In experiments in which dura mater was physically removed from calvaria and then co-cultured with the calvaria, it was demonstrated that dura mater secreted soluble, heparin-binding factors that were required for maintaining suture patency (Opperman et al., 1996). An effective way to begin to understand the regulatory mechanisms that maintain sutures in their unossified state, while allowing them to function as bone growth sites, is to look at systems in which these regulatory mechanisms are interrupted. It is now known that several growth factor receptor and transcription factor mutations are associated with craniosynostosis and that interaction of these factors with one another and with a host of other growth factors is necessary for maintenance of normal sutures.
Several recent reviews and a new book have very eloquently described the clinical pathologies associated with the known mutations (Webster and Donoghue, 1997; Wilkie, 1997; Elmslie and Reardon, 1998; Jabs, 1998; Nuckolls et al., 1999; Sperber, 1999; Cohen, 2000). These reviews give excellent descriptions of the types of mutations that occur and describe how some mutations appear to manifest very different phenotypes. Typically, the nature of the mutations is not completely described. For example, mutations of FGF receptor and MSX2 genes generally are described as activating or gain of function mutations, whereas mutations in TWIST are considered as loss of function; but the “function” that is affected is often not clear.
Localization of Growth and Transcription Factors and Their Receptors Within Sutural and Perisutural Tissues
It is essential to look at the distribution and localization of factors known to be involved in suture morphogenesis to begin to understand how suture morphogenesis is regulated and to explore what goes wrong during premature fusion of sutures (Fig. 4). It is clear that before suture formation, factors known to be involved in epithelio-mesenchymal signaling, such as BMP-4, BMP-7, FGF-9, MSX1 and MSX2, as well as TWIST, are present in the presumptive sutural mesenchyme, the underlying dura and the approaching bone fronts (Fig. 4A, shades of green, red, and shades of orange, respectively). Also present are FGF receptors 1–3, with FGFR2 absent from the suture and dura, but highly expressed in the approaching bone fronts. TGF-β1, 2, and 3 are present in the dura and approaching bone fronts, but are absent from the suture mesenchyme.
Figure 4. Diagrammatic representation of a presumptive suture (A), a fully formed suture (B), and a fusing suture (C). The color-coded regions of the sutures (green), osteogenic bone fronts (orange), and bone (blue) are given in the accompanying key. Periosteum is colored pink. The key lists the growth and transcription factors, receptors, and extracellular matrix components known to be present in each of the stages of suture morphogenesis presented. (1Opperman et al., 1997, 2Most et al., 1998, 3Rice et al., 2000, 4Johnson et al., 2000, 5Marks et al., 1999, 6Rice et al., 1999, 7Roth et al., 1997a, 8Roth et al., 1997b, 9Kim et al., 1998, 10Iseki et al., 1997, 11Iseki et al., 1999, 12Liu et al., 1999, 13Lemmonier et al., 2000, 14Zhou et al., 2000.)
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During the process of approximation and overlap of the bone fronts (Fig. 4B), factors involved in pattern formation, such as SHH, PTC, and ID are present, whereas TWIST and MSX2 are down-regulated. At the same time, differential distribution of extracellular matrix components can be seen, with CBFA1, osteopontin (BSP-I), bone sialoprotein (BSP-II), type I collagen, osteonectin (ON), and alkaline phosphatase (AP) found in the bone and type I collagen and type III collagens being expressed in the bone fronts, along with FGFR1-3 and TGF-β1, 2, and 3. The suture matrix shows mostly type III collagen expression and still expresses FGF-9, MSX1, and begins to express FGFR1.
To characterize a fusing suture (Fig. 4C), data were taken from both normally fusing sutures, such as the interfrontal suture of rats and mice and from abnormally fusing sutures either experimentally induced or as a product of activity of mutant genes. Factors associated with bone formation, such as type I collagen, TGF-β1, TGF-β2, FGFR1, FGFR2, and BSP-I are up-regulated in the suture matrix, whereas CBFA1, FGF-2, and IGF-1 become expressed in the bone fronts. MSX1, ID, SHH, PTC, and FGF-9 are all down-regulated and in the completely fused suture, the suture is indistinguishable from bone.
The Nature of Mutations Associated With Abnormal Suture Development and Growth
Although the first mutation to be associated with craniosynostosis was found in the MSX2 gene (Jabs et al., 1993), subsequent mutations were identified in the FGF receptor genes (Jabs et al., 1994; Muenke et al., 1994; Reardon et al., 1994; Shiang et al., 1994). Boston-type craniosynostosis is the only craniosynostotic syndrome associated with mutations in the gene for MSX2 (Jabs et al., 1993). MSX2 mutation enhances DNA binding of the MSX2 protein to its DNA binding sequence (Ma et al., 1996). This produces an autosomal dominant defect resulting in fused cranial sutures, a finding supported by studies in which overexpression of MSX2 results in suture fusion (Liu et al., 1995, 1999).
The converse of premature fusion occurs with functional haploinsufficiency of MSX2 in humans, resulting in delayed suture formation and wide-open fontanels (Wilkie et al., 2000). MSX2-deficient mice showed similar delayed suture formation and marked endochondral growth plate defects, with narrowing of the cranial base synchondroses and long bone growth plates and reduced cancellous bone and cortical bone thickness. This finding is not surprising, because MSX2 abrogates cellular responsiveness to FGF-2 (Newberry et al., 1997a,1997b). It is tempting to speculate that the cranial vault defect seen in MSX2 haploinsufficiency is due mainly to a shortening of the cranial base synchondroses. The open fontanels would then be a result of rapid expansion of mesenchymal tissue within the suture to compensate for failure of growth in the cranial base and to accommodate the growing brain. This is best demonstrated in hydrocephalus, for which abnormal expansion of the neurocranium is accommodated by rapid expansion of the bones of the cranial vault (Schendel and Shuer, 1994).
Mutations in FGFR2 associated with the Crouzon phenotype result in disulfide-bonded receptor dimers that are constitutively activated, leading to increased kinase activity (Galvin et al., 1996). In contrast to these mutations, the mutations associated with Apert and Jackson-Weiss phenotype exhibit a selective decrease in FGF-2 dissociation kinetics from mutant FGFR (Anderson et al., 1998). All mutations result in fusion of cranial vault sutures and mid-face hypoplasia, with Crouzon syndrome also exhibiting shortening of the cranial base. A similar phenotype is noted in the Bey mutant mouse, in which an insertional mutation at the FGF-3/FGF-4 locus produces a mouse with facial shortening, increased interorbital distance, and premature craniosynostosis (Carlton et al., 1998). Expression of both FGF-3 and FGF-4 is up-regulated in this mutant mouse, indicating that locally elevated levels of growth factors can produce similar phenotypes to those seen with mutant growth factor receptors. Interestingly, Coffin and co-workers (1995) showed that transgenic mice over-expressing FGF-2 exhibit markedly shortened long bones associated with growth plate defects, as well as “calvaria enlarged over the occipital bones,” which is usually associated with either cranial base shortening or with suture fusion. However, these authors did not report on whether the sutures were patent and did not report looking at the cranial base. This phenotype appears to be similar to the cranial bossing seen in achondroplasia, in which mutations result in FGFR3 receptor dimerization and ligand-independent stimulation of kinase activity. This produces inappropriate cartilage growth plate differentiation, with abnormally shortened long bones and cranial base (Webster and Donoghue, 1996). These data were supported by the findings of Naski et al. (1998), who showed that mice with an activated FGFR3 transgene have shortened long bones and cranial occipital bossing.
This role for FGFR3 in cartilage growth plate development was confirmed by knockout experiments (Deng et al., 1996), in which disruption of FGFR3 expression led to prolonged endochondral bone growth and concomitant elongated long bones. However, no abnormalities in the intramembranous bones of the craniofacial skeleton were found, indicating that osteoblast function appeared unaffected by the presence or absence of FGFR3. Muenke craniosynostosis, which is the result of a P250R mutation in FGFR3, does present with unilateral or bilateral coronal craniosynostosis (Muenke et al., 1997). However, it is unclear whether this is a consequence of a primary defect at the suture or is secondarily induced by changes in the cranial base.
Recently, (Zhou et al., 2000) demonstrated that mice carrying a P250R substitution in FGFR1, orthologous to the Pfeiffer mutation in humans, exhibited fusion of interfrontal, sagittal and coronal sutures. This mutation resulted in transiently increased cell proliferation and dramatically increased CBFA1 expression at the suture site. CBFA1 is a protein required for activation of osteoblast differentiation. Analysis of the cranial base revealed no premature closure of the synchondroses, supporting the idea that craniosynostosis can develop as a primary defect, without involvement from the cranial base (Moss, 1960; Smith and Tondury, 1978). These data contradict the long-stated hypothesis that the cranial base is the primary site of defects in human craniosynostosis (Cohen, 1993; Moss, 1959).
Role of Transcription Factors, Growth Factors, and Their Receptors in Regulating Cell Proliferation and Cell Differentiation Within Sutures
The cell assay system used to establish that Crouzon mutations result in constitutively activated receptors also showed increased numbers of foci of transformed cells, suggesting that the mutations result in increased cell proliferation (Galvin et al., 1996). However, little is known about how the mutation affects cell proliferation or cell differentiation. In experiments in which osteoblasts were isolated from Apert patients and cultured, Fragale et al. (1999) demonstrated that these osteoblasts showed low levels of cell proliferation accompanied by elevated markers for differentiation, such as AP, BSP-I, and BSP-II. This was similar to the findings of De Pollak et al. (1996), who cultured cells isolated from patients with nonsyndromic craniosynostosis. Experiments by Lomri et al. (1998) demonstrated that osteoblasts with FGFR2 mutations resulting in the Apert phenotype appeared to have normal proliferative responses to the addition of FGF-2. However, these cells showed accelerated differentiation and bone formation in the presence of FGF-2.
Increased presence of FGF-2 has been demonstrated to be associated with both normal (Most et al., 1998) and induced (Iseki et al., 1997) suture closure. Iseki et al. (1999) found that FGF-2-induced suture closure was associated with a localized decrease in cell proliferation, a change from FGFR2 to FGFR1 expression by osteoblasts in the bone fronts, and increased expression of BSP-I, a marker for osteoblast differentiation. These findings were confirmed by Lemonnier et al. (2000), who showed that the Apert S252W mutation did not alter cell proliferation, but up-regulated collagen type I, osteocalcin (OC), and BSP-I, associated with protein kinase C-independent down-regulation of FGFR2. Importantly, it has been noted that immature osteoblasts respond to FGF-2 by proliferating, whereas differentiating osteoblasts respond by becoming apoptotic (Mansukhani et al., 2000). Osteoblasts transfected with Apert S252W or Crouzon C342Y mutant FGFR2 showed inhibited differentiation, with dramatically elevated levels of apoptosis (Mansukhani et al., 2000), suggesting that the mutation likely affects maturation of osteoblastic cells at the suture and that apoptosis may be a feature of craniosynostosis.
In contrast to the pathology of these FGFR mutations, the P250R mutation in FGFR1, producing Pfeiffer phenotype when introduced in mice, results in transiently increased levels of cell proliferation before visible suture fusion (Zhou et al., 2000). This increase in cell proliferation is accompanied by increased expression of CBFA1, BSP-I, and OC. Zhou et al. (2000) demonstrated that CBFA1 is downstream of FGFR1 and suggest that mutated FGFR1 causes up-regulation of CBFA1, which activates expression of its target genes, BSP-I and OC. These authors found no changes in expression of STAT1, STAT5a, STAT5b, or MSX2, supporting that MSX2 is likely not downstream of FGF. Another mutation that results in premature suture fusion preceded by a transient increase in proliferative activity of the osteoblastic cells lining the osteogenic bone fronts (Liu et al., 1999) is one occurring in MSX2. MSX2 is expressed mainly in the suture mesenchyme, where it apparently binds to the promoter region of collagen type I and of OC (Towler et al., 1994; Dodig et al., 1996), inhibiting their transcription. Although it is counterintuitive that MSX2 inhibits osteoblast differentiation, it may be that MSX2 allows cells to go through additional proliferative cycles, increasing the total number of cells ultimately available for differentiation. Conversely, mice deficient for the MSX2 gene show wide open fontanels in the cranial vault, similar to the defect seen in the human condition induced by MSX2 haploinsufficiency (Satokata et al., 2000; Wilkie et al., 2000). This finding could be a result of reduction of numbers of cells available to become osteoblasts, due to reduced cell proliferation.
TWIST is a transcription factor specifically associated with Saethre-Chotzen syndrome (el Ghouzzi et al., 1997, 1999). In contrast to mutations in the MSX2 gene, for which overexpression of MSX2 results in craniosynostosis, mutations in the TWIST gene produce haploinsufficiency, resulting in craniosynostosis. This haploinsufficiency can result from several mutations, with insertion of premature stop codons, frameshifts, and nonsense mutations all resulting in a lack of functional protein product being translated (Paznekas et al., 1998; el Ghouzzi et al., 1999). TWIST is found in developing suture mesenchyme and has been shown to decrease osteoblast differentiation and FGFR2 in sutures (Johnson et al., 2000; Rice et al., 2000). FGF-2 induces up-regulation of TWIST expression (Johnson et al., 2000; Rice et al., 2000), which could be associated with normal suture development. However, because MSX2 decreases FGF-2 effects, it is possible that MSX2 overexpression or MSX2 mutations result indirectly in decreased TWIST expression by down-regulating FGF-2, producing the equivalent effect of TWIST haploinsufficiency and resulting in suture fusion (Fig. 5).
Figure 5. Schematic representation of the known (solid long arrows) and some potentially interesting unknown (dashed arrows) associations between factors linked with craniosynostosis. Numbers of short arrows show degree of up-regulation () or down-regulation () of each factor known to result in craniosynostosis. Asterisks show factors that are known to result in craniosynostosis when perturbed independently of upstream regulators. Factors shown more than once show alternate pathways or pathways where possible links are not yet known.
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It is known that FGF-2 levels are highly elevated in fusing sutures (Most et al., 1998) and that addition of FGF-2 to sutures will induce fusion (Iseki et al., 1997). Interestingly, normal human osteoblasts increase TGF-β2 production during prolonged exposure to FGF-2, accompanied by increased OC production and matrix mineralization (Debiais et al., 1998). Recent studies demonstrated that TGF-β2 induced suture obliteration in cultured fetal rat calvaria accompanied by elevated levels of cell proliferation (Opperman et al., 2000) similar to that seen when dura mater was removed from calvaria before culture (Opperman et al., 1998). However, rescue of sutures from obliteration was achieved by removal of TGF-β2 activity with neutralizing antibodies, without altering cell proliferation. Rescue of coronal sutures from obliteration was also achieved by addition of TGF-β3 to cultured calvaria, with increased concentrations of TGF-β3 resulting in decreased levels of cell proliferation (Opperman et al., 2000). In converse experiments, where sutural obliteration was induced by removal of TGF-β3 activity by neutralizing antibodies, it was found that sutural obliteration was preceded by elevated levels of cell proliferation.
These data provide good evidence for abnormally elevated cell numbers being a contributory factor to premature obliteration of cranial sutures. Support for the hypothesis that cell proliferation is associated with premature suture closure is provided by evidence that addition of FGF-4 to cultured fetal mouse calvaria induces premature suture fusion associated with elevated levels of cell proliferation (Kim et al., 1998), as did overexpression of MSX2 (Liu et al., 1999). Recent data provide several good lines of evidence that MSX2 and TGF-βs regulate suture patency in part through regulating cell proliferation. However, it is apparent that most of the FGFR mutations do not affect proliferative activity, but rather alter cell differentiation. Increased cell proliferation at the suture, contributing more cells to the bone cell lineage and accelerated osteoblast differentiation both result in increased bone formation. Therefore, up-regulation of either would be sufficient to induce premature suture obliteration.
Transcription Factor and Growth Factor Regulation of Apoptosis in the Suture
Because osteoblasts transfected with either Apert or Crouzon FGFR2 mutant genes exhibit dramatically elevated levels of apoptosis (Mansukhani et al., 2000), it is likely that apoptosis is a feature of craniosynostosis. Rice et al. (1999) showed that apoptosis occurs during normal suture morphogenesis in the cells lining the bone fronts and particularly at the leading edges of the overlapping bones within the suture. They proposed that apoptosis is a part of normal suture development and suggested that increased apoptosis could be associated with delayed suture closure, as occurs in cleidocranial dysplasia, whereas decreased apoptosis could result in premature suture fusion. That apoptosis slowed bone formation at the suture was confirmed by data showing that increased numbers of apoptotic cells were present in sutures both during normal suture maintenance and during rescue of sutures from obliteration with TGF-β3 (Opperman et al., 2000). Furthermore, low numbers of apoptotic cells were found in sutures induced to fuse by removal of dura mater or by addition of TGF-β2 (Opperman et al., 2000).
Furtwangler et al. (1985) hypothesized that apoptotic cells should be found at the edges of bone fronts that become too closely approximated, thereby preventing sutural obliteration. This idea was supported by the finding that abundant apoptotic cells were present along the bone fronts of sutures not undergoing fusion (Opperman et al., 2000). However, this was not sufficient to prevent obliteration, because apoptotic cells were also found along the bone fronts of fusing sutures. Current evidence indicates that to maintain suture patency, a critical number of cells within the sutural matrix must become apoptotic. If this does not happen, the number of cells within the suture will exceed critical density, triggering cell differentiation, ultimately leading to bony obliteration of the suture. This concept is supported by the data of Frenkel et al. (1990, 1992) who showed that using in vitro cell culture techniques that increased cell density is associated with differentiation.