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
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