The canonical Wnt/ß-catenin signaling pathway regulates Fgf signaling for early
Yongping Wang1, Lanying Song1, Chengji J. Zhou⁎
Department of Cell Biology and Human Anatomy, University of California, Davis, Sacramento, CA 95817, USA
Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children-Northern California, 2425 Stockton Blvd., Sacramento, CA 95817, USA
a b s t r a c ta r t i c l e i n f o
Received for publication 30 May 2010
Revised 1 November 2010
Accepted 2 November 2010
Available online 9 November 2010
Anterior neural ridge (ANR)
The canonical Wnt/ß-catenin signaling pathway has implications in early facial development; yet, its function
and signaling mechanism remain poorly understood. We report here that the frontonasal and upper jaw
primordia cannot be formed after conditional ablation of ß-catenin with Foxg1-Cre mice in the facial ectoderm
and the adjacent telencephalic neuroepithelium. Gene expression of several cell-survival and patterning
factors, including Fgf8, Fgf3, and Fgf17, is dramatically diminished in the anterior neural ridge (ANR, a rostral
signaling center) and/or the adjacent frontonasal ectoderm of the ß-catenin conditional mutant mice. In
addition, Shh expression is diminished in the ventral telencephalon of the mutants, while Tcfap2a expression
is less affected in the facial primordia. Apoptosis occurs robustly in the rostral head tissues following
inactivation of Fgf signaling in the conditional mutants. Consequently, the upper jaw, nasal, ocular and
telencephalic structures are absent, but the tongue and mandible are relatively developed in the conditional
mutants at birth. Using molecular biological approaches, we demonstrate that the Fgf8 gene is
transcriptionally targeted by Wnt/ß-catenin signaling during early facial and forebrain development.
Furthermore, we show that conditional gain-of-function of ß-catenin signaling causes drastic upregulation of
Fgf8 mRNA in the ANR and the entire facial ectoderm, which also arrests facial and forebrain development.
Taken together, our results suggest that canonical Wnt/ß-catenin signaling is required for early development
of the mammalian face and related head structures, which mainly or partly acts through the initiation and
modulation of balanced Fgf signaling activity.
© 2010 Elsevier Inc. All rights reserved.
The highly complex vertebrate face arises from several facial
primordia that consist of an epithelial cover (developed from the
ectoderm externally and the endoderm internally), the neural crest-
derived mesenchyme (which mainly gives rise to the facial skeleton),
and the mesodermal core (which develops facial muscles) (Chai and
Maxson,2006;Creuzetet al.,2005;MinouxandRijli, 2010;Nodenand
Francis-West, 2006). Outgrowth and fusion of the paired maxillary
prominences (maxp), paired medial and lateral nasal prominences
(mnp and lnp), and a single midline frontonasal mass (fnm) establish
the mid and upper face, including the upper jaw, upper lip, nose, and
forehead (Szabo-Rogers et al., 2010). The maxp is developed from the
first pharyngeal arch, while lnp, mnp and fnm originate from a single
primordium, the frontonasal process or prominence (fnp). The
mandibular prominences (manp) derive from the first pharyngeal
arch and form the lower jaw. Several key morphogenetic signaling
pathways, such as Wnt, Fgf, Shh, and Bmp, other pathways such as
endothelin and Dlx signaling, as well as an increasing array of
molecules, are involved in dynamic facial morphogenesis at various
developmental sites and stages (Clouthier and Schilling, 2004; Depew
et al., 2005; Jiang et al., 2006; Nie et al., 2006a,b; Szabo-Rogers et al.,
2010; Wilkie and Morriss-Kay, 2001).
The role of the Wnt/ß-catenin signaling pathway in craniofacial
morphogenesis has been revealed mainly through studying the develop-
ment of neural crest cells and their craniofacial derivatives (Christiansen
et al., 2003). Indeed, conditional deletion of ß-catenin in neural crest cells
with Wnt1-Cre resulted in failure of craniofacial development in mice
(Brault et al., 2001). ß-catenin is a key intracellular molecule in the
canonical Wnt signaling pathway (Mosimann et al., 2009; Willert and
Nusse, 1998). On binding of Wnt ligands to Fzd and Lrp receptors, the
intracellular ß-catenin is stabilized by inactivation of a ß-catenin
degradation complex (which consists of Gsk3ß, APC, Axin, CK1 and
others), and then translocated into the nucleus to activate Tcf/Lef
transcription factors binding to the promoter region of the Wnt target
genes. The canonical Wnt/ß-catenin signaling pathway plays important
roles in development and disease (Logan and Nusse, 2004; MacDonald
et al., 2009). Unique activation patterns of several Wnt signaling
Developmental Biology 349 (2011) 250–260
⁎ Correspondence author. 2425 Stockton Blvd., SHCNC-602B, Sacramento, CA 95817,
USA. Fax: +1 916 453 2288.
E-mail address: firstname.lastname@example.org (C.J. Zhou).
0012-1606/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
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components and signaling reporters are found in the facial prominences
Vendrell et al., 2009). Lef1 and Tcf4 double knockout mice exhibit
abnormal mid face (Brugmann et al., 2007), and mutations in human
WNT3 and mouse Wnt9b genes have been associated with cleft lip/palate
(Carroll et al., 2005; Niemann et al., 2004). We have recently
demonstrated that Lrp6-mediated Wnt signaling is required for the
growthand fusionof facialprimordia, and that genetic disruptionof Lrp6
signaling in mice leads to cleft lip with cleft palate, which resembles a
common birth defect in humans (Song et al., 2009). Yet, the role of the
Wnt/ß-catenin signaling pathway in early facial development, particu-
larly in the formation and patterning of facial primordia remains almost
been suggested as critical for facial morphogenesis in chick models
(Hu and Marcucio, 2009; Hu et al., 2003; Marcucio et al., 2005). To
address the related mechanisms and the role of the Wnt/ß-catenin
used loxP-floxed ß-catenin conditional gene-targeting mouse line
(Brault et al., 2001; Grigoryan et al., 2008) and a well-established
Foxg1kiCremouse line (Hebert and McConnell, 2000). We show that
ß-catenin in the facial ectoderm and/or telencephalic neuroepithelium
processes at the earliest stage of facial development. Combined with
molecular biological approaches and gain-of-function analyses, we
early facial morphogenesis mainly or partly through regulating Fgf
Materials and methods
This study used the following genetically modified mice: condi-
tional ß-catenin loss-of-function (LOF) Catnb1lox(ex2-6)(Brault et al.,
2001) (Jackson Laboratory stock no. 004152), conditional ß-catenin
gain-of-function (GOF) Catnb1lox(ex3)(Harada et al., 1999), Foxg1kiCre
(Hebert and McConnell, 2000) (Jackson Laboratory stock no. 004337),
and Cre reporter Rosa26lacZ (Soriano, 1999) (Jackson Laboratory
stock no. 002292). Mice were housed in the vivarium of the UC Davis
School of Medicine (Sacramento, CA, USA). The day of conception was
designated embryonic day 0 (E0). All research procedures using mice
were approved by the UC Davis Animal Care and Use Committee and
conformed to NIH guidelines.
Skeletal and H&E stains
E18.5 embryos were dissected in PBS, fixed in 95% ethanol for one
week, and then stained with Alcian Blue and Alizarin Red for bone and
cartilage according to a published protocol (McLeod, 1980). For
general histological analyses, embryos were fixed in 4% paraformal-
dehyde (PFA), frozen-embedded in O.C.T. compound (Sakura Finetek
USA), sectioned with a Leica cryostat, and then stained with H&E
solutions according to standard protocols.
Wholemount in situ hybridization, X-gal staining, immunofluorescence,
These experiments were carried out as described previously (Song
et al., 2009). Embryos were fixed in 4% PFA for most experiments.
Wholemount in situ hybridization was performed according to
standard protocols using digoxigenin-labeled antisense RNA probes
(Zhou et al., 2008; Zhou et al., 2004). X-gal staining (Wang et al.,
2008) was carried out overnight on embryos after brief fixation with
2% PFA. The wholemount embryos after in situ hybridization or X-gal
staining were sectioned at 50 μm with a vibrotome. Immunofluores-
cence was carried out on 7- to 10-μm frozen or paraffin-wax
embedded tissue sections using appropriate primary antibodies and
Alexa fluorescence-conjugated secondary antibodies (Molecular
Probes). Rabbit anti-phospho-histone H3 (pHH3, Cell Signaling) and
rabbit anti-ß-catenin (Santa Cruz Biotech) were used in this study.
TUNEL assays were performed using the Dead End Fluoreometric
TUNEL system (Promega), following the manufacturer's instructions.
RNA isolation and real-time quantitative RT-PCR
Total RNA was isolated from tissue dissected from the rostral head
region (including both orofacial and forebrain primordia) of E9.5
embryos (wild-type littermate controls and mutants or LiCl/NaCl-
treated embryos). Semiquantitative PCR was carried out as described
previously (Song et al., 2009). The mRNA levels of ß-catenin, Fgf8, Fgf3,
Fgf17, Fgf4, and Fgf10 were normalized to the mRNA levels of Gapdh
(Glyceraldehyde-3-phosphate dehydrogenase) to allow comparisons
among different experimental groups using the ΔCtmethod (Goydos
and Gorski, 2003). Primers for these genes are listed in Table 1.
Chromatin immunoprecipitation (ChIP)
In vivo ChIP was carried out as described previously (Song et al.,
2009). Briefly, rostral heads including facial and forebrain tissues were
dissected from E9.5 embryos in ice-cold PBS. After pipetting and cross-
linking with 2% formaldehyde, chromatin extraction and immunopre-
cipitations were performed with a ChIP assay kit according to the
manufacturer's protocols (Upstate Biotechnology). Rabbit anti-ß-
catenin antibody (Santa Cruz Biotechnology) was used to pull down
the ß-catenin/Tcf/DNA complex; rabbit IgG was used as a negative
control. The following PCR primers against the Fgf8 promoter region
were used: Tcf/Lef-binding site 1 (BS1) (Fig. 8A), 5′-TGCTTGCC
TCTCTTTAGCC (forward), 5′-ATTTTTGAAGACCAGGTGGC (reverse);
BS2, 5′-TACCTGTGTCTTGTGACTC (forward), 5′-AGCATATGAGATACT-
CAGG (reverse); and BS3, 5′-TGATTGCAGATTCGAGGAAAC (forward),
Luciferase reporter assay
The luciferase reporter assay was carried out according to a
procedure similar to one we described previously (Song et al., 2009).
The 334-bppromoter regionofthemouseFgf8 genebetween−2290bp
and −2624bp, which contains a conserved Tcf/Lef-binding site (BS3)
(Fig. 8), and thesamepromoter region without BS3 were amplified and
cloned into the luciferase reporter O-Fluc upstream of a minimal c-fos
promoter. The resulting plasmids were designated pFgf8-Luc and pFgf8-
mut-Luc, respectively, and transfected into L cells with Lipofectamine
2000 reagent following the manufacturer's instructions (Invitrogen).
The cells were treated with 0–4 ng Wnt3a or 25 mM LiCl. L cells were
also transiently transfected with pFgf8-Luc/pFgf8-mut-Luc in combina-
constructs of Lef1 and constitutively active ß-catenin (aß-catenin)
(Lin et al., 2007). Luciferase activity was assayed using the Dual-
Luciferase assay kit (Promega) after 24-hour transfection.
Primers for real-time PCR.
Y. Wang et al. / Developmental Biology 349 (2011) 250–260
Maternal administration of lithium chloride
Pregnant dams were injected with 30 μl of a 600-mM LiCl or NaCl
processed by in situ hybridization or real-time RT-PCR as described
Sample size and statistical analysis
Two to four samples per genotype were used for each experiment
(detailed numbers were indicated in the figure legends). A minimum
of three samples, or triplicates, were used for quantitative analyses.
Student's t-test was used for statistical comparisons when appropri-
ate, and differences were considered significant at Pb0.05.
Conditional gene-targeting of ß-catenin with Foxg1-Cre in the facial
ectoderm and telencephalic neuroepithelium
To study the role of ß-catenin signaling in early facial development,
we used Foxg1-Cre knockin (Foxg1kiCre) mice and the loxP-flanked
ß-catenin exons 2–6 Catnb1lox(ex2-6)mice for conditional loss-of-
function (LOF) analysis of ß-catenin in the Foxg1-expressing tissues.
be weak in the anterior neural ridge (ANR) and rostral head region at
examine the recombination pattern of Foxg1kiCrein the mixed 129 and
C57BL mouse strain background used in the current study, we crossed
Foxg1kiCrewith the Cre reporter Rosa26lacZ mice in the 129 and C57BL
mixed background. We foundstrongX-galstaining(that represents the
recombination activity) in the ANR, facial ectoderm, and adjacent
telencephalic neuroepithelium at E8.75 (with 12 to 14 somite pairs)
(Fig. 1A, B, and data not shown). This data indicates that Foxg1kiCreis a
powerful tool for conditional gene-targeting, not only in the forebrain
but also in the facial ectoderm. Indeed, we found that ß-catenin-
immunoreactivity was drastically lost in the facial ectoderm and
telencephalic neuroepithelium, but relatively conserved in the facial
mesenchyme (Fig. 1C, D). Real-time RT-PCR for ß-catenin mRNA from
the rostral head tissue at E9.5 also confirmed the successful deletion of
ß-catenin (Fig. 1E).
Facial deformation in the ß-catenin-LOF mutants
The mutant embryos of ß-catenin-LOF mice represented 25% of
total embryos recovered, which matches the Mendelian ratio, and
they survived until birth. All of the mutants displayed striking facial
defects, including the absence of nasal, upper jaw, and ocular
structures (Fig. 2). The upper lip and nasal primordia, including the
medial nasal prominence, lateral nasal prominence, and maxillary
prominence were well-developed in the normal embryos at E10.5
(Song et al., 2009); all of these structures were absent in the mutants
(Fig. 2A, B and data not shown). The optic cup and telencephalic
vesicle in the mutants were also not developed at this early stage.
At E14.5, the facial defects in the mutants were further evident
(Fig. 2C, D). In contrast, the tongue and mandible were formed in the
mutants. Skeleton preparations for E18.5 heads clearly showed the
absence of the premaxillary and maxillary bones and the presence of
Meckel's cartilage in the mandible of the mutant embryos (Fig. 2E, F).
H&E staining of sagittal head sections at E18.5 revealed that, although
the upper jaw and nasal structures in the face and the olfactory bulb
and telencephalon in the forebrain were absent, the basal bone of the
skull dividing the facial andthe forebrain structures was formed in the
mutants (Fig. 2G, H). These data suggest that the basal bone-forming
mesenchymal cells located between the surface ectoderm and
forebrain neuroepithelium are more or less unaffected in ß-catenin-
LOF mutants, which is quite consistent with the recombination
pattern of Foxg1-Cre in the forehead region.
Fig. 1. Recombination activity and conditional inactivation of ß-catenin by Foxg1-Cre in the rostral head. (A, B) A whole head and its representative sagittal section after X-gal
staining (blue) show the recombination activation sites in the embryo of Rosa26lacZ;Foxg1kiCre/+at E8.75 (with 14 somite-pairs) (n=2). (C–E) Immunofluorescence and real-time
RT-PCR demonstrate the site-specific deletion of ß-catenin by Foxg1-Cre in the rostral head at E9.5 (n=3). Arrows indicate the facial ectoderm (FE). FB, forebrain; LOF, loss-of-
function (standing for ß-cateninlox(Ex2-6)/(Ex2-6);Foxg1kiCre/+mice in this study); ME, mesenchyme; NE, neuroepithelium; WT, wild-type littermate control. *Pb0.05.
Y. Wang et al. / Developmental Biology 349 (2011) 250–260
Severe disruption of Fgf signaling in the ANR and facial ectoderm, with
less-affected Shh and Tcfap2a signaling in the rostral head primordia of
the ß-catenin-LOF mutants
To address the mechanism of facial defects in the ß-catenin-LOF
mutants, we first examined several key signaling genes, particularly
those involved in Fgf signaling that are critical for both facial and
forebrain development. At E9.5, Fgf8 was expressed in both facial
ectoderm and adjacent telencephalic neuroepithelium at the rostral
midline-signaling center ANR region, but Fgf8 was expressed only in
the ectoderm around the future nasal pit and pharyngeal arches in the
wild-type control embryo (Fig. 3A–F). These Fgf8 expression domains,
particularly in the ANR and adjacent frontonasal ectoderm, were
dramatically diminished in the ß-catenin-LOF mutants (Fig. 3G–I). In
contrast, expression of Fgf8 in the mutant brain was relatively
conserved in a well-known signaling center, the mid-hindbrain
boundary (MHB). At E10.5, the nasal pit was evident by the formation
of the medial and lateral nasal prominences in the wild-type embryos.
Fgf8 was expressed in the ectodermal edge of the nasal prominences
along the nasal pit in the control embryo, but neither Fgf8-expressing
nasal prominences nor maxillary prominence formed in the mutants
at E10.5 (Fig. 3J, K). In contrast, ectodermal expression of Fgf8 was
relatively conserved in the mandibular prominence of the mutants.
These findings indicate that the defective upper jaw and telenceph-
alon in the ß-catenin-LOF mutants might be caused by loss of Fgf8
mRNA in the ANR and frontonasal ectoderm. Real-time RT-PCR
revealed that Fgf8, Fgf3, and Fgf17, but not Fgf10 or Fgf4, were
significantly downregulated in the rostral head tissues of the
ß-catenin-LOF mutants at E9.5 (Fig. 4A). Additionally, wholemount
in situ hybridization demonstrated the absence of Fgf3 and Fgf17
expression in the mutant ANR at E9.5 (Fig. 4B, C). These findings
suggest that ß-catenin is critical for multiple Fgf ligands, particularly
Fgf8 expression in the ANR and facial ectoderm.
On the other hand, Shh in the anterior ventral midline tissue and
Tcfap2a in neural crest cells both play important roles in facial and/or
forebrain development. Wholemount in situ hybridization revealed
that Shh expression in a ventral telencephalic domain, but not in other
brain regions, was apparently diminished in the mutants at E9.5
(Fig. 5A–D). In contrast, Tcfap2a expression in the facial region was
notdiminished, oreven upregulated in the mutantsat E9.5 (Fig.5E, F).
These results suggest that the altered expression of Shh, but not
Tcfap2a, in the developing rostral head may also contribute to the
severe defects seen in the mutants.
Drastic apoptosis followed the inactivation of Fgf8 expression in the
facial and forebrain primordia of ß-catenin-LOF mutants
Inactivated Fgf8 expression in the ANR and the facial ectoderm was
the most notable change in the gene alterations of the ß-catenin-LOF
mutants at E9.5. Because Fgf8 is a critical survival factor for various
precursor cells (Chi et al., 2003; Grieshammer et al., 2005; Trumpp
et al., 1999), we examined both proliferation and apoptosis in the
rostral head of the mutants. We evaluated proliferation by immuno-
labelingfor pHH3 at E9.5 and found no apparentalterations of the rate
of the pHH3(+) mitotic cells in the facial ectoderm, adjacent
mesenchyme, and telencephalic neuroepithelium of the ß-catenin-
LOFmutants compared withwild-typelittermatecontrols(Fig. 6A–C).
We then assessed programmed cell death by TUNEL assays and found
a marked increase of the apoptotic rate in the mutants (Fig. 6D–F),
with particularly high rates of apoptosis in the facial ectoderm and
telencephalic neuroepithelium, where Fgf8 expression was lost in the
mutants. These results suggest that cell death is a consequence of
diminished Fgf8 expression in the mutants. The architecture of
the telencephalic neuroepithelium was also dramatically affected,
which was shown to be a result of defective cell-cell adhesion in the
ß-catenin-LOF mutants (Junghans et al., 2005). The TUNEL(+) facial
Fig. 2. Dorsal face and upper jaw are absent in the conditional ß-catenin-LOF mutant mice. (A–D) Sagittal views of whole embryos or heads show the defective structures in the
rostral head region (red arrows and the area circled by the white-dashed line) of the mutants at E10.5 and E14.5. (E, F) Skeletal stain at E18.5 demonstrates the absence of the dorsal
facial structures including the upper jaw, nose, and eye (e). The tongue (t) and mandible (man) are apparently formed. (G, H) H&E stain on sagittal head sections at E18.5 more
clearly shows the defective facial and forebrain structures in the mutant. Arrowheads indicate the formation of the basisphenoid-like basal bone (bp) of the skull that divides the
facial and the forebrain regions in the mutants. bo, basioccipital bone; exo, exoccipital bone; F, frontal bone; max, maxilla; P, parietal bone; pmax, premaxilla; pt, palate; ul, upper lip.
Y. Wang et al. / Developmental Biology 349 (2011) 250–260
mesenchymal cells were also highly increased in the area adjacent
to the telencephalic neuroepithelium in the mutants (Fig. 6E),
suggesting a secondary effect of the defective Fgf8-expressing neuro-
epithelium at this developmental stage.
To clarify the causal relationship between the diminished Fgf8
expression and apoptosis in the mutants, we examined Fgf8 expression
and TUNEL at E8.75 (Fig. 7). We found a dramatic decrease of the Fgf8
expression domain specifically in the ANR, but not in the MHB or
branchial arches in the ß-catenin-LOF mutants at this early stage
(Fig. 7A–D). In contrast, no significant changes in TUNEL(+) cells were
E8.75 (Fig. 7E, F). These results suggest that cell death is a consequence
of diminished Fgf8 expression in the ß-catenin-LOF mutants during
early facial development.
Fgf8 was transcriptionally regulated by Wnt/ß-catenin signaling
To determine the molecular mechanism of the diminished Fgf8
expression in the ß-catenin-LOF mutants, we examined a 3-kb putative
promoter region upstream of the mouse Fgf8 gene and found three
evolutionarily conserved Tcf/Lef binding sites, the Wnt-responsive
elements (Fig. 8A). ChIP assays were performed on the extracts from
region (Fig. 8B). Luciferase reporter assays were carried out to
investigate the functional significance of BS3 (Fig. 8C, D). The luciferase
between -2290bp and -2624bp, which contains BS3. The reporter was
Wnt signaling activator (Fig. 8D). Significantly, the Fgf8-promoter
reporter was directly activated by the expression constructs of Lef1 and
the dominantly active ß-catenin (Fig. 8E). Conversely, these luciferase
activities were significantly diminished when BS3 was deleted in the
Fgf8-promoter construct, demonstrating that Fgf8-promoter reporter
activation was dependent on the Tcf/Lef consensus site 3. These results
strongly suggest that Fgf8 is a direct target gene of Wnt/ß-catenin
signaling during early facial development.
Fgf8 was ectopically upregulated in vivo by gain-of-function of
To further demonstrate that Fgf8 is regulated by Wnt/ß-catenin
signaling in vivo, we examined the reverse effect of Fgf8 expression by
gain-of-function (GOF) analyses. To activate Wnt/ß-catenin signaling
Fig. 3. Loss of Fgf8 expression in the anterior neural ridge (ANR) and facial ectoderm of the ß-catenin-LOF mutants. (A–F) The normal expression pattern of Fgf8 in the wild-type head
at E9.5. Sagittal (A) and oblique frontal (B) views of a whole head show the Fgf8 mRNA signals in the ANR (arrows) and its adjacent nasal eminence (arrowhead), maxillary (maxp)
and mandibular (man) processes, and mid-hindbrain boundary (MHB). (C) Illustration of the Fgf8 expression domain in the frontal face and the approximal positions for the sagittal
sections in panels D–F. (D) Fgf8 mRNA is present in both facial ectoderm (arrowhead) and its tightly adjacent neuroepithelium (arrow) at the midline region. (E) Fgf8 mRNA is found
in both facial ectoderm (arrowhead) and neuroepithelium (dashed arrow), which are divided by a non-Fgf8-expression mesenchymal layer on a proximal lateral section. (F) Fgf8
mRNA is present only in the facial ectoderm (arrowhead) on a distal lateral section. (G–I) Fgf8 mRNA is absent in both facial ectoderm (arrowhead in I) and neuroepithelium (arrow in
I) of the ANR (arrows in G,H) and nasal eminence (arrowhead in H) (n=4). Asterisks indicate the Fgf8 mRNA signals in the MHB and mandibular process. (J,K) Frontal facial views of
the wild-type and mutant embryos with Fgf8 expression signals at E10.5 (n=3). Red arrow in J indicates Fgf8 mRNA in the developed ANR. Arrowheads in J indicate the restricted
expression domain of Fgf8 at the edge of the medial nasal process. Dashed arrow in K indicates Fgf8 expression in a non-ANR brain domain in the mutant. FE, facial ectoderm; lnp,
lateral nasal process; manp, mandibular process; maxp, maxillary process; mnp, medial nasal process; NE, neuroepithelium.
Y. Wang et al. / Developmental Biology 349 (2011) 250–260
in developing tissue, pregnant mice were injected with LiCl, which
inhibits Gsk3ß and, in turn, stabilizes ß-catenin for its transcriptional
function in the nuclei. After daily maternal administration of LiCl from
E7.5, the Fgf8 mRNA level was upregulated in the ANR and facial
region of the normal embryos at E9.5 (Fig. 9A–C). To further verify
that Fgf8 is upregulated in vivo by Wnt/ß-catenin signaling, we
analyzed Cre/loxP-mediated ß-catenin-GOF by conditionally deleting
the ß-catenin exon 3 (encoding for a Gsk3ß-phosphorylation site),
which results in constitutive ß-catenin stabilization or activation in
Cre-expressing cells (Harada et al., 1999). Strikingly, the Fgf8
expression domain in the ANR and facial ectoderm was expanded
ectopically in the rostral head after conditional activation of ß-catenin
with Foxg1-Cre at E9.5 (Fig. 9D, F, E, G). At E10.5, the orofacial
primordia, including the lateral/medial nasal prominences and
maxillary prominence did not form (Fig. 9H vs. Fig. 3J), the Fgf8
expression domains remained ectopic and clustered horizontally
in the upper jaw region and vertically in the head midline of the
ß-catenin-GOF mutants (Fig. 9I vs. Fig. 3J). These results indicate that
Fgf8 expression can be efficiently upregulated by GOF of Wnt/ß-
catenin signaling in vivo, and that the over-activated Fgf8 signaling by
ß-catenin may also prevent orofacial development.
Conditional gene-targeting with Foxg1kiCrefor early facial development
The current study has clearly shown that conditional deletion of ß-
frontonasal (including fnm, mnp, and lnm) and maxillary primordia,
recombination pattern of Foxg1kiCremice was previously reported in the
mapping approach, we have demonstrated the specific and intensive
recombination activity of Foxg1kiCrein both facial ectoderm and
telencephalic neuroepithelium at E8.75 (about 12- to14-somite-pair
stage). Another group has also shown Foxg1kiCreactivity in both cell
lineages at E9.5 (Kawauchi et al., 2005). The recombination pattern of
Foxg1kiCrematches well with the gene expression pattern of endogenous
Foxg1 (formerly BF1) that has been previously demonstrated in the
presumptive facial ectoderm first, as early as the 4-somite-pair stage, and
subsequently in the adjacent neuroectoderm (the future telencephalic
neuroepithelium) before the closure of anterior neural folds (Shimamura
and Rubenstein, 1997). Also, although numerous studies used Foxg1 as a
clearly show expression of Foxg1 in both facial ectoderm and forebrain
primordia at E8.5 or E9.5 in mice (Acampora et al., 2001; Filosa et al.,
ectopic recombination patterns in some, but not all, mouse strain
backgrounds (Hebert and McConnell, 2000). For instance, BALB/c and
in non-Foxg1-expressing cell lineages, while the 129SvJ strain shows a
and found consistent and reliable results of the Foxg1kiCrerecombination
and the facial phenotypes in the conditional mutants. Taken together,
these data indicate that the Foxg1kiCremouse line is a critical tool for
conditional gene-targeting in both facial ectoderm and forebrain
neuroepithelium in the early developmental stage. However, particular
attention needs to be paid to the mouse strain background and use of
identical strains in both genetic fate mapping and conditional gene-
Fgfs as the target genes of the Wnt/ß-catenin signaling pathway
The current study provides strong evidence that the expression of
several critical morphogenetic signaling molecules, particularly Fgfs,
Fig. 4. Alterations of additional Fgf members in the mutant ANR of ß-catenin-LOF at
E9.5. (A) Real time RT-PCR results for selective Fgf family members expressed in the
rostral head of the normal and mutant embryos. The mRNA level of each gene was
normalized against the mRNA level of Gapdh. Data were obtained from three
independent experiments. *Pb0.05. (B) Frontal heads or sagittal sections show the
defective Fgf3 expression in the neuroepithelium of the mutant ANR (arrows) (n=3).
(C) Defective expression of Fgf17 expression in the mutant ANR (arrows) (n = 3).
Fig. 5. Alterations of Shh and Tcfap2a in the mutant head of ß-catenin-LOF at E9.5.
Sagittal (A, B) and ventral (C, D) views of the whole heads show the decrease of the
ventral telencephalic domain (arrows) of Shh mRNA in the mutants (n=3).
(E, F) Tcfap2a mRNA was unlikely diminished in the facial region (arrows) but even
might be upregulated in the rostral head of the mutants (n = 3).
Y. Wang et al. / Developmental Biology 349 (2011) 250–260
is dramatically diminished in the rostral head primordia of ß-catenin-
LOF mutants. This may subsequently prevent proper facial develop-
ment in mutants due to conditional deletion of ß-catenin with
Foxg1kiCrein the facial ectoderm and telencephalic neuroepithelium.
Fgfs are evolutionarily conserved polypeptide growth factors playing
various important roles in embryonic development (Goldfarb, 1996;
Itoh and Ornitz, 2008; Thisse and Thisse, 2005). At E9.5 and E10.5 of
mice, genes encoding multiple Fgf ligands (including Fgf3, Fgf8, Fgf9,
Fgf10, Fgf15, Fgf17, and Fgf18) and two Fgf receptors, Fgfr1 and Fgfr2,
are expressed in the facial primordia (Bachler and Neubuser, 2001).
Our results suggest that Fgf8 is a direct target gene regulated by
ß-catenin signaling in the ANR and facial ectoderm. This is partially
supported by a recent study that a Tcf/Lef binding site located in the
intron 3 of the Fgf8 gene is responsive to Wnt/ß-catenin activation
during tooth development (Wang et al., 2009). In contrast, we
determined a different Wnt-responsive element in the 5′ promoter
region of Fgf8 in the early developing rostral head, which can be
activated by Wnt3a and LiCl, or directly activated by constitutively
active ß-catenin and Lef1 constructs. The different Wnt-responsive
elements in the Fgf8 gene in early facial and tooth development
suggest the age- and lineage-dependent roles of Fgf8 activation by
the Wnt/ß-catenin signaling pathway. Future transgenic studies may
helpto determine ifthese Wnt-responsiveelementscan actuallydrive
reporter gene activation in respective tissue sites. In addition, we
found that Fgf3 and Fgf17 are also regulated by Wnt/ß-catenin
signaling in the ANR of early mouse embryos. Interestingly, Fgf4 in the
mouse tooth bud (Kratochwil et al., 2002), Fgf10 in the developing
mouse heart (Cohen et al., 2007), and Fgf20 in cancer cells or Xenopus
embryos (Chamorro et al., 2005) have previously been determined as
the downstream target genes of Wnt/ß-catenin signaling pathway.
Taken together, these data suggest that a set of different Fgf ligands is
regulated by the canonical Wnt/ß-catenin signaling pathway in a
wide range of biological processes.
Fig. 6. Proliferation and apoptosis in the ß-catenin-LOF mutants at E9.5. (A-C) Proliferation was assessed by immunofluorescence for pHH3. The proportion of pHH3(+) cells is not
significantly changed in the facial ectoderm (FE), facial mesenchyme (ME), and telencephalic neuroepithelium (NE) of the mutants. Asterisk in B indicates the disorganized
neuroepithelium in the mutants. (D-F) TUNEL(+) apoptotic cells are dramatically increased in the facial ectoderm and telencephalic neuroepithelium as well as in facial
Fig. 7. Loss of Fgf8 precedes programmed cell death in the anterior neural ridge (ANR) of
the ß-catenin-LOF mutants at E8.75. Sagittal head (A,B) and frontal views show the
dramatic decrease of Fgf8 mRNA signals in the ANR (arrows) but not in mid-hindbrain
boundary (MHB) orbranchial arches (BA).(E,F) No significantchanges of TUNEL(+)cells
in the facial ectoderm (FE, arrows) and adjacent neuroepithelium (NE) between the
controls and mutants at E8.75 (n=2 per genotype). Asterisks indicate some clustered
TUNEL(+) cells in the ventral region of both normal and mutant telencephalon.
Y. Wang et al. / Developmental Biology 349 (2011) 250–260
Wnt/ß-catenin signaling activates Fgf signaling for cell survival and
Among the 22 Fgf ligands, Fgf8 is a key molecule in the developing
head/brain signaling centers that are required for facial and brain
development (Liu and Joyner, 2001; Suzuki-Hirano and Shimogori,
2009; Szabo-Rogers et al., 2010). The current study shows that Fgf8
expression is dramatically diminished in the ANR and facial ectoderm
of the ß-catenin-LOF mice; this may be a major cause of facial defects
in these mutants. In support of this, conditional deletion of Fgf8 with
Fig. 8. Fgf8 is a target gene of Wnt/ß-catenin signaling during facial development. (A) Sequence analysis revealed three Tcf/Lef binding sites (BS1-BS3) within the 3-kb putative
promoter region of the mouse Fgf8. (B) ChIP assays were performed on the tissue extracts from the rostral head of E9.5 wild-type mice. ß-catenin formed a transcriptional complex
with Tcf/Lef at the BS3 in the putative Fgf8 promoter region. (C-E) Luciferase assay results for L cells transfected with pFgf8-Luc or pFgf8-mut-Luc constructs, stimulated by Wnt3a
(C), lithium (D), or co-transfected with pcDNA3 or Lef1 and constitutively active ß-catenin expression constructs (E). The luciferase activity of pFgf8-Luc in the control assay was
defined as one unit. Luciferase assay results were obtained from three independent experiments, and each was performed in triplicate. *Pb0.05.
Fig. 9. Fgf8 is upregulated by gain-of-function (GOF) of Wnt/ß-catenin signaling during facial development. (A–C) Wholemount in situ hybridization and real-time RT-PCR demonstrate
the increase of Fgf8 mRNA signals in the anterior neural ridge (arrows) of E9.5 wild-type embryos after maternal administration with the Wnt activator lithium (n=3). *Pb0.05.
(D–H) Conditional ß-catenin-GOF with Foxg1-Cre causes dramatic upregulation of Fgf8 expression in the facial ectoderm (arrows) and anterior neural ridge (asterisks) at E9.5 (n=2), and
lateral and medial nasal processes and maxillary process, which formed a horizontal line with intensive Fgf8 expression (I) in the ß-catenin-GOF mutants at E10.5. fb, forebrain; manp,
Y. Wang et al. / Developmental Biology 349 (2011) 250–260
Nes-Cre1 mice in the ectoderm of the first pharyngeal or branchial
arch 1 (BA1) causes extensive cell death in the BA1 mesenchyme and
results in severe defects of BA1 derivatives (such as mandible)
(Trumpp et al., 1999). The hypomorphic Fgf8 mutant mice exhibit
defective craniofacial development with significantly increased
apoptosis in the ANR and other Fgf8-expressing regions or migrating
neural crest cells (Abu-Issa et al., 2002).
Significantly, conditional deletion of exon 5 in the Fgf8 gene with
AP2a-Cre (also called Tcfap2a-Cre) in facial ectoderm and neural crest
cells leads to the developmental failure of entire facial structures,
including both upper jaw and lower jaw, probably due to massive cell
death in the mutant pharyngeal arches and other facial primordia
(Macatee et al., 2003). This is consistent with the findings in the
current study, except that the lower jaw and tongue are formed but
the eye is absent in the Catnb1lox(ex2-6);Foxg1kiCremutants. The similar
facial deformation in these mutants may be due to the fact that the
recombination pattern of Foxg1-Cre and AP2a-Cre are largely over-
lapped in the facial ectoderm. The different mandibular and ocular
phenotypes in these mutants are likely caused by Foxg1kiCre
recombination being less active in the pharyngeal arches but highly
activated in the telencephalic neuroepithelium, including the optic
vesicle, compared to AP2a-Cre recombination. Collectively, these
results suggest that Fgf8 activated by Wnt/ß-catenin signaling in the
facial ectoderm is critical for cell survival in facial ectoderm and facial
mesenchyme during the development of the facial primordia. This is
supported further by a recent study conducted by Trevor Williams'
group. They use their newly developed ectodermal Cre transgenic
mice with a promoter derived from Tcfap2a (or AP2a) to conditionally
delete ß-catenin in the facial ectoderm, which resultsin similardefects
in entire facial primordia associated with diminishedexpressionof Fgf
signaling in the facial ectoderm (personal communication from T.
Williams). Some minor differences between these studies may be a
Cre mouse lines used.
Conditional deletion of exons 2 and 3 in the Fgf8 locus with Foxg1-
Cre mice results in defective nasal and other craniofacial structures
(Kawauchi et al., 2005). However, a small and short snout is formed
in the mutant mouse at birth. The less-severe facial defects in the
Fgf8lox(exons2,3);Foxg1kiCremice compared with the extremely severe
facial defects in the Fgf8lox(exon5);AP2a-Cre or the Catnb1lox(ex2-6);
Foxg1kiCremice indicate that Cre-mediated deletion of Fgf8lox(exons2,3)
may create a hypomorphic allele, and that dose-dependent LOF of
Fgf8 downstream of ß-catenin signaling may lead to a spectrum of
craniofacial disorders with corresponding severities. Related clinical
significance is demonstrated by the association of FGF8 mutations
with cleft lip/palate in humans (Riley et al., 2007). We have recently
found no dramatic alterations of Fgf8 expression in the craniofacial
primordia of the Wnt coreceptor Lrp6-null mice that exhibit full-
penetrant cleft lip/palate (Song et al., 2009). However, we cannot
exclude that there may be subtle alterations in Fgf8 expression
during lip fusion processes or that Lrp6 and Lrp5 are functionally
redundant upstream of ß-catenin in the regulation of Fgf8 signaling.
Because the double-null mice of Lrp6 and Lrp5 die at gastrulation
(Kelly et al., 2004), Lrp6 conditional gene-targeting mice (Zhou et al.,
2010) in combination with the viable/fertile Lrp5-null mice (Kato
effects of Wnt signaling on Fgf8 activation and craniofacial birth
The relationship between facial and forebrain development
We show that a Shh expression domain in the ventral telenceph-
alon is diminished in conditional Catnb1lox(ex2-6);Foxg1kiCremutants at
E9.5, suggesting that Shh signaling may also be regulated by the Wnt/
ß-catenin signaling pathway directly or indirectly in this region. Shh
signaling plays important roles in both facial and forebrain develop-
ment. Mutations in the human SHH gene are a cause of holoprosen-
cephaly (HPE), a common midline defect in the forebrain and face
(Belloni et al., 1996; Bendavid et al., 2010; Dubourg et al., 2004;
Roessler et al., 1996,2009). The completely arrested frontonasal
primordia and telencephalic/optic vesicles in the Catnb1lox(ex2-6);
Foxg1kiCremutants may resemble a severe form of HPE, which also
suggests a close relationship between facial and forebrain develop-
ment by sharing a common signaling mechanism. Wnt signaling may
induce both Fgf and Shh signaling pathways in the rostral head
signaling centers for facial and forebrain development.
In the chick model, Shh and Fgf8 are expressed in a complementary
manner and form a boundary structure in both frontonasal ectoderm
and telencephalic neuroepithelium, which is required for both facial
and forebrain patterning and growth (Hu et al., 2003; Schneider et al.,
2001). Inhibition of Shh signaling in the neuroectoderm of chick
embryos not only alters the dorsoventral patterning of the telen-
cephalon, but also disrupts a signaling center in the frontonasal
ectoderm, and subsequently affects the facial development (Marcucio
et al., 2005). Ectopic Shh signaling in the forebrain splits a single
frontonasal ectodermal zone of the chick beak primordium into two
separated domains that resemble the paired medial nasal promi-
nences seen in the E10.5 mouse embryo (Hu and Marcucio, 2009).
From these studies, the Helms group suggests that the forebrain is not
only a structural base supporting facial primordia, but also a source of
signals modulating facial development.
However, the signaling-mediating mechanisms from the forebrain
dramatically diminished in the forebrain neuroepithelium first and
then in the nasal pit by the ectopic activation of Shh signaling in the
chick forebrain (Hu and Marcucio, 2009), suggesting a stepwise
repressive role of Shh on Fgf8 expression in the ANR, and
subsequently in the facial ectoderm. In the current study, we have
observed a progressive activation pattern of Fgf8 that is expressed
specifically in the ANR at E8.75 (after closure of the anterior neural
tube), subsequently expands to the facial ectoderm adjacent to ANR at
E9.5, and is finally restricted to the nasal pit at E10.5 during normal
development. These observations suggest that Fgf8 may first be
induced by Wnt/ß-catenin signaling in the ANR, then subsequently
Fgf8 regulate its own expression in the facial ectoderm. However,
ß-catenin in the facial ectoderm may also modulate Fgf8 activation or
expansion through its roles in both Wnt signaling and cell adhesion.
Notably, it has been proposed that ß-catenin-mediated cell adhesion,
but not the Wnt signaling, is critical for cell survival and organization
during forebrain development (Junghans et al., 2005). In disagree-
ment with the conclusion by Junghans et al., the current study has
demonstrated that the transcriptional function of ß-catenin signaling
is critical for Fgf signaling activation at the ANR, which subsequently
modulates cell survival and patterning in forebrain development. As
direct evidence, combinatorially deleting Fgf receptors (Fgfr1, Fgfr2,
and Fgfr3) with Foxg1kiCremice demonstrated a dose-dependent effect
of Fgf signaling on cell survival and telencephalic development
(although no information on facial development was available) (Paek
et al., 2009). Interestingly, Paek et al. also observed diminished Shh
expression in the ventral telencephalon of the triple Fgfrs1,2,3;
Foxg1kiCreconditional mutants. Together with our findings of the
diminished Shh expression in the same region of Catnb1lox(ex2-6);
Foxg1kiCremutants, these results suggest that Shh signaling may also
be regulated by Fgf signaling downstream of Wnt/ß-catenin signaling
for forebrain and facial development. However, the dual functions of
ß-catenin in cell adhesion and Wnt signaling may act simultaneously
during facial and forebrain development. Our double conditional
gene-targeting approaches of Lrp6 and Lrp5 discussed above may
clarify these questions in future studies.
In conclusion, the current study demonstrates that conditional
inactivation of ß-catenin in the Foxg1-expressing facial ectoderm and
neuroectoderm severely impairs the development of the mid/upper
Y. Wang et al. / Developmental Biology 349 (2011) 250–260
face and the telencephalic/ocular structures in a form that may
resemble a severe type of HPE. These defects are mediated by
alterations of a set of signaling genes required for cell survival and
patterning during early development. Our results provide genetic
evidence that the canonical Wnt/ß-catenin signaling pathway is a key
upstream factor that initiates and modulates a balanced Fgf signaling
activity in the rostral signaling center ANR and the adjacent facial
ectoderm required for both early facial and forebrain development.
Kemler for the Catnb1lox(ex2-6), and S. McConnell for the Foxg1-Cre
mice, and S. Evans for the active ß-catenin and Lef1 expression
constructs. We are grateful to T. Williams for sharing unpublished
data; M. Hrachova, K. Wang, Y. Li and J. Chan for technical assistance;
B. Guo and D. Cortes for critical reading and editing of the manuscript;
A. Molotkov for some preliminary studies; Y.Z. Wang, Q. Gan, T. Zhao,
and T. Yamagami for general help or discussion. This work was
supported by the Shriners Hospitals for Children (research grants
87500 and 86100 to C.Z. and a postdoctoral fellowship to L.S.) and UC
Davis School of Medicine (a research award to C.Z.).
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