1 Institute of Genetic Medicine, Department of Pediatrics, The Johns Hopkins
University School of Medicine, 733 North Broadway, Baltimore, MD 21205,
USA
2 Department of Comparative Medicine, The Johns Hopkins University School of
Medicine, 733 North Broadway, Baltimore, MD 21205, USA
3 Department of Medicine and Plastic Surgery, The Johns Hopkins University
School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA
4 Center for Craniofacial Development and Disorders, The Johns Hopkins
University School of Medicine, 733 North Broadway, Baltimore, MD 21205,
USA
5 Department of Biomedical Engineering, The Johns Hopkins University, 3400 N.
Charles Street, Clark 106, Baltimore, MD 21218, USA
6 Departments of Cell Biology/Microinjection and Microchemistry, The Jackson
Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA
7 Department of Anthropology, The Pennsylvania State University, 409 Carpenter
Building, University Park, PA 16802, USA
8 Department of Molecular Biology and Pharmacology, Washington University
Medical School, 660 S. Euclid Avenue, St Louis, MO 63110, USA
Author for correspondence (e-mail:
ejabs1{at}jhem.jhmi.edu)
Accepted 13 May 2005
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SUMMARY |
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Key words: Apert syndrome, Fibroblast growth factor receptor 2, Mouse, Cartilage, Bone, Mesenchyme, Neural crest, Skull, Suture, Craniosynostosis
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Introduction |
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The midline interfrontal suture is neural crest derived, whereas, at the
midline sagittal suture, there are neural crest cells present between the
mesodermally derived parietal bones. The coronal suture lies at the interface
of the neural crest-derived frontal bones and the mesoderm of the parietal
bones (Jiang et al., 2002). At
these sutures, a proportion of these cells are recruited to differentiate into
osteoblasts and make new bone (Cohen and
Kreiborg, 1993
; Opperman,
2000
). A cartilaginous layer underlies the lower part of the
parietal bone and coronal sutures proximate to the endochondral bones at the
base of the skull (Iseki et al.,
1999
). By contrast, there is no compelling evidence that cartilage
is normally formed at the superior part of the parietal bone and midline
sutures of the membranous neurocranium.
In humans one of the most severe conditions involving abnormal sutural
development and skull growth is Apert syndrome
(Cohen, 2000). In infancy, a
patent midline defect of the metopic and sagittal sutures, and synostosis
(premature fusion) of the coronal suture, are present. During childhood, the
sagittal and lambdoid sutures become synostosed
(Kreiborg and Cohen, 1990
).
Other abnormalities of the skeleton include severe syndactyly, and internal
organs are also affected, but less frequently
(Table 1).
|
Normally, the ligand-binding characteristics of FGFR2 vary depending on its
isoform. Alternative splicing of exons IIIb and IIIc encoding the
immunoglobulin-like III domain results in spliceforms that are expressed
predominantly by epithelial or mesenchymal cells, respectively
(Miki et al., 1992;
Orr-Urtreger et al., 1993
).
Distinct ligands bind to the IIIb and IIIc isoforms. Ligands FGF7 and FGF10
activate FGFR2 IIIb, whereas FGFs 2, 4, 6, 8 and 9 activate FGFR2 IIIc. An Alu
insertion flanking exon IIIc in an Apert syndrome patient was found to affect
splicing and cause the ectopic expression of FGFR2 IIIb in
fibroblasts from the patient (Oldridge et
al., 1999
).
FGFR2, as well as the other three FGFR tyrosine kinases and their 22 FGF
ligands, is known to play a crucial role in the control of cell migration,
proliferation, differentiation and survival, by activating two primary
pathways, the mitogen-activating protein kinase pathway
(Kouhara et al., 1997;
Ornitz and Itoh, 2001
) and
protein kinase C pathway (Debiais et al.,
2001
; Lemonnier et al.,
2001
). Signaling through FGFR2 regulates stem cell proliferation,
affecting different lineages such as osteoblasts and chondroblasts
(De Moerlooze et al., 2000
;
Eswarakumar et al., 2002
;
Iseki et al., 1999
;
Ornitz and Marie, 2002
).
Chen et al. created a transgenic mouse with an
Fgfr2+/S250W mutation
(Chen et al., 2003). (Although
the correct conserved serine at position 252 was targeted, it was incorrectly
designated as residue 250 Chu-Xia Deng, personal communication.) Their
mice were found to have several features similar to Apert syndrome, including
a small body size, brachycephaly, midface hypoplasia, short presphenoid bone,
wide-spaced eyes, and malocculsion. The long bones revealed few abnormalities.
The heights of the growth plates correlated with the smaller size of the
mutants, and the column of proliferating chondrocytes was slightly short in
older mice (postnatal day 10, P10). Most importantly, Chen et al. performed
detailed studies of the transverse coronal sutures where they found premature
closure, decreased bone formation, and increased apoptosis, but no obvious
change in cell proliferation and differentiation. Thus, they suggest that
dysregulated apoptosis plays an important role in the pathogenesis of Apert
syndrome-related phenotypes.
In this study, we explored the pathogenesis of other Apert syndrome
malformations, including the midline sutural abnormalities and those of the
internal organs, by introducing the Fgfr2 S252W mutation into the mouse
genome. Our resulting heterozygotes have phenotypic features consistent with
the human Apert syndrome. This mutation alters proliferation and
differentiation of osteoblasts at the midline calvarial sutures. It is also
responsible for ectopic cartilage formation in the neurocranium and
cartilaginous abnormalities in several organs including the long bones. These
findings highlight the delicate balance of migration, proliferation and
differentiation that orchestrate individual suture and bone development
(Marie et al., 2002).
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Materials and methods |
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We identified two clones with the targeted mutant allele, and germline chimeric mice were generated from each clone (Fig. 1B). Male chimeras were mated with C57BL/6J females to achieve germline transmission of the mutant allele. The presence of the neo cassette interfered with mutant allele expression. Therefore, F1 heterozygotes with neo (+/S252Wflox) were crossed with CMV or EIIA promoter Cre transgenic C57BL/6J mice (CMV-Cre, Transgenic Core Facility of the Johns Hopkins University School of Medicine; EIIA-Cre, The Jackson Laboratory) to remove the neo cassette. Care and use of mice for this study were in compliance with the relevant animal welfare guidelines approved by the Johns Hopkins University Animal Care and Use Committee.
Mutant allele genotyping and RT-PCR expression analysis
Genotypes of tail DNA from the resulting progeny were determined by PCR
analysis using primers from within Fgfr2 intron 6 (forward primer F,
5'-CTCGGAGTGTAACGTGTTC-3'), intron IIIa (reverse primer R,
5'-CAACAGGAAATCAAAGACC-3') and the neo cassette (forward
primer Fn, 5'-GATGTTTCGCTTGGTGGTC-3')
(Fig. 1A,C), and their identity
was confirmed by DNA sequencing. We identified Fgfr2 mutant allele
expression in total RNA isolated from various organs and tissues from
Fgfr2+/S252W mice by using the RNAqueousTM-4PCR Kit
(Ambion) and the Omniscript RT Kit (QIAGEN)
(Fig. 1D). RT-PCR amplification
was performed using primers corresponding to the Fgfr2-coding
sequence of exons 5 and 10 (forward primer,
5'-CAACACCGAGAAGATGGAG-3' and reverse primer,
5'-CCATGCAGGCGATTAAGAAG-3'). Products were digested with
SfiI to distinguish the mutant from the wild-type allele.
Image analyses of the skull from the Fgfr2+/S252Wflox/S252W mouse
Adult mice were euthanized by halothane inhalation and X-rays were
performed. After removing the skin, mice were decapitated and the heads were
fixed in 4% paraformaldehyde. Micro-computed tomography scans were acquired by
the Center for Quantitative Imaging at the Pennsylvania State University.
Using eTDIPS software
(http://www.cc.nih.gov/cip/software/etdips/),
3D reconstructions were made using the surface reconstruction module of
eTDIPS, and 24 biological landmarks were located twice on each skull and
mandible. The means of the three-dimensional coordinates of these landmarks
from each mouse were used to analyze morphological differences between mice. A
quantitative comparison of the linear distances estimated among the 24
landmarks using Euclidean Distance Matrix Analysis, or EDMA
(http://oshima.anthro.psu.edu),
provided precise measures of localized differences between mutant and control
littermates.
Bone and cartilage staining, and measurements of skeleton
Skeletal staining with Alizarin Red S and Alcian Blue was performed
(McLeod, 1980). Skull
measurements, including skull and nose lengths, skull height and width,
mandibular and maxillary lengths, and inner canthal distance
(Richtsmeier et al., 2000
),
and limb measurements, including the length of the humerus, radius, ulna,
femur, tibia and fibula, were taken with digital calipers (Fisher
Scientific).
Histological analysis and special staining of the skeleton
Whole heads of embryos at E14.5 to P1 were dissected. Histological sections
(5 µm) were prepared from selected tissues that had been fixed in 4%
paraformaldehyde and embedded in paraffin. Sections were stained with
Hematoxylin and Eosin (HE) for histology. Alkaline phosphatase (ALP, specific
for osteoblasts) and tartrate resistant acid phosphatase (TRAP, specific for
osteoclasts) in skull sutures and growth plates were stained using the TRACP
and ALP double-stain kit (TaKaRa Bio).
Immunohistochemical and TUNEL assays of skeleton
Skull sutures, sectioned as above, were deparaffinized and hydrated through
a xylene and graded alcohol series. The immunohistochemical assays for Ki67
(Novocastra) and collagen II Ab-2 (Lab Vision) were performed using the VECTOR
M.O.M Immunodetection Kit (Vector Laboratories). To assess cell proliferation,
the slides were incubated with anti-Ki67 antibody, and visualized with
biotinylated anti-mouse IgG (Vector) followed by the peroxidase reaction
(brown color for Ki67-positive nuclei). Cell proliferation was analyzed by
counting the number of Ki67-positive cells in more than 100 cells from a
defined area, including and between the osteogenic fronts in three serial
sections from four mutant and four control littermates for each developmental
stage. The counts for Ki67-positive cells in mutant and wild-type embryos were
compared by the t-test. To assess apoptosis, the TUNEL assay was
performed, using the In Situ Cell Death Detection Kit, POD (Roche Applied
Science) for detection of apoptotic cell death by light microscopy.
In situ hybridization
In situ hybridization was performed on sections as described by Wilkinson
(Wilkinson, 1992) with
modifications. The mouse osteonectin (ON), osteopontin (OP) and Sox9 cDNA
fragments were each cloned into the pCR®II-TOPO® Vector. The plasmids
were linearized, and antisense and sense single-stranded RNA probes were
generated with the T7 and SP6 RNA polymerase.
In vitro culture
Cells were obtained from limbs of newborn Fgfr2+/S252W
mutant and wild-type mice. The long bones were dissected and connective tissue
was removed. The bone samples from the middle third of the shaft were washed
and centrifuged with phosphate-buffered saline (PBS, GIBCO). The samples were
then digested in 1 mg/ml sterile collagenase D (Boehringer Mannheim) solution
at 37°C for 2 hours, centrifuged and washed with PBS before resuspension
in standard culture medium [Dulbecco's modified Eagle's medium (DMEM; GIBCO),
10% fetal bovine serum, 100 unit/ml penicillin and 100 µg/ml streptomycin].
Cells were plated onto a flask and medium changes occurred first after 3 days
to allow cell attachment and then three times a week until confluency.
Expanded cells were then placed in 3D culture
(Elisseeff et al., 2005) and
incubated in standard osteogenic medium for 3 weeks to evaluate
differentiation. Osteogenic medium consisted of high-glucose DMEM, 100 nM
dexamethasone (Sigma-Aldrich Co.), 50 µg/ml ascorbic acid 2-phosphate, 10
mM ß-glycerophosphate, 10% fetal bovine serum, 100 unit/ml penicillin and
100 µg/ml streptomycin. Medium was changed every 2-3 days. At the end of 3
weeks in culture, wet and dry weights from all constructs for normalization of
extracellular matrix content were obtained after 48 hours of lyophilization.
ALP and calcium quantification, Von Kossa staining for mineralization
(Pittenger et al., 1999
), and
immunohistochemical analyses for collagen type I and II (Research Diagnostics,
rabbit polyclonal antibodies), were performed. For quantitative ALP assay,
tissue cultures or constructs were homogenized in 0.75 M 2-amino-2-methyl
propanol (Sigma; pH 10.3) solution and the supernatants were collected for ALP
assay using Sigma ALP Determination Kits (Sigma Diagnostics 245), following
the manufacturer's protocol. For quantitative calcium measurement, lyophilized
tissue constructs were homogenized in 0.5 M HCl and vigorously vortexed for 16
hours at 4°C. The supernatant was collected for calcium assay by following
the manufacturer's protocol (Sigma Diagnostics 587). Three samples from each
group were harvested for each assay.
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Results |
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Skeletal and other organ abnormalities in Fgfr2+/S252W mutant mice
Autopsy with skeletal preparations of mutant mice revealed multiple
malformations that resembled those of human Apert syndrome
(Fig. 2). A total of 33
Fgfr2+/S252W mutant mice were analyzed at P1
(Table 1). The skull length,
skull height and upper jaw length were significantly reduced when compared
with control littermates [+/S252W (n=4) versus +/+ (n=4):
average skull length 9.415 mm versus 10.450 mm, P=0.006; skull height
6.135 mm versus 6.570 mm, P=0.028; upper jaw length 5.399 mm versus
6.200 mm, P=0.018]. Their skull width, nose length, inner canthal
distance, and lower jaw measurements did not differ from those of controls
(P=0.052 to 0.842).
Other skeletal system abnormalities included increased cartilage of the basicranium, malformation of the palate, thickened nasal cartilage, fusion of joints separating the zygomatic arch bones, fusion of bones of the sternum, and complete cartilage sleeve of the trachea (Fig. 2). Although the mutant mice were smaller, the upper and lower limb lengths were proportional between mutant and control littermates (ratio of humerus/radius in +/S252W versus +/+: 0.836 versus 0.838, P=0.975; ratio of femur/tibia in +/S252W versus +/+: 0.616 versus 0.595, P=0.790). None of the mutants exhibited syndactyly.
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Skull abnormalities in the Fgfr2+/S252Wflox/S252W mutant mice
We analyzed the only two mutant mice with the hypomorphic
S252Wflox allele that survived to adulthood. In these mice, the
neo cassette was not completely removed in all tissues
(Fig. 1C, lane 7). The
expression levels of both mutant Fgfr2 IIIb and IIIc
alternative transcripts in hypomorphic mutant mice were generally reduced by
16% to 41%, when compared with those of the normal transcripts in multiple
tissues examined, including lung, liver, kidney and stomach (see Table S1 in
the supplementary material). At 10 months, these mutants had an abnormal head
shape, with a body weight that was 29% (mutant 10.63 g versus +/+ 36.35 g) and
a length that was 68% (nasal tip to base of tail, mutant 62.69 mm versus +/+
92.27 mm) of those of their littermate controls
(Fig. 3). Micro-CT of the adult
skull revealed multiple abnormalities with an interfrontal sutural defect
(Fig. 3C, arrow), dysmorphology
of the nasal bones, and obliteration of the fronto-premaxillary,
premaxillary-maxillary, and nasal-frontal sutures. The structures of the face
and palate were 40-50% smaller in the mutant mouse than in the control mouse,
while structures of the neurocranium and basicranium were reduced by 30-40%.
Linear distances measuring the width of the skull were affected less than the
length was, resulting in a very brachycephalic skull shape. The frontal
process of the maxilla showed an abnormally large sinus and bowing of the
medial wall and the zygomatic process of the maxilla was more bowed laterally.
The interparietal bone was compressed superiorly at the lambdoid suture
whereas the parietal bones showed an obvious superior bulge. The inner surface
of the calvarium showed `thumb printing' or fine indentations presumed to be
secondary to increased intracranial pressure. The mandible was very small with
a dysmorphic angular process.
|
The midline interfrontal suture showed an obvious gap between the
osteogenic fronts of the frontal bones in the mutant mice when compared with
control littermates (Fig.
4G,H). The widely separated osteogenic fronts correlate with the
patent midline defect observed macroscopically in skeletal preparations
(Fig. 2B) and CT scans of
mutant mice (Fig. 3C, see
arrow). These results in mice are consistent with the midline sutural defect
found in all Apert syndrome infants
(Kreiborg and Cohen,
1990).
The transverse coronal (Fig.
4I,J) and lambdoid sutures
(Fig. 4K,L) in P1 mutants
showed synostosis with osteoid deposition. Our results are consistent with the
coronal synostosis found in P1 to P18 Apert syndrome mutant mice reported by
Chen et al. (Chen et al., 2003)
and on CT scans of Apert syndrome patients
(Kreiborg and Cohen,
1990
).
Abnormal osteoblastic proliferation and differentiation at the midline sutures in mutant mice
The effects of the Fgfr2 +/S252W mutation on proliferation, differentiation
and apoptosis were investigated at the sagittal suture from E16.5 to P1. Cell
proliferation was investigated by counting Ki67-positive cells from a
designated area in the sagittal sutures using serial sections. At E16.5, no
difference in the number or distribution of Ki67-positive cells was apparent
between mutants and controls (data not shown). At E18.5 and P1, there were
twice as many proliferating Ki67 cells at the sagittal suture in mutants
(E18.5 +/S252W versus +/+: 98.0±5.0 positive cells versus
45.5±10.6 positive cells, P<0.023; P1 94.5±10.6
versus 26.5±0.7, P<0.018;
Fig. 5A,B). These mutant
Ki67-positive cells were abnormally distributed in the sutural space between
the osteogenic fronts, as well as being localized at the fronts where they are
present in controls.
Osteoblast differentiation was assessed using osteonectin (ON), osteopontin (OP) and alkaline phosphatase (ALP) markers. At E16.5, no differences were apparent in the expression of these markers between mutants and controls (data not shown). At E18.5, ON, OP and ALP were expressed in the developing bone and in cells extending from the margin of the bone plates into the mid-sutural area in mutants (Fig. 5C-H). These results suggest abnormal osteoblastic differentiation, because this latter area normally contains mesenchymal or osteoprogenitor cells, but no preosteoblasts. Apoptosis at the sagittal suture was studied by TUNEL staining. There was no apparent difference in apoptosis between mutants and controls at E16.5, E18.5 and P1 (Fig. 5I,J; E18.5 is shown).
We also examined proliferation, differentiation and apoptosis at the midline interfrontal suture. In mutants, there was a significant increase in the number of Ki67-positive cells between the osteogenic fronts (E18.5 +/S252W versus +/+: 181.0±4.2 positive cells versus 111.0±8.5 positive cells, P<0.01). The mutant Ki67 cells had a pronounced alteration in their distribution. Rather than being enriched at the osteogenic fronts, Ki67-positive cells in the mutant extended from the widely separated margins of the osteoid plates almost into the mid-sutural space (Fig. 6C,D). In situ hybridization with ON and OP revealed an obvious lack of differentiating osteoblasts between the osteogenic fronts in mutants (Fig. 6E-H). This difference could be detected at E16.5 and became more obvious at E18.5. Similar to our results at the sagittal suture, there was no obvious difference in apoptosis at the interfrontal suture of mutants and controls from E16.5 to E18.5 (data not shown).
Abnormal cartilage at the midline sagittal suture in mutant mice
In Fgfr2+/S252W mice, there was ectopic cartilage at
the sagittal suture (n=20 mutants, n=26 controls;
Fig. 7). Normally, with
intramembranous bone formation at the sagittal suture, there is no
intermediate cartilaginous stage. At E16.5, cartilage was detected
consistently throughout most of the entire length of the sagittal suture of
mutants. At E18.5, cartilage was present along more than half of the length of
the sagittal suture (0.45±0.11 mm of 0.71± 0.05 mm, as
determined by 5 µm serial sections), beginning at the junction between the
parietal and interparietal bones (see Fig. S1 in the supplementary material).
At P1, the cartilage resembled that at E18.5 in the sagittal suture of
mutants. Two chondrogenic markers, Sox9 and collagen type II, were expressed
in these cells (Fig. 7E-L). No
chondrocytes or cartilage was detected histologically, and no Sox9 expression
was present in mutant sagittal sutures at earlier stages of E14.5 or E15.5
(data not shown) or in controls from E14.5 to E18.5. At the later P1 stage,
cartilage was detected near the junction of the parietal and interparietal
bones at the sagittal suture in only one out of the 26 controls.
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Discussion |
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We confirmed the results of Chen et al.
(Chen et al., 2003), including
the presence of coronal synostosis in mutant mice. In addition, our analysis
focused on the midline sagittal and interfrontal sutures, where a skull defect
is present in all human Apert syndrome newborns. Although, severe syndactyly
is a key feature of Apert syndrome in humans, none of the mutant mice from
either laboratory exhibited syndactyly by gross inspection and histopathology.
The absence of this feature is consistent with the possibility that this
mutation causes less severe digital abnormalities in humans when compared with
the Apert FGFR2 P253R mutation (Slaney et
al., 1996
; von Gernet et al.,
2000
). Others speculate that there is no human phenotypic
difference for these two mutations (Park
et al., 1995
). Thus, the absence of syndactyly in our mice may be
a consequence of species-specific modifications or of alternative splicing
affecting levels of expression of receptors
(Oldridge et al., 1999
).
Abnormal bone development at the midline calvarial sutures in Apert syndrome
Previously in the literature there has been few, if any, histopathological
studies of the midline sutures, especially the interfrontal suture, in
craniosynostosis mouse models. For the Fgfr2+/S252W
mutant, the interfrontal suture had increased proliferation, but abnormally
localized, decreased or delayed differentiation of osteoprogenitor cells to
preosteoblasts. Therefore, abnormal proliferation and differentiation of
osteoblasts, not increased apoptosis, may underlie the metopic sutural defect
in Apert syndrome.
At the mutant sagittal suture, the number of proliferating cells and the expression of osteogenic markers were mislocalized and increased, suggesting that the observed synostosis results from increased proliferation and abnormal differentiation. In mutant mice, the osteogenic markers are expressed abnormally in cells extending from the margin of bone plates to the mid-sutural area. It is possible that the Fgfr2 +/S252W mutation may cause abnormal differentiation at the developing sagittal sutures by altering the fate of mesenchymal cells.
|
Abnormal cartilage development in Apert syndrome
We provide strong evidence that abnormal cartilage formation is involved in
the pathogenesis of Apert syndrome. In humans, although cartilage
abnormalities have been reported in this condition, as well as in other
craniosynostosis syndromes such as Crouzon and Pfeiffer
(Cohen and Kreiborg, 1992;
Cohen and Kreiborg, 1993
;
Kreiborg et al., 1993
;
Kreiborg et al., 1999
),
abnormal cartilage formation has not previously been recognized as being
significant. In our mouse model, the Fgfr2 +/S252W mutation has a
generalizable effect on the development of cartilage. Chondrogenic markers and
increased cartilage were found in the mutant sagittal suture at embryonic
stages when normally they are not present. These findings suggest that either
there was a significant increase in cartilage proliferation at the junction of
the parietal and interparietal bones, where it normally may be present, or
that there is ectopic cartilage formation from progenitor cells. We also
observed increased cartilage at the basicranium, nasal turbinates, trachea and
long bones of our mutant mice. At the long bone growth plates, abnormal
cartilage formation was detected histologically in vivo, and this was further
supported by our in vitro cultures. These results indicate that the Fgfr2
+/S252W mutation significantly enhances the formation of cartilage in some
organs, and we speculate that abnormal chondrogenesis may occur.
Interestingly, our results differ for the two midline sutures. At the
sagittal suture, there is ectopic cartilage and synostosis, and at the
interfrontal suture there is a defect where no cartilage was found. The
distinct tissue origins of the sagittal and interfrontal sutures may reflect
their mutant osteogenic potentials. The sagittal suture is formed from neural
crest cells and mesoderm, whereas the interfrontal suture is neural crest
derived (Jiang et al., 2002).
The cranial neural crest cells can give rise to either cartilage or bone
(Le Douarin et al., 1993
;
Noden, 1983
;
Selleck et al., 1993
). In
vitro studies have shown that mutant FGFRs can induce ectopic cartilage from
premigratory neural crest cells early in development. Petiot et al.
demonstrated that transfection of activating FGFR1 K656E or FGFR2 C278F
mutations can induce cartilage differentiation when electroporated into quail
premigratory neural crest cells (Petiot et
al., 2002
), but this effect is drastically reduced if transfection
is carried out after the onset of neural crest migration. Therefore, the
observed abnormal development of cartilage in Apert syndrome may be due to the
mis-migration or mis-positioning of neural crest cells at an early embryonic
stage.
Of note, there have been previous reports of abnormal cartilage formation
in the sagittal suture in pathologic states. In mutant mice overexpressing
Msx2, cartilage has been demonstrated to underlie the midline sagittal suture
(Liu et al., 1999). Mice,
exposed to retinoic acid at E10, develop cartilage in the parietal region
concomitant with the decrease in the intramembranous ossification
(Jiang et al., 2002
),
suggesting that neural crest or mesenchymal cell fate may be sensitive to Msx2
dosage. Rice et al. reported that cartilaginous rods are occasionally and
transiently seen in the sutural mesenchyme around the time of birth
(Rice et al., 2000
), and this
may be a tissue reaction to mechanical irritation.
The complexity in the precise temporospatial definition of each developing
suture in Apert syndrome is underscored by the variable effects of the Fgfr2
+/S252W mutation, differences in tissue origins, and potential genetic and
environmental modifiers. These distinctions cannot be easily studied in human
surgical or cultured cells because of the lack of specimens at different
developmental stages, sites, and controls. In fact, previous studies on human
samples have differing results. Lomri et al.
(Lomri et al., 1998) and
Lemonnier et al. (Lemonnier et al.,
2000
) concluded that there was increased osteoblast
differentiation of calvarial cells without making a distinction between
sagittal, metopic and coronal synostosed sutures in Apert syndrome fetuses and
infants. Mansukhani et al. (Mansukhani et
al., 2000
) noted an opposite effect of inhibition of
differentiation, and increased apoptosis in immortalized human osteoblasts,
not specifically derived from sutures, introduced with the FGFR2 S252W
mutation.
Insights into the mechanism of the Fgfr2 +/S252W mutation
Mutant mice with loss of function or dominant-negative mutations in Fgfr2
showed abnormalities in some of the same organs that are affected in our Apert
syndrome mouse model. Mice homozygous for null alleles of Fgfr2 die at E10.5
with multiple defects in organogenesis, and chimeras between this mutant and
wild-type had abnormal epithelial-mesenchymal interactions, respiratory
failure and intramembranous and endochondral bone formation
(Arman et al., 1998;
Arman et al., 1999
;
Xu et al., 1998
). A mouse with
a specific deletion and a dominant-negative loss of Fgfr2 IIIb isoform
function resulted in craniofacial (wide cleft palate, reduced maxillary bone,
absent otic capsule, rudimentary inner ear structures) and truncating limb
anomalies, dysgenesis of several visceral organs (thymus, glandular stomach,
pancreas, kidney), and agenesis of the tooth bud, salivary gland, thyroid,
pituitary and lung (Celli et al.,
1998
; De Moerlooze et al.,
2000
).
Mice created by a specific deletion of Fgfr2 IIIc function associated with
exon switching more closely resemble our Apert syndrome mice. Hemizygotes were
affected with neonatal growth retardation and death, coronal synostosis,
ocular proptosis with fused joints of the zygomatic arch bones, precocious
sternal fusion, and abnormalities in secondary branching in several organs,
such as the lungs, kidneys and lacrimal glands
(Hajihosseini et al., 2001).
With a splice-switch mechanism, Fgfr2 IIIb expression was increased in
calvarial sutures and the zygomatic joints allowing cells, which usually
express Fgfr2 IIIc, to respond to a broader set of ligands. Similarity between
this mouse mutant and the Apert syndrome mouse suggests that the Fgfr2 +/S252W
mutation is neomorphic as supported by the observed change in ligand-binding
specificity (Yu et al., 2000
),
rather than being a `simple' gain-of-function mutation
(Anderson et al., 1998
).
Mice created by conditional inactivation of Fgfr2 with
Dermo1cre/+, specifically targeting Cre expression to disrupt
signaling in the chondrocyte or osteocyte lineages, gave further insight into
the potential mechanism of the Apert mutation
(Yu et al., 2003). These
mutant mice had a few features, such as a domed-shaped skull with a midline
interfrontal sutural defect, that are similar to our Apert syndrome model.
They also had a dwarfism phenotype with a shortened axial and appendicular
skeleton that differed from our mice by a reduced hypertrophic chondrocyte
zone, vertebral abnormalities with non-ossified gap in the dorsal midline of
both cervical and thoracic vertebrae, absence of the spinous processes, and a
lack of tarsal joints because of failure of cavitation of the cartilaginous
anlage. They demonstrated that Fgfr2 is essential for the proliferation of
osteoprogenitors and for the maintenance of osteoblast anabolic function, but
that it is not required for osteoblast differentiation. The phenotype of their
mice is different from our Apert syndrome mice because the mechanism of the
Fgfr2 +/S252W mutation is not inactivation. Analysis of our mouse model
establishes a potential link between abnormal proliferation and
differentiation, and possibly altered cell fate determination of progenitor
cells with developmental abnormalities in Apert syndrome. Abnormal
chondrogenesis may play a role in the pathogenesis of craniosynostosis
syndromes. The Apert mouse model provides an in vivo system for future studies
of determinants of cell fate for the chondrocyte or osteoblast lineages, which
are important in the larger context of bone cell biology.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/15/3537/DC1
* These authors contributed equally to this work
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