Prevention of Premature Fusion of Calvarial Suture in GLI-Kruppel Family Member 3 (Gli3)-deficient Mice by Removing One Allele of Runt-related Transcription Factor 2 (Runx2)*

Background: Gli3-deficient mice (Gli3Xt-J/Xt-J) show premature suture closure (craniosynostosis). Results: Gli3Xt-J/Xt-J mice have aberrant cell proliferation and osteogenic differentiation in the sutures. Reducing the dosage of Runx2 (Gli3Xt-J/Xt-J;Runx2+/− mice) rescues the abnormality through canonical Bmp-Smad signaling. Conclusion: Gli3 represses bone formation Bmp-Smad signaling, which integrates Dlx5/Runx2-II cascade. Significance: Targeting Runx2 might provide an attractive way of preventing craniosynostosis in patients. Mutations in the gene encoding the zinc finger transcription factor GLI3 (GLI-Kruppel family member 3) have been identified in patients with Grieg cephalopolysyndactyly syndrome in which premature fusion of calvarial suture (craniosynostosis) is an infrequent but important feature. Here, we show that Gli3 acts as a repressor in the developing murine calvaria and that Dlx5, Runx2 type II isoform (Runx2-II), and Bmp2 are expressed ectopically in the calvarial mesenchyme, which results in aberrant osteoblastic differentiation in Gli3-deficient mouse (Gli3Xt-J/Xt-J) and resulted in craniosynostosis. At the same time, enhanced activation of phospho-Smad1/5/8 (pSmad1/5/8), which is a downstream mediator of canonical Bmp signaling, was observed in Gli3Xt-J/Xt-J embryonic calvaria. Therefore, we generated Gli3;Runx2 compound mutant mice to study the effects of decreasing Runx2 dosage in a Gli3Xt-J/Xt-J background. Gli3Xt-J/Xt-J Runx2+/− mice have neither craniosynostosis nor additional ossification centers in interfrontal suture and displayed a normalization of Dlx5, Runx2-II, and pSmad1/5/8 expression as well as sutural mesenchymal cell proliferation. These findings suggest a novel role for Gli3 in regulating calvarial suture development by controlling canonical Bmp-Smad signaling, which integrates a Dlx5/Runx2-II cascade. We propose that targeting Runx2 might provide an attractive way of preventing craniosynostosis in patients.

Mutations in the gene encoding the zinc finger transcription factor GLI3 (GLI-Kruppel family member 3) have been identified in patients with Grieg cephalopolysyndactyly syndrome in which premature fusion of calvarial suture (craniosynostosis) is an infrequent but important feature. Here, we show that Gli3 acts as a repressor in the developing murine calvaria and that Dlx5, Runx2 type II isoform (Runx2-II), and Bmp2 are expressed ectopically in the calvarial mesenchyme, which results in aberrant osteoblastic differentiation in Gli3-deficient mouse (Gli3 Xt-J/Xt-J ) and resulted in craniosynostosis. At the same time, enhanced activation of phospho-Smad1/5/8 (pSmad1/5/8), which is a downstream mediator of canonical Bmp signaling, was observed in Gli3 Xt-J/Xt-J embryonic calvaria. Therefore, we generated Gli3;Runx2 compound mutant mice to study the effects of decreasing Runx2 dosage in a Gli3 Xt-J/Xt-J background. Gli3 Xt-J/Xt-J Runx2 ؉/؊ mice have neither craniosynostosis nor additional ossification centers in interfrontal suture and displayed a normalization of Dlx5, Runx2-II, and pSmad1/5/8 expression as well as sutural mesenchymal cell proliferation. These findings suggest a novel role for Gli3 in regulating calvarial suture development by controlling canonical Bmp-Smad signaling, which integrates a Dlx5/Runx2-II cascade. We propose that targeting Runx2 might provide an attractive way of preventing craniosynostosis in patients.
A suture is a fibrous connection between adjacent craniofacial bones, which functions as an articulation, a site of bone deposition, and a shock absorber. Craniosynostosis is the premature fusion of one or more cranial or facial sutures, and ϳ30% of those cases are induced by known gene mutations in For sutures to operate as growth sites, they must remain patent. Bone must be formed at the correct location, in the bones, and at the osteogenic fronts, but osteogenesis also must be repressed in between the bone ends in the midsutural mesenchyme. The processes of osteoblastic cell differentiation and proliferation have to be fine-tuned to occur at the correct speed, at the correct location, and at the appropriate time. The size of the osteoprogenitor cell population is important for intramembranous bone growth, and Bmp proteins acting through the transcription factors Msx1 and Msx2 regulate the size of this population (4,5). The fine balance between osteoprogenitor cell proliferation and the rate of differentiation are regulated by Fgf signaling, specifically by the temporal-spatial expression of Fgf receptors (6,7). The Forkhead transcription factor Foxc1 sits at the nexus between Bmp and Fgf signaling during calvarial bone development. Foxc1 is induced by Fgf and regulates the effects of Bmp signaling. It does this independently of the Bmp post-transcriptional repressor, Noggin, which is itself critical for suture patency (8,9). Foxc1 Ϫ/Ϫ mice exhibit defective calvarial osteogenesis with the calvarial bones remaining rudimentary at the sites of initial osteogenic condensations (10).
We have reported previously that Gli3-deficient mice (Gli3 Xt-J/Xt-J ) develop craniosynostosis during embryogenesis because of the aberrant enhancement of Runx2 and reduced Twist1 expression in the mid-sutural mesenchymal cells (11). The localization of the phenotype in Gli3 Xt-J/Xt-J mice to the interfrontal and lambdoid sutures can be explained by the location specificity of Gli3 expression. In addition, we have shown that the premature ossification in the lambdoid suture can be rescued in vitro by applying FGF2-soaked beads, which normalized Twist1 expression. This rescue was based on the ability of Twist1 to repress Runx2 and thereby stop the excessive differentiation of osteoprogenitors in the suture (11).
Several reports have indicated a close connection between Gli family proteins and bone development. Shimoyama and coworkers (12) have demonstrated the physical interaction between Gli2 and Runx2 and that Indian Hedgehog (Ihh) promotes osteoblast differentiation through enhancement of Runx2 expression by Gli2 but not Gli3. In contrast, Ohba and co-workers have shown that reduction of the repressor form of Gli3 (Gli3 R ) in Ptch1 ϩ/Ϫ (Patched1) osteoblasts results in accelerated osteoblast differentiation, and demonstrated the indirect and competitive inhibitory effect of Gli3 against DNA binding by Runx2 in vitro (13). Finally, they concluded that Hh signaling through Ptch plays a critical role in postnatal bone homeostasis (13).
Runx2 is a key regulatory factor in the differentiation of osteoblasts and chondrocytes (14,15). Runx2 absolutely is required for the initial stage of osteoblast differentiation but must be down-regulated to permit osteoblast maturation (16). Additionally, Runx2 plays a critical role in linking cell fate, cell proliferation, and control of cell growth by regulating genes transcribed by RNA polymerase II and repressing RNA polymerase-mediated ribosomal RNA synthesis (17). Homozygous Runx2-null mice (Runx2 Ϫ/Ϫ ) show complete lack of endochondral and membranous bone (14,15), and their teeth are misshapen and severely hypoplastic (18). Heterozygotes Runx2 ϩ/Ϫ mice mimic human cleidocranial dysplasia exhibiting hypoplasia of clavicles, delayed ossification of cranial bones, wide anterior and posterior fontanels, and wide cranial sutures (19).
Runx2 has two alternative promoters, the distal P1 promoter and the proximal P2 promoter. The P1 promoter encodes the Runx2-II isoform, also known as Til-1 G1 and Osf2/Cbfa1, whereas the P2 promoter encodes the Runx2-I isoform (Pebp2␣〈). Runx2-I is expressed in immature osteoprogenitor cells, whereas Runx2-II is expressed in more mature osteoblasts (20). Mice that have a selective loss of Runx2-II unexpectedly form axial, appendicular, and craniofacial bones with the exception of cranial bones derived from endochondral ossification. The expression of Runx2-I is compensatory up-regulated in Runx2-II Ϫ/Ϫ mice, but they fail to complete osteogenesis (21). Furthermore, the Runx2-I isoform is strongly expressed in mesenchymal cells of sagittal suture and the osteogenic fronts of the parietal bones. In contrast, the Runx2-II isoform expression is limited to the osteogenic fronts and the parietal bones (20). This mutually exclusive expression of the Runx2 isoforms indicates that they have distinct functional significance in the development of the cranial suture. Interestingly, Dlx5 (Distalless homeobox 5) has been reported to regulate the expression of Runx2-II isoform but not Runx-I in BMP2-induced osteogenic differentiation (22).
In vertebrates, Hh signal transduction is initiated by the binding of Hh ligands to the cell surface receptor Ptch1 (23). In the absence of the Hh ligand, Ptch1 blocks a signal transducer Smoothened. Binding of Hh ligand to Ptch1 releases this repression and inhibits the truncation of Gli to produce the repressor form (Gli R ) and promotes the production of fulllength activator form (Gli A ). In vertebrates, Gli family consists of Gli1, Gli2, and Gli3. Gli3 and to a lesser extent Gli2 can be proteolytically processed into short Gli R in the absence of Hh ligands. Gli2 acts mainly as Gli A inducing Gli1 and Ptch expression, which is regarded as readout of Hh signaling as it is also up-regulated by Hh (23).
Our aim in this study was to test the hypothesis that Gli3 signaling is important in keeping Runx2 in check and that Runx2 dosage is important in maintaining the correct balance of osteogenesis in the developing suture. We generated Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ compound mutant mice to reduce the dosage of Runx2. We discovered that Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ mice have no craniosynostosis or ectopic ossification centers in either the lambdoid or interfrontal sutures.
From the analysis of different stages of osteoprogenitor differentiation in the lambdoid suture revealed that the mRNA expression of Dlx5, Runx2-II, and Bmp2 were increased in the mid-sutural mesenchymal cells of Gli3 Xt-J/Xt-J but decreased in Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ compound mutant mice. The expression of Noggin, which is induced by Bmp2 administration (8), was slightly up-regulated at the interfrontal suture of Gli3 Xt-J/Xt-J mice indicating the enhancement of Bmp2 expression there. The lambdoid and interfrontal sutures of Gli3 Xt-J/Xt-J mice showed high proliferative activity across the broad suture area, whereas low proliferative activity was observed in Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ mice predominantly at the osteogenic front and was similar to that of WT mice. Finally, phosphorylation of Smad1/ 5/8 in both calvarial tissue and mid-sutural mesenchymal cells in Gli3 Xt-J/Xt-J mice was increased, whereas in Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ mice, this was normalized. These data suggest that Gli3 has an important role in cranial suture development through the canonical Bmp-Smad pathway involving a Dlx5-Runx2-II cascade. The data also show that targeting Runx2 activity using siRNA might provide an attractive way of preventing craniosynostosis in patients.

EXPERIMENTAL PROCEDURES
Generation of Gli3;Runx2 Compound Mutant Mice-All animal experiments were approved by the University of Helsinki, Helsinki University Hospital and the Southern Finland Council animal welfare and ethics committees. Gli3 ϩ/Xt-J mice in C57BL/6 background and Runx2 ϩ/Ϫ mice were maintained and PCR genotyping was performed using tail or skin DNA as described previously (24,25). We generated Gli3;Runx2 compound mutant mice by mating Gli3 ϩ/Xt-J Runx2 ϩ/Ϫ mice with Gli3 ϩ/Xt-J Runx2 ϩ/Ϫ mice as described in Table 1. WT littermates were used as a control.
In Situ Hybridization-Preparation of 35 S-labeled uridine 5Ј-triphosphate and digoxigenin-UTP-labeled riboprobes, in situ hybridization, and image processing have been described previously (26). The Runx2-I probe used in in situ hybridization was prepared from a 639-bp fragment of murine Runx2 cDNA isolated from C3H10T1/2 cells by RT-PCR with the specific primers: 5Ј-CGGGATCCTCTCAGCTTTAGCGTCGTCA-3Ј for the forward primer and 5Ј-GCTCTAGACCGCAAGGG-ACTTGAAGTT-3Ј for the reverse primer. The PCR products were digested with BamHI/XbaI and subcloned into pBluescript II KS(-).
Skeletal Staining-For skeletal preparations, the skin of embryos was removed, and the embryos were fixed with cold 96% ethanol overnight. Skeletal elements were stained with 0.3% Alcian blue and 0.03% Alizarin Red-S solution, followed by clarification of the tissues by a glycerol/KOH series.
Cell Proliferation Analysis in Calvarial Sutures by Assessment of BrdU Incorporation-Pregnant female mice (E15.5) were injected intraperitoneally with BrdU solution (10 l/g body weight, Invitrogen). Two hours after injection, embryos were removed and fixed with 10% neutral buffered formalin. Tissues were processed through alcohol dehydration, embedded in paraffin, and sectioned at 7-m intervals. The BrdUincorporated cells were detected using biotinylated monoclonal anti-BrdU antibody and visualized with streptavidin-biotin staining system following the manufacturer's protocol (Invitrogen). Sections were counterstained with hematoxylin. Quantification of BrdU-positive cells in each suture was performed as described previously (11). Results are reported as mean Ϯ S.D. The significance of differences between means was assessed by analysis of variance followed by Mann-Whitney U test with Bonferroni (multiple comparisons). The p value of Ͻ 0.05 was considered as significant.

Hh Signaling in Osteogenic Front of Calvarial Sutures-We
analyzed the presence of Gli3 R in WT calvaria at E15.5 (Fig. 1A) by Western blot analysis using a Gli3 antibody that recognizes both Gli3-full and Gli3 R forms. As shown in Fig. 1A, the truncated Gli3 R form was detected dominantly in the intact calvarial tissue as well as limb and brain tissue, indicating that processing of the Gli3-full into Gli3 R is occurring in calvarial tissue (Fig.  1A). We have shown previously that Gli3 and Ptch1 are expressed in the developing calvaria (11). Ptch1 is a down-  (arrows in B). C and D, Runx2-II was expressed in the osteogenic front and in the mature bone areas of the frontal, parietal, and interparietal bones of WT calvaria. However, in Gli3 Xt-J/Xt-J calvaria, Runx2-II was expressed intensely across the lambdoid suture and in the middle of interfrontal suture area (arrows in D). E and F, Runx2-I was specifically expressed in the osteogenic front and sutural-mesenchymal cells especially around the frontal and parietal bones in WT mice. In Gli3 Xt-J/Xt-J tissue, Runx2-I was expressed in the sutural-mesenchymal cells similarly to WT tissue but with less intensity in the osteogenic fronts of the parietal bones (insets in A, C, and E). Samples hybridized with sense control probes. f, frontal bone; if, interfrontal suture; ip, interparietal bone; ls; lambdoid suture; p, parietal bone; ss, sagittal suture. Scale bar in A, 1 mm. stream target of Hh and is considered as readout of Hh signaling. Transcript of Ptch1 was observed clearly at the osteogenic fronts of the frontal, parietal, and interparietal bones, by whole mount in situ hybridization in WT E15.5 tissue, indicating active Hh signaling at this location (Fig. 1, B, b panel). In addition to our previous report, the expression of Gli2 and Gli3 mRNA were observed at the osteogenic fronts of the parietal, interparietal, and frontal bones (Fig. 1, C and D), and Ihh mRNA was observed clearly at the osteogenic front of these bones (Fig.  1, C and D).
Dlx5 and Runx2-II Isoform Are Aberrantly Expressed in Lambdoid Sutures of Gli3 Xt-J/Xt-J Mice-To further elucidate the mechanism of craniosynostosis in Gli3 Xt-J/Xt-J mice, we performed whole mount in situ hybridization using riboprobes recognizing Dlx5, Runx2-I, and Runx2-II. We analyzed tissue at E15.5, which is just prior to the onset of craniosynostosis in Gli3 Xt-J/Xt-J mice. In WT calvaria, the transcription factor Dlx5 lies downstream of Bmp2 and is important in the regulation of Runx2 by Bmp2 (27), especially the Runx2-II isoform (22). Also, Dlx5 is important in post-proliferative osteoblasts for the initiation of Oc (Osteocalcin) gene transcription (28). Dlx5 transcripts were localized in the osteo-genic fronts of WT frontal, parietal, and interparietal bones, but not in the mid-sutural mesenchymal area at E15.5 ( Fig.  2A). Runx2-II was expressed in osteogenic fronts and in a more differentiated bone site (Fig. 2C). However, Runx2-I was expressed specifically in the osteogenic fronts and sutural mesenchymal cells some distance from osteogenic fronts (Fig. 2E). Interestingly, Dlx5 and Runx2-II were ectopically expressed in the central mesenchyme of Gli3 Xt-J/Xt-J lambdoid and interfrontal sutures (Fig. 2, B and D, arrows), whereas Runx2-I expressed in the osteogenic front and sutural mesenchymal cells of Gli3 Xt-J/Xt-J calvaria (Fig. 2F).

Increased Proliferation in Interfrontal and Lambdoid Sutures of Gli3 Xt-J/Xt-J Embryos Was Normalized in Gli3
Xt-J/Xt-J Runx2 ϩ/Ϫ Embryos-To test whether reduced Runx2 dosage would rescue the increased proliferation observed in Gli3 Xt-J/Xt-J mice, we pulsed Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ embryos with BrdU. We analyzed tissue at E15.5, which is just prior to the craniosynostosis. In WT embryos, BrdU-labeled cells were predominantly detected at the osteogenic front and periosteal surface of the frontal, parietal, and interparietal bones (Fig. 4A). In contrast, in Gli3 Xt-J/Xt-J embryos, a significant number of BrdU-incorporated cells were observed in mid-sutural mesenchymal cell layer of interfrontal and lambdoid sutures (Fig. 4, A  and B). This aberrant cell proliferation was specific to the interfrontal and lambdoid sutures but not to coronal suture of Gli3 Xt-J/Xt-J embryos (Fig. 4, A and B). In Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ embryos, a significant decrease in the number of BrdU-incorporated cells in all sutures assayed was detected compared with Gli3 Xt-J/Xt-J embryos (Fig. 4, A and B).  , and i panels) and not in the mid-sutural mesenchymal cells. B, graphs represent the number of BrdU-positive nuclei as a percentage of the total nuclei counted as described previously (11). In Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ interfrontal and lambdoid sutures, significant smaller numbers of BrdU-positive cells were detected than in Gli3 Xt-J/Xt-J sutures. Results are reported as mean Ϯ S.D. with p Ͻ 0.05 considered statistically significant (*, p Ͻ 0.05). f, frontal bone; of, osteogenic front; p, parietal bone; IHC, immunohistochemistry. Scale bars in A, a, d, and g panels, 100 m.

Ectopic and Up-regulated Expression of Osteoblast Differentiation-related Genes Was Normalized in Gli3
Xt-J/Xt-J Runx2 ϩ/Ϫ Mice-To clarify the mechanism in establishing patent lambdoid sutures and a lack of heterotopic bones in interfrontal suture in Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ embryos, we performed in situ hybridization using Dlx5, Runx2 I/II, and Osteocalcin riboprobes (Fig. 5). In addition to the ectopic expression of Dlx5 and Runx-II mRNA in Gli3 Xt-J/Xt-J in the central of mesenchyme (Fig. 5, A, b and h panels, and B, b and h panels), Oc (Osteocalcin) mRNA was also expressed there (Fig. 5, A, k panel,  and B, k panel). Dlx5 has been reported to be down-regulated in Runx2 ϩ/Ϫ mice (29), so the normalization of Dlx5 in mid-sutural mesenchymal cells in Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ mice might contribute to reduced expression of Runx2 and its downstream targets. We analyzed the expression of Bmp2 and its down-stream target Msx2 and the antagonist Noggin in Gli3 Xt-J/Xt-J lambdoid and interfrontal sutures (Fig. 6). Bmp2 transcripts were expressed ectopically in the sutural mesenchyme of Gli3 Xt-J/Xt-J mice (Fig. 6, A, b panel, and B, b panel, arrows), and this was normalized in Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ mice (Fig. 6, A, c  panel, and B, c panel). The expression level of Msx2 in sutural mesenchymal cells on WT, Gli3 Xt-J/Xt-J , and Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ mice were similar (Fig. 6, A, d-f panels, and B, d-f  panels). However, the expression of Noggin, which is induced by Bmp2 administration (8), was slightly up-regulated at the interfrontal suture of Gli3 Xt-J/Xt-J mice, indicating the enhancement of Bmp2 expression (Fig. 6-B-h).
Lack of Gli3 R Leads to Activation of Bmp Signaling through Canonical Smad Pathway in Mid-sutural Mesenchyme Resulting in Bone Formation-As we found Dlx5 to be up-regulated in Gli3 Xt-J/Xt-J sutures and as it has been reported previously that Bmp will induce Dlx5 in avian calvaria (30), we analyzed the distribution of pSmad1/5/8, which is a regulator of Bmp signaling, by Western blot analysis using calvarial tissue from WT (n ϭ 4) and Gli3 Xt-J/Xt-J mice (n ϭ 5) (Fig. 7, A and B). Higher phosphorylation of Smad 1/5/8 was detected in calvaria from Gli3 Xt-J/Xt-J mice than WT with statistically difference (Fig. 7C). Additionally, we analyzed the phosphorylation of Smad1/5/8 by immunohistochemistry (Fig. 7D). Intensive immune-reactive cells were observed in the osteogenic front and in the periosteum of the frontal, parietal, and interparietal bones but not in the mid-sutural mesenchyme in WT embryos (E15.5) (Fig. 7,  D, a, d, and g panels). However, in Gli3 Xt-J/Xt-J mice, considerable numbers of pSmad1/5/8-positive cells were spread across the sutural mesenchyme of the interfrontal and lambdoid sutures (Fig. 7, D, b and h panels), but not coronal suture (Fig. 7,   D, e panel). These abnormalities were corrected in Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ specimens (Fig. 7D, c and i panels). As pSmad1/5/8 distribution correlated with expression of Bmp2, Bmp4 (data not shown), the Bmp antagonist Noggin and the Bmp target Msx2 in the sutures of WT, Gli3 Xt-J/Xt-J , and Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ calvaria; these results indicate that Gli3 R regulates suture development by keeping Bmp-induced canonical Smad cascade under control ( Fig. 7 and data not shown).

DISCUSSION
In this study, we generated Gli3;Runx2 compound mutant mice to correct aberrant osteoblastic cell proliferation and dif- ferentiation in Gli3 Xt-J/Xt-J suture mesenchymal cells and to rescue the craniosynostosis in these mice (11). Craniosynostosis is an infrequent but important feature of Grieg cephalopolysyndactyly syndrome caused by mutations in GLI3 (2,3). Most forms of craniosynostosis, including those in Grieg cephalopolysyndactyly syndrome, occur prior to birth and are not detected until birth. Targeting this type of early craniosynostosis is therefore difficult. Treatment often involves the postnatal resection of the prematurely fused sutures and reshaping the calvaria to allow for proper skull growth. However, instead of staying open and allowing normal growth, the affected sutures often re-fuse, committing the child to repeat operations. Targeting RUNX2 may slow down the re-fusion process and reduce the number of operations required. As RUNX2 is a master regulator of osteogenesis, this type of therapy might by applicable to many types of craniosynostosis not just those caused by mutations in GLI3.
We have reported that FGF2-soaked beads applied to the lambdoid suture of Gli3 Xt-J/Xt-J embryos will prevent synostosis, and this is by restoring Twist1 expression which, in turn, inhibits Runx2 (11,26). In this study, we found that Gli3 Xt-J/Xt-J mice showed higher expression of Dlx5 in mid-sutural mesenchymal cells in the interfrontal and lambdoid sutures followed by ectopic expression of Runx2-II but not Runx2-I. Although there is no report about direct association between Gli3 and Dlx5, indirect interaction between Shh signaling and Dlx5 through Fgf7 signaling has been shown during palate development and during vestibular morphogenesis through Wnt signaling (31,32). Dlx3, Dlx5, and Runx2 are expressed by post-proliferative osteoblasts and activate the Oc promoter with the role of Dlx5 being more dominate than Dlx3 in mature osteoblasts at the mineralization stage of differentiation (28). Hassan and co-workers (33) also show that administration of BMP2 to osteogenic fibroblasts induces Dlx3 and Dlx5 expression resulting in the activation of Runx2. Others have reported that Dlx5 specifically transactivates the Runx2-II driven by "bone-related" P1 promoter (22). We suggest that Hedgehog signaling has an effect on bone development through Dlx gene function. Interestingly, the aberrant expression of Dlx5 and Runx2-II in Gli3 Xt-J/Xt-J mice calvaria overlapped with the expression of Gli3 (11). These results suggest that Gli3 participates in controlling Dlx5 transcription and that this mechanism contributes to the craniosynostosis in Gli3 Xt-J/Xt-J .
Many signaling cascades have been reported to have important roles in calvarial suture development, including Fgf, Bmp, Wnt, Ephrin-Eph, and Hh signaling (4,10). We focused on Bmp signaling in Gli3 Xt-J/Xt-J calvarial sutures and found ectopic expression of Bmp2 in mid-sutural mesenchymal cells. To evaluate functional Bmp signaling, we performed Western blot analysis and immunohistochemistry using pSmad1/5/8. We showed higher phosphorylation of Smad1/5/8 in Gli3 Xt-J/Xt-J calvarial tissue. Furthermore, in WT calvaria, pSmad1/5/8 was expressed at the osteogenic front and periosteum, but not at mid-sutural mesenchymal cells, meaning that Bmp signaling is occurring at the place of new bone apposition. The processing of Gli3 and expression of Bmp4 protein has been reported to mediate both cell survival and programmed cell death in the developing limb bud in a position-dependent manner (34).
Thus, the absence of Gli3, especially the lack of the repressor form of Gli3 up-regulates Bmp2 and subsequently pSmad1/5/8 in Gli3 Xt-J/Xt-J calvarial sutures. Taken together, up-regulation of Bmp signaling contributes to the intramembranous ossification abnormalities in Gli3 Xt-J/Xt-J embryos.
It is intriguing that only the interfrontal and lambdoidal sutures exhibit craniosynostosis/heterotopic bones. In patients with craniosynostosis only, some sutures are affected, and only very rarely are all sutures involved. In Gli3 Xt-J/Xt-J mice, location specificity of the phenotype is the result of the temporal-spatial expression pattern of Gli3 (11). Here, we show a mechanism which underlies these abnormalities. This involves aberrant osteoprogenitor proliferation in the interfrontal and lambdoidal sutures but not in the coronal suture, which is not affected. We have shown more severe hypoplasia of calvarial bones in Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ compared with Runx2 ϩ/Ϫ mice. Additionally, Gli3 Xt-J/Xt-J Runx2 ϩ/Ϫ mice displayed smaller clavicles than either Gli3 Xt-J/Xt-J or Runx2 ϩ/Ϫ . It has been reported that Runx2 controls the cell fate, proliferation, and growth through regulating ribosomal biogenesis (17,35). In Gli3 Xt-J/Xt-J mice, cells fated into mature osteoblasts are abundant in the interfrontal and lambdoidal sutures. Proliferation of these ectopic differentiated osteoblasts might be reduced specifically because of loss of Runx2. Additionally, genetic interaction between Gli3 and Runx2 in morphogenesis of membranous bone might contribute to this matter.
In conclusion, Gli3 R plays a key role in repressing bone formation in the suture, thus maintaining suture patency and normal calvarial development. Gli3 inhibits osteogenesis by three different mechanisms. Twist1, which can be induced by FGF2, lies downstream of Gli3 and directly inhibits Runx2 (11,26,36). Gli3 also represses Runx2 via a Bmp2/Dlx5 mechanism, specifically targeting Runx2-II (22,30). Gli3 represses Oc directly thereby inhibiting the action of Runx2 (13). Within the sutural system, the importance of the repression of Runx2 and Oc is that osteogenesis occurs at the correct time and location to ensure proper growth and morphogenesis.