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Histological development and dynamic expression of Bmp2 У6 mRNAs in the embryonic and postnatal mousecranial base.

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THE ANATOMICAL RECORD PART A 288A:1250–1258 (2006)
Histological Development and Dynamic
Expression of Bmp2–6 mRNAs in the
Embryonic and Postnatal Mouse
Cranial Base
PÄIVI KETTUNEN, XUGUANG NIE, INGER HALS KVINNSLAND,
AND KEIJO LUUKKO*
Department of Biomedicine, University of Bergen, Bergen, Norway
ABSTRACT
The cranial base is formed by endochondral ossification and is characterized by the presence of the synchondrosis growth centers. The aim of
this study was to describe the histological development of the mouse midsagittal cranial base area from embryonic day 10 (E10) to the postnatal
age of 2 months. The Bmp family of signaling molecules serves important
functions in embryo and bone development and may therefore play a significant role in the early formation of the cranial base. To investigate this,
we analyzed the mRNA pattern of expression of Bmp2–6 in the mouse cranial base from E10 to 5 days postnatally using radioactive in situ hybridization. We found that the formation of the mouse cranial base corresponds
to that of rat and proceeds in a caudorostral sequence. Moreover, all Bmps
studied showed distinct and overlapping developmentally regulated expression domains. Bmp2, Bmp5, and Bmp6 were expressed in the early
mesenchymal condensations. Later, Bmp2, Bmp3, Bmp4, and Bmp5 were
detected in the perichondrium and in the adjacent mesenchyme. Subsequently, Bmp2 and Bmp6 expressions were confined to hypertrophic chondrocytes, while Bmp3, Bmp4, and Bmp5 were expressed in the osteoblasts
of the trabecular bone and bone collar. Interestingly, Bmp3 was uniquely
expressed postnatally in the resting zone of the synchondrosis growth center, suggesting a role in the regulation of cranial base growth. These results
suggest that Bmp signaling may serve specific and synergistic functions at
different key stages of cranial base development and growth. Anat Rec Part
A, 288A:1250–1258, 2006. Ó 2006 Wiley-Liss, Inc.
Key words: bone morphogenetic proteins; skeletogenesis; endochondral bone; hard tissue; growth; synchondrosis
The cranial base, which is located between the brain
and the facial bones, forms the floor of the neurocranium
and is established by the coordinated development and
growth of several skeletal elements. The human midline
cranial base is composed of the basioccipital, sphenoid,
ethmoid, and frontal bones (Scott, 1958). The cranial base
is an important growth center of the head and is believed
to play a significant role in the integration of craniofacial
development and growth.
In contrast to the bones of the cranial vault and the face,
which are formed by intramembranous ossification, the midline cranial base bones, like the long bones, the vertebrae, and
the ribcage, are formed by endochondral ossification. However,
Ó 2006 WILEY-LISS, INC.
Grant sponsor: The Norwegian Cancer Society; Grant sponsor:
the L. Meltzer Foundation; Grant sponsor: the University of Bergen; Grant sponsor: the Research Council of Norway.
The first two authors contributed equally to this work.
*Correspondence to: Keijo Luukko, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. Fax: 47-55-58-63-60. E-mail: keijo.luukko@biomed.uib.no
Received 2 September 2006; Accepted 11 September 2006
DOI 10.1002/ar.a.20402
Published online 25 October 2006 in Wiley InterScience (www.
interscience.wiley.com).
Bmps IN DEVELOPING CRANIAL BASE
the frontal bone is formed by intramembranous ossification.
The anterior cranial base, rostral to the sella turcica, is formed
from neural crest-derived ectomesenchymal cells, whereas the
cells forming the posterior cranial base derive from paraxial
mesoderm (Couly et al., 1993). The mesenchymal cells condense and differentiate into chondrocytes, which proliferate and subsequently differentiate into hypertrophic chondrocytes. The cartilage model is gradually replaced by
bone except for the synchondroses, which are the sites
where the postnatal growth and expansion of the cranial
base take place. The synchondrosis is a bidirectional
growth site; it is structurally analogous to the growth
plate of the long bones, which allow the growth of the
bone. In the midsagittal region of the human cranial base,
two synchondroses, the spheno-occipital and the intersphenoidal (midsphenoidal), are present (Scott, 1958; Melsen, 1974). The spheno-occipital synchondrosis is considered to be particularly important: in addition to providing
most of the growth of the cranial base, it remains active
into adolescence (Ingervall and Thilander, 1972; Thilander
and Ingervall, 1973; Roberts and Blackfood, 1983; Kjaer,
1990). The enlargement of the brain has also been speculated to contribute to the growth of the cranial base by
affecting the proliferation and differentiation of the chondrocytes in the synchondrosis (Ingervall and Thilander,
1972; Wang and Mao, 2002a, 2002b; Tang and Mao, 2006).
While there has been significant progress in understanding the molecular regulation of long bone and cranial vault
development, little is known concerning that of the cranial base (see the reviews in Opperman, 2000; Wilkie and
Morriss-Kay, 2001; Karsenty and Wagner, 2002; Ornitz
and Maria, 2002). Cranial base defects have been identified in many human syndromes caused by gene mutations.
For instance, abnormal cranial bases in achondroplasia
and Apert, Crouzon, and Muenke syndromes are caused by
mutations in FGFR, indicating that FGF signaling is not
only important to the formation of the cranial vault, but
also for the cranial base (Horowitz, 1981; Cohen et al.,
1985; Kreiborg et al., 1993, 1999; Wilkie and Morriss-Kay,
2001; Reinhart et al., 2003). Similar defects have been
observed in FGFR transgenic mouse strains (Chen et al.,
2001; Eswarakumar et al., 2002, 2004). Mutations in
RUNX2 and TWIST give rise to cleidocranial dysplasia
and Saethre-Chotzen syndromes, respectively, which are
characterized by defects in the cranial base (Evans and
Christiansen, 1976; Kreiborg et al., 1981; Howard et al.,
1997; Lee et al., 1997). In addition, abnormalities in the
cranial base have been reported in syndromes with chromosome abnormalities, such as Cri-du-Chat, Down’s,
Meckel, Seckel, Turner, and Williams, as well as in human
triploid fetuses (Jensen, 1985; Kjaer et al., 1997, 1999, 2001;
Andersen et al., 2000; Quintanilla et al., 2002; Axelsson
et al., 2005). Abnormal cranial bases have also been
described in anencephalia and hydrocephalus (Kantomaa
et al., 1987; Lomholt et al., 2004). Together, these results
suggest that several signaling pathways are involved in the
regulation of the formation of the cranial base. Interestingly,
recent experimental in vitro studies have shown that the
proliferation and differentiation of the chondrocytes in the
synchondrosis are modulated by mechanical loading, which
influences the gene expression in the synchondrosis (Wang
and Mao, 2002a, 2002b; Tang and Mao, 2006).
Bone morphogenetic proteins (Bmps) are a family of
more than 20 secreted signaling molecules that belong to a
larger transforming growth factor b (Tgfb) superfamily (for
1251
review, see Ducy and Karsenty, 2000). Bmps signal through
heteromeric complexes of transmembrane type I and type
II serine/threonine kinase receptors, which transfer signals
from the cell surface through the Smad pathway to the nucleus (for review, see Hoffmann and Gross, 2001). Bmp signaling is modulated by interaction with antagonists such
as Chordin, Dan, Follistatin, Gremlin, Noggin, and Sclerostin (for review, see Canalis et al., 2003; Avsian-Kretchmer
and Hsueh, 2004; Yanagita, 2005). The first Bmps were
identified by their ability to induce ectopic bone formation
in vivo (Wozney et al., 1988). Later they were found to serve
broader signaling functions in embryo and organ formation
(for review, see Hogan, 1996; Chen et al., 2004). In vitro
experiments and the analysis of transgenic mouse strains
have shown that Bmps regulate skeletal patterning and
bone development, but that their functions are largely
redundant. Bmp7 null mutation leads to multiple developmental defects in the skeleton, including polydactyly, defective basisphenoid bone and xyphoid cartilage, fused ribs
and vertebrae, and retarded ossification of bones (Luo
et al., 1995; Jena et al., 1997). Deletion of Bmp3, Bmp5,
and Bmp6 in mice resulted in mild abnormalities in bones
(Kingsley et al., 1992; King et al., 1994; Solloway et al.,
1998; Daluiski et al., 2001). Bmp2/7 and Bmp5/7 double
heterozygotes have normal skeletons, while Bmp4/7 double heterozygotes showed minor defects in the rib cage and
digits (Katagiri et al., 1998). Disruption of Bmp2, Bmp4,
and Bmpr1A (Alk3) did not produce much information
about their roles in skeletogenesis due to early embryonic
lethality (Mishina et al., 1995; Winnier et al., 1995; Zhang
and Bradley, 1996). Defects in bone development have been
reported in Bmp receptor and antagonist mutant mice such
as ActR1 (Alk2), Bmpr1B (Alk6), and Noggin (Brunet et al.,
1998; Yi et al., 2000; Dudas et al., 2004).
The histological development and growth of the cranial
base have been described earlier for rat (Baume, 1968; Dorenbos, 1973; Roberts and Blackwood, 1983) and humans
(Baume, 1968). As an increasing number of transgenic
mice are now produced for use as in vivo models for human
syndromes and developmental disorders, this prompted us
to investigate whether mouse cranial base development
and ossification are similar to that in humans and rat. In
addition, knowledge of the gene expression patterns is
essential for unraveling the molecular control of cranial
base formation. In the present study, therefore, we systematically investigated the histological development of
the mouse cranial base in midsagittal tissue sections
between embryonic day 10 (E10) and the postnatal age of
2 months. Bmp signaling is important for skeletal development and therefore most likely also plays a significant
role in cranial base formation. Although expression domains of Bmp receptor mRNAs have been previously
reported in the developing cranial base (Dewulf et al.,
1995; Verschueren et al., 1995), the expression patterns of
Bmps have not been analyzed systematically. We therefore
compared the expression of Bmp2–6 mRNAs during cranial base development in mice from before the visible
onset of its development from E10 until the presence of
functional synchondroses 5 days postnatally (5PN).
MATERIALS AND METHODS
Preparation of Tissues and Histology
The study was approved by the Animal Welfare Committee of the Biomedical Institute, University of Bergen.
1252
KETTUNEN ET AL.
NMRI mice were mated overnight and the appearance of a
vaginal plug was taken as day 0 of embryogenesis (E0).
Delivery of NMRI mice takes place at E19, which corresponds to the newborn (NB) stage. Embryonic mouse
heads between E10 and E18, NB heads, and 5-day-old
(PN5), 9-day-old, and 2-month-old cranial bases were used
in this study. The adult mice were killed by cervical dislocation. Embryos and postnatal mice were killed by decapitation. The age of the embryos was determined by the day
of appearance of the vaginal plug and further confirmed by
morphological criteria according to Theiler (1989). Heads
and cranial bases were dissected in Dulbecco’s phosphatebuffered saline (PBS) and fixed in 4% paraformaldehyde
(PFA) overnight at 48C. Heads and cranial bases older
than E14 were decalcified with 12.5% EDTA/2.5% PFA in
PBS. They were then rinsed in PBS, dehydrated in ethanol, cleared in xylene, and embedded in paraffin.
For histological analysis, 7 mm midsagittal sections of
E10–E14, E16, NB, PN5, PN9, and 2-month-old tissues
were cut with a microtome (Leica Microsystems, Wetzlar,
Germany). The sections were deparaffinized in xylene,
rehydrated in graded ethanol, and stained with hematoxylin and eosin (Shandon instant hematoxylin and eosin;
Thermo Electron, Waltham, MA) for 2 and 5 min, respectively, according to the manufacturer’s instructions. The
sections were then dehydrated, cleared in xylene, and
mounted with DePeX (BDH, Poole, U.K.).
In Situ Hybridization
We used 7 mm midsagittal sections of E10–E14, E16, NB,
and PN5 tissues for in situ hybridization analysis of Bmp2–
6 mRNA expression. The mouse Bmp2–6 cDNAs, which
were a kind gift of Dr. Wozney of the Genetics Institute
(Cambridge, MA), have been described earlier in Vainio
et al. (1993) and Aberg et al. (1997). In vitro transcription
and in situ hybridization were performed as described in
Luukko et al. (1996). In brief, the cDNA fragments containing plasmids were linearized, and antisense and sense RNA
probes were generated using 35S-labeled UTP (Amersham,
Buckinghamshire, U.K.) and appropriate RNA polymerases. Alkaline hydrolysis was omitted as the length of the
probes was considered to be appropriate for hybridization.
The probes were passed through Sephadex G-50 NICK columns (Pharmacia Biotech, Uppsala, Sweden) according to
the manufacturer’s instructions, ethanol-precipitated, airdried, and dissolved in 1 M dithiothreitol (DTT; Sigma
Chemical, Dorset, U.K.) and Wilkinson’s hybridization
buffer at 1:9. The probes were diluted to the final concentration of 5 3 104 cpm/ml, and 50–100 ml of the hybridization
buffer with the labeled cRNA was added to each slide.
Hybridization was carried out for 15–20 hr at 528C and the
glasses were washed with 60% formamide (Merck, Darmstadt, Germany) and 30 mM of DTT at 658C. For autoradiography, the dehydrated slides were coated with NTB-2
emulsion (Eastman Kodak, New Haven, CT), dried, and
exposed for 3 weeks at 48C. The sections were developed in
Kodak D-19, fixed with Unifix (Eastman Kodak), counterstained with hematoxylin (Thermo Electron, Waltham,
MA), and mounted with DePeX (BDH). Photomicrography
was performed using a Zeiss Axioskop 2 microscope (Carl
Zeiss, Jena, Germany) and a SPOT Insight digital camera
(Diagnostic Instruments, Sterling Heights, MI). Brightand dark-field images were taken from hybridized sections.
Digital images were processed with Adobe Photoshop 7.0
software (Adobe Systems, San Jose, CA). Silver grains
were selected from the dark-field images, colored red, and
superimposed onto the bright-field images.
RESULTS
Histological Development of Mouse
Cranial Base
We used midsagittal sections to investigate the histological development of the mouse cranial base during embryonic and early postnatal development (E10 to 5PN).
We further examined the histology of the spheno-occipital synchondrosis of 9-day-old and 2-month-old mice. In
our analysis, we focused particularly on the basioccipital and sphenoid bones and the spheno-occipital synchondrosis.
At E10, the notochord, which first defines the midline
axis, was observed in the area between the developing hindbrain and foregut. In addition, Rathke’s pouch was observed
at the roof of the foregut, but no sign of cell condensation
was detected in the loose mesenchyme in the presumptive
cranial base area between the foregut epithelium and the
developing brain (Fig. 1A). Mesenchymal cell condensation
of the developing cranial base began to be visible at E11 in
the caudal end of the future basioccipital bone (Fig. 1B).
During subsequent development, the mesenchymal condensations of the anterior part of the presumptive basioccipital,
basisphenoid, and presphenoid bones appeared (Fig. 1C). At
E13, the chondrocranium was largely formed and chondrocyte differentiation had started in the middle of each cartilage template. Perichondral mesenchyme condensation surrounding the cartilage models was clearly visible on the
brain side, whereas on the oral side the perichondrium was
not fully developed along the whole chondrocranium (Fig.
1D). At E14, the sites of the future synchondroses began to
be recognizable. Terminally differentiated hypertrophic
chondrocytes were present in the caudal and middle part of
basioccipital cartilage, but were not detected in the basisphenoid and presphenoid cartilages at this stage (Fig. 1E). At
E16, the areas of hypertrophic chondrocytes had expanded
in the presumptive basioccipital and basisphenoid bones.
However, differentiation of hypertrophic chondrocytes was
not yet observed in the presphenoid cartilage. At E16, bone
formation was detected in the perichondrium of the future
basioccipital and basisphenoid bones. In addition to the bone
collar, trabecular bone formation was visible in the developing basioccipital bone. At this stage, the angle between the
anterior and posterior cranial base was greater compared to
earlier stages, and the straight shape of the skull base was
observed (Fig. 1F).
During postnatal development, the future cranial base
became increasingly ossified except in the areas of the synchondroses (Fig. 1G–J). In the middle part of the synchondrosis, there was a narrow band of resting cells, surrounded by the proliferation, prehypertrophic, and hypertrophic zones on each side. At PN5, as at the earlier
stages, the intersphenoidal synchondrosis was still broader
than the spheno-occipital synchondrosis. In the young
adult mouse (2 months), the basisphenoidal and intersphenoidal synchondroses had become a thin band of chondrocytes embedded in cartilage matrix (Fig. 1K).
Expression of Bmp2–6 mRNAs in Developing
Cranial Base
We systematically compared the expression of Bmp2–6
at consecutive developmental stages between E10 and
Bmps IN DEVELOPING CRANIAL BASE
Fig. 1. Histological development of the embryonic (E10–E14 and
E16) cranial base (A–F) as well as newborn (G), 5-day and 9-day postnatal and 2-month-old spheno-occipital synchondrosis (I–K). Midsagittal sections stained with hematoxylin and eosin. Higher magnifications
of the developing E13, E14, E16, and NB spheno-occipital synchondrosis (marked with a black asterisk) are provided in smaller photomicrographs (D–F, H). The intersphenoidal synchondrosis is marked with a
turquoise asterisk. b, brain; ba1, first branchial arch; bo, basioccipital
1253
cartilage; bob, basioccipital bone; bs, basisphenoid cartilage; bsb,
basisphenoid bone; cm, calcified cartilaginous matrix; e, erosive zone;
h, hypertrophic chondrocytes; m, mesenchymal cell condensate; n,
notochord; p, perichondrium; pe, periosteum; pi, pituitary gland; pm,
perichondral mesenchyme condensation; pr, proliferating chondrocytes; pre, prehypertrophic chondrocytes; ps, presphenoid cartilage;
rc, resting chondrocytes; r, Rathke’s pouch; t, trabecular bone; to,
tongue. Scale bars ¼ 200 mm.
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KETTUNEN ET AL.
Fig. 2. Expression of mRNAs for Bmp2, Bmp3, and Bmp4 in
E10–14 and E16 (A–F, I–N, Q–V) developing cranial base as well as
in newborn (G, O, W) and 5 days postnatal (H, P, X) developing
spheno-occipital synchondrosis. Midsagittal sections. The expression
of Bmp2–4 is also seen in the developing dura mater (arrowheads).
Scale bars ¼ 200 mm in A (applies to A, I, Q and G, H, O, P, W, X) and
200 mm in B (applies to B–F, J–N, R–V).
PN5. At E10, before any sign of cranial base development,
Bmp2 mRNAs were detected in the loose mesenchyme
next to the foregut epithelium in the prospective cranial
base area (Fig. 2A). At E11, Bmp2 expression was seen
in the first mesenchymal condensations marking the
sites of the future basioccipital and basisphenoidal bones
(Fig. 2B). At E12, its expression extended to the anterior
cranial base. Interestingly, in the caudal end of the basioccipital cartilage, Bmp2 mRNAs were downregulated in
the differentiating chondrocytes, while the expression
persisted in the surrounding mesenchymal condensate
(Fig. 2C), and by E13 transcripts were mostly confined to
Bmps IN DEVELOPING CRANIAL BASE
the perichondrium (Fig. 2D). At E14, Bmp2 expression
was clustered in hypertrophic chondrocytes in the future
basioccipital bone (Fig. 2E). Between E16 and PN5, transcripts were mostly seen in the hypertrophic chondrocytes
in the spheno-occipital synchondrosis and in the perichondrium next to the developing synchondrosis (Fig. 2F–H).
Bmp3 showed restricted expression in the epithelium
of the foregut adjacent to the future anterior cranial base
at E10 (Fig. 2I). From E11 onward, transcripts became
confined to the perichondrium (Fig. 2J–M). At E16, Bmp3
mRNAs also appeared in the developing trabecular bone
in the middle of the basioccipital cartilage (Fig. 2N). At
birth, Bmp3 was expressed in the osteoblasts of trabecular bone and periosteum (Fig. 2O). Interestingly, a weak
expression was also observed in the future site of the
resting zone of the synchondrosis, which became more
prominent by PN5 (Fig. 2P).
Bmp4 expression was detected in the foregut epithelium
and had a diffuse presence in the adjacent mesenchyme in
the cranial base area at E10 (Fig. 2Q). From E11 onward,
expression was found in the mesenchyme surrounding the
cartilage templates (Fig. 2R–U). From E16 onward, Bmp4
mRNAs were observed in the trabecular bone and in the
perichondrium (Fig. 2V–X).
Bmp5 was seen in the notochord of the E10 cranial
base area (Fig. 3A). At E11, expression appeared in condensed mesenchyme and, 1 day later, also in the prospective perichondrium (Fig. 3B and C). At E13 and later
stages, Bmp5 expression was confined to the perichondrium and subsequently to the periosteum (Fig. 3D–G).
In addition, some transcripts were observed in the trabecular bone at E16 and later stages (Fig. 3E–G).
Bmp6 expression first appeared in the developing cranial
base at E11. Transcripts were seen in the mesenchymal condensations of the caudal part of the cranial base during E11
and E12, but not at the rostral end (Fig. 3H–J). At E13,
transcripts were observed in the early hypertrophic chondrocytes (Fig. 3K). From E16 onward, Bmp6 expression
was confined to the hypertrophic chondrocytes of the developing synchondrosis (Fig. 3L–N). In addition, some transcripts were seen in the trabecular bone and in the perichondrium/periosteum at NB and PN5 (Fig. 3M and N).
DISCUSSION
The cranial base is an important growth center of the
head, integrating the growth of the cranial vault and facial bones. However, the molecular mechanisms of its formation and growth have remained largely unknown. In
this study, we have described the histological formation
of the mouse cranial base in the midsagittal area between
E10 and 2 months after birth. We found that the histological development of the embryonic and early postnatal
mouse cranial base corresponds to that of rats (Baume,
1968; Dorenbos, 1973; Roberts and Blackwood, 1983).
Mouse cranial base development was observed to start at
the caudal end as a mesenchymal condensation and, as in
humans and rats, proceeded in a caudorostral sequence
(Scott, 1958; Baume, 1968; Dorenbos, 1973; Roberts and
Blackwood, 1983; Kjaer, 1990). Moreover, analysis of the
expression of Bmp2–6 mRNAs in embryonic and early
postnatal stages using radioactive in situ hybridization
demonstrated that all the genes studied had a distinct,
developmentally regulated expression domain. This sug-
1255
Fig. 3. Expression of mRNA for Bmp5 and Bmp6 in E10–E13 (A–D,
H–K) and E16 (E, L) developing cranial base as well as in NB (F, M) and
PN5 (G, N) spheno-occipital synchondrosis. Midsagittal sections. In
addition to the cranial base, the expression of Bmp5 and Bmp6 is seen
in the developing dura mater (arrowheads). Scale bars ¼ 200 mm in A
and H; 200 mm in N (applies to B–G, I–N).
gests that Bmp signaling plays an important role at different stages of cranial base development and growth.
Possible Functions of Bmps in Condensation
of Cranial Base Mesenchyme
The first event of the skeletogenesis is the migration of
undifferentiated mesenchymal cells to the areas destined to
become bone. This is followed by the condensation of the
cells, which determines the site, size, and shape of future
bones (Ducy and Karsenty, 1998; Hall and Miyake, 2000).
During the initial stages of mouse cranial base development, two types of mesenchyme condensations were
observed. One was the mesenchymal condensation for cartilage templates; the other was the perichondral mesenchymal condensation that surrounds the cartilage templates.
Bmp signaling has been shown to be an important patterning signal during body development (for review, see Hogan,
1256
KETTUNEN ET AL.
1996) and may be involved in prepatterning of neural crest
skeletal lineage (Kanzler et al., 2000). We found that Bmp2
and Bmp4 showed restricted expression in the mesenchymal area of the cranial base before the condensations
appeared. Subsequently, at E11, Bmp2 expression continued in the condensate where Bmp5 and Bmp6 were also
upregulated, while Bmp3 and Bmp4 were expressed in the
surrounding mesenchyme. Bmp2 and Bmp5 have been
reported in the mesenchymal condensates prefiguring the
future skeleton (Ducy and Karsenty, 2000), and their signaling appears to regulate the size and shape of the condensations (King et al., 1994; Zou et al., 1997; Pizette and Niswander, 2000). Bmp signaling, including Bmp2, regulates
Sox9, which is essential for formation of the mesenchymal
condensation and subsequent cartilage formation (Bi et al.,
1999, 2001; Healy et al., 1999). In vitro, the Bmp antagonist
Noggin inhibits aggregation of mesenchymal cells into prechondrogenic condensations (Pizette and Niswander, 2000).
On the other hand, Bmp3 has been shown to antagonize
osteogenic Bmps (Daluiski et al., 2001). Thus, the combined
signaling activity of Bmp2 and Bmp5, and probably Bmp3,
Bmp4, and Bmp6, may serve patterning functions in cranial
base development by regulating the site, size, and shape of
the mesenchymal condensates. This may take place through
activation of Sox9 in the developing cranial base anlage (Nie
et al., 2005) and through coordinated action with Shh, which
is a patterning signal for the head that includes the cranial
base and facial bones and, like Sox9, is expressed in the
developing cranial base area (Chiang et al., 1996; Zeng
et al., 2002; Jeong et al., 2004, Nie et al., 2005). Mouse
embryos lacking Hh signaling or Sox9 in the neural crest
cells show similar patterning defects in the cranial base
(Mori-Akiyama et al., 2003; Jeong et al., 2004). Interestingly,
we observed Bmp6 in the mesenchymal condensation of the
presumptive basioccipital bone, which derives from the paraxial mesoderm, but no expression was seen in the basisphenoid and presphenoid condensations derived from the neural
crest (Couly et al., 1993). Thus, the early Bmp6 expression
may reflect the differing origin of the cranial base bones.
Possible Roles of Bmps in Chondrocyte
Proliferation and Differentiation
in Cranial Base
After the mesenchymal condensation, the cells in the
condensate differentiate into chondrocytes, which form the
cartilage templates for the cranial base bones. There is a
substantial body of in vitro and genetic evidence that Bmp
signaling is involved in the regulation of chondrocyte development and differentiation (Zou et al., 1997; Ducy and
Karsenty, 2000; Pizette and Niswander, 2000; Yi et al.,
2000; Minina et al., 2002; Shum et al., 2003). We found
that expression patterns of the studied Bmps in the developing cartilage templates of the cranial base were largely
similar to those of other developing bones after condensation and prior to osteogenesis (Duprez et al., 1996; Solloway et al., 1998; Minina et al., 2001). The perichondrium
and the surrounding mesenchyme showed Bmp2, Bmp3,
Bmp4, and Bmp5 expression. Bmp2 and Bmp6 expressions
were gradually confined to the hypertrophic chondrocytes.
Thus, these results suggest that Bmp signaling, possibly in
interaction with Fgfs and Ihh/Pthlh, regulates chondrocyte
differentiation, proliferation, maturation, and hypertrophy
during cranial base development, as has also been proposed in relation to long bones (Minina et al., 2002;
Kobayashi et al., 2005). Recently, Ihh, which controls chondrocyte proliferation and differentiation, was shown to
transduce mechanical signals and upregulate Bmp2/4 in
chondrocytes (Wu et al., 2001). Moreover, mechanical loading was proposed to induce cartilage formation in the mesenchymal tissue in vivo by downregulating BMP3 (Aspenberg et al., 2000). In addition, mechanical stress was
shown to promote the proliferation of the cranial base cartilage in vivo (Wang and Mao, 2002b). Thus, Bmps may be
involved in mediation of the possible mechanical influence
of the expanding brain on cartilage formation during cranial base development and growth.
Possible Roles of Bmps in Ossification
of Cranial Base
The developing cranial base is formed through two types
of ossification processes. The cartilage templates of the cranial base bones undergo endochondral ossification, whereby
hypertrophic chondrocytes are replaced by invading osteoprogenitor cells that subsequently differentiate into osteoblasts and produce bone. The periosteum lining the bone
proper forms through intramembranous ossification,
whereby the mesenchymal cells differentiate directly into
osteoblasts (Karsenty and Wagner, 2002). Bmps were originally identified by their ability to induce ectopic bone formation (Wozney et al., 1988), but their actual in vivo roles and
the molecular basis of their functions in bone formation
have remained largely unknown, partly due to the fact that
several Bmp mutant mouse strains have only mild skeletal
patterning and joint formation anomalies or early lethality
(Karsenty and Wagner, 2002). However, targeted inactivation of the antiproliferative protein gene Tob in transgenic
mice resulted in greater bone mass due to an increased
number of osteoblasts, providing indirect evidence that
Bmp/Smad signaling controls osteoblast proliferation and
differentiation in vivo (Yoshida et al., 2000). We found the
studied Bmps in sites or adjacent to sites that contain osteoblast precursors and osteoblasts that largely correspond to
their reported expression patterns in the long bones and
ribs (Duprez et al., 1996; Solloway et al., 1998; Minina et al.,
2001). Bmp2 and Bmp4 share the same receptors and apparently regulate bone formation (Yoshida et al., 2000; Minina
et al., 2001; Wright et al., 2002). We found Bmp2 and Bmp4
mRNAs in the perichondrium and adjacent mesenchyme,
respectively. In addition, the developing trabecular bone
showed Bmp4 transcripts. Thus, the proteins might regulate osteoblast proliferation and differentiation and subsequent ossification in the cranial base. In contrast, Bmp3,
transcripts of which were found in the developing periosteum and trabecular bone, and which acts as an antagonist
to osteogenic Bmps, especially to Bmp2 (Daluiski et al.,
2001), might modulate the activity of osteogenic Bmps and
negatively regulate the density of the cranial base bones as
shown for the long bones (Daluiski et al., 2001).
Possible Roles of Bmps in Postnatal Growth
of Cranial Base
The postnatal lengthening of the cranial base takes
place predominantly in the spheno-occipital synchondrosis. Earlier Bmp signaling has been shown to control
chondrocyte proliferation in cultured cranial bases
(Shum et al., 2003). Interestingly, we observed Bmp3
expression in the resting zone of the spheno-occipital syn-
Bmps IN DEVELOPING CRANIAL BASE
chondrosis from the newborn stage onward. The resting
zone of the growth plate in the long bones has been suggested to contain stem cell-like cells or precursor cells of
proliferating chondrocytes (Abad et al., 2002). The in vivo
function of Bmp3 in chondrocytes is unknown, but recent
in vivo data suggest that it inhibits cell differentiation
during bone formation (Aspenberg et al., 2000; Daluiski
et al., 2001). Therefore, it is tempting to propose that
Bmp3 may act to keep resting zone chondrocytes and adjacent proliferating chondrocytes undifferentiated, which
would control the proliferation rate of the chondrocytes
and thereby influence the growth of the cranial base.
ACKNOWLEDGMENTS
The authors thank Kjellfrid Haukanes and Helen
Olsen for their excellent technical assistance.
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