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. 2010 Jun 22;19(17):3457–3467. doi: 10.1093/hmg/ddq258

Gli3Xt−J/Xt−J mice exhibit lambdoid suture craniosynostosis which results from altered osteoprogenitor proliferation and differentiation

David PC Rice 1,2,3,4,*,†, Elaine C Connor 2,3,†, Jacqueline M Veltmaat 5,6, Eva Lana-Elola 2,3, Lotta Veistinen 1, Yukiho Tanimoto 1, Saverio Bellusci 5,7, Ritva Rice 2,3
PMCID: PMC2916710  PMID: 20570969

Abstract

Gli3 is a zinc-finger transcription factor whose activity is dependent on the level of hedgehog (Hh) ligand. Hh signaling has key roles during endochondral ossification; however, its role in intramembranous ossification is still unclear. In this study, we show that Gli3 performs a dual role in regulating both osteoprogenitor proliferation and osteoblast differentiation during intramembranous ossification. We discovered that Gli3Xt−J/Xt−J mice, which represent a Gli3-null allele, exhibit craniosynostosis of the lambdoid sutures and that this is accompanied by increased osteoprogenitor proliferation and differentiation. These cellular changes are preceded by ectopic expression of the Hh receptor Patched1 and reduced expression of the transcription factor Twist1 in the sutural mesenchyme. Twist1 is known to delay osteogenesis by binding to and inhibiting the transcription factor Runx2. We found that Runx2 expression in the lambdoid suture was altered in a pattern complimentary to that of Twist1. We therefore propose that loss of Gli3 results in a Twist1-, Runx2-dependent expansion of the sutural osteoprogenitor population as well as enhanced osteoblastic differentiation which results in a bony bridge forming between the parietal and interparietal bones. We show that FGF2 will induce Twist1, normalize osteoprogenitor proliferation and differentiation and rescue the lambdoid suture synostosis in Gli3Xt−J/Xt−J mice. Taken together, we define a novel role for Gli3 in osteoblast development; we describe the first mouse model of lambdoid suture craniosynostosis and show how craniosynostosis can be rescued in this model.

INTRODUCTION

Craniosynostosis, the premature fusion of cranial sutures, is a disfiguring condition occurring in approximately one in 2500 live births (1). Sutures are the main sites of osteogenesis in the calvaria and have to remain patent to function as growth sites. If a suture fuses, bone growth ceases at that location and this restriction is compensated for by excessive growth at other, still patent, sutures. However, this unregulated development leads to deformity in the shape of the skull and to neurological problems (2).

Craniosynostosis can occur in single or multiple sutures and may not be restricted to the calvaria but affect the facial skeleton (3). Isolated synostosis of the lambdoid suture is of unknown etiology. The effects of lambdoid craniosynostosis on skull development can be profound with unilateral synostosis causing posterior plagiocephaly with flattening of the affected occipitoparietal area and compensatory bossing of the contralateral parietal and sometimes frontal areas. There is ipsilateral occipitomastoid bulging and ipsilateral inferior displacement of the ear (2). Bilateral lambdoid synostosis is often combined with sagittal suture fusion, and this results in a characteristic head shape with frontal bossing, turribrachycephaly, biparietal narrowing, occipital concavity and inferior displacement of the ears (4).

Approximately 30% of craniosynostosis cases are caused by known gene mutations (5). These include mutations in Ephrin-B1, Fibroblast Growth Factor Receptors 1, 2 and 3 and the transcription factors MSX2 and TWIST1. Hedgehog (Hh) growth factor signaling has key roles in the development of multiple organs, in adult homeostasis and in the control of stem cell proliferation, and its abnormal activation causes multiple different tumors (6). In vertebrates, Hh signaling is mediated by three Gli transcription factors and regulated by several feedback loops. Gli transcription and protein activity are controlled at several different levels by Hh signaling. When levels of Hh ligand are low Gli2 and Gli3 are processed into truncated N-terminal forms which repress Hh target genes (7–9). Full-length Gli2 and Gli3 can act as activators of downstream targets, though Gli3 only weakly (8,10). High concentrations of Hh ligand downregulate Gli3 at the transcriptional level and also suppress the processing of Gli2 and Gli3 into truncated repressor forms (11).

The ligand, Indian hedgehog (Ihh), and its receptor components Patched1 (Ptch1) and Smoothened are expressed by osteoblasts (12,13) and targeted disruption of Ihh results in impaired osteoblastogenesis (14–16). Gli2 and Gli3 are important mediators of Hh signaling during bone development and bone homeostasis. The genetic loss of either Gli2 or Gli3 results in bone abnormalities (17,18). Ihh acts through Gli2 to regulate the osteoblast specific transcription factor Runx2 (19). During endochondral ossification, Gli3 repressor acts downstream of Ihh in regulating two distinct steps of chondrocytes differentiation (20). The repressor form of Gli3 has been shown to attenuate Runx2 transcriptional activity by competitively inhibiting its DNA binding and this has been suggested as a mechanism to account for the increased bone mass seen in Ptch1 heterozygote mice (21). The basic helix–loop–helix transcription factor Twist1 directly inhibits Runx2 to control osteoblast differentiation (22–24). Consistent with these functions in mice, haploinsufficiency of RUNX2 causes cleidocranial dysplasia characterized by delayed calvarial bone formation leading to widened sutures in humans (25). Also, haploinsufficiency of TWIST1 results in the Saethre–Chotzen syndrome which has craniosynostosis as a central feature (26,27).

Mutations that result in a loss of GLI3 function cause Greig cephalopolysyndactyly syndrome (GCPS) (MIM 175700) (28,29). GCPS is characterized by craniofacial abnormalities including macrocephaly and scaphocephaly (long and narrow cranial vault) (30–32). Craniosynostosis has been reported in patients with GCPS but it is not a common feature (30). The limbs exhibit soft tissue syndactyly, postaxial polydactyly in the hands and preaxial polydactyly in the feet (30,32). Extra-toes mutant mice (Gli3Xt−J/Xt−J) have an intragenic deletion of the Gli3 gene that results in a null allele and have been put forward as a model for GCPS (17). They exhibit polydactyly as well as defects in the craniofacial region (17,33). Although patients with GCPS do not generally exhibit craniosynostosis, patients with Carpenter syndrome do. Carpenter syndrome, a recessive condition characterized by craniosynostosis and polysyndactyly, is caused by mutations in RAB23 which result in a loss of protein function (34). Rab23 encodes a putative vesicle transport protein that acts as a cell autonomous negative regulator of Hh signaling. Additionally, Rab23 may have a role in promoting the generation of truncated form of Gli3 (35).

In the calvaria, bone is formed by intramembranous ossification directly from mesenchyme. In sutures, all stages of osteoblast differentiation can be observed with no cartilage scaffold forming to complicate the picture (36). In this paper, we have used the developing calvaria as a model of osteoblast development and have studied the role Gli3 plays during intramembranous ossification and in the pathogenesis of craniosynostosis. We have used Gli3Xt−J/Xt−J mice as a tool for studying aberrant Hh signaling in the calvaria and discovered that ectopic Ptch1 expression results in a localized reduction of Twist1 and ectopic Runx2 expression which leads to abnormal osteoprogenitor proliferation and differentiation and ultimately in craniosynostosis in the lambdoid suture. The abnormalities in the lambdoid suture are normalized by the local application of FGF2 which upregulates Twist1 which then, we predict, inhibits Runx2. Taken together, we define a novel role for Gli3 in osteoblast development, describe the first mouse model of lambdoid suture craniosynostosis and show how craniosynostosis can be rescued in this model.

RESULTS

Gli3Xt−J/Xt−J mice exhibit synostosis of the lambdoid suture and other craniofacial malformations

All Gli3Xt−J/Xt−J heads analyzed at E18.5 exhibited bilateral premature fusion of the parietal and interparietal bones across the lambdoid sutures (Fig. 1A–F). In Wt mice, this suture remains patent throughout life. The suture fused between E16.5 and E18.5. At E16.5, all mutant lambdoid sutures were patent, whereas at E17.5 synostosis was detected in either both lambdoid sutures or in just one. If at this age one suture was patent, the sutural space between the parietal and interparietal bones was reduced (data not shown). The bony fusion was confirmed in tissue sections. Sections through E17.5 sutures were analyzed (Fig. 1G–V). In Gli3Xt−J/Xt−J tissues, osteogenic matrix was observed between the parietal and interparietal bones (Fig. 1O and S), and this matrix was mineralized at multiple locations (Fig. 1P, Q, T and U). In general, tissues were thicker in Gli3Xt−J/Xt−J specimens and this was most notable in the cartilage ectocranial to the lambdoid suture (Fig. 1R and V).

Figure 1.

Figure 1.

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Gli3Xt−J/Xt−J mice exhibit synostosis of the lambdoid suture and multiple other craniofacial abnormalities (A–F) Wt and Gli3Xt−J/Xt−J E18.5 heads stained with Alizarin red to show mineralized bone and with Alcian blue to show cartilage. (A and D) Lateral view. Gli3Xt−J/Xt−J skulls are dome-shaped with an acute angle between the nasal and frontal bones. The lambdoid suture is fused in Gli3Xt−J/Xt−J mice (arrow). (B and E) Apical view (cranial base and mandible removed). The shape of the calvaria in Gli3Xt−J/Xt−J skulls is rectangular when compared with the rounded shape of Wt littermates. The lambdoid sutures in Gli3Xt−J/Xt−J skulls are fused. (C and F) High magnification of the left lambdoid suture. Bony bridging across the normally patent suture (arrow). (G–J) Serial sections through Wt and Gli3Xt−J/Xt−J lambdoid sutures, E17.5. (K–N) Enlargements of (G–J). (S–V) Enlargements of (O–R). (G and K) Hematoxylin-and-eosin-stained sections showing the osteogenic fronts (of) of the parietal and interparietal bones. (O and S) The matrix of the parietal and interparietal bone in the Gli3Xt−J/Xt−J lambdoid sutures are continuous with no distinct osteogenic fronts. The asterisks (*) in (H) and (I) show the mineralized matrix some distance from the osteogenic fronts, compare with G and K. (L and M) The sutural mesenchyme in Wt samples is devoid of mineralized matrix. (T and U) Mineralization within the Gli3Xt−J/Xt−J lambdoid suture (arrowheads). (J, N, R and V) Alcian blue-stained sections showing thicker cartilage endocranial to the lambdoid suture in Gli3Xt−J/Xt−J specimens. f, frontal bone; if, interfrontal suture; ip, interparietal bone; of, osteogenic fronts; p, parietal bone; s, sagittal suture. Scale in (A) and (C): 1 mm. Scale in (G) and (K): 100 µm. The following groups of images are of the same magnification: A, B, D and E; C and F; G–J and O–R; K–N and S–V.

In addition to the lambdoid suture phenotype, the interfrontal and sagittal sutures display abnormalities, these sutures were widened initially reflecting an expanded forebrain. This finding is analogous to the wide frontal and sagittal sutures that are characteristic of patients with GCPS. Later, islands of ossification formed in the interfrontal suture. Also, the overall calvarial shape was abnormal. In lateral view, the calvaria was dome-shaped with an acute angle between the frontal and nasal bones. In apical view, the Gli3Xt−J/Xt−J calvaria was less rounded possibly in part due to the lambdoid suture synostoses. It is difficult to estimate the contribution the synostosis has on the abnormal skull shape as this is dependent on many factors including the function of the cranial base and the shape of the developing eyes and brain; both of these latter structures are deformed in Gli3Xt−J/Xt−J mice (33). As abnormalities in the cranial base can have secondary effects on the shape of the calvaria, we analyzed the cranial base of E18.5 Gli3Xt−J/Xt−J mice. We found that the cranial base was of normal length and width and the synchondroses patent as in Wt littermates (data not shown).

Gli3 transcripts are specifically localized to the developing lambdoid and interfrontal sutures

mRNA expression of Hh ligands Indian (Ihh), Sonic (Shh) and Desert hedgehog (Dhh), the Hh the transmembrane receptor Patched 1 (Ptch1) and the transcription factor Gli3 were studied in Wt lambdoid sutures at E16.5 and compared with the osteoblast-specific transcription factor Runx2. Indian hedgehog (Ihh) was expressed in the Runx2-positive mesenchymal condensations at the edge of the parietal and interparietal bones (osteogenic fronts) (Fig. 2A and B). We did not detect Shh or Dhh. As Ptch1 is a target of Hh, the level of Ptch1 transcription is commonly used as a functional readout of the level Hh signaling (37). Similar to Ihh, Ptch1 and Gli3 were expressed in the osteogenic fronts (Fig. 2C and D). We also examined the expression of Gli3 in E15.5 whole-mount tissue and compared it with the osteoblast marker Bone sialoprotein (Bsp). Interesting, Gli3 transcripts were highly expressed in the interparietal side of the lambdoid suture and in the interfrontal suture (Fig. 2E and F). This restricted expression pattern explains why abnormalities occur at these locations in Gli3Xt−J/Xt−J mice.

Figure 2.

Figure 2.

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Ihh, Ptch1 and Gli3 are expressed by osteoprogenitors in the osteogenic fronts of Wt interparietal and parietal bones. (A) Runx2 is used as a marker of osteoprogenitors and also of mature osteoblasts. At E16.5, Runx2 transcripts are located in the osteogenic fronts (condensing osteogenic mesenchyme) of the parietal (arrow) and interparietal (arrowhead) bones, and also within parietal and interparietal bones. (B and C) Like Runx2, Ihh and Ptch1 are expressed in the parietal (arrow) and interparietal (arrowhead) bone osteogenic fronts. (D) Gli3 is expressed in the osteogenic fronts in a pattern broader than that of Ihh (arrows). Its expression covers a slightly larger area and is more highly expressed in the interparietal side of the suture compared with the parietal (arrowhead). (E and F) Bsp and Gli3 expression in E15.5 whole mount calvaria. Gli3 expression is highly localized to the lambdoid (arrowheads) and interfrontal sutures (asterisks) compared with the coronal and sagittal sutures. ep, epithelium; f, frontal bone; ip, interparietal bone; of, osteogenic front; p, parietal bone. Scale bars: (A), 200 µm; (E), 1 mm. The following groups of images are of the same magnification: A–D; E and F.

Ectopic osteoblast differentiation contributes to the synostosis of the lambdoid suture in Gli3Xt−J/Xt−J mice

In order to understand the etiology underlying the lambdoid craniosynostosis in Gli3Xt−J/Xt−J mice, we studied serial tissue sections of the lambdoid suture at E16.5, just before the synostosis occurs (Fig. 3). The sutural mesenchyme, between the parietal and interparietal bones, was thicker in Gli3Xt−J/Xt−J mice compared with their Wt littermates (Fig. 3A–F). By in situ hybridization, we investigated the mRNA expression of Twist1 and Patched1 (Ptch1) and compared these with the expression of the osteoblast markers Bsp and Runx2. In Wt tissue, Ptch1 is expressed in the parietal and interparietal osteogenic fronts (Figs 2C and 3G and H). While in Gli3Xt−J/Xt−J mice, Ptch1 was ectopically expressed in the mid-sutural mesenchymal domain, suggesting that Hh signaling is elevated at this location (Fig. 3I and J). We have previously shown that Twist1 is expressed in the sutural mesenchyme and suggested that Twist1 is a negative regulator of osteogenesis and acts to keep sutures patent (24). Partial loss of Twist1 function results in coronal suture synostosis in mice and in the craniosynostosis syndrome, Saethre–Chotzen, in humans (38,39). In Wt mice, Twist1 transcripts were detected in a thin strip across the lambdoid suture mesenchyme with the highest intensity close to the osteogenic fronts. Runx2 is expressed in a reciprocal pattern to Twist1, being mainly restricted to the osteogenic fronts and the bones. In Gli3Xt−J/Xt−J mice, Twist1 and Runx2 expression was abnormal. Twist1 was downregulated in a domain stretching across the lambdoid suture and Runx2 was ectopically upregulated in the same domain (Fig. 3K–R).

Figure 3.

Figure 3.

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Ectopic osteoblastic differentiation in Gli3Xt−J/Xt−J lambdoid sutures. (A–R) Para sagittal sections through E16.5 Wt and Gli3Xt−J/Xt−J lambdoid sutures. (A–D, G, H, K, L, O and P) Wt and (E, F, I, J, M, N, Q and R) Gli3Xt−J/Xt−J. The images for each genotype are serial sections from a single specimen. The second column for each genotype is a high magnification image of the parietal bone osteogenic front highlighted by the box in (A). (A and B) The architecture of the lambdoid suture—the parietal bone, interposed sutural mesenchyme and the interparietal bone—is illustrated by showing the expression of bone sialoprotein (Bsp) in mature osteoblasts. (C–F) Hematoxylin-and-eosin-stained sections reveal that the lambdoid suture mesenchyme in Gli3Xt−J/Xt−J mice is thickened (asterisks). (G–J) In Wt and Gli3Xt−J/Xt−J mice tissue, the Hh receptor Ptch1 is expressed in the osteogenic fronts of both the parietal and interparietal bones (arrowheads). In Gli3Xt−J/Xt−J lambdoid sutures, Ptch1 is additionally expressed in the sutural mesenchyme some distance from the parietal and interparietal bones (arrow). (N and R) At the same location as this ectopic Ptch1 expression, Twist1 is downregulated and the osteoblast specific transcription factor Runx2 is upregulated (arrows). ip, interparietal bone; of, osteogenic front; p, parietal bone. Scale bars: 200 µm. The following groups of images are of the same magnification: A, C, G, K, O, E, I, M and Q; B, D, H, L, P, F, J, N and R.

These data show that when Gli3 function is downregulated Ptch1 is ectopically expressed in the mid-sutural mesenchyme and reciprocally Twist1 expression is downregulated at the same location. Thus, the suppression of osteoblast differentiation by Twist1 in the lambdoid suture is lost and this allows the master regulatory switch, Runx2, to be expressed in the undifferentiated mesenchyme, and this ultimately results in bone formation and craniosynostosis.

Increased cell proliferation in Gli3Xt−J/Xt−J lambdoid sutures

We performed a cell proliferation assay (BrdU incorporation) on Wt and Gli3Xt−J/Xt−J tissue. Gli3Xt−J/Xt−J E16.5 sutures had a larger number of BrdU-positive cells as percentage of the total number of cells Gli3Xt−J/Xt−J (BrdU-positive cells/total cells 164/545, 30.1%), compared with Wt (BrdU-positive cells/total cells 53/367, 14.4%) (P < 0.001) (Fig. 4A, C and E). Increased cell proliferation was not restricted to one tissue compartment but widespread and throughout the sutural tissue.

Figure 4.

Figure 4.

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FGF2 rescues craniosynostosis in Gli3Xt−J/Xt−J mice by normalizing calvarial proliferation and osteoprogenitor differentiation. (A, C and E) Cell proliferation, assayed by BrdU incorporation, is increased in E16.5 Gli3Xt−J/Xt−J mutant lambdoid sutures compared with Wt littermates. Wt BrdU-positive cells/total cells 53/367 (14.4%), Gli3Xt−J/Xt−J BrdU-positive cells/total cells 164/545 (30.1%) (P < 0.001). (F, H and J) Elevated levels of cell proliferation in Gli3Xt−J/Xt−J calvaria are normalized by exogenous FGF2 in E15.5 calvarial explants. (H and J) Proliferation levels in Gli3Xt−J/Xt−J calvaria are reduced by FGF2 from 30.1–11.3% of total cells (#). The percentage of BrdU-positive cells in Wt tissue treated with FGF2 is 12.7%. (B, D, G and I) Alizarin red-stained E15.5 Wt and Gli3Xt−J/Xt−J calvarial explants cultured for 96 h. (B) Wt lambdoid sutures treated with either BSA (blue beads) (10 out of 10) or FGF2 (red beads) (6/6). (D) Gli3Xt−J/Xt−J lambdoid suture explants fuse whether untreated or treated with BSA beads (14/15) (arrowheads). (G) Gli3Xt−J/Xt−J lambdoid suture explants treated with FGF10 beads (red) (5/5) fuse (arrowheads). (I) FGF2 beads (red) placed in the midsutural mesenchyme prevents lambdoid suture fusion in Gli3Xt−J/Xt−J explants (5/6) (arrow). (K) Whole-mount in situ hybridization for Twist1 of E15.5 Gli3Xt−J/Xt−J calvarial explant cultured for 48 h. FGF2-impregnated beads (red) placed in the lambdoid suture induce Twist1, whereas BSA beads (blue) in the contralateral suture do not. ip, interparietal bone; p, parietal bone. Scale bars in (E–H) 1 mm and in (K) 500 µm. The following groups of images are of the same magnification: A, C, F and H; B, D, G and I. Error bars: standard deviation. Blue beads, BSA-impregnated; red beads, either FGF2- or FGF10-impregnated.

FGF2 rescues the craniosynostosis in Gli3Xt−J/Xt−J mice by normalizing osteoprogenitor proliferation and differentiation

In several organs, including the developing midbrain, hindbrain and mammary gland, Fgfs lie downstream from Gli3 in a signaling cascade which then regulate tissue patterning, differentiation and growth, and the application of exogenous FGF10 can rescue defective mammogenesis in Gli3Xt−J/Xt−J mice (40,41). We have previously shown that FGF2 and FGF10 together with their specific receptors are expressed in the developing mouse calvaria (24,42). We therefore hypothesized that FGF signaling could act downstream of Gli3 in the developing calvaria. In addition, we have previously shown that exogenous FGF2 can induce Twist1 in mouse calvarial mesenchyme and hypothesized that the upregulation of Twist1 and the consequent inhibition of Runx2 could rescue the craniosynostosis in Gli3Xt−J/Xt−J mice (24). We therefore selected FGF2 and FGF10 as good candidates to correct the deregulated osteogenesis seen in the Gli3Xt−J/Xt−J mice lambdoid suture.

FGF2- or FGF10-impregnated beads were placed onto the center of the right lambdoid suture at E15.5 of Gli3Xt−J/Xt−J mice and Wt littermates and cultured for 24–96 h. BSA beads were placed on the left lambdoid suture of the same specimen. FGF2 normalized elevated levels of cell proliferation in the Gli3Xt−J/Xt−J lambdoid suture: 30.1% (BrdU-positive cells/total cells = 164/545) to 11.3% (53/468) of total cells. The percentage of BrdU-positive cells in Wt tissue treated with FGF2 was reduced from 14.4% (53/367) to 12.7% (41/323) (Fig. 4E, F, H and J). All Wt lambdoid sutures, whether treated with BSA (10 out of 10) or FGF2 (6/6) or FGF10 (5/5), remained patent (Fig. 4B, data not shown). Gli3Xt−J/Xt−J lambdoid sutures whether untreated or treated with BSA fused in a manner similar to that seen at E17.5 in vivo (14/15) (Fig. 4D). Gli3Xt−J/Xt−J mutant lambdoid sutures treated with FGF10 fused (5/5) (Fig. 4G). Gli3Xt−J/Xt−J lambdoid sutures treated with FGF2 stayed patent (5/6) (Fig. 4I). Also, we performed in situ hybridization for Twist1 on E15.5 Gli3Xt−J/Xt−J calvarial explants cultured with FGF-impregnated beads. FGF2 beads but not BSA beads induced Twist1 in the lambdoid suture mesenchyme (Fig. 4K).

Taken together, we hypothesize that FGF2 rescued Gli3Xt−J/Xt−J lambdoid sutures from fusion by first normalizing previously elevated levels of mesenchymal proliferation and secondly by reinstating Twist1 expression and thereby maintaining osteoprogenitors in an undifferentiated state.

DISCUSSION

Normal development of the calvaria and maintenance of suture patency involve coordinated osteoblastic cell turnover, differentiation and function. These processes are regulated by interacting growth factor signaling networks that include TGFβ superfamily signaling, FGF signaling and as we show here signaling through Gli3 (39). The balance and timing of calvarial osteoblastic proliferation and differentiation is critical in determining the level of osteogenesis at a given location and normal suture function (43–46). In this study, we demonstrate that signaling through Gli3 controls both calvarial osteoblast proliferation and differentiation and that craniosynostosis caused by the loss of Gli3 can be rescued with the local application of FGF2.

In Gli3Xt−J/Xt−J mice, Gli3 null allelic, we show how patency across the lambdoid sutures is lost. In Wt sutures, Twist1 maintains mesenchymal cells in an undifferentiated state by negatively regulating Runx2, a critical osteoblast differentiation transcription factor (22,24,47). Normally, Twist1 expression spans across the suture and therefore prevents the calvarial bones from fusing. This allows the suture to function as a bidirectional growth site. In Gli3Xt−J/Xt−J lambdoid sutures (E16.5), two abnormalities are evident. First, proliferation levels are increased. Secondly, Twist1 expression is lost from a narrow strip of mesenchyme that stretches across the suture. This second abnormality heralds the start of osteoblastic differentiation and is accompanied by an elevated level of Runx2 expression. Ectopic bone in the sutures is formed, which eventually results in synostosis. Thus, in Gli3Xt−J/Xt−J calvaria, we propose that the increase in proliferation leads to an increase in the pool of osteoprogenitors and that this combined with enhanced osteoblastic differentiation ultimately results in craniosynostosis.

In the developing long bone, Ihh is transiently required for the specification of progenitor cells into Runx2-positive osteoblastic precursors (14,48). Ihh null allele mice fail to produce osteoblasts in endochondral bones; however, the removal of Hh activity late in osteoblast development does not prevent the formation of mature osteoblasts. We have previously reported, using whole-mount in situ hybridization, that Shh is expressed in the late embryonic (E18.5) and early postnatal calvaria (49). In the current study, we show that Ihh is expressed by calvarial osteoblastic progenitors but we could detect neither Shh nor Dhh transcripts in calvarial tissue sections between E13.5 and E16.5. In the axial and appendicular skeleton of Ihh−/− mice, osteoblasts fail to differentiate and function. Although Ihh−/− mice exhibit a delay in calvarial osteogenesis, calvarial osteoblasts still develop (14,50). What compensates for the lack of Ihh in the developing calvaria is unknown. It is possible that Shh has a role in calvarial development but it has not been possible to study this as Shh null allele mice die before skeletogenesis begins.

In addition to regulating the early stages of osteoblast differentiation, Ihh also controls growth plate vascularization, chondrocyte proliferation and maturation (14,51). Interestingly, the short limb phenotype exhibited by Ihh−/− mice can be partially rescued by deactivating Gli3. The explanation for this normalization of Ihh−/− mice is that in the absence of Hh ligand the truncated repressor form of Gli3 is generated. Consequently, in Ihh−/−;Gli3−/− compound mice, the repression by Gli3 is removed and the phenotype restored (52,53). With regard to osteoprogenitor proliferation and differentiation in the lambdoid suture, Gli3 would appear to be acting as a repressor as in Gli3Xt−J/Xt−J mice they are both enhanced. Gli3 repressor can control osteoblastic differentiation by attenuating Runx2 transcriptional activity as a result of competitively inhibiting its DNA binding to the OSE2 (osteoblast-specific cis-acting element) site in the osteocalcin promoter (21). We suggest that in the lambdoid suture Gli3 may also regulate osteoblastic differentiation through a Twist1-dependent mechanism. Twist1's anti-osteogenic function is mediated by a novel domain, the Twist box, which interacts with the Runt DNA-binding domain in the Runx2 protein to inhibit its function (22). Twist1+/−;Gli3+/Xt-J compound mutant mice display a more severe limb phenotype (polydactyly) than that seen in either Twist1+/− or Gli3+/Xt-J single mutant mice, suggesting that there is a genetic interaction between Twist1 and Gli3 activity (54). Further evidence supporting the proposition that Gli3Xt−J/Xt−J mice have a loss of Gli3 repressor function in the calvaria comes from the work of Ruther and coworkers (55,56). They have generated a mouse model for the Pallister–Hall syndrome which carries a targeted mutation 3′ of the zinc finger encoding domain at the Gli3 locus and this mutation predicts a loss of Gli3 activator. As mice homozygotic for this mutation (Gli3Δ699/Δ699) have normal lambdoid sutures, the lambdoidal phenotype in Gli3Xt−J/Xt−J mice must be due to the loss of Gli3 repressor.

Gli3 is required for the correct development of many different organs and is known to act upstream of Fgf signaling. In the developing midbrain and hindbrain, Fgf8 has been shown to be a downstream mediator of Gli3 signaling (40). Gli3 regulates patterning and growth of the cerebellum and isthmus by repressing Fgf8 expression and regulating cell proliferation and cell viability. During formation of the mammary gland placodes, Gli3 has been shown to lie in a signaling cascade upstream of Fgf10. This in turn activates Fgfr2-IIIb which leads to the activation of canonical Wnt signaling and the correct induction and patterning of the mammary placodes. Fgf10 binds to Fgfr2-IIIb with a high affinity. In Gli3Xt−J/Xt−J mice, somatic levels of Fgf10 are reduced and the correct induction and patterning of mammary gland primordia is impaired. Mammary gland development is rescued by the application of recombinant FGF10 in Gli3Xt−J/Xt−J mice (41). Multiple Fgfs and Fgfrs are known to be expressed in the developing suture (24,42,49,57,58). As Gli3 regulates Fgf signaling in several embryonic organs, we tested the effects of applying Fgf10 or Fgf2 to Gli3Xt−J/Xt−J calvarial explants. Only the application of exogenous FGF2 could by-pass the abnormal Gli3 signaling, restore sutural Twist1 expression and rescue the lambdoid suture phenotype. In cell and tissue culture, FGF2 decreases calvarial mesenchymal proliferation and inhibits osteoblastic differentiation in a stage-specific manner, specifically affecting immature calvarial osteoblasts (24,59–62). We suggest that in the calvaria of Gli3Xt−J/Xt−J mice, FGF2 normalizes both elevated osteoblastic progenitor proliferation and that it normalizes osteoblastic differentiation through Twist1-mediated repression of Runx2.

The effects of FGF signaling on osteoblast development, and therefore suture patency, differ depending on the stage of osteoblast differentiation targeted (59,60). We have previously shown that exogenous FGF applied to mouse calvarial osteogenic fronts will accelerate suture closure (49). At the osteogenic fronts, FGF acts on cells that are partially differentiated into osteoblasts. In contrast, exogenous FGF applied to the mid-sutural mesenchyme, where undifferentiated mesenchymal cells are located, does not accelerate suture closure (49), and we show in this study that FGF2 can prevent craniosynostosis in a Gli3Xt−J/Xt−J mice. Taken together, in the Gli3Xt−J/Xt−J suture, FGF2 inhibits osteoblastic progenitor proliferation and osteoblastic differentiation in sutural cells and this rescues the synostosis phenotype.

We hope that Gli3Xt−J/Xt−J mice will provide a valuable model of lambdoid suture synostosis. The sutures fuse at a late embryonic stage which correlates well with patients with lambdoid suture synostosis (4). The model is 100% predictable with the sutures fusing in all mice analyzed. Also, the fusion of the Gli3Xt−J/Xt−J lambdoid sutures can be followed in organ culture which may obviate the immediate need for experimentation on live animals.

MATERIALS AND METHODS

Mutant mice and tissue preparation

Gli3Xt−J (Gli3+/−) mice (The Jackson Laboratory, USA, stock no. 000026) were maintained on a pure C57BL/6J background. The age of the embryos was determined by the day of the appearance of the vaginal plug (day 0) and by morphological criteria. Tissues were processed as described previously (43). Wt littermates were used as controls in all experiments. Tissues for skeletal preparations had the skin removed and were fixed in 95% ethanol overnight.

Histology

Embryonic calvaria were fixed in 10% neutral buffered formalin (pH 6.8) for 24 h at 4°C, dehydrated in an ethanol series, embedded in paraffin and sectioned at 7 µm intervals. After dewaxing and rehydrating, sections were stained with hematoxylin and eosin. To detect bone mineral, von Kossa staining was performed by adding 5% silver nitrate solution, exposed to a 60 W lamp for 1 h and stopped by treatment with 5% sodium thiosulfate and counterstained by nuclear fast red. Also to detect the bone mineral, Alizarin red staining was performed with 2% Alizarin red S solution (pH 4.2) for 2 min. To detect cartilage, Alcian blue staining was performed with 1% Alcian blue 8GX solution (pH 2.5) for 30 min at room temperature and counterstained by nuclear fast red.

Organ culture

Calvaria were dissected free from the overlying skin and underlying brains from mice and placed on Nuclepore polycarbonate filters in a Trowell-type organ culture system. DMEM (Sigma) supplemented with glutamax, penicillin/streptomycin (Sigma) and 10% bovine calf serum was used.

Bead assays: heparin-coated acrylic beads (Sigma) were incubated in 25 ng/µl recombinant human FGF2, FGF10 (R&D Systems) or bovine serum albumin (BSA) at 37°C for 40 min and stored at 4°C before being placed on the explant. Bead assays were cultured for 24–96 h.

In situ hybridization

Preparation of Bsp, Dhh, Gli3, Ihh, Ptch1, Runx2, Shh and Twist1 35S-UTP and digoxigenin-UTP labeled riboprobes, in situ hybridization and image analysis have all been described previously (24,63,64). The Gli3 probe used in whole-mount in situ hybridization was prepared from a 581 bp fragment of murine Gli3 cDNA, which was isolated from C3H10T1/2 cells by RT–PCR using the specific primer pairs (5′-cgggatcctgcccatcagctactcagtg-3′ for upstream, 5′-gctctagatggtatggtccccatcatct-3′ for downstream). The amplified products were digested with BamHI/XbaI and were subcloned into pBluescript II KS.

Skeletal staining

Twenty-eight Gli3Xt−J/Xt−J mice heads (E15.5–E18.5) and a similar number of Wt heads from littermates were stained with Alizarin red and Alcian blue as previously described (43). E16.5 calvarial explants were also stained.

Proliferation assay and cell counting

Cell proliferation was assessed by pulsed BrdU incorporation. Mice were injected intraperitoneally with 10 µl/g body weight BrdU (Zymed) and sacrificed after 2 h. Explants were cultured for 20–96 h before BrdU was added to the medium (1:200) for 3 h prior to fixation. Zymed's streptavidin–biotin staining system was used according to the manufacturer's instructions and sections were counterstained with hematoxylin.

In the lambdoid suture studies, 0.375 mm2 counting grids were placed over the tip of each osteogenic front. Each grid consisted of two rows of three squares with the two squares covering the osteogenic front and the four remaining squares projecting into the sutural mesenchyme. Skin, cartilage and dural cells were not included in the assay area. Data from both sides of each suture were pooled.

FUNDING

This work was supported by grants from the MRC (D.P.C.R.), EU/Marie Curie (E.L.-E.), American Lung Association (S.B.), NIH RO1 HL074832-01 (S.B.), California Breast Cancer Research program (S.B. and J.M.V.), European Commission 5th Framework (J.M.V.), Childrens Hospital LA (J.M.V.), Biocentrum Helsinki (D.P.C.R.), Helsinki University (D.P.C.R., Y.T.), Jusélius Foundation (D.P.C.R., L.V.) and Academy of Finland (D.P.C.R.).

ACKNOWLEDGEMENTS

We thank Stalin Kariyawasa, Heide Olsen and Airi Sinkko for their technical assistance. We thank Prof. Irma Thesleff for her support during this project.

Conflict of Interest statement. None declared.

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