Uncoupling fibroblast growth factor receptor 2
ligand binding specificity leads to Apert
Kai Yu and David M. Ornitz*
Department of Molecular Biology and Pharmacology, Washington University Medical School, Campus Box 8103, 660 South Euclid Avenue,
St. Louis, MO 63110
tor receptors (FGFRs) 1–3 are the etiol-
ogy of many craniosynostosis (premature
fusion of the cranial sutures) and chon-
drodysplasia (dwarfism) syndromes (1–3).
Mutations in Fgfr2 cause craniosynostosis
syndromes including Crouzon syndrome,
Pfeiffer syndrome, and Apert syndrome
(AS). The article in this issue of PNAS by
Hajihosseini et al. (4) presents the first
animal model for a mutation in Fgfr2 that
is associated with AS, one of the most
severe of the human craniosynostosis
FGFRs are transmembrane receptor ty-
rosine kinase proteins that are activated
by many of the 22 members of the FGF
family (5, 6). The extracellular region of
the FGFR contains two or three Ig-like
domains and mediates ligand binding (7).
heparan sulfate, ligand binding induces
receptor dimerization and subsequent ac-
tivation (5, 8–10). Importantly, the affin-
ity and specificity of FGFRs are regulated
by tissue-specific alternative splicing. The
paper by Hajihosseini et al. (4), and two
additional studies (11, 12), show that mu-
tations that cause AS circumvent the bio-
chemical and developmental regulatory
mechanisms that are normally imposed by
tissue-specific alternative splicing of Fgfr2
and result in ectopic ligand-dependent
Alternative splicing in the region en-
coding the carboxyl-terminal half of Ig
domain III creates receptor isoforms with
distinct ligand binding specificity by incor-
porating either a b or a c exon (Fig. 1A).
For FGFR2, this splicing event is very
tissue-specific with b exon usage in epi-
thelial tissue and c exon usage in mesen-
chymal tissue (13). Directional epithelial-
mesenchymal signaling is maintained
because mesenchymally expressed li-
gands, such as FGF7 and FGF10, can
activate only epithelially spliced FGFR2b.
Similarly, ligands such as FGFs 2, 4, 6, 8,
9 and 17, which tend to be expressed in
P253R) in the highly conserved region
ominant missense mutations in the
genes encoding fibroblast growth fac-
epithelial tissues activate mesenchymally
spliced FGFR2c (14–16). The reciprocal
nature of this signaling mechanism is most
eloquently illustrated in the developing
limb where FGF10 is a required mesen-
chymal signal that induces formation of
the apical ectodermal ridge and FGF8
(and possibly FGFs 4, 9, and 17) is an
epithelial factor that signals to distal mes-
enchyme (Fig. 1B) (17–21).
The importance of FGFR2 signaling in
organogenesis is illustrated by gene tar-
geting studies. Mouse embryos lacking
Fgfr2 die at stages before the limbs or
lungs develop (22, 23). Embryos in which
only the b isoform of Fgfr2 has been
deleted survive until birth but also fail to
develop limb buds, lung, and other organs
(24). These phenotypes are remarkably
similar to those seen in mice lacking Fgf10
(25–27) and demonstrate the importance
of the mesenchymal to epithelial FGF
signaling pathway. The effects of loss of
FGFR2 signaling in mesenchymal tissues
are obscured by the early embryonic le-
thality and the agenesis of the limbs and
other structures in the null mutant.
Biochemical studies show that many of
these mutations result in ligand indepen-
dent FGFR activation, often involving the
formation of an intermolecular disulfide
bond. AS, although allelic with other cra-
niosynostosis syndromes, is much more
severe and is characterized by premature
fusion of the coronal sutures, severe syn-
dactyly in the hands and feet, brain mal-
formations, and mental retardation. The
severe syndactyly and neurological disor-
ders are not associated with other cranio-
synostosis syndromes. These phenotypic
differences suggest that the signals trans-
duced by receptors harboring AS muta-
tions differ from those in other craniosyn-
ostosis syndrome mutations. Differences
could lie in the intensity of the signal or in
the spatial and temporal patterns of re-
The vast majority of AS patients harbor
one of two missense mutations (S252W or
28). Biochemical analysis revealed that
these genetic alterations render the mes-
enchymally expressed mutant FGFR2c
abnormally susceptible to activation by
mesenchymally expressed ligands such as
FGF7 and FGF10 and the epithelially
expressed mutant FGFR2b abnormally
susceptible to activation by epithelially
expressed ligands such as FGF2, 6, and 9,
thus circumventing the normal epithelial-
mesenchymal signaling restrictions (Fig.
1C) (12). The mechanism by which these
mutations affect receptor signaling is fun-
damentally different from that of other
mutations that activate FGFR2, which
generally cause ligand independent recep-
tor dimerization, stabilized by intermolec-
ular disulfide bonds (1, 3).
Recently Oldridge et al. (11) have iden-
tified two cases of AS (of 260) that do not
have missense mutations in Fgfr2. Inter-
estingly, these patients were found to have
de novo Alu-insertions upstream or within
the c exon (exon 9) of Fgfr2. Molecular
analysis showed that these Alu insertions
affect alternative splicing of Fgfr2, result-
ing in the ectopic expression of FGFR2b
in tissues that normally would express
FGFR2c (Fig. 1D). Thus mesenchymal
tissue from these patients coexpresses
both FGFR2c and FGFR2b. Interestingly,
those authors also have identified patients
with Pfeiffer syndrome with a mutation in
of the c exon. The phenotype of these
patients is more severe than that of
Pfeiffer syndrome patients with different
mutations in FGFR2 and suggests that
alternative splicing may be affected and
could contribute to the more severe
Hajihosseini et al. (4) have modeled
these types of mutations in the mouse by
specifically deleting the c exon of FGFR2
See companion article on page 3855.
*To whom reprint requests should be addressed. E-mail:
March 27, 2001 ?
vol. 98 ?
no. 7 ?
(Fgfr2?c). The consequence of this dele-
tion is the utilization of the alternative
exon b in tissues where c normally would
be used exclusively (Fig. 1D). Similar to
the Alu insertions in humans, this is a
dominant mutation in the mouse, and
heterozygous mice (Fgfr2?/?c) develop
skeletal and visceral defects resembling
those seen in patients with AS and more
severe cases of Pfeiffer syndrome. Taken
together, the biochemical studies and the
mouse and human mutations suggest that
the primary pathology in AS arises from
ligand-dependent activation of FGFR2,
predominantly in mesenchymal tissue.
Cartilage is formed through differen-
tiation of condensed mesenchyme during
early stages of embryonic development.
Fgfr2c is highly expressed in precartilage
cell condensations, and Fgf7 is highly
expressed in the surrounding loose mes-
enchyme (29–32). Although this ligand-
receptor pair is normally not functional,
AS mutations or aberrations in the nor-
mal splicing pattern of FGFR2 could
permit ectopic signals between different
mesenchymal cell populations resulting
in aberrant mesenchymal growth and
differentiation. This explains why the
rare Alu insertion mutations in humans
and the mouse FGFR2?cmutation de-
velop similar phenotypes to patients with
AS missense mutations.
The AS limb defects include osseous
fusion of the digits and phalangeal joints
and ectopic cartilage in periarticular tis-
sues and flexor tendons (33). The skel-
etal defects observed by Hajihosseini et
al. (4) in the Fgfr2?/?cmice include pre-
mature fusion of the coronal sutures and
precocious sternal fusion. Although limb
abnormalities are not found in the mu-
tant mice, abnormal thickening of the
sternebra and ectopic ossification of in-
tersternebral cartilage is observed.
These phenotypic differences could be
species specific or could result from dif-
ferences in receptor signaling in epithe-
lial tissue. For example, in the apical
ectodermal ridge, missense mutations
are predicted to cause increased auto-
crine signaling. In contrast, alternative
splicing mutations would not be expected
to affect signaling in epithelial tissues
that already express FGFR2b.
Crouzon syndrome, unlike AS, is not
associated with limb abnormalities. Most
Crouzon syndrome mutations involve the
gain or loss of a cysteine residue within Ig
domain III of FGFR2. The consequence
of this class of mutation on the function of
FGFR activity is to create an unpaired-
cysteine residue, which facilitates the for-
mation of intermolecular disulphide
bonds, causing ligand-independent dimer-
ization, phosphorylation, and signaling
(34). The mechanism of this mutation
suggests that uniformly elevated FGFR
signaling in one cell type is not sufficient
to induce ectopic differentiation or that
the intensity of receptor activation for this
type of mutation is not sufficient to affect
skeletal limb development. This mecha-
nism supports the concept that signaling
between adjacent cell layers within mes-
enchyme is an essential component of the
pathogenesis of AS.
Hajihosseini et al. (4) also observed
visceral defects in mutant mice in tissues
such as lungs, kidneys, and lacrimal
glands. As in the limb, these tissues
also require extensive epithelial-mes-
enchymal interactions for normal devel-
opment. In lung development, chemotac-
tic signals (FGF10) originating from
mesenchyme regulate epithelial branch-
ing. Interestingly, in mice lacking Fgf9,
mesenchymal tissue is deficient and ab-
normal lung branching ensues (J. Colvin
and D.M.O., unpublished work). Inter-
estingly, in the Fgfr2?/?cmice increased
lung mesenchyme and defects in lung
development are observed, suggesting
that precise signaling within mesenchy-
mal tissue is essential for normal devel-
Most gene targeting studies result in
loss of function. The neomorphic prop-
linked (s-s) Ig loops (I, II, III), a transmembrane domain (TM) and an intracellular tyrosine kinase domain
(TK). The striped region in the carboxyl- terminal half of Ig loop III is subject to alternative utilization of
either exon b or exon c. These correspond to exons 8 and 9 of the Fgfr2 gene. (B) Schematic diagram of
mesenchymal condensation. Sites of Fgf and Fgfr expression are shown. Limb bud initiation and out-
growth is regulated by a reciprocal signaling loop in which FGF10 signals to FGFR2b and FGFs 4, 8, 9, and
17 signal to FGFR1c (solid arrows). FGFR2c is prominently expressed in the mesenchymal condensation;
(dashed arrows). (D) Alu element insertions and the hemizygous deletion of exon c allow FGFR2b
expression in mesenchymal tissue, effectively mimicking the missense mutations by allowing ectopic
receptor activation by ligands such as FGF7 (dashed arrow).
FGF signaling pathways in limb development. (A) Structure of the FGFR showing three disulfide
www.pnas.org?cgi?doi?10.1073?pnas.081082498 Yu and Ornitz
erties of the Fgfr2 allele generated by Download full-text
Hajihosseini et al. (4) provides a unique
opportunity to explore the molecular
pathogenesis of AS. This engineered mu-
tation also has provided unique insight
into the role of FGF-FGFR signaling
between epithelial and mesenchymal
We thank G. Martin and I. Boime for re-
viewing this manuscript. This work was
funded by National Institutes of Health Grant
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