expression in this region.
To test directly whether Foxc1 negatively regulates Msx2, we
used siRNA to knock down Foxc1 in cultured cells (Fig. 2H). In
initial experiments, we used C3H10T1/2 cells, which are
multipotent mesenchymal cells capable of differentiating into
muscle cells, chondrocytes, adipocytes and osteoblasts (Pinney
and Emerson, 1989; Katagiri et al., 1990). Because the
mesenchyme of the supraorbital ridge at the level of the frontal
bone rudiment is derived from cranial neural crest (Jiang et al.,
2002; Ishii et al., 2003), we also used a cranial neural crest (CNC)
cell line, O9-1, that we developed recently (Ishii et al., 2012). This
cell line was derived from Wnt1-Cre; R26R-EGFP-expressing
cells from the head region of E8.5 mouse embryos. We established
culture conditions that allow O9-1 cells to be grown as multipotent
stem-like cells, maintaining an ability to differentiate into
osteoblasts, chondrocytes, smooth muscle cells and glial cells. O9-
1 cells can be propagated and passaged indefinitely, and can
contribute to bone and smooth muscle after injection into mouse
embryos (Ishii et al., 2012). O9-1 cells undergo differentiation into
osteoblasts with high efficiency after being placed into an
Fig. 1. Expansion of frontal bone
osteogenic domain in the
supraorbital ridge of Foxc1 mutant
embryos. (A-E? ?)Control (B-E) and
Foxc1ch/chmutant (B?-E?) embryos at
E12.5 were sectioned in the indicated
(coronal) plane (A) and stained for ALP
to mark osteogenic cells (B-C?) or were
subjected to in situ hybridization for
Msx2 (D-E?). Boxed areas are shown at
higher magnification on the right.
(F)Increased ALP-positive osteogenic
progenitor domain and Msx2 mRNA
signal inside the boxed area in Foxc1
mutant embryos (Student’s t-test,
P<5.00×10–5). Error bars indicate s.d.
We transfected either a control siRNA or a Foxc1 siRNA into
10T1/2 and O9-1 cells, and assessed endogenous Foxc1 and Msx2
mRNA levels by real-time PCR (Fig. 2H). In 10T1/2 cells, Foxc1
mRNA declined to 50% of its level in control cells, and Msx2
transcript levels increased approximately twofold, consistent with
Foxc1 exerting an inhibitory effect on Msx2 expression. In O9-1
cells, Foxc1 siRNA caused a reduction of Foxc1mRNA to ~30% of
its level in control siRNA-treated cells by 24 hours after
transfection, and caused Msx2 transcript levels to increase by
approximately twofold. To rule out off-target effects, we used
siRNAs against distinct sequences in the Foxc1transcript (Fig. 2H).
These siRNAs (a mixture of four, none of which had sequences that
overlapped with the siRNA used in the initial experiments) caused
a similar reduction of Foxc1 mRNA and increase in Msx2 mRNA
in both 10T1/2 (Fig. 2H) and O9-1 cells (data not shown). These
results support the hypothesis that Foxc1 negatively regulates Msx2
in the supraorbital ridge.
We next asked whether knockdown of Foxc1 augmented the
osteogenic differentiation of O9-1 cells (Fig. 2I). We assessed the
Foxc1-mediated regulation of Msx2
expression of the osteogenic markers ALP, Runx2 and osteocalcin
in Foxc1-knockdown and control O9-1 cells cultured in osteogenic
medium. We found that the expression of each marker was
increased relative to controls, suggesting that knockdown of Foxc1
not only upregulates Msx2, but also accelerates the osteogenic
differentiation of O9-1 cells. These data support the hypothesis that
reduced Foxc1 function can increase the rate of osteogenic
differentiation of cranial neural crest cells.
Ectopic osteogenesis and Msx2 expression
correlate with ectopic Bmp signaling in calvarial
rudiments of Foxc1 mutants
We assessed the expression of Bmp2, Bmp4, P-Smad1/5/8 and the
extracellular Bmp inhibitor Noggin in the calvarial rudiments and
supraorbital ridge of Foxc1ch/chmutant and control embryos (Fig. 3).
We found that Bmp2 and Bmp4 expression domains were expanded
at E12.5 and E13.5, similar to ALP (arrowheads, Fig. 3B?,C?,E?,G?;
quantitation in 3M). In control embryos, Noggin transcripts were
located ventral to the Bmp/ALP/Msx2domain (arrows, Fig. 3I,K). In
Fig. 2. Foxc1 inhibits Msx2 expression in
the supraorbital ridge and in cultured
10T1/2 and O9-1 cranial neural crest
cells. (A,A? ?,B,B? ?,E,E? ?,F,F? ?) Control and
Foxc1ch/chembryos at E12.5 or E13.5 were
sectioned as in Fig. 1A and subjected to in
situ hybridization for Msx2 and Foxc1
mRNAs simultaneously. Msx2 is in green;
Foxc1 in red. (C-D? ?) Sections from the same
embryo were stained for ALP. Boxed areas
are shown at higher magnification on the
right. (G)The area of Msx2 and Foxc1 co-
expression, indicated by the yellow color in
B and B?, is significantly larger
(P<5.00×10–4) in Foxc1 mutants. (H)Effect of
siRNA-mediated knockdown of Foxc1 on
Msx2 mRNA levels in C3H10T1/2 cells and
O9-1 cranial neural crest cells. mRNA levels
of Foxc1 and Msx2 were measured by qPCR
and normalized to Gapdh. siRNA against
EGFP provided a negative control. (I)We
assessed the influence of Foxc1 knockdown
on the rate of osteogenic differentiation of
cultured O9-1 cells. Cells were treated with
siRNA against Foxc1, and qPCR was used to
measure levels of Msx2, ALP, Runx2 and
osteocalcin mRNAs. Significant (P<0.05)
upregulation of each of these osteogenic
differentiation markers was observed after
48 hours of Foxc1 siRNA treatment.
Significance was assessed by Student’s t-
test. Error bars indicate s.d.
mutants, Noggin expression declined substantially (arrowheads,
Fig. 3I?,K?). P-Smad1/5/8, the downstream effector of canonical Bmp
signaling (Massagué, 1998), was distributed in a pattern that largely
coincided with the Bmp/ALP/Msx2domain (Fig. 3L,L?). Expression
expanded ventrally in the mutant (star, Fig. 3L?), consistent with a
net increase in Bmp signaling in the area of ectopic ALP expression.
Reduced Msx2 gene dose rescues ectopic
osteogenesis and Bmp signaling in Foxc1 mutants
To determine whether Msx2 has a functional role downstream of
Foxc1 in the regulation of osteogenic precursor differentiation, we
asked whether reduced Msx2dosage mitigated ectopic osteogenesis
and Bmp signaling in Foxc1mutants (Fig. 4). We crossed Msx2and
Foxc1 mutants, producing embryos with the genotype Foxc1ch/+;
Msx2+/−and Foxc1ch/ch; Msx2+/−. We did not recover double
homozygous null embryos from double heterozygous matings
(n=59), consistent with this genotype causing lethality prior to E13.
We assessed the size and growth of the calvarial rudiments by ALP
staining of serial sections through the supraorbital ridge at E12.5
(Fig. 4A-F). Foxc1ch/+embryos exhibited a subtle but reproducible
expansion of ALP, suggesting that this phenotype is Foxc1 dose
dependent at early developmental stages (supplementary material
Fig. S1). However, this deficiency was transient, as frontal bone
Development 140 (5)
development was indistinguishable from wild type at E17 and P0
(Rice et al., 2003; J.S., M.I., M.-C.T. and R.M., unpublished).
The ectopic ALP staining evident in E12.5 Foxc1ch/+and
Foxc1ch/chmutant embryos was reduced or absent in Foxc1/Msx2
double mutants (Fig. 4B-F). Quantitation of the ectopic ALP signal
(e.g. Fig. 4H) by enumerating ALP pixels revealed reductions of
50% in both Foxc1ch/+; Msx2+/−and Foxc1ch/ch; Msx2+/−
combinations. Similarly, ectopic P-Smad1/5/8 activity was reduced
(Fig. 4J-N). These results suggest that the ectopic expression of
Msx2 resulting from loss of Foxc1 function is causally related to
ectopic osteogenesis and to Bmp signaling in the supraorbital ridge.
Because Foxc1ch/+animals do not have a frontal bone phenotype at
late stages of embryogenesis or in early postnatal life, we were
unable to ask whether reduced Msx2 dose rescued such animals. In
addition, reduced Msx2 dose did not rescue the hydrocephalus
defect in Foxc1ch/chembryos, precluding examination of such
embryos at late stages.
Foxc1 acts through an upstream Msx2 enhancer to
regulate the Msx2 expression domain and Bmp
responsiveness in the supraorbital ridge
We next asked whether Foxc1 influences Msx2 expression via an
effect on an upstream Msx2 enhancer (Fig. 5). ChIP experiments in
Fig. 3. Elevated Bmp signaling activity
in the supraorbital ridge of Foxc1
mutant embryos. (A-G? ?,M) We detected
Bmp4 and Bmp2 expression by in situ
hybridization in coronal sections of
embryos at E12.5 and E13.5 wild-type and
Foxc1 mutant embryos. There is Bmp4
expression at the apex of the osteogenic
area and in the meninges of wild-type
embryos (arrows, B and E), and in the
rudiment of Foxc1 mutants (arrowheads,
B? and E?). Bmp2 expression is increased
in the rudiment in Foxc1 mutants
(arrowheads, C? and G?). The increases in
both Bmp4 and Bmp2 expression in these
areas were statistically significant at E12.5
(Student’s t-test, P<1.00×10–8, M). Error
bars indicate s.d. (H-K? ?) Expression of a
Bmp antagonist, Noggin, assessed by in
situ hybridization at E12.5 and E13.5. ALP
was used to visualize the signal at E12.5
because it provided a more robust signal
than fluorescent detection with extended
developing time. Noggin transcripts are
present in the rudiment and in an
adjacent cartilage in wild-type embryos
(arrows, I and K), but reduced in Foxc1
mutants (arrowheads, I? and K?).
(L,L? ?) Immunodetection of P-Smad 1/5/8
served to indicate the overall activity of
the canonical Bmp pathway. The number
of P-Smad1/5/8-positive nuclei is
increased in Foxc1 mutant (asterisk, L?).
ES cells and adult heart tissue showed that this enhancer is enriched
for the transcription co-activator p300, as well as histone marks
associated with active enhancers (Santos-Rosa et al., 2002;
Bernstein et al., 2005; Chen et al., 2008; Visel et al., 2009) (Encode
Project, UCSC Genome Browser).
We crossed mice carrying the 560bpMsx2-hsplacZtransgene with
Foxc1mutants (Foxc1ch/+) and assessed its expression in the area of
the frontal and parietal bone rudiments. lacZ expression expanded,
similar to endogenous Msx2 expression (Fig. 5D-E?). Therefore the
560 bp enhancer is sufficient to respond to a loss of Foxc1function.
Foxc1 negatively regulates Msx2 expression and Msx2 is an
immediate early target of the Bmp signaling pathway (Brugger et
al., 2004) suggesting that Foxc1 might inhibit the Bmp
responsiveness of Msx2. To test this hypothesis, we implanted
BMP2-soaked beads in the supraorbital ridge of explants of
embryonic heads of E12.5 embryos (Fig. 5F). We cultured the
explants and, after 2 days, carried out in situ hybridization with a
probe for Msx2 (Fig. 5G-H?). We found that the BMP2 beads
elicited a greater increase in Msx2 expression in Foxc1 mutant
embryos than in control embryos.
This result is in contrast to the findings of Rice et al. (Rice et al.,
2003) who found that a BMP2-soaked bead implanted in the dorsum
of the head of E15 embryos elicited a reduced Bmp response in
Foxc1-mediated regulation of Msx2
Foxc1 mutants compared with wild-type embryos. We repeated
their experiment and obtained closely similar results; i.e. a reduction
in Bmp responsiveness of Msx2 (supplementary materialFig. S3).
Therefore, the difference between our findings in the supraorbital
ridge and those of Rice et al. (Rice et al., 2003) in the head
mesenchyme is likely a consequence of the difference in the sites
and developmental stages of bead implantation.
To further test the hypotheses that Foxc1 modulates the Bmp
responsiveness of Msx2, and that the 560 bp enhancer is sufficient
for this effect, we carried out a BMP2 bead implantation experiment
in embryonic heads (Fig. 5I-J?) and limbs (data not shown) of mice
carrying the 560bpMsx2-hsplacZ transgene. Staining for β-
galactosidase activity revealed that the 560 bp enhancer was indeed
sufficient for the increase in the Bmp responsiveness of Msx2 in
Foxc1 mutants in the supraorbital ridge.
Finally, we used both gain- and loss-of-function approaches in
cultured cells to assess the influence of Foxc1 on the Bmp-
responsiveness of Msx2 (Fig. 5K-M). We transfected a construct
bearing the 560 bp enhancer driving tk-luciferase (560bpMsx2-tk-
luc) into 10T1/2 cells and O9-1 CNC cells, and tested the effect of
overexpressing Foxc1 on the Bmp-inducibility of 560bpMsx2
(Fig. 5K,L). In both 10T1/2 and O9-1 cells, Bmp inducibility was
more than halved (P<0.05). Reciprocally, transfection of siRNA
Fig. 4. Reduced dose of Msx2 rescues ectopic
osteogenesis and Bmp signaling in the
supraorbital ridge of Foxc1 mutant embryos.
(A-F)ALP expression in the supraorbital ridge in E12.5
wild-type, Foxc1, Msx2 and compound mutant
embryos. There is near-normal expression of ALP in
the Foxc1ch/+; Msx2+/−compound heterozygous
mutant (E) versus the Foxc1 heterozygous mutant (B).
Also note the partial rescue in the Foxc1ch/ch; Msx2+/−
mutant (F) compared with the Foxc1 homozygous
mutant (C). (G)Numbers of embryos harvested for
this experiment. *N/A, not applicable. (H)We
measured the level of ALP expression in the
supraorbital ridge by counting the purple pixels in
two areas: p (primordium) and e (ectopic). (I)The
influence of Msx2 dose on ALP expression in Foxc1
mutants was statistically significant in area e (*P<0.05,
**P<0.005). (J-M)Bmp signaling activity in E13
embryos was detected by P-Smad1/5/8
immunohistochemistry. P-Smad1/5/8 expansion is
partially rescued: compare the area spanned by the
white arrow from p to e in K with the corresponding
area in M. (N)Significant reduction of P-Smad1/5/8
signal in area e in Foxc1ch/+; Msx2+/−mutant embryo in
comparison with Foxc1ch/+mutant (*P<5.00×10–6).
Significance was assessed by Student’s t-test. Error
bars indicate s.d.
against Foxc1 caused an increase in 560bpMsx2 Bmp inducibility
in O9-1 cells of approximately twofold (P<0.05, Fig. 5M). These
data support the hypothesis that Foxc1 regulates the Bmp
responsiveness of Msx2 through an effect on the 560 bp upstream
Foxc1 is associated with the Bmp responsive
element within the Msx2 560bp upstream
We next performed ChIP experiments to determine whether Foxc1
interacts with the endogenous Msx2 locus in the area of the 560 bp
enhancer when induced by BMP2 (Fig. 6). Using primers that flank
the genomic region containing the 560 bp enhancer, and control
primers against the β-actin promoter, we performed ChIP with a
Foxc1 antibody on chromatin extracts derived from control or
BMP2-treated O9-1 CNC and 10T1/2 cells (Fig. 6A). Treatment of
both O9-1 cranial neural crest and 10T1/2 cells with BMP2 resulted
in a substantial (approximately two- to threefold) enrichment of
Development 140 (5)
Foxc1 at the endogenous Msx2locus but not at the β-actin promoter
(P<0.001). Quantitative RT-PCR showed that Foxc1 mRNA is not
increased upon treatment of O9-1 or 10T1/2 cells with BMP2
(Fig. 6B). The increase in Foxc1 at the Msx2 enhancer is thus not
controlled at the level of transcript accumulation.
To test whether Foxc1 interacts with the 52 bp Bmpre, we
transfected into 10T1/2 cells a plasmid carrying only the 52 bp
Bmpre driving lacZ(52bpMsx2-hsplacZ). ChIP assays with primers
hybridizing with the plasmid sequence adjacent to the Bmpre (red
arrows, Fig. 6C) showed that Foxc1 was enriched substantially at
the Bmpre and that its occupancy at this site increased upon BMP2
treatment (P<0.005, Fig. 6D).
The AT-rich domain of the Msx2 Bmpre has both an
Antennapedia superclass recognition sequence (AATTAA) and an
overlapping partial Fox site (AGCAATT, underlines) that matched
the consensus (G/A)(T/C)(C/A)AA(T/C)A in 5/7 positions (Gao et
al., 2003; Benayoun et al., 2011). To determine whether the
interaction of Foxc1 with the Bmpre requires the AT-rich domain,
Fig. 5. Foxc1 inhibits the Bmp responsiveness
of Msx2 in embryos and cultured cells.
(A)Msx2 locus showing 560 bp Msx2 upstream
enhancer. (B-E? ?) ALP and lacZ staining was
performed on adjacent sections of E12.5
560bpMsx2-hsplacZ transgenic embryos carrying
either the wild-type (B-E) or Foxc1chmutant allele
(B?-E?). Boxed areas are shown at higher
magnification on the right. The 560bpMsx2
transgene is sufficient to respond to loss of Foxc1
function. (F-J? ?) Influence of BMP2-soaked beads
on endogenous Msx2 (G-H?) assessed by in situ
hybridization and 560bpMsx2-hsplacZ expression
(I-J?). The signal intensity in the supraorbital ridge
is increased in both the endogenous Msx2 (H
versus H?) and the transgene (J versus J?) in Foxc1
mutants compared with control embryos.
(K-M)We tested the effect of overexpression (K,L)
and siRNA-mediated knockdown (M) of Foxc1 on
the Bmp inducibility of the 560bpMsx2-tk-luc
construct in 10T1/2 and O9-1 cranial neural crest
cells. The insets show the fold change in
luciferase expression. The results represent
means of three biological replicates. Error bars
show one s.d. Significance was assessed using
we tested the effect of a mutation (52bpMsx2-mut-hsplacZ) in this
domain on the association of Foxc1 with the 52 bp element. We
transfected mutant and control constructs into 10T1/2 cells and
asked whether we could detect Foxc1 by ChIP. As is evident in
Fig. 6D, Foxc1 occupancy was reduced substantially relative to that
of the control construct (P<0.005). Together, our findings support
the view that Foxc1 acts directly on a Bmpre in the 560 bp enhancer
upstream of Msx2 to attenuate its Bmp responsiveness in the
mesenchyme of the supraorbital ridge.
Foxc1 functions to exclude P-Smad1/5/8 from the
Msx2 Bmp responsive element
A simple mechanism that could explain the increased Bmp
responsiveness of Msx2 in Foxc1 mutants is that Foxc1 attenuates
the interaction of P-Smad1/5/8 with the Msx2 Bmpre. Thus, loss of
Foxc1 should result in an increase in P-Smad1/5/8 levels within the
560 bp enhancer. We used ChIP to assess P-Smad1/5/8 levels within
the 560 bp enhancer in O9-1 cranial neural crest cells in which
Foxc1 was knocked down by siRNA. Consistent with our
hypothesis, we found that in Foxc1 knockdown cells, P-Smad1/5/8
Foxc1-mediated regulation of Msx2
levels were substantially higher than in control cells (P<0.01,
Here, we demonstrate a role for Foxc1 in regulating the influence of
Bmp signaling on the expression of Msx2 and the specification of
osteogenic precursor cells in the developing skull vault. Using both
mouse embryos and a cranial neural crest cell line, we show that
Foxc1 acts directly on a Bmp-responsive enhancer to reduce the
occupancy of P-Smad1/5/8 and thus restrict Msx2 expression to an
osteogenic zone fated to become the developing frontal bone. We
propose that within the supraorbital ridge, Foxc1 functions through
Msx2 to set a threshold level of Bmp signaling activity, thus
controlling the differentiation of osteogenic precursor cells and the
development of the calvarial bones.
Rice and colleagues first suggested a relationship between Foxc1
and Msx2 (Rice et al., 2003). These authors showed that BMP2
beads implanted in the head mesenchyme of E15 Foxc1 mutants
stimulated expression of Msx2 to a lesser extent than in wild-type
mice, leading to the conclusion that Bmp-induced expression of
Fig. 6.Foxc1 interacts with a Bmp-responsive
element in the 560 bp Msx2enhancer and
inhibits the recruitment of P-Smad1/5/8.
(A)Chromatin immunoprecipitation experiments in
C3H10T1/2 and O9-1 cranial neural crest cells. We
performed ChIP on control and BMP2-treated cells
with an antibody against Foxc1 or rabbit IgG as a
control. qPCR was used to amplify the 560 bp Msx2
enhancer as well as a control β-actin promoter
region. We show results of a single experiment
(three PCR amplifications) representative of three
independent experiments. Student’s t-test was used
to evaluate the strength of the difference between
starred groups. Error bars represent one s.d. Foxc1 is
enriched on the Msx2enhancer. (B)Effect of a 24-
hour BMP2 treatment on Foxc1and Msx2mRNA
level was evaluated by qPCR in both 10T1/2 and
O9-1 cells. (C)The 52 bp Msx2Bmp-responsive
element (Bmpre) (Brugger et al., 2004). Smad1
binding sites (blue) flank an AT-rich sequence
(green). A partial Fox consensus binding site,
AGCAATT [matching the consensus
(G/A)(T/C)(C/A)AA(T/C)A in 5/7 positions
(underlined)], overlapping the AT-rich sequence is
boxed in red. (D)10T1/2 cells were transfected with
plasmids containing wild type (52bpMsx2-hsplacZ)
or a mutant (52bpMsx2-mut-hsplacZ) in which the
Fox site was mutated, and treated with BSA or BMP2
for 30 minutes. They were analyzed by ChIP with an
anti-Foxc1 antibody or rabbit IgG as a control. qPCR
was used to amplify a 200 bp hsp68fragment
immediately downstream of the 52 bp Msx2(red
arrows). We show results from at least three
independent transfections. The interaction of Foxc1
with the Bmpre was almost completely abrogated
by the mutation in the Fox/AT-rich element. (E)O9-
1 cranial neural crest cells were treated with an
siRNA against Foxc1 or EGFP. Cells were then treated
with BSA or BMP2 and subjected to ChIP with an
anti-P-Smad1 antibody or rabbit IgG. qPCR was
used to amplify a 350 bp fragment in the 560 bp
Msx2enhancer. Student’s t-test was used to
evaluate the strength of the difference between
asterisked groups. Error bars represent one s.d.
Msx2 requires Foxc1. We were thus surprised at first by our finding
that Foxc1 negatively regulates Msx2. We note, however, that Rice
et al. (Rice et al., 2003) implanted Bmp beads in the dorsum of the
head of E15 embryos, whereas we implanted them in the
supraorbital ridge of E12.5 embryos. We repeated the bead
implantation performed by Rice et al. (Rice et al., 2003) and
obtained similar results (supplementary material Fig. S3). Thus, our
finding of increased Msx2 expression and Bmp responsiveness in
the supraorbital ridge of Foxc1 mutants is not in conflict with the
findings of Rice et al. (Rice et al., 2003), but instead is likely a result
of the different site or developmental stage of bead implantation.
While this work was under review, Mirzayans and colleagues,
working in several mouse and human cell lines, reported that
FOXC1 positively regulates Msx2 (Mirzayans et al., 2012). They
identified a 480 bp proximal promoter fragment that contains a
FOXC1-binding site and is sufficient to respond to FOXC1. This
fragment is distinct from the 560 bp Msx2 enhancer. These findings
underline our conclusion that the nature of the interaction between
Foxc1 and Msx2 is highly dependent on cellular context. The
molecular details of how Foxc1 represses Msx2gene activity in one
context and activates it in another remain obscure, but are likely to
reflect the ability of Foxc1 to recruit either co-activators or co-
repressors to target promoters. It is thus interesting that in zebrafish
podocytes, Foxc1a can both activate and repress the Podocalyxin
promoter, depending on the dose of Foxc1 relative to an interacting
transcription factor, Wt1 (O’Brien et al., 2011).
The supraorbital ridge domain is crucial for the development of
the skull vault. DiI labeling and recent Cre-based lineage-tracing
experiments (Yoshida et al., 2008; Ting et al., 2009; Roybal et al.,
2010; Deckelbaum et al., 2012) show that at E11.5 undifferentiated
precursor cells of the frontal and parietal bones, as well as the
coronal suture, are located in the supraorbital ridge. These cells
subsequently migrate apically and contribute to the growing bones
and suture. These cells are defined functionally by their ability to
Development 140 (5)
contribute to the ALP-positive frontal and parietal bone rudiments
and the ALP-negative coronal suture between them. Our data show
that the distribution of neural crest and mesoderm within the
supraorbital ridge is not grossly changed in Foxc1 mutants
compared with controls. Given this, our finding that the frontal bone
rudiment begins to develop in Foxc1 mutants but does not elongate
suggests that the deficiency in Foxc1mutants is not in the migration
of the precursors into the supraorbital ridge, but in their
development during the apical growth phase of calvarial
development. The expansion of ALP across the ridge strongly
suggests a mechanism that could slow or prevent elongation – the
depletion of precursors by differentiation. That knockdown of
Foxc1 in cultured neural crest cells accelerates the differentiation
of such cells also supports this model. We note that Rice and
colleagues documented a reduction in proliferation of cells within
the rudiments of Foxc1mutants (Rice et al., 2003). Such a reduction
could also contribute to the slowed calvarial growth. We point out,
finally, that as Foxc1 homozygous mutants do not survive into late
embryogenesis and heterozygotes do not have a skull phenotype,
we do not yet know the consequences of the premature
differentiation of osteogenic precursor cells in supraorbital ridge on
the later development of skull vault. Addressing this issue will have
to await the conditional inactivation of Foxc1 in prospective
calvarial tissues, an approach made possible by the recent
generation of a conditional Foxc1 mutant allele (Sasman et al.,
It is well established that Bmp signaling controls the
differentiation of osteogenic precursor cells in the frontal and
parietal bone rudiments (Kim et al., 1998). There is also evidence
that reduced dose of Msx1/2 results in reduced numbers of
osteogenic precursor cells in calvarial bone rudiments, and reduced
calvarial bone growth (Ishii et al., 2003; Han et al., 2007). Given
that Msx genes are known to be transcriptional effectors of Bmp
signaling (Vainio et al., 1993; Graham et al., 1994; Bei and Maas,
Fig. 7. Schematic model showing role of Foxc1 regulating Bmp signaling and osteoprogenitor development in the supraorbital ridge. Foxc1 is
expressed in the meninges and adjacent mesenchyme (red in A). Osteogenic cells are marked with purple shading. (A)In wild-type embryos, Foxc1
negatively regulates Msx2 and Bmp2/4, and positively regulates Noggin, thereby maintaining levels of Bmp signaling appropriate for the migration of
progenitor cells into the frontal bone rudiment and their differentiation into osteoblasts. (A? ?)In Foxc1 mutants, loss of Foxc1 results in upregulation of
Msx2, which initiates a positive-feedback loop between Msx2 and Bmp2/4. The result is the propagation of a wave of Msx2 and Bmp2/4 expression from
medial to lateral across the supraorbital ridge, and the lateral displacement of the boundary between ALP-expressing cells of the frontal bone rudiment
and adjacent non-ALP expressing mesenchyme.
1998), our finding that Foxc1 negatively regulates the Bmp
inducibility of Msx2 in the mesenchyme of the supraorbital ridge
and in cultured cranial neural crest cells suggests that Foxc1 controls
a Bmp threshold response of Msx2. Reduced Foxc1 activity lowers
the threshold (i.e. makes Msx2 more sensitive to Bmp induction);
increased Foxc1 activity raises it. This threshold, we propose,
regulates the balance between the maintenance of an
undifferentiated precursor cell population on the one hand, and the
differentiation of osteogenic precursors as they move into the high
Bmp environment of the frontal bone rudiment, on the other.
Our data suggest that the expression domain of Foxc1 has only a
narrow region of overlap with that of Msx2, yet the influence of
Foxc1 on Msx2 expression spans a much wider area. The
overlapping region includes parts of the dura and the mesenchyme
immediately adjacent to the dura. We were surprised to find that in
Foxc1 mutants, Msx2 is upregulated over the major part of the
supraorbital ridge, not just in the Foxc1-Msx2 overlapping region.
ALP, Bmp2 and Bmp4 expand similarly across this region. Thus,
loss of Foxc1 results in a non-autonomous cascade of changes in
gene expression across the supraorbital ridge. We propose that the
expansion of Msx2, as well as that of ALP, Bmp2 and Bmp4, is the
result of a positive-feedback loop between Bmp signaling and Msx2:
in the absence of Foxc1, Msx2 is upregulated in the Foxc1 domain,
resulting in upregulation of Bmp genes in this same domain. Bmp
proteins are able to signal adjacent cells, resulting in upregulation
of Msx2, and in turn a further expansion of Bmp gene expression.
The net result is a wave of Msx2 and Bmp expression that spreads
from medial to lateral across the supraorbital ridge. This, we
suggest, results in the ectopic differentiation of supraorbital
mesenchyme to ALP-positive osteogenic cells.
Direct functional evidence for a Foxc1-Msx2-Bmp loop comes
from the genetic reduction of Msx2 activity in the Foxc1 mutant.
This results not only in a rescue of Msx2 expression, but also of P-
Smad1/5/8 activity. Thus, our results show that Msx2 is required
downstream of Foxc1 to maintain cells of the supraorbital ridge in
an undifferentiated state. It is intriguing that loss of Foxc1 also
results in reduced Noggin expression and increased P-Smad1/5/8
activity in a localized area medial to the growing rudiment. We do
not know that fate of these Noggin-expressing cells, but speculate
that they may be osteogenic precursors. DiI labeling experiments
show such precursor cells are present in the supraorbital ridge
(Yoshida et al., 2008; Ting et al., 2009).
The Foxc1-Msx2-Bmp regulatory loop has some features of a
bistable system (Ferrell, 2002). Such systems contain positive or
double-negative feedback loops whose component genes typically
respond to their upstream regulators in an ultrasensitive manner
(Furtado et al., 2008). Bistable systems function to convert graded
inputs into switch-like responses (Ferrell, 2002). In the case of the
Foxc1/Msx2/Bmp axis, we propose that a positive loop between
Bmp and Msx2 is modulated by negative regulation of Msx2 by
Foxc1. The net result of this Foxc1-dependent regulation of the
Msx2-Bmp loop is a sharp boundary between differentiating
osteogenic cells of the rudiments and undifferentiated cells of the
mesenchyme lateral to the rudiments (Fig. 7). An apparently graded
Bmp input is thus converted into a sharp functional boundary. Loss
of Foxc1 function results in a blurring of the boundary.
Experiments in cultured CNC cells and 10T1/2 cells support the
view that Foxc1 can act directly on Msx2. These experiments show:
(1) that Foxc1 is associated with chromatin in the area of a 52 bp
Bmp-responsive element within an upstream Msx2 enhancer; and
(2) that the Foxc1 interaction is abrogated by a mutation that targets
both a Foxc1 consensus site and an overlapping AATTAA site.
Foxc1-mediated regulation of Msx2
Moreover, loss of Foxc1 results in increased Bmp-inducibility of
endogenous Msx2. That we obtained similar results in 10T1/2 cells
and in the O9-1 cranial neural crest cell line strengthens the
argument for a direct, negative regulatory interaction between
Foxc1 and Msx2.
Our data suggest a molecular mechanism by which reduced
Foxc1 activity could influence Msx2 transcription. We have found
that loss of Foxc1 function in cranial neural crest and 10T1/2 cells
results in an increase in P-Smad1/5/8 in the region of the 560 bp
enhancer. Given the crucial function of P-Smad1/5/8 in Bmp-
dependent transcription (Massagué, 1998; Massagué, 2000), this
increase could explain the enhanced Bmp inducibility of Msx2when
Foxc1 activity is reduced genetically or by siRNA. How this
reciprocal relationship between Foxc1 and P-Smad1/5/8 enhancer
occupancy might work on a molecular level is not yet clear. Foxc1
and Smad1 are not known to interact. However, FoxO proteins can
interact with Smad4 to modulate Tgfβ signaling (Seoane et al.,
2004; Gomis et al., 2006). Moreover, Foxc1 can associate with
Smad4, which is also required for Bmp-dependent signaling and is
found in a transcriptional complex with Smad1 (Lagna et al., 1996;
Kretzschmar et al., 1997). Thus, loss of Foxc1 could act through
Smad4 to affect indirectly the ability of Smad1 to participate in a
complex on the enhancer.
J.S. thanks Dr Mamoru Ishii for the hypothesis that led to her fellowship from
the California Institute of Regenerative Medicine. We also thank Dr Deborah
Johnson for advice on siRNA and members of Dr Michael Stallcup’s laboratory
for help with ChIP and qPCR analyses.
This work was supported by grants from the National Institutes of Health
[R01DE016320 and R01DE019650 to R.M.]. J.S. was supported by a
fellowship from the California Institute of Regenerative Medicine [T100004].
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material available online at
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