Although human genetic analyses have linked craniosynostosis to mutations in the FGFR
family (Burke et al., 1998
(Jabs et al., 1993
) and TWIST
genes (el Ghouzzi et al., 1997
; Howard et al., 1997
), they only account for ~20% of clinical cases (Wilkie and Morriss-Kay, 2001
). The molecular mechanisms underlying suture development remain largely unknown. Here, we show that disruption of Axin2
in mice induces premature suture closure, a phenotype resembling craniosynostosis in humans. Craniosynostosis is the major developmental disorder of the skull vault and is often associated with difficulties in vision, hearing and breathing (Wilkie and Morriss-Kay, 2001
). In the adult Axin2 mutants, we have frequently noticed eye abnormalities that might be caused by the skull deformity with increased intracranial pressure. The spatial and temporal expression pattern of Axin2 in skull development correlates with craniofacial abnormalities caused by its ablation. Axin2 is expressed in osteogenic fronts and periosteum of the developing suture during skull formation. However, its expression gradually diminishes before the metopic suture has initiated its normal fusion processes. This might permit the suture fusion to occur slowly at early postnatal stages. By contrast, the lack of Axin2 greatly accelerates the fusion processes. A recent study, which linked mutations in AXIN2
to familial tooth agenesis/oligodontia in humans, also suggests that modulating the intensity of cellular signaling is necessary during specific stages of organ development (Lammi et al., 2004
). Aulehla et al. (Aulehla et al., 2003
) have proposed that the role of Axin2 as a negative feedback-regulator of Wnt/β -catenin signaling is crucial for segmentation during vertebrate development. However, our results show that the ablation of Axin2 has no discernible event on segmentation.
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 β -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 β -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 β -catenin in the absence of Wnts. Based on the analyses in TOPGAL mice, β -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 β -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/β -catenin signaling in craniofacial bone development remained elusive. Our present study shows that stimulation of β -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 β -catenin signaling, implying a novel role for this signaling pathway in cranial skeletogenesis. Indeed, the β -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 β -catenin signaling is stimulated during late embryogenesis. As the expression of Axin2 is enhanced at early postnatal stages, β -catenin signaling becomes inactivated. Furthermore, activation of β -catenin signaling is necessary and sufficient to induce intramembranous ossification. Although it remains to be determined whether stimulation of β -catenin signaling leads to craniosynostosis in mice, these results strongly support the hypothesis that the presence of Axin2 antagonizes β -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 β (TGFβ) 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 TGFβ superfamily (H.-M.Y. and W.H., unpublished). This raises the possibility that Axin2 interacts with the TGF-β /BMP pathways. It has been suggested that Axin1/Axin2 binds directly to Smad2/3 to stimulates TGFβ 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.