Title: Syndromic FGFR Gain-of-Function Mutations Suppress Noggin: Implications for Cranial Suture
Biology
Authors: Stephen M. Warren, MD, Aris N. Economides, PhD, Lisa J. Brunet, PhD, Richard M. Harland
PhD, Kenton D. Fong, MD, and Michael T. Longaker, MD
Coordinated growth of the brain and skull is achieved through a series of tissue interactions within the
cranial sutures. These interactions couple the expansion of the brain to growth of the calvarial plates.
Both genetic and environmental perturbations can cause premature cranial suture fusion
(craniosynostosis). Craniosynostosis can result in abnormal skull shape, midface hypoplasia,
hydrocephalus, deafness, blindness and mental retardation; syndromic forms are often accompanied by
anomalies of the digits1. Although non-syndromic sagittal synostosis is most common, more than 150
craniosynostotic syndromes have been identified2. Positional cloning and candidate approaches have
linked gene mutations with many of the craniosynostotic syndromes. For example, recent studies have
demonstrated that gain-of-function mutations in three of the four known fibroblast growth factor
receptors (FGFR) are associated with Apert, Crouzon, Pfeiffer, and Jackson-Weiss syndromes3, 4.
Despite the identification of these genetic mutations, downstream molecular mechanisms remain
unknown.
We are using murine models to study the molecular mechanisms mediating premature cranial suture
fusion. In these models, the posterior frontal (PF) cranial suture fuses while all other sutures, including
the sagittal (SAG) and coronal (COR) remain patent. Previous work from our laboratory indicates that
suture-specific dura mater initiates PF suture fusion via multiple paracrine (e.g. TGF-1 and FGF-2)
signaling pathways. Numerous fate-based studies have demonstrated that cranial suture fusion is
controlled by these pro-osteogenic paracrine factors and, moreover, we have found that these same proosteogenic cytokines are absent in the patent SAG and COR dura mater. We had long suspected that
cranial suture patency was a passive process (i.e. insufficient levels of osteogenic cytokines), however,
we have recently demonstrated that Noggin, a BMP antagonist, is actively expressed in the SAG and
COR sutures. In this study, using gene chip analysis, we analyze BMP levels in PF and SAG sutures.
Moreover, we use in situ hybridization to screen for additional BMP antagonists. Using a model of COR
craniosynostosis, we have previously demonstrated that FGF-2 overexpression causes Noggin
suppression in the COR suture and pathologic suture fusion. In this study, we link our murine model
with human Apert and Crouzon FGFR gain-of-function FGFR2 mutations by examining the effects of
these mutated receptors on Noggin expression in chimeric dura mater and osteoblasts.
Total RNA was isolated and pooled from PF and SAG sutures (n=20/timepoint) with associated dura
mater from Sprague-Dawley rats (harvested on days 5, 10, 15, 20, and 30 of life). For each time point,
cDNA synthesis, in vitro transcription and biotin-labeling of cRNA, and hybridization to the R34U A
Chips (Affymetrix) were performed according to Affymetrix protocols. In situ hybridization screening
was performed on mouse cranial sutures (days 15, 25, 35, and 45) for the BMP antagonists: DCR4,
DCR5, DCR6, DCR7, Gremlin, Cerberus, and Noggin. The calvaria of transgenic beta-galactosidase/
Noggin mice were harvested at similar time points, treated with an X-gal solution and histologically
examined. PF, SAG and COR dural cell cultures were established and Western blots were performed.
Chimeric gain-of-function FGFR2 mutated dura mater and osteoblasts created using retroviral vectors.
A Noggin over-expression adenovirus was injected into the PF suture of 22 day-old calvaria in vitro or 3
day-old mice in vivo.
Gene chip analysis demonstrated markedly elevated, but similar, levels of BMP-4 in both PF and SAG
sutures (Figure 1). This was surprising since BMP-4 is a bone forming protein, we expected to find it
preferentially expressed in the fusing PF suture. Our previous results suggested that BMP-4 activation
1
might be differentially controlled by suture-specific Noggin expression. Next, we investigated the
expression of other BMP antagonists. In situ hybridization demonstrated that Noggin is the only BMP
antagonist present in murine cranial sutures. In a previous study, we found that X-gal staining of
transgenic mice demonstrated no Noggin expression in the PF suture complex before, during or after
suture fusion. In contrast, SAG and COR sutures expressed high levels of Noggin throughout the period
of predicted suture fusion. PF dural cultures did not produce Noggin. In contrast, SAG and COR dural
cultures constitutively produced high levels of Noggin. In a model of COR craniosynostosis, we found
that FGF-2 overexpression dramatically suppressed COR Noggin expression and lead to pathologic
suture fusion. Therefore, in order to link our murine findings to human Apert and Crouzon gain-offunction FGFR2 mutations were tested the effects of this receptor mutation on Noggin expression.
Interestingly Apert and Crouzon gain-of-function FGFR2 mutations suppressed constitutive SAG dura
mater Noggin expression (Figure 2) and BMP-4 induced Noggin expression in chimeric osteoblasts
(Figure 3)
Since normal cranial suture patency depends on BMP antagonism, some forms of craniosynostoses may
result from inappropriate FGF-mediated down-regulation of Noggin expression. Ongoing studies are
investigating the effects of a Pfeiffer (Pro250Arg) FGFR1 transgenic mutation on Noggin expression in
vivo.
References
1. McCarthy JG, Epstein FJ, Wood-Smith D. Craniosynostosis. In: McCarthy JG, ed. Plastic Surgery.
Philadelphia: W.B. Saunders Co.; 1990:3013-3053.
2. Cohen MM, Jr. Craniosynostoses: phenotypic/molecular correlations. Am J Med Genet.
1995;56:334-339.
3. Wilkie AOM. Craniosynostosis: genes and mechanisms. Hum Mol Genet. 1997;6:1647-1656.
4. Kan SH, Elanko N, Johnson D, et al. Genomic screening of fibroblast growth-factor receptor 2
reveals a wide spectrum of mutations in patients with syndromic craniosynostosis. Am J Hum
Genet. 2002;70:472-486.
rc_AA799981_at
SAG PF
rc_AA866259_at
rc_AA925506_s_at Gng7
Affymetrix Genome U34A
Chip
X13549_s_at Rps10
U47312_s_at
AB003357_at Serine/threonine kinase 2
rc_AA875431_at
E07296cds_s_at
rc_AI229620_s_at Cox5b
rc_AI231354_g_at SAPK
rc_AI639477_at
D88034_at Pdi3
SAG PF
rc_AI235358_at
U75928UTR#1_s_at
rc_AA944324_at Arf6
SAG PF
BMP-6
X5 8 8 3
0_at
Bmp 6
X58830_at Bmp6
5.0
E
x
p
r
e Epr
s esi
s
I
o
n
E
x
p
r
e
s
s
i
o
n
5.0
4.0
rc_AA875261_at
3.0
2.5
2.0
P F
C o nd i ti on
P F
rc_AA875047_at
rc_AI176726_at Bcdo
S49491_s_at proenkephalin
L07074_at p47B
rc_AA891255_at
J03627_at S100A10
rc_AA891812_g_at
M15882_at Clta
rc_AI171462_s_at Cd24
rc_AI638990_at
AF017637_at Carboxypeptidase Z
L32132_at Lipopolysaccharide binding protein
M18853_g_at
rc_AA891260_at
rc_AI638950_at
rc_AA924198_s_at Mvk
L81136cds_f_at Rps2
AF031880_at Neurofilament, light polypeptide
J00777_at
X83735exon#2_at FBPase-2
rc_AI009132_at
J02962_at Lgals3
X53377cds_s_at
rc_AA849769_g_at Fstl
3.0
2.5
...
2.0
...
1.5
1.2...
1.5
1.2
Condition CONTROL
Condition SAG
Condition PF
U44845_at Vtn
rc_AI233219_at Pg25
X55572_at Apod
...
1.0
0.9
0.8
...
0.7 ... ...
...
0.6 ...
...
0.5 ...
...
0.4 .........
0.3...... ...
0.2... ......... ......
... ...
0.1
...
... ... ......
0.0
...
1.0
U51898_at Pla2g6
BMP-2
L20678_at Bmp2
0.9
L 2 06 7 8 _a t
Bmp 2
rc_AA818604_s_at Hspa1a
rc_AI172064_at Lgals1
0.8
M11266_at Otc
rc_AA799997_at
0.7
AF087839mRNA#1_s_at SUR2
rc_AA892391_at
0.6
L23148_g_at Id1
rc_AA893235_at
rc_AA859954_at
X98377_at Emd
U47316_s_at
rc_AA800803_at
J05166_at Slc4a2
rc_AA894312_at
J02752_at RATACOA1
rc_AA875072_at
J02852_at Cyp2a3a
U66470_at
rc_AA799418_at
rc_AA799942_at
rc_AA892897_at
U15550_at
X91810_at Stat3
AF004218_s_at Opioid receptor, sigma 1
0.5
...
0.4
0.3
0.2
Condition CONTROL
0.1
Condition SAG
Condition PF
AF084186_s_at Spna2
X06889cds_at Rab3a
rc_AA818097_at Glp1r
0.0
Trust
Con
d i ti o
n
rc_AA859983_at
4.0
M33025_s_at Ptms
D13623_at RBP34
Condition PF
rc_AA875025_at
BMP-4
Z22607_at Bmp4
Z2 26 0 7 _a t
Bmp 4
AB004278_at
L16995_at
C o nd i ti on
P F
AF087945_at
K02111_at
rc_AA817854_s_at Cp
rc_AA875629_at
M89945mRNA_at
AB016425_at Occludin
U09401_s_at
U52948_at
AA685376_f_at
rc_AA866221_at
AF004017_at Slc4a4
Figure 1: Using an Affymetrix gene chip we analyzed over 8000 genes. Focusing in on BMPs,
surprisingly, we found markedly elevated, but similar, levels of BMP-4 in both the patent SAG and
fusing PF suture complexes.
M64301_at Mapk6
AF041082_at Robo1
Condition CONTROL
Condition SAG
Condition PF
2
-
C342Y FGFR2:
S252W FGFR2:
WT FGFR2:
+
+
-
+
-
Noggin
Figure 2: Apert and Crouzon FGFR2 syndromic mutations suppress sagittal dura mater Noggin
production. Noggin western analysis on sagittal dura mater transduced with a retrovirus
containing a wild-type FGFR2 (WT FGFR2), Apert mutated FGFR2 (S252W/FGFR2) or
Crouzon mutated FGFR2 (C342Y/FGFR2).
C342Y FGFR2:
S252W FGFR2:
WT FGFR2:
rhBMP-4:
-
+
+
+
+
+
+
+
Noggin
Figure 3: Apert and Crouzon FGFR2 syndromic mutations suppress osteoblast Noggin production.
Noggin western analysis on Bmp-4 (25 ng ml-1) treated osteoblasts transduced with a
retrovirus containing a wild-type FGFR2 (WT FGFR2), Apert mutated FGFR2
(S252W/FGFR2) or Crouzon mutated FGFR2 (C342Y/FGFR2).
3