1241
Development 125, 1241-1251 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV1213
FGF-, BMP- and Shh-mediated signalling pathways in the regulation of cranial
suture morphogenesis and calvarial bone development
Hyun-Jung Kim*,†, David P. C. Rice*, Päivi J. Kettunen and Irma Thesleff‡
Institute of Biotechnology and Institute of Dentistry, PO Box 56, University of Helsinki, FIN 00014, Finland
*Both authors have contributed equally to this work
†Present address: School of Dentistry, Chung-Gu Dong-in Dong 2Ga, Daegu, Republic of Korea
‡Author for correspondence (e-mail: [email protected])
Accepted 13 January; published on WWW 26 February 1998
SUMMARY
The development of calvarial bones is tightly co-ordinated
with the growth of the brain and needs harmonious
interactions between different tissues within the calvarial
sutures. Premature fusion of cranial sutures, known as
craniosynostosis, presumably involves disturbance of these
interactions. Mutations in the homeobox gene Msx2 as well
as the FGF receptors cause human craniosynostosis
syndromes. Our histological analysis of mouse calvarial
development demonstrated morphological differences in
the sagittal suture between embryonic and postnatal stages.
In vitro culture of mouse calvaria showed that embryonic,
but not postnatal, dura mater regulated suture patency. We
next analysed by in situ hybridisation the expression of
several genes, which are known to act in conserved
signalling pathways, in the sagittal suture during
embryonic (E15-E18) and postnatal stages (P1-P6). Msx1
and Msx2 were expressed in the sutural mesenchyme and
the dura mater. FGFR2(BEK), as well as Bmp2 and Bmp4,
were intensely expressed in the osteogenic fronts and Bmp4
also in the mesenchyme of the sagittal suture and in the
dura mater. Fgf9 was expressed throughout the calvarial
mesenchyme, the dura mater, the developing bones and the
overlying skin, but Fgf4 was not detected in these tissues.
Interestingly, Shh and Ptc started to be expressed in
patched pattern along the osteogenic fronts at the end of
embryonic development and, at this time, the expression of
Bmp4 and sequentially those of Msx2 and Bmp2 were
reduced, and they also acquired patched expression
INTRODUCTION
Co-ordinate growth of the brain and skull is achieved through
a series of tissue interactions between the developing brain, the
growing bones of the skull and the sutures that unite the bones.
These interactions couple the expansion of the brain to the
growth of the bony plates at the sutures (Liu et al., 1995).
Cranial sutures develop initially by a wedge-shaped
proliferation of cells at the periphery of the extending bone
field, called the osteogenic front (OF), whose cells undergo
patterns. The expression of Msx2 in the dura mater
disappeared after birth.
FGF and BMP signalling pathways were further
examined in vitro, in E15 mouse calvarial explants.
Interestingly, beads soaked in FGF4 accelerated sutural
closure when placed on the osteogenic fronts, but had no
such effect when placed on the mid-sutural mesenchyme.
BMP4 beads caused an increase in tissue volume both when
placed on the osteogenic fronts and on the mid-sutural
area, but did not effect suture closure. BMP4 induced the
expression of both Msx1 and Msx2 genes in sutural tissue,
while FGF4 induced only Msx1. We suggest that the local
application of FGF on the osteogenic fronts accelerating
suture closure in vitro, mimics the pathogenesis of human
craniosynostosis syndromes in which mutations in the FGF
receptor genes apparently cause constitutive activation of
the receptors. Taken together, our data suggest that
conserved signalling pathways regulate tissue interactions
during suture morphogenesis and intramembranous bone
formation of the calvaria and that morphogenesis of mouse
sagittal suture is controlled by different molecular
mechanisms during the embryonic and postnatal stages.
Signals from the dura mater may regulate the maintenance
of sutural patency prenatally, whereas signals in the
osteogenic fronts dominate after birth.
Key words: Tissue interaction, Sagittal suture, Msx1, Msx2, FGF4,
FGF9, FGFR2(BEK), BMP2, BMP4, Shh, Ptc, Intramembranous
bone formation, Mouse
differentiation into osteoprogenitor cells that ultimately lay
down the bone matrix (Johansen and Hall, 1982; Decker and
Hall, 1985).
Craniosynostosis, the premature fusion of cranial sutures, is
a birth defect that results in a profoundly abnormal skull shape
(Virchow, 1851). Recently, mutations in the genes encoding for
Msx2, TWIST and fibroblast growth factor receptors (FGFRs)
have been identified as causes of craniosynostoses (Jabs et al.,
1993, 1994; Muenke et al., 1994; Wilkie et al., 1995; El
Ghouzzi et al., 1997; Howard et al., 1997) suggesting
1242 H.-J. Kim and others
involvement of these genes in the signalling pathways
regulating cranial suture maintenance. The functions of these
genes in cranial suture development are still unclear and, in
general, the molecular basis of signalling during suture
morphogenesis remains to be analysed.
There is increasing evidence that the shapes of individual
bones and the relative proportions of the skeleton are regulated
by complex cascades involving multiple signals, their receptors
and transcription factors (Kingsley, 1994; Erlebacher et al.,
1995). FGFs and their receptors have been frequently
implicated in the coordination of proportional bone growth and
development (Givol and Yayon, 1992; Muenke and Schell,
1995). Their significance has been recently supported by the
demonstrations of several FGFR mutations in dominantly
inherited human skeletal disorders (Shiang et al., 1994;
Tavormina et al., 1995; Cohen, 1995; Bellus et al., 1996). The
four FGFRs, which are tyrosine kinases in type, have several
splice forms and interact with varying specificity and affinity
to at least ten FGF ligands, FGF1 to 10 (Yamaguchi and
Rossant, 1995; Green et al., 1996; Yamasaki et al., 1996). FGFs
regulate various cellular processes, most notably the
proliferation and differentiation of cells of mesenchymal and
neuroectodermal origin (Ullrich and Schlessinger, 1990; Cobb
et al., 1991).
Bone morphogenetic proteins (BMPs) were originally
identified by their ability to induce ectopic bone formation
(Wozney et al., 1988). Since then, they have been shown to be
important signalling molecules in a range of developmental
processes, including mesoderm induction, odontogenesis, limb
development and skeletal patterning (Kingsley et al., 1992;
Vainio et al., 1993; Francis et al., 1994; Northrop et al., 1995).
Recently, both BMP2 and BMP4 were shown to be involved
in determining the size and shape of skeletal elements in chick
limbs (Duprez et al., 1996).
The vertebrate hedgehog gene family consists of three
members, Sonic (Shh), Desert and Indian hedgehog (Ihh)
(Riddle et al., 1993; Krauss et al., 1993). Shh is expressed
in several organising centres, such as notochord, the zone of
polarising activity and the enamel knot, regulating the
dorsoventral patterning of neural tube, limb polarity and
crown pattern of tooth (Yamada et al., 1993; Tabin, 1995;
Vaahtokari et al., 1996a). Shh also plays a critical role in
growth of forebrain as well as in establishing craniofacial
midline structures (Marti et al., 1995; Chiang et al., 1996).
Ihh, on the contrary, is a regulator of chondrogenesis
(Vortkamp et al., 1996). The hedgehog signalling pathway
has been conserved in Drosophila and vertebrates. Hedgehog
regulates the transcription of target genes by antagonising
the action of the segment polarity gene patched (Ptc), a
multipass transmembrane protein, which has been identified
as the receptor of hedgehog (Stone et al., 1996; Marigo et
al., 1996). Dpp in Drosophila and its homologues, BMP2
and 4, in vertebrates are induced in several tissues in
response to hedgehog (Laufer et al., 1994; Bitgood and
McMahon, 1995).
Msx1 and Msx2 are homeobox-containing transcription
factors, expressed mostly in overlapping patterns at multiple
sites of tissue interactions during vertebrate development
(Mackenzie et al., 1992; Jowett et al., 1993; Catron et al.,
1996). In particular, they have been associated with epithelialmesenchymal interactions during organogenesis, they are
targets of BMP and FGF signalling (Satokata and Maas,
1994). For instance, in vitro BMP2 and BMP4 induce the
upregulation of Msx gene expression in tooth explants as well
as in rhombomeres (Vainio et al., 1993; Graham et al., 1994;
Chen et al., 1996) and several FGFs induce the expression of
Msx1 in dental mesenchyme (Kettunen and Thesleff, 1998).
Msx1 and Msx2 have also been associated with the
differentiation of neural crest-derived intramembranous bones
in the skull (Takahashi and Le Douarin, 1990; Takahashi et
al., 1991), as well as with the formation of the dorsal part of
the vertebral cartilage forming the spinous process (Watanabe
and Le Douarin, 1996). Furthermore, the Boston-type
craniosynostosis results from a mutation in the Msx2 gene
(Jabs et al., 1993).
The available data therefore suggest that the FGF, BMP and
Hh pathways form complex signalling networks regulating
tissue interactions in developing vertebrate embryos. In this
study, we have tested the hypothesis that they also regulate the
tissue interactions that are responsible for the formation and
subsequent maintenance of the sagittal suture during calvarial
development. We demonstrate developmentally regulated
expression patterns of Msx1, Msx2, Bmp2, Bmp4, FGFR2,
Fgf9, Shh and Ptc. In addition, we show that the local exposure
of sutural tissue to FGF4 and BMP4 affects suture morphology.
Notably, FGF accelerates the approximation of the parietal
bone margins. Furthermore, FGF4 stimulates Msx1 gene
expression and cell proliferation, whereas BMP4 induces both
Msx1 and Msx2 genes. These data suggest specific roles of the
FGF, BMP and Shh signalling pathways in membranous bone
growth and cranial suture development.
MATERIALS AND METHODS
Preparation of tissues
Calvaria of mice (CBA + NMRI) aged between E15 and P6 were
dissected in Dulbecco’s phosphate-buffered saline (pH 7.3) under a
stereomicroscope, and they were fixed overnight at 4°C in 4%
paraformaldehyde (PFA) in PBS. The age of the embryos was
determined by the day of the appearance of the vaginal plug (day 0)
and confirmed by morphological criteria. Whole-mount material was
denude of skin, dehydrated in a methanol series and stored in absolute
methanol at −20°C until use. Material for tissue sections, which
consisted of parietal bones and the interposed sagittal suture, was
dehydrated and embedded in paraffin. Sections of 5 µm were mounted
on silanized slides, dried overnight at 37°C and stored at 4°C.
Postnatal material for tissue sections was decalcified in 12.5%
ethylenediaminetetracetic acid/2.5% PFA in PBS, changed every 4th
day, for about 7-10 days, at 4°C.
Culture of calvaria
Calvaria were dissected from mice aged both E16.5 and P1 mice for
the dura mater experiment, and from mice aged E15.5 for the bead
experiment. In the dura mater experiment, the dura mater was
removed from the sagittal suture. All explants were cultured in a
Trowell-type organ culture system. Explants were placed on 0.1 µm
pore size Nuclepore filtres (General Electron), supported by metal
grids and cultured for 24-96 hours in Dulbecco’s minimal essential
medium supplemented with 10% foetal bovine serum and
penicillin/streptomycin in a humidified atmosphere of 5% CO2 in air
at 37°C. 100 µg/ml of ascorbic acid was supplemented daily and
culture medium was changed every other day. Explants were cultured
for 24-48 hours (20 explants with FGF4 beads; 20 with BMP4 beads
Signalling in cranial suture development 1243
and 40 with BSA beads), and in the dura mater experiments explants
were cultured for 96 hours (15 with dura and 25 without).
Treatment of beads
Beads incubated in recombinant human BMP4 protein (100 ng/µl),
FGF4 protein (25 ng/µl) or bovine serum albumin (BSA) (same
concentrations as for BMP4 and FGF4) were prepared as previously
described by Vainio et al. (1993) and Vaahtokari et al. (1996b). Before
use, beads were rapidly washed in culture media, then 2 or 3 beads were
placed with a mouth-controlled capillary pipette onto the OFs or the
mesenchyme of sagittal suture of freshly dissected calvaria explants.
Preparation of probes and in situ hybridisation
The preparation of the following RNA probes has previously been
described: Msx1 and Msx2 (Jowett et al., 1993); Bmp2, Bmp4 and
Shh (Vaahtokari et al., 1996a), Fgf4 and Fgf9 (Kettunen and Thesleff,
1998). The FGFR2(BEK) probe was prepared as follows. The
corresponding cDNA was cloned by RT-PCR from E15 mouse molar
tooth mRNA. The PCR product was subcloned into the pAMP1 vector
(Cloneamp System, Gibco BRL); this was sequenced and confirmed
to be the desired cDNA by comparison to a previously published
sequence (Orr-Urtreger et al., 1993). The plasmid DNA was digested
with EcoRI or BamHI producing a 139 bp fragment, then transcribed
with SP6 or T7 RNA polymerase yielding antisense and sense
riboprobes, respectively. The Ptc probe was prepared from a 841 bp
fragment of murine Ptc cDNA in pBluescript II KS+; this was
digested with BamHI or HindIII and transcribed with T3 or T7 RNA
polymerase for antisense and sense riboprobes, respectively.
In situ hybridisation on tissue sections was performed using 35SUTP-labelled riboprobes as described by Vainio et al. (1991).
Following in situ hybridisation, the sections were stained with
Delafield’s haematoxylin and mounted with DePeX (BDG). In situ
hybridisation on whole mounts was performed using digoxigeninUTP-labelled riboprobes according to the protocol described by
Wilkinson and Green (1990) with modifications. Briefly, explants
were pretreated with proteinase K (Sigma), refixed in fresh 4%
PFA/0.2% glutaraldehyde in PBST and then prehybridised for 5 hours
at 55°C in a hybridisation buffer including 50% formamide. After
hybridisation, tissues were washed in
high-stringency
conditions
and
preblocked in antibody blocking solution,
then incubated with preabsorbed
antibody. Colour development was
performed in NBT/BCIP in NMT
solution. Following visualisation of the
stain, the tissues were postfixed and
cleared in 50% glycerol before
photography.
RESULTS
Changes in sagittal suture morphology during
development
The histological appearance of sagittal suture morphogenesis
was analysed from frontal sections of calvaria from E15 to P3
(Fig. 1). At the initiation stage, the OFs of parietal bones in the
presumptive sagittal suture are widely separated by intervening
mesenchyme which is abundant (Fig. 1A). The OFs of parietal
bones are pointing toward the underlying dura mater and the
suture appeared tear-drop in shape. This morphological picture
continues through embryonic development (Fig. 1A-C). After
birth, the morphology of sagittal suture is altered, notably, the
ends of opposite OFs confront each other in an end-to-end
fashion and the amount of mesenchyme between OFs is
reduced (Fig. 1D,E).
The effect of dura mater on cranial suture
development
To evaluate the role of dura mater on mouse sagittal suture
development, we dissected calvaria from E16.5 and P1 mice
and cultured them in vitro both with and without the underlying
dura mater (Fig. 2). After 24 hours in culture, the OFs of
parietal bones had approached each other slightly, but no
differences could be detected in the morphology between the
explants with and without dura mater (data not shown). After
48 hours, the E16.5 calvarial explants showed clear difference:
when cultured in the absence of dura mater, the OFs of the
parietal bones had approached each other more rapidly than
those cultured with dura mater, and they appeared to form
contacts (Fig. 2C,D). After 72 hours, the explants cultured
without dura mater appeared partially fused (Fig. 2F), whereas
with intact dura mater the sagittal suture was still open (Fig.
2E). Histological analysis of the explants after 72 hours of
culture supported these findings (Fig. 2G,H). However, in
Cell proliferation assay
Whole-mount tissues were labelled with
bromodeoxyuridine (BrdU, BoehringerMannheim, 1:1000) for 1 hour, fixed in
MeOH overnight at −20°C, and detected
by immunoperoxidase staining using a
Vector ABC Elite kit.
Histological staining
Serial coronal sections of calvaria
showing sagittal sutures from mice, aged
between E15 and P3, were stained with
Harris’ haematoxylin and eosin.
Transverse sections of sagittal suture
explants from E16.5 or NB mice, cultured
for 72 or 96 hours, respectively, were
stained with van Gieson stain.
Fig. 1. Morphogenesis of the mouse sagittal suture from E15 to P1 as seen in frontal sections.
(A) The osteogenic fronts (OFs) of parietal bones are widely separated and point down toward the
dura mater at E15. (B) The suture then narrows at E17. (C) Higher magnification of B. (D) The
opposing OFs confront each other in a ‘butt joint’ at P1. (E) Higher magnification of D. d, dura
mater; e, epidermis; h, hair follicle; m, mesenchyme; of, osteogenic front; p, parietal bone. Scale
bar 200 µm.
1244 H.-J. Kim and others
expressed at the same location but its intensity was
diminished (Fig. 3E,G). Interestingly at P6 these areas of
expression appeared to join above and below the mid-sutural
mesenchyme, possibly indicating the forming periosteum
sheathing this area. At E15 Fgf9 was expressed with high
intensity in the dural layers, the calvarial mesenchyme and
the overlying epidermis (Fig. 3B). By E17 transcripts were
most notably located in the dura mater and endocranial
portion of the mesenchyme and dermis (Fig. 3D). Postnatally,
expression was still noted in the calvarial mesenchyme at a
diminished level (Fig. 3F). Fgf4 was not present in these
tissues between E15 and P6 (Fig. 3H). Msx1 was expressed
in the mesenchyme of sagittal suture and the dura mater
during embryonic (Fig. 4A,C,) and postnatal stages (Fig.
4E,G). Msx2 was intensely expressed in the sutural
mesenchyme and the dura mater during embryonic stages
(Fig 4B,D). Interestingly, after birth, the expression of Msx2
was dramatically diminished in the mesenchyme and it
completely disappeared from the dura mater (Fig. 4F,H).
Furthermore, analysis of serial sections revealed that the
expression of Msx2 was no longer continuous after birth
around the OFs, indicating a patched pattern of expression.
Msx1 and Msx2 transcripts were intensely expressed in hair
follicles (Fig. 4D-H). It should be noted, however, that the
hair pigment also contributed an additional component to the
authentic expression. Hybridisation with sense probes gave
negative results in all cases (data not shown).
Fig. 2. Effect of dura mater on closure of the sagittal suture. Mouse
embryonic calvaria (E16.5) were cultured, in vitro, with or without
dura mater. (A,C,E,G) Macroscopic views of calvarial explants
cultured with dura mater. The osteogenic fronts (OFs) of the parietal
bones have approximated after 48 and 72 hours; however, the midsutural mesenchyme is still present between the bones (arrows).
(B,D,F,H) Calvarial explants cultured without dura mater. The OFs
approximate each other more rapidly and the interposed mesenchyme
is greatly reduced (arrowhead) after 48 hours (D) and, after 72 hours,
the edges of parietal bones are partially fused (F) (arrowhead).
(G,H) Histological sections of explants cultured for 72 hours with
(G) and without (H) dura mater confirming macroscopic findings
(E,F). of, osteogenic front; p, parietal bone; s, sagittal suture. Scale
bar 1 mm (A-F), 100 µm (G,H).
cultures of postnatal calvaria, no differences were detected
between explants with and without underlying dura mater (data
not shown). These data therefore confirm the important role of
the embryonic dura mater in the maintenance of cranial sutures
as suggested by Opperman et al. (1995).
Expression of FGFR2 (BEK), Fgf4, Fgf9, Msx1 and
Msx2 during sagittal suture development
To address the roles of FGFR2 (BEK), FGF4, FGF-9 (known
ligands of the BEK receptor) and the homeobox-containing
Msx genes in calvarial development, we examined their
expression patterns in serial sections of sagittal sutures by in
situ hybridisation. BEK, a splicing alternative of FGFR2, was
intensely expressed in the OFs of parietal bones of E15 and
E17 mouse embryos, and transcripts were also detected in the
superficial dermis of skin (Fig. 3A,C). Postnatally BEK was
Expression of Bmp2 and Bmp4 during sagittal
suture development
During embryonic stages, Bmp2 mRNA was detected the OFs
and weakly in parietal bones (Fig. 5A,C). After birth, the
expression of Bmp2 was greatly reduced (Fig. 5E). Bmp4
mRNA, on the contrary, was localised in the OFs at high levels
and at a lower level in mesenchyme of sagittal suture until E17
(Fig. 5B). At E15 Bmp4 was also expressed in the developing
falx cerebri and in the dura (Fig. 5B). From the end of
embryonic stage (E18), the expression of Bmp4 was decreased
and the analysis of serial sections showed that expression was
not continuous in the OFs, indicating a patched pattern (Fig.
5D,F). Both Bmp2 and 4 transcripts were expressed in
epidermis of skin and from E16 in hair follicles, however, as
with Msx probes the hair pigment also contributed somewhat
to the authentic expression (Fig. 5A-F). Hybridisation with
sense probes gave negative results in all cases (data not
shown).
Expression of Shh and Ptc during cranial suture
development
The spatiotemporal expression patterns of Shh and Ptc were
analysed by whole-mount in situ hybridisation. The first weak
signs of Shh gene expression were found at E17 in
posteromedial OFs of the parietal bones (data not shown).
From E18 onwards, Shh transcripts were expressed in a
patched pattern in the OFs in sagittal suture as well as in
metopic suture (Fig. 6A,C). Expression of Ptc, recently
identified as Shh receptor (Stone et al., 1996), was not detected
by whole-mount in situ hybridisation until E18 (Fig. 6B). From
E18 onwards, Ptc transcripts were localised in the OFs in
sagittal and metopic sutures (Fig. 6B,D). The patterns of Ptc
and Shh expression were remarkably similar. Control tissues
Signalling in cranial suture development 1245
hybridised with sense probes gave negative results (Fig. 6E,F).
Interestingly, neither Shh nor Ptc was expressed in the area of
the coronal suture during these stages (Fig. 6A-D).
Stimulation of suture closure by local application of
FGF4
To analyse the role of FGFs in cranial suture development,
we added FGF4 protein locally, using heparin acrylic beads,
on sutural tissue of E15.5 mouse in vitro (Figs 7, 8). When
the FGF4 beads were placed directly on OFs, acceleration of
sutural closure was evident after 24 hours (Fig. 7E),
compared to explants without beads (Fig. 7B), or with
control beads soaked in BSA (Fig. 7H). After 48 hours in
culture, a partial fusion of the suture was observed in the
explants cultured with FGF4 beads placed on the OFs (Fig.
7F). However, when FGF4 beads were placed on the midsutural mesenchyme, no acceleration of suture
closure was observed, only a local increase in
tissue thickness (Fig. 7J-L). Histological analysis
of tissue sections showed increased bone growth
of the OFs and partial fusion in explants where
FGF4 beads were placed on the OFs (Fig. 8A-C).
Cell proliferation was analysed by BrdU
incorporation of explants as whole mounts. After
24 hours of culture, FGF4 beads located on both
the OFs (Fig. 8D) and the mid-sutural
mesenchyme (data not shown) increased cell
proliferation around beads. BSA beads showed no
signs of activity (Fig. 8E).
Effect of local application of BMP4 on
suture morphology
Similar experiments to those with FGF4 beads were
performed with beads soaked in BMP4 recombinant
protein. There was no difference in the width of
sagittal suture between BMP and BSA bead explants
after 24 hours of culture (Fig. 9B,E,H,K). A small
increase in tissue volume around the BMP4 beads
was observed after 24 hours of culture (Fig. 9B,H)
as compared to BSA control beads (Fig. 9E,K). This
increase continued, so that after 48 hours the tissue
volume had increased considerably around beads
located on both the OFs and in the mid-sutural
mesenchyme (Fig. 9C,I). In some instances, this
greater tissue volume masked the appearance of the
OFs making interpretation more taxing. Control
beads showed no such effects (Fig. 9D-F,J-L). The
effect of BMP4 beads on tissue volume is illustrated
in Fig. 10E,F as a dramatic increase in tissue
thickness.
Induction of Msx1 expression by FGF4 and
of Msx1 and Msx2 expression by BMP4
In situ hybridisation analysis of explants after 48
hours culture showed that, FGF4 beads induced
the expression of Msx1 (Fig. 10A), but not Msx2
(Fig. 10B) in the mesenchymal cells around the
beads, both when placed on the OFs and the midsutural area (data not shown). BMP4 beads placed
both on the OFs and the mid-sutural mesenchyme
(data not shown) induced expression of both Msx1
and Msx2 around the beads (Fig. 10E,F). No signals were
seen around the control BSA beads (Fig. 10C,D,G,H).
Hybridisation with sense probes gave negative results in all
cases (data not shown).
DISCUSSION
In this study, we have analysed the expression of several
genes, involved in conserved signalling pathways, during
mouse cranial suture development (Fig. 11). In addition, we
have used an in vitro model system to examine the effects of
the dura mater, as well as the local application of FGF and
BMP proteins on sutural closure, parietal bone growth and
gene expression. Our results support the hypothesis that
sutural morphogenesis is regulated by interactions between at
Fig. 3. FGFR2(BEK), Fgf9 and Fgf4 expression in the developing mouse
sagittal suture. In situ hybridisation analysis of tissue sections. (A,C,E,G)
FGFR2(BEK) transcripts are detected in the osteogenic front of the parietal
bones (arrows) and in the superficial dermis of skin (arrowhead). FGFR2(BEK)
mRNA expression is most intense at E15 (A). By P6 (G), the expression is less
intense, the expression at the two osteogenic fronts appear to coalesce to form a
continuous line spanning above and below the mid-sutural mesenchyme.
(B,D,F) Fgf9 expression. (B) At E15, Fgf9 transcripts are seen with great
intensity in the dural layers, the calvarial mesenchyme, and the overlying
epidermis. (D) At E17, transcripts are most notably located in the dura mater
(arrow) and endocranial portion of the mesenchyme and dermis. (F) Postnatally
(P4), expression is still noted in the calvarial mesenchyme at a diminished level.
(H) E15 tissue section hybridised with Fgf4 riboprobe c.f. A and B. It should be
noted that, after birth, the pigment in the developing hair follicles appears bright
in dark-field illumination and gives a false positive (E,F). Scale bar 200 µm.
1246 H.-J. Kim and others
Fig. 4. Msx1 and Msx2 expression in the mouse sagittal suture from
E15 to P5. In situ hybridisation analysis of tissue sections.
(AC,E,G) Msx1 mRNA is present in the mid-sutural mesenchyme
and in the dura mater during all embryonic and postnatal stages.
(B,D) Msx2 mRNA is present in the mid-sutural mesenchyme and
the dura mater during embryonic stages. (F,H) After birth, Msx2
expression has completely disappeared from the dura mater. In the
mid-sutural mesenchyme Msx2 expression is reduced (arrow). Some
of the reaction in the hair follicles in E-H is false positive due to
pigment. Scale bar 200 µm.
least three tissues: the sutural mesenchyme, the OFs of
calvarial bones and the dura mater. The data also indicate that
the same signalling pathways that are used widely in
developmental regulation and which have been conserved in
evolution may be involved in the regulation of suture
development.
Involvement of BMP signalling in cranial suture
development and intramembranous bone growth
There is increasing biochemical and genetic evidence
indicating that BMPs are key signalling molecules in the
initiation of bone and cartilage formation, and that they
regulate the early commitment of mesenchymal cells to the
chondrogenic and osteogenic lineages (Wozney, 1992). The
composite expression patterns of different BMPs as well as
experimental studies affecting their expression suggest that
they are likely to control the basic form and pattern of the
vertebrate skeleton, and that induction of Msx genes may be
involved in the mediation of these effects (Kingsley, 1994;
Katagiri et al., 1994; Davidson, 1995). In line with these
findings, our in situ hybridisation analysis demonstrated that
Bmp2 and Bmp4 was expressed in the OFs. In addition, Bmp4
Fig. 5. Bmp2 and Bmp4 expression in the mouse sagittal suture from
E15 to P3. In situ hybridisation analysis of tissue sections.
(AC) Bmp2 mRNA is shown in the osteogenic fronts (OFs) during
embryonic stage (arrows). (E) Bmp2 expression in the OFs is greatly
reduced after birth. (B) Bmp4 is intensely expressed in the OFs and
at lower level in the mid-sutural mesenchyme until E17 (arrowhead).
At E15, Bmp4 mRNA is also present the developing falx cerebri (fc)
and in the dura (d). (D,F) From E18 onwards, Bmp4 gene expression
in the OFs is absent (D) or reduced (arrows in F). Not all serial
sections show Bmp4 expression, possibly indicating a patched
pattern. Scale bar 200 µm.
was expressed in the sutural mesenchyme. Msx1 and Msx2
were expressed in the intervening mesenchyme of sagittal
suture. We showed that BMP4 protein, when applied locally
by beads on the OFs and sutural mesenchyme, induced the
expression of Msx1 and Msx2 and contributed to an increase
in tissue mass around beads. This induction maybe a direct
effect or a result of an increased number of mesenchymal
cells/osteoprogenitors that express Msx1 and Msx2. Msx2 and
Msx1 are known to be present in vitro in osteoblastic cells
(Hodgkinson et al., 1993; Towler et al., 1994a). There is
evidence that Msx2 is involved in the regulation of collagen 1
and osteocalcin, genes expressed by terminally differentiated
osteoblasts (Towler et al., 1994b; Dodig et al., 1996; Hoffmann
et al., 1996; Newberry et al., 1997). Interestingly, the MSX2
craniosynostosis phenotype appears to be caused by a gain-offunction gene mutation (Jabs et al., 1993; Liu et al., 1995;
Semenza et al., 1995; Ma et al., 1996).
We suggest that the BMPs may be involved in regulating the
balance between the undifferentiated and differentiated states
of osteogenic cells, that Msx genes are involved in this
signalling pathway and, furthermore, that FGFs may act later
on committed osteoblasts.
The function of FGF signalling pathway in the
calvarial suture morphogenesis
The identification of FGF receptor mutations as causes of
several dominantly inherited skeletal disorders, in particular
Signalling in cranial suture development 1247
Fig. 6. Shh and Ptc expression in mouse calvaria from E18 to P3. In
situ hybridisation analysis of whole mounts. (A,C) Shh mRNA is
detected in patched pattern lining the osteogenic fronts of the sagittal
and metopic sutures from E18 onwards (arrows). (B,D) From E18,
Ptc mRNA is expressed in a similar patched pattern.
(E,F) Hybridisation with sense probes showing no signs of activity. c,
coronal suture; f, frontal bone; m, metopic suture; p, parietal bone; s,
sagittal suture. Scale bar 1 mm.
craniosynostosis, reflects functions of FGF signalling in bone
development (Shiang et al., 1994; Cohen, 1995). The FGFR2
mutation causing Crouzon syndrome produces FGF
Fig. 7. The effect, in vitro, of the local application of
FGF4 on the development of mouse calvarial explants.
(A-C) Control explants of E15.5 calvaria cultured for
48 hours. (D-F) FGF4 beads placed on the osteogenic
fronts (OFs) of the sagittal suture accelerate the
approximation of the parietal bones resulting in the
partial obliteration of the mid-sutural space (arrow).
(G-I) BSA beads placed on the OFs have no
detectable effects. (J-L) FGF4 beads placed on centre
of the sutural mesenchyme and cultured for 48 hours
cause a local increase mesenchymal thickness
(arrowhead). of, osteogenic front; p, parietal bone; s,
sagittal suture. Scale bar 1 mm.
independent, constitutive activation of the receptor due to
aberrant intermolecular disulphide-bonding (Neilson and
Friesel, 1995; Galvin et al., 1996). Interestingly signalling
through FGFRs can upregulate an element in the osteocalcin
promoter (Newberry et al., 1996). Hence, in our in vitro
experiments, the local application of FGF4, one of the FGF
ligands binding to FGFR2 (Green et al., 1996), apparently
mimicked the effects of the FGFR mutation in the Crouzon
syndrome. Interestingly, we found that beads releasing FGF4
protein accelerated the approximation of the OFs resulting in
the closure of sutural space only when they were placed on
the OFs. Application on the mid-sutural mesenchyme did not
affect the speed of sutural closure. Cell proliferation was,
however, stimulated around the FGF4-releasing beads in both
locations. One possible explanation for these different
responses is that, in the mid-sutural mesenchyme, FGF4 may
stimulate the proliferation of undifferentiated mesenchymal
cells, whereas, in the OFs, the stimulation of the proliferation
of differentiating osteoblasts may increase the number of
osteoblasts and the production of bone matrix. BMP4 did not
accelerate suture closure. The effect of FGF4 on the OFs
would appear to be related to the high expression of the BEK
in the OFs as shown in our in situ hybridisation analysis. BEK
is known to bind to several ligands including FGF4 and FGF9
(Ornitz et al., 1996). We suggest that it is valid to use FGF4
in in vitro experiments, as it can activate the FGF receptor
and thereby mimic the action of an in vivo ligand. Our
evidence is suggestive that one natural ligand of the BEK
receptor in the OF is FGF9, but this maybe one of many.
Indeed, it is known that the FGFRs bind to a wider range of
ligands, including heparan sulphate proteoglycans and neural
cell adhesion molecules (Green et al., 1996). Iseki and
1248 H.-J. Kim and others
Fig. 8. The effect of FGF4 beads on the histological appearance
and cell proliferation of sutural tissue in vitro (E15.5).
(A) Calvarial E15.5 explant with an FGF4 bead placed on the
osteogenic fronts and cultured for 48 hours. (B,C) Tissue sections
corresponding to the dotted lines on the calvarial explants in A
show partial fusion of the parietal bones. (D) Whole-mount
analysis of BrdU incorporation indicates stimulated cell
proliferation around FGF4 beads (arrows). (E) Control BSA beads
have no effect on cell proliferation. p, parietal bone; s, sagittal
suture. Scale bar, 1 mm (A,D,E), 100 µm (B,C).
coworkers (1997) have suggested that FGF2 is another such
ligand.
Different signalling pathways may regulate suture
maintenance prenatally and postnatally
Macroscopic and microscopic analysis of calvarial explants
revealed that the embryonic, but not postnatal, dura mater
stimulated the maintenance of the overlying cranial suture. The
dura mater is a thin sheet of cells separating the forming
calvarial bones from the brain tissue. Based on studies on
cultured rat coronal sutures, a role for the dura mater, mediated
by soluble factors, in the maintenance of calvarial sutures has
earlier been suggested (Opperman et al., 1995). We performed
similar experiments with the mouse sagittal suture using a
different type of organ culture system.
Our in situ hybridisation analysis showed that the expression
of Msx2 and Bmp4 is correlated with the prenatal activity of
dura mater. Postnatally, neither of these genes were detected in
this location.
Although the expressions of Msx1 and Msx2 were
overlapping, they showed clear differences and, in vitro, BMP4
induced the expression of both Msx1 and Msx2, whereas FGF4
induced the expression of Msx1 only. Our recent experiments
indicate that, in dental mesenchyme also, FGFs preferentially
regulate Msx1, but not Msx2 (Kettunen and Thesleff 1998).
These findings are in line with a recent report by Catron et al.
(1996) showing that the functions of Msx1 and Msx2 genes are
modulated differentially by their non-conserved N-terminal
regions. However, it was shown that Msx1 and Msx2 have
similar DNA binding and transcriptional properties suggesting
redundant functions of the two genes.
Fig. 9. The effect of BMP4 on E15.5 mouse calvarial explants. No
difference is seen in the sagittal suture width between explants
cultured for 48 hours with BMP4 beads on the osteogenic fronts
(OFs) (A-C) or on the mid-sutural mesenchyme (G-I), or with BSA
beads on the OFs (D-F) or on the mid-sutural mesenchyme (J-L). An
increase in tissue volume around the BMP4 beads is observed after
24 hours culture (B,H) after 48 hours, tissue volume increased
greatly around the BMP4 beads located on both the OFs (C) and the
mid-sutural mesenchyme (I), making it more difficult to observe the
OFs (arrowheads). Control explants with BSA beads show no such
effects (D-F, J-L). of, osteogenic front; p, parietal bone; s, sagittal
suture. Scale bar, 1 mm.
Our whole-mount in situ hybridisation analysis showed that
Shh as well as its receptor, Ptc, started to be expressed at the
end of embryonic development. Their expression appeared as
patches on the OFs of the midline sutures. This indicates that,
firstly, the target tissue for Shh signalling is in the OF and,
secondly, there are site-specific differences in Shh signalling in
the calvaria, which may reflect the difference in sutural
architecture. The coronal suture whose OFs are overlapping
apparently lacked Shh expression, whereas as Shh was seen in
end-to-end type midline sutures. We suggest that the Shh
signalling may be involved in regulating cranial suture
development and intramembranous bone formation. It is
possible that Shh has an analogous effect on intramembranous
bone development as has been shown for Ihh, another
hedgehog family member, in endochondral bone formation,
where Ihh controls the differentiation of hypertrophic
chondrocytes (Vortkamp et al., 1996). The patched expression
pattern of these genes may be related to the subsequent
development of the serrated morphology of bone surface at the
sutures.
There are several examples of interactions between Shh and
Signalling in cranial suture development 1249
Fig. 10. FGF4 beads induce Msx1 expression,
while BMP4 beads induce both Msx1 and Msx2 in
calvarial explants. Mouse calvarial explants
(E15.5) were cultured for 48 hours with beads
incubated with either FGF4, BMP4 or BSA, and
analysed by in situ hybridisation. (A,B) FGF4
beads induce Msx1, but not Msx2 expression.
(E,F) BMP4 beads induce the expression of both
Msx1 and Msx2, and cause an increase in tissue
thickness around beads. (C,D,G,H) BSA control
beads. b, bone, m, mesenchyme. Scale bar 200
µm.
BMP signalling pathways. (Bitgood and McMahon, 1995).
Analysis of serial tissue sections indicated that postnatally the
expression of Bmp2, Bmp4 and Msx2 was discontinuous along
the OFs apparently reflecting a patched pattern thus resembling
the expression of Shh and Ptc. We speculate that Shh may
interact with Bmps in the OF through a Ptc-dependent pathway
which maybe involved in the prevention of precocious sutural
closure. It should be highlighted that our evidence for this
model is correlative, and that additional functional data will be
required to elucidate these speculations.
We propose that the maintenance of suture patency during
postnatal calvarial development may be controlled by
signalling events at the OFs, with Shh playing an important
role. Thus signalling pathways regulating cranial suture
development during the embryonic and postnatal stages
would differ, with the relative importance of each varying
with time.
Fig. 11. Schematic diagram to illustrate the genes active prenatally
and postnatally in the developing mouse sagittal suture. At E15,
Bmp4 is also expressed in the central portion of the mid-sutural
mesenchyme and in the dura. Postnatally, Msx2 expression is
reduced and seen in a patched pattern. Also conversely, Shh and its
receptor Ptc are present at P1 but absent at E15. For clarity, Ffg9 is
not shown in this illustration, it is present in the calvarial
mesenchyme postnatally and with great intensity prenatally, this
prenatal expression is most noteworthy in the dural layers. of,
osteogenic front; s, skin; p, parietal bone periosteum; d, dura mater;
m, mesenchyme.
1250 H.-J. Kim and others
We thank Maire Holopainen, Kaija Kettunen, Sirpa Kyllönen,
Marjatta Kivekäs, Merja Mäkinen and Riikka Santalahti for their
excellent technical assistance and Thomas Åberg for his help with the
illustrations. We thank J. Wozney for the Bmp2 and Bmp4 cDNAs
and BMP4 protein, M. Scott for the Ptc cDNA, T. Edlund for the Shh
cDNA and C. Basilico for the Fgf4 cDNA. This research was
supported by the Finnish Academy, the Wellcome Trust and the
European Orthodontic Society.
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