1 Center for Oral Biology, Department of Biomedical Genetics, Abs Institute of
Biomedical Sciences, School of Medicine and Dentistry, University of
Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
2 Max Delbruck Center for Molecular Medicine, Robert-Rossle-Strasse 10, 13122
Berlin, Germany
3 Department of Orthopedics, School of Medicine and Dentistry, University of
Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
4 Department of Genetics and Development, College of Physicians and Surgeons,
Columbia University, 701 West 168th Street, New York, NY 10032, USA
* Author for correspondence (e-mail: wei_hsu{at}urmc.rochester.edu)
Accepted 14 February 2005
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SUMMARY |
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Key words: Axin, Axin2, Wnt, Neural crest, Craniosynostosis
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Introduction |
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The cranial skull consists of the neurocranium and the viscerocranium that
are formed from cranial skeletogenic mesenchyme derived from both mesoderm and
neural crest (Jiang et al.,
2002). The cranial skeletogenic mesenchyme undergoes
intramembranous ossification to form the skull bones during calvarial
morphogenesis (Hall, 1990
).
Expansion of the brain is accommodated by calvarial growth. This developmental
process is regulated by cranial sutures, which serve as growth centers for
osteogenesis. Individuals with craniosynostosis develop abnormal skull shapes
due to premature fusion of the cranial sutures. Craniosynostosis is one of the
most common human congenital craniofacial deformities affecting one in
approximately 2,500 individuals (Cohen and
MacLean, 2000
). Premature suture closure results in cranial
dysmorphism, which can be familial or sporadic in origin
(Cohen and MacLean, 2000
).
Although linkage analyses have shown that the Fgfr
(Burke et al., 1998
),
Msx2 (Jabs et al.,
1993
) and Twist (el
Ghouzzi et al., 1997
; Howard
et al., 1997
) genes are associated with craniosynostosis-related
syndromes, the mechanisms underlying suture development remains largely
unknown. Therefore, identification of genes and signaling pathways that
mediate calvarial morphogenesis is critical for deciphering the pathogenesis
of craniosynostosis.
Axin1, which regulates embryonic axis determination by modulating the
canonical Wnt pathway, was first identified in a mouse mutant strain
(Zeng et al., 1997).
Substantial evidence has established that Axin1 and its homolog
Axin2/conductin/Axil plays a central role in regulating the stability of
ß-catenin, which is a crucial event in cellular response to Wnt signaling
(Kikuchi, 2000
;
Miller et al., 1999
;
Moon et al., 2002
;
Peifer and Polakis, 2000
).
Axins serve as scaffold proteins directly associating with several Wnt
signaling molecules, including disheveled, the serine/threonine kinase GSK-3,
ß-catenin, adenomatous polypopsis coli (APC) and the serine/threonine
protein phosphatase 2A (PP2A) (Behrens et
al., 1998
; Fagotto et al.,
1999
; Hedgepeth et al.,
1999
; Hsu et al.,
1999
; Itoh et al.,
1998
; Julius et al.,
2000
; Kishida et al.,
1998
; Sakanaka et al.,
1998
). In the absence of a Wnt signal, the Axin-dependent complex
mediates ß-catenin degradation, while Wnt signals perturb formation of
this complex (Farr et al.,
2000
; Li et al.,
1999
; Smalley et al.,
1999
; Yanagawa et al.,
1995
). Therefore, ß-catenin is accumulated and binds to
LEF/TCF family proteins to activate target genes
(Behrens et al., 1996
;
Brannon et al., 1997
;
Molenaar et al., 1996
).
Wnt signaling controls early craniofacial morphogenesis
(Parr et al., 1993). Wnt1 and
Wnt3a are both expressed in the dorsolateral region of the neural tube that
gives rise to CNC (McMahon et al.,
1992
). Although inactivation of either Wnt1 or Wnt3a gene did not
cause defects in craniofacial development
(McMahon and Bradley, 1990
;
Takada et al., 1994
), mice in
which both the Wnt1 and Wnt3a genes are inactivated showed a
marked deficiency in CNC derivatives
(Ikeya et al., 1997
).
Furthermore, downstream components of the Wnt signaling pathway, including
Lrp6, APC and ß-catenin, have also been implicated in craniofacial
development (Brault et al.,
2001
; Hasegawa et al.,
2002
; Mitchell et al.,
2001
). Nevertheless, the importance of the Wnt pathway in
intramembranous ossification during mammalian skull formation remains
unclear.
In this study, we have investigated the involvement of Axin2 in cranial skeletogenesis. Targeted disruption of Axin2 did not cause obvious embryonic abnormalities, although Axin2 is highly expressed in CNC. However, our data demonstrate that Axin2 is required for skull development at early postnatal stages. The inactivation of Axin2 in mice induces craniosynostosis, a common human congenital defect. The premature fusion of cranial sutures is mediated by alterations in intramembranous ossification in the mutants. The neural crest dependent skeletogenesis is particularly sensitive to the loss of Axin2 that stimulates ß-catenin signaling in the developing calvarium. These findings demonstrate not only the importance of Axin2, but also a novel role of the canonical Wnt pathway, in calvarial morphogenesis and craniosynostosis.
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Materials and methods |
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Histology, skeletal preparation and ß-gal staining
Skulls were fixed in formaldehyde/formic acid (Cal-Rite, Richard-Allan
Scientific) and paraffin embedded. Samples were sectioned, and stained with
Hematoxylin/Eosin/Orange G for histological evaluation. Staining for
ß-galactosidase activity in cranial skulls
(Whiting et al., 1991) and
mouse skeletal preparation (Selby,
1987
) was performed as described. The stained skulls were
photographed for whole-mount analyses and then processed for analyses in
sections.
Primary osteoblast isolation, culture and differentiation
Primary osteoblast precursors were isolated from P1 mouse calvaria as
described (Mansukhani et al.,
2000). Isolated osteoblasts were cultured in
MEM media
containing 10% fetal calf serum. Only the first passage cells were used.
Primary osteoblasts (2x104) were seeded in 12 well plates for
48 hours. To induce maturation of the osteoblasts, cells were maintained in
differentiation media containing 50 µg/ml ascorbic acid and 4 mM
ß-glycerophosphate.
Alkaline phosphatase and mineralized bone matrix formation assays
For histochemical staining in situ, cells were fixed in 10% formalin.
Alkaline phosphatase staining was performed according to manufacturer's
protocol (Pierce). For liquid assays, cell lysates were prepared by M-PER
(Pierce). Protein concentrations were determined (BioRad) and alkaline
phosphatase activities were analyzed with solutions containing 0.5 mg/ml of
p-nitrophenylphosphate (Sigma-Aldrich) in AMP buffer (0.5 M
2-methyl-1, 2-aminopropanol and 2 mM magnesium chloride, pH 10.3) at 37°C
for 20-30 minutes. The reaction was stopped by a solution containing 300 mM
sodium phosphate (pH 12.3). The differentiated culture was monitored by
mineralized nodule formation with the standard von Kossa staining method.
Immunoblot and immunohistochemistry
Tissue sections were subject to immunological staining with
avidin:biotinlylated enzyme complex as described
(Hsu et al., 2001). Protein
extracts were subject to immunoblotting as described
(Hsu et al., 1999
). Bound
primary antibodies were detected with horseradish peroxidase-conjugated
secondary antibodies, followed by ECL mediated visualization (Amersham) and
autoradiography. Mouse monoclonal antibodies
-ABC
(van Noort et al., 2002
),
-Actin (Lab Vision) and
-BrdU (Lab Vision); rabbit monoclonal
antibody
-Ki67 (Lab Vision); or rabbit polyclonal antibodies
-Cyclin D1 (Lab Vision),
-ß-catenin (Lab Vision) and
-FGFR1 (Santa Cruz) were used as primary antibodies.
Isolation of RNA and real time RT-PCR
RNA was isolated using TRIzol (Invitrogen). Total RNA concentration was
determined by ultraviolet spectroscopy at 260 and 280 nm. cDNA was synthesized
with RNase H-free reverse transcriptase (Invitrogen) using oligoT primers
(0.03 A260 units/reaction, Sigma) in 20 µl for 1 hour at
42°C. The cDNA was then amplified by PCR (45 cycles, 94°C for 15
seconds, 55°C for 20 seconds and 72°C for 20 seconds) with SYBR Green
Master Mix (Applied Biosystems) in 20 µl buffered solution containing 1
µl of the diluted (1:5) reverse transcription product in the presence of 20
pmoles each of the sense and antisense primers specific for the various target
sequences. The primers were: osteopontin
(5'-tcccggtgaaagtgactgattct-3' and
5'-catcatcgtcatcatcgtcgtcca-3'), osteo-calcin
(5'-cttgaagaccgcctacaaac-3' and
5'-gctgctgtgacatccatac-3'), fgf4
(5'-tactgcaacgtgggcatcggatt-3' and
5'-aggcttcgtaggcgttgtagttgt-3'), or fgf18
(5'-tggtactagcaaggagtgcgtgtt-3' and
5'-tcgcagtttcctcgttcaagtcct-3'). In the same PCR reaction,
expression of ß-actin (5'-agatgtggatcagcaagcag-3' and
5'-gcgcaagttaggttttgtca-3') was analyzed as an internal
control.
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Results |
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|
|
|
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We next examined if the CNC-dependent osteogenesis is particularly defective in the Axin2/ mice (Fig. 4C). The activity of alkaline phosphatase was determined in liquid assays during the course of osteoblast differentiation (days 0, 3, 6, 9 and 12) in vitro. In three independent experiments, our results demonstrated that the stimulation of osteoblast differentiation caused by the Axin2 mutation occurs predominantly in the anterior calvarium (Fig. 4D). Cells isolated from the Axin2/ nasal/frontal bones showed an average 13-fold increase in alkaline phosphatase activity on differentiation days 6, 9 and 12, compared with controls. However, Axin2+/+ and Axin2/ parietal osteoblasts, which originate from the mesoderm, showed similar alkaline phosphatase activities. These data suggest that osteoblasts derived from different calvarial regions possess distinct cellular and developmental properties.
To further examine the effects of the Axin2 mutation on developmental
stages of the differentiated cell, the temporal expression patterns of two
late-stage osteoblast specific markers, osteopontin and osteocalcin, were
determined quantitatively by real time RT-PCR analyses
(Fig. 5A,B). In
Axin2/ cells, the expression of osteopontin
was prematurely elevated between 6 and 7.5 fold during the course of
differentiation (day 3 to day 9). The level of osteocalcin was unaffected by
the mutation in the early stages of the differentiation, but on
differentiation day 9, it was abruptly activated in the
Axin2/ cells (3-fold induction). The
results, which agree with the data on alkaline phosphatase activity, suggest
that inactivation of Axin2 promotes maturation of the CNC-derived osteoblasts.
Furthermore, the developmental properties of mesoderm-derived osteoblasts are
not affected significantly by the Axin2 ablation
(Fig. 4D; data not shown).
|
Expression of Axin2 in cranial sutures
To investigate the role of Axin2 in cranial skull development, we then
examined the expression of Axin2 using the Axin2lacZ
allele (Fig. 6). ß-Gal
staining revealed that Axin2 is expressed in the cranial suture and bone
during calvarial morphogenesis. At late embryonic and early postnatal stages,
Axin2 was expressed strongly in nasal cartilages, nasal bones and cranial
sutures (Fig. 6A,B,E,F). In the
suture regions, Axin2 expression was mostly restricted to the osteogenic
fronts and periosteum, which are enriched with pre-osteoblasts and osteoblasts
by E16.5 (Fig. 6I) and P4
(Fig. 6J). The expression of
Axin2 was maintained at elevated levels in the nasal bone at these stages. On
day 14, Axin2 was highly expressed in the developing nasal and frontal bones,
as well as the osteogenic fronts (Fig.
6C,G,K,L). By day 31, very little Axin2 expression could be
detected after the metopic suture had initiated the fusion process
(Fig. 6D,H). This spatial and
temporal expression pattern of Axin2 correlates with the onset of premature
suture closure in the Axin2/ mice
(Fig. 2), suggesting an
inhibitory role of Axin2 in osteoblast development. These results demonstrate
an important role of Axin2 in skull morphogenesis.
|
|
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|
Finally, we investigated if stimulation of ß-catenin signaling is
sufficient to promote osteoblast differentiation. A pharmacological compound
BIO (Meijer et al., 2003) was
added to the calvarial osteoblast cultures in vitro. BIO is a potent GSK-3
specific inhibitor, which has been shown to induce axis formation in frogs
(Meijer et al., 2003
) and
maintain pluripotency in mouse and human embryonic stem cells
(Sato et al., 2004
) through
activation of Wnt signaling. The control culture without BIO showed a gradual
increase in alkaline phosphatase activity during the course of differentiation
(Fig. 9E). However, the
addition of BIO greatly accelerated osteoblast differentiation, as alkaline
phosphatase activity was elevated at
three to fourfold on days 3, 6 and 9
(Fig. 9E). Therefore,
stimulation of ß-catenin signaling is sufficient to promote osteoblast
differentiation. Our studies strongly support a significant role of the
Wnt-Axin regulatory network in cranial skeletogenesis and
craniosynostosis.
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Discussion |
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The expression of Axin2 in the pre-osteoblasts and osteoblasts is consistent with its role in intramembranous bone development. Targeted disruption of Axin2 enhances expansion of osteoprogenitors and facilitates osteoblast differentiation in vivo and in vitro, suggesting a mechanism of craniosynostosis. Based on the expression and the loss-of-function analyses in vivo and in vitro, we demonstrate that Axin2 is required for proper development of the calvarial osteoblast. Furthermore, stimulation of ß-catenin signaling is not only necessary but also sufficient to promote osteoblast maturation. As a negative regulator in the osteoblasts, Axin2 prevents their development and premature suture closure, thereby controlling the timing of these developmental processes. This raises the possibility that therapeutic Axin2 could be exploited to correct defects in postnatal skull development.
The enhanced ß-catenin signaling induced by the Axin2 mutation during
cranial suture and bone development suggests a novel role of the canonical Wnt
pathway in skull morphogenesis. Axin2 has been suggested to function as a
negative-feedback regulator for Wnt (Jho
et al., 2002; Lustig et al.,
2002
). This raises the issue of whether Axin2 is regulating the
response to a Wnt signal, or the stability of ß-catenin in the absence of
Wnts. Based on the analyses in TOPGAL mice, ß-catenin signaling is active
during calvarial morphogenesis, even though there is a lack of information on
the expression of Wnts. Axin2 is constantly present between E16.5 and P4 in
both metopic and coronal sutures. However, TOPGAL transgene was transiently
active in the coronal suture, and never showed activity in the metopic suture.
These differences might imply suture specificity in skull development.
Therefore, it is possible that Axin2 is modulating the response to a Wnt
signal in the coronal suture, whereas this mechanism seems unlikely to occur
in the metopic suture.
The neural crest dependent skeletogenesis is particularly sensitive to the loss of Axin2. Inactivation of Axin2 apparently stimulates development of the CNC-derived osteoblasts. By contrast, mesoderm-derived osteoblasts of the parietal bone are not significant affected by the Axin2 mutation. This region-specific effect on calvarial osteoblast development suggests that Axin2 is crucial for the CNC dependent skeletogenesis. Even though Axin2 is highly activated in migratory neural crest cells in the cranial and trunk regions (W.H., unpublished), it seems to be dispensable for most of the CNC development. This could be due to a redundant function of Axin1 and Axin2. When the Axin2/ mice were crossed into the AxinTg1 (a Axin1-null) background, a genetic interaction between these two genes in early craniofacial morphogenesis was revealed (B.J., W.H., F.C. and W.B., unpublished). Axin2/ embryos in the Axin1+/ background exhibited severe abnormalities in craniofacial regions. Together with the present study, our results reveal the importance of Axin1 and Axin2 in CNC development. Craniofacial development, especially the neural crest derived tissues and structures, is particularly sensitive to the loss of the Axin family genes.
The canonical Wnt pathway is intimately involved in the CNC and
craniofacial development during early embryogenesis, as demonstrated by
mutations affecting ß-catenin signaling
(Brault et al., 2001;
Hasegawa et al., 2002
;
Ikeya et al., 1997
;
Mitchell et al., 2001
). Using
a genetic labeling system, CNC was further shown to derive from the
Wnt1-expressing neural progenitor (Chai et
al., 2000
; Jiang et al.,
2000
). However, the role of Wnt/ß-catenin signaling in
craniofacial bone development remained elusive. Our present study shows that
stimulation of ß-catenin signaling occurs not only in the
Axin2/ suture displaying premature fusion,
but also in the Axin2/ cells undergoing
intramembranous ossification. These data suggest that the inhibition of suture
closure and osteoblast development by the Axin family genes is mediated
through the regulation of ß-catenin signaling, implying a novel role for
this signaling pathway in cranial skeletogenesis. Indeed, the ß-catenin
and LEF/TCF mediated transcription is highly elevated during normal skull
formation. Interestingly, activation of this signaling pathway seems to occur
in a temporally and spatially restricted pattern that is the reverse of the
Axin2 expression pattern. In the anterior cranium, Axin2 is expressed in the
area immediately adjacent to where ß-catenin signaling is stimulated
during late embryogenesis. As the expression of Axin2 is enhanced at early
postnatal stages, ß-catenin signaling becomes inactivated. Furthermore,
activation of ß-catenin signaling is necessary and sufficient to induce
intramembranous ossification. Although it remains to be determined whether
stimulation of ß-catenin signaling leads to craniosynostosis in mice,
these results strongly support the hypothesis that the presence of Axin2
antagonizes ß-catenin signaling to inhibit intramembranous ossification
and prevent suture closure.
In addition to the origin of calvarial osteoblasts, the region-specific
effect may be attributed to differences in fundamental properties between
anterior and posterior parts of the cranium. The metopic suture fuses in the
first 45 days of life, whereas the sagittal suture remains patent. It has been
suggested that differential activation of FGF2, which inhibits the bone
morphogenetic protein (BMP) antagonist Noggin, might be responsible for normal
closure of the metopic suture (Warren et
al., 2003). BMP belongs to the transforming growth factor ß
(TGFß) superfamily, which plays an important role in bone morphogenesis
(McCarthy et al., 2000
;
Serra and Chang, 2003
).
Targeted disruption of Axin2 apparently interferes with cellular signaling of
the TGFß superfamily (H.-M.Y. and W.H., unpublished). This raises the
possibility that Axin2 interacts with the TGF-ß/BMP pathways. It has been
suggested that Axin1/Axin2 binds directly to Smad2/3 to stimulates TGFß
signaling (Furuhashi et al.,
2001
). Wnt signaling also has been shown to coordinately regulate
expression of the BMP target gene Msx2
(Hussein et al., 2003
). As
activation of Msx2 has been associated with craniosynostosis, inactivation of
Axin2 might induce this synergistic effect of Wnt and BMP. Finally, as direct
targets of Wnt, FGF4 (Kratochwil et al.,
2002
) and FGF18 (Shimokawa et
al., 2003
) are stimulated by the Axin2 inactivation. The former is
required for the downstream events mediated by Wnt in odontogenesis
(Kratochwil et al., 2002
),
whereas the latter is important for osteogenesis and chondrogenesis
(Liu et al., 2002
;
Ohbayashi et al., 2002
). It
remains to be elucidated whether FGF singling mediates the effect of Axin2
during calvarial morphogenesis. Future studies focused on delineating the
interplay of these cellular signaling pathways promise new insights into the
calvarial morphogenetic regulatory mechanism, and the molecular basis of
craniosynostosis.
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ACKNOWLEDGMENTS |
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