Fibroblast growth factors lead to increased Msx2 expression and fusion in calvarial sutures.

Craniosynostosis, the premature fusion of the skull bones at the sutures, represents a disruption to the coordinated growth and development of the expanding brain and calvarial vault and is the second most common birth defect that affects the craniofacial complex. Mutations in the human homeobox-containing gene, Msx2, have been shown to cause Boston type craniosynostosis, and we have shown that overexpression of Msx2 leads to craniosynostosis in mice. Activating mutations in fibroblast growth factor (FGF) receptors are thought to cause craniosynostosis in Crouzon, Apert, Jackson-Weiss, Beare-Stevenson, and Muenke syndromes. To mimic activated signaling by mutated FGF receptors, we used heparin acrylic beads to deliver FGF ligands to mouse calvaria and demonstrated increased Msx2, Runx2, Bsp, and Osteocalcin gene expression, decreased cell proliferation, and suture obliteration and fusion. FGF2 elicited the greatest increase in Msx2 expression, and FGF1 was most likely to cause suture obliteration and fusion. Of the three sutures studied, the coronal suture exhibited the greatest increase in Msx2 expression and was the most likely to undergo obliteration and fusion. These results are intriguing because the coronal suture is the most commonly affected suture in syndromic craniosynostosis. These results suggest that Msx2 is a downstream target of FGF receptor signaling and that increased FGF signaling leads to osteogenic differentiation by sutural mesenchyme in mouse calvaria. These results are consistent with the hypotheses that increased Msx2 expression and activated signaling by mutated FGF receptors lead to craniosynostosis.     

Smad7 Modulates TGFβ Signaling During Cranial Suture Development to Maintain Suture Patency

Craniosynostosis, the premature fusion of one or more sutures between the calvarial bones, is a common birth defect. Mutations in genes encoding receptors for the transforming growth factor‐beta (TGFβ) family of signaling molecules have been associated with craniosynostosis, but how TGFβ signaling is regulated during suture development is not known. In the present study, we found that expression of Smad2 and Smad3, intracellular mediators of canonical TGFβ signaling, gradually increases during early postnatal suture development in rat in both the coronal suture (CS), which remains patent throughout life, and the posterior frontal suture (PFS), which undergoes programmed closure by postnatal day 22. The amounts of phosphorylated Smad2 and Smad3 proteins showed a similar gradual increase in the PFS and CS, but in the CS, Smad2/3 activation was suppressed after neonatal day 10. The suppression of Smad2/3 activation in the CS correlated with upregulation of Smad7 expression. We demonstrate that siRNA‐mediated knockdown of Smad7 caused increased phosphorylation of Smad2 and Smad3 and induced osseous obliteration of the CS from postnatal days 10 to 22. The Smad7 siRNA‐induced CS closure was associated with significantly increased levels of Fgf10 and phosphorylated ERK1/2 in the suture mesenchyme. Moreover, addition of the Erk1/2 inhibitor U0126 partially blocked Smad7‐siRNA–induced CS closure. These findings suggest that canonical TGFβ signaling induces suture closure at least in part through activation of FGF and ERK signaling and that Smad7 plays an important role in maintaining suture patency by suppressing canonical TGFβ signaling during suture development. © 2014 American Society for Bone and Mineral Research.     

Interrelationship of Cranial Suture Fusion, Basicranial Development, and Resynostosis Following Suturectomy in Twist1+/? Mice, a Murine Model of Saethre-Chotzen Syndrome

The interrelationships among suture fusion, basicranial development, and subsequent resynostosis in syndromic craniosynostosis have yet to be examined. The objectives of this study were to determine the potential relationship between suture fusion and cranial base development in a model of syndromic craniosynostosis and to assess the effects of the syndrome on resynostosis following suturectomy. To do this, posterior frontal and coronal suture fusion, postnatal development of sphenooccipital synchondrosis, and resynostosis in Twist1(+/+) (WT) and Twist1(+/-) litter-matched mice (a model for Saethre-Chotzen syndrome) were quantified by evaluating μCT images with advanced image-processing algorithms. The coronal suture in Twist(+/-) mice developed, fused, and mineralized at a faster rate than that in normal littermates at postnatal days 6-30. Moreover, premature fusion of the coronal suture in Twist1(+/-) mice preceded alterations in cranial base development. Analysis of synchondrosis showed faster mineralization in Twist(+/-) mice at postnatal days 25-30. In a rapid resynostosis model, there was an inability to fuse both the midline posterior frontal suture and craniotomy defects in 21-day-old Twist(+/-) mice, despite having accelerated mineralization in the posterior frontal suture and defects. This study showed that dissimilarities between Twist1(+/+) and Twist1(+/-) mice are not limited to a fused coronal suture but include differences in fusion of other sutures, the regenerative capacity of the cranial vault, and the development of the cranial base.     

Toward an understanding of nonsyndromic craniosynostosis: altered patterns of TGF-beta receptor and FGF receptor expression induced by intrauterine head constraint

Although the etiology of nonsyndromic forms of craniosynostosis remains uncertain, recent experiments from our laboratory have demonstrated that fetal head constraint induces cranial suture fusion in mice through a process associated with altered patterns of transforming growth factor beta (TGF-beta) isoform expression. Other recent studies have highlighted the role of secreted signaling molecules, including members of the TGF-beta superfamily and the fibroblast growth factors (FGFs), as well as their receptors, in regulating suture development and fusion. The purpose of these experiments was to examine the potential role of TGF-beta receptors and FGF receptor 2 (FGFR2) in nonsyndromic craniosynostosis by determining their temporospatial patterns of expression during development complicated by intrauterine head constraint. This study consisted of two groups of C57BI/6J mice: an experimental group subjected to intrauterine constraint and a control unconstrained group. Fetal head constraint was induced by performing uterine cerclage on day 17.5 of gestation and allowing intrauterine fetal growth to continue 24 and 48 hours beyond the normal gestational period. Control animals underwent hysterotomy on day 17.5 and the nonconstrained pups were allowed to continue intra-abdominal fetal growth 48 hours beyond normal gestation. Expression of TGF-beta receptor types I and II, and FGFR2 in the calvarial tissue was determined by immunohistochemical analysis. In the unconstrained control animals, there was minimal immunoreactivity for both TGF-beta receptors and FGFR2 within the coronal suture. After 24 hours of constraint, however, there was a marked increase in immunoreactivity of TGF-beta receptors and FGFR2 in the osteoblasts along the osteogenic fronts and in the dural cells. After 48 hours, there was continued expression of both type I and type II receptors and FGFR2 within the midsutural mesenchyme of the coronal suture, in the osteoblasts, and in the dura. The authors demonstrated substantial upregulation of TGF-beta receptor types I and II and FGFR2 in coronal sutures subjected to in utero constraint. These results suggest an important role for TGF-beta/TGF-beta receptor, and FGF/FGFR signaling in the pathogenesis of constraint-induced craniosynostosis.     

Animal models of craniosynostosis

BackgroundVarious animal models mimicking craniosynostosis have been developed, using mutant zebrafish and mouse. The aim of this paper is to review the different animal models for syndromic craniosynostosis and analyze what insights they have provided in our understanding of the pathophysiology of these conditions.Material and methodsThe relevant literature for animal models of craniosynostosis was reviewed.ResultsAlthough few studies on craniosynostosis using zebrafish were published, this model appears useful in studying the suture formation mechanisms conserved across vertebrates. Conversely, several mouse models have been generated for the most common syndromic craniosynostoses, associated with mutations in FGFR1, FGFR2, FGFR3 and TWIST genes and also in MSX2, EFFNA, GLI3, FREM1, FGF3/4 genes. The mouse models have also been used to test pharmacological treatments to restore craniofacial growth.ConclusionsSeveral zebrafish and mouse models have been developed in recent decades. These animal models have been helpful for our understanding of normal and pathological craniofacial growth. Mouse models mimicking craniosynostoses can be easily used for the screening of drugs as therapeutic candidates.     

A Pro253Arg mutation in fibroblast growth factor receptor 2 (Fgfr2) causes skeleton malformation mimicking human Apert syndrome by affecting both chondrogenesis and osteogenesis

Apert syndrome is one of the most severe craniosynostosis that is mainly caused by either a Ser252Trp(S252W) or Pro253Arg(P253R) mutation in fibroblast growth factor receptor 2 (FGFR2). As an autosomal dominant disorder, Apert syndrome is mainly characterized by skull malformation resulting from premature fusion of craniofacial sutures, as well as syndactyly, etc. A P253R mutation of FGFR2 results in nearly one-thirds of the cases of Apert syndrome. The pathogenesis of Apert syndrome resulting from P253R mutation of FGFR2 is still not fully understood. Here we reported a knock-in mouse model carrying P253R mutation in Fgfr2. The mutant mice exhibit smaller body size and brachycephaly. Analysis of the mutant skulls and long bones revealed premature fusion of coronal suture, shortened cranial base and growth plates of long bones. In vitro organ culture studies further revealed that, compared with wild-type littermates, the mutant mice have prematurely fused coronal sutures and retarded long bone growth. Treatment of the cultured calvaria and femur with PD98059, an Erk1/2 inhibitor, resulted in partially alleviated coronal suture fusion and growth retardation of femur respectively. Our data indicated that the P253R mutation in Fgfr2 directly affect intramembranous and endochondral ossification, which resulted in the premature closure of coronal sutures and growth retardation of long bones and cranial base. And the Erk1/2 signaling pathway partially mediated the effects of P253R mutation of Fgfr2 on cranial sutures and long bones.     

Rap1b Is an Effector of Axin2 Regulating Crosstalk of Signaling Pathways During Skeletal Development

Recent identification and isolation of suture stem cells capable of long‐term self‐renewal, clonal expanding, and differentiating demonstrate their essential role in calvarial bone development, homeostasis, and injury repair. These bona fide stem cells express a high level of Axin2 and are able to mediate bone regeneration and repair in a cell autonomous fashion. The importance of Axin2 is further demonstrated by its genetic inactivation in mice causing skeletal deformities resembling craniosynostosis in humans. The fate determination and subsequent differentiation of Axin2+ stem cells are highly orchestrated by a variety of evolutionary conserved signaling pathways including Wnt, FGF, and BMP. These signals are often antagonistic of each other and possess differential effects on osteogenic and chondrogenic cell types. However, the mechanisms underlying the interplay of these signaling transductions remain largely elusive. Here we identify Rap1b acting downstream of Axin2 as a signaling interrogator for FGF and BMP. Genetic analysis reveals that Rap1b is essential for development of craniofacial and body skeletons. Axin2 regulates Rap1b through modulation of canonical BMP signaling. The BMP‐mediated activation of Rap1b promotes chondrogenic fate and chondrogenesis. Furthermore, by inhibiting MAPK signaling, Rap1b mediates the antagonizing effect of BMP on FGF to repress osteoblast differentiation. Disruption of Rap1b in mice not only enhances osteoblast differentiation but also impairs chondrocyte differentiation during intramembranous and endochondral ossifications, respectively, leading to severe defects in craniofacial and body skeletons. Our findings reveal a dual role of Rap1b in development of the skeletogenic cell types. Rap1b is critical for balancing the signaling effects of BMP and FGF during skeletal development and disease. © 2017 American Society for Bone and Mineral Research.     

Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3

From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis.     

生长因子FGF在颅缝细胞培养中作用的研究

目的:研究生长因子FGF在颅缝闭合中的调控作用.方法:以颅缝早闭动物模型(SD鼠)的颅缝为研究模型,采用细胞生物学技术、组织化学技术,研究颅缝闭合期间,生长因子bFGF作用下,细胞胶原及骨钙素分泌情况;观察生长因子bFGF,对分离培养的颅缝细胞增殖与代谢影响.结果:在大鼠颅缝分离培养细胞中,bFGF促进颅缝分离培养细胞分泌胶原、骨钙素,加快细胞增殖生长曲线平台期出现,并有效促进大鼠颅缝分离培养细胞S期增殖(p＜0.05).结论:在体外器官培养中,bFGF能促进大鼠分离培养颅缝细胞的活性(p＜0.05).     

新致病基因在颅缝早闭Crouzon综合征中的初步机制研究

目的研究Rbp4(Retinol binding protein 4)、Gpc3(Glypican family of growth factor binding protein 3)、C1qtnf3(Collagenous repeat-containing sequence of 26 KDa protein)等新致病基因在颅缝早闭症中的发病机制,为疾病非手术治疗奠定理论基础。方法以Crouzon综合征Fgfr2cC342Y/+小鼠为实验模型,应用MicroCT和组织学染色,研究小鼠颅缝闭合模式;以Fgfr2cC342Y/+模型,RT-qPCR研究Rbp4、Gpc3、C1qtnf3等新致病基因的表达差异,初步探讨其在颅缝闭合过程中的调控作用;以体外培养的Fgfr2cC342Y/+小鼠颅缝细胞为模型,研究基因突变动物细胞增殖与代谢改变。结果获得Fgfr2cC342Y/+小鼠后额缝、冠状缝、人字缝、矢状缝等颅缝闭合模式,随颅骨发育、颅缝闭合,OC(Osteocalcin)、ALP(Alkalinephosphatase)表达增加,目的基因Rbp4、Gpc3、C1qtnf3表达下降,Msx2(Muscle segment homeobox gene 2)表达增加,杂合子与野生型小鼠之间均存在显著统计学差异,与前期人颅缝组织Microarray研究结果一致。Gpc1(Glypican family ofgrowth factor binding protein 1)、FliI(Flightless I)在颅缝闭合中的表达较恒定,野生型与杂合子之间未见明显统计学差异。体外培养Fgfr2cC342Y/+小鼠颅缝细胞,CellTiter96 MTS和Quant-iT Picogreen dsDNA细胞增殖与代谢分析结果显示,Fgfr2功能获得性突变可促进冠状缝细胞的增殖,从而导致成骨增加,颅缝早闭。结论 Fgfr2cC342Y/+模型新致病基因表达趋势与前期人颅缝组织Microarray研究结果一致,Rbp4、Gpc3、C1qtnf3可能在颅缝早闭中具重要调控作用。     

Cranial deformation in craniosynostosis. A new explanation.

Skull growth after premature fusion of a single suture was described by Virchow in 1851. He observed that growth was restricted in a plane perpendicular to a fused suture. However, he failed to predict the compensatory growth patterns that produce many of the deformities recognized as features of individual craniosynostosis syndromes. The deformities resulting from premature closure of a coronal, sagittal, metopic, or lambdoid suture can be predicted by the following observations: (1) cranial vault bones that are prematurely fused act as a single bone plate with decreased growth potential; (2) asymmetrical bone deposition occurs mainly at perimeter sutures, with increased bone deposition directed away from the bone plate; (3) sutures adjacent to the stenotic suture compensate in growth more than those sutures not contiguous with the closed suture; and (4) enhanced bone deposition occurs along both sides of a nonperimeter suture that is a continuation of the prematurely closed suture. These four rules were derived by critically examining the clinical deformities observed with each form of craniosynostosis. These rules assume that cranial sutures have the capacity to compensate by depositing bone asymmetrically along their edges. Unequal growth patterns have been demonstrated in the frontonasal suture of rabbits by Selman and Sarnat. In addition, unequal bone deposition has also been demonstrated along the parieto-interparietal suture in albino rats by Baer. Human studies to determine if asymmetrical bone deposition actively occurs along cranial vault sutures in response to a stenotic suture have not been performed, however. It is also unclear whether these four guidelines apply to cranial base abnormalities observed with craniosynostosis. As new radiologic techniques develop to define the configuration of the skull in intricate detail, a skull pattern of growth explaining the pathogenesis of all deformities created by premature fusion of a cranial vault suture may become apparent.     

Smad4‐Shh‐Nfic signaling cascade–mediated epithelial‐mesenchymal interaction is crucial in regulating tooth root development

Transforming growth factor β (TGF‐β)/bone morphogenetic protein (BMP) signaling is crucial for regulating epithelial‐mesenchymal interaction during organogenesis, and the canonical Smad pathway–mediated TGF‐β/BMP signaling plays important roles during development and disease. During tooth development, dental epithelial cells, known as Hertwig's epithelial root sheath (HERS), participate in root formation following crown development. However, the functional significance of HERS in regulating root development remains unknown. In this study we investigated the signaling mechanism of Smad4, the common Smad for TGF‐β/BMP signaling, in HERS in regulating root development. Tissue‐specific inactivation of Smad4 in HERS results in abnormal enamel and dentin formation in K14‐Cre;Smad4^fl/fl mice. HERS enlarges but cannot elongate to guide root development without Smad4. At the molecular level, Smad4‐mediated TGF‐β/BMP signaling is required for Shh expression in HERS and Nfic (nuclear factor Ic) expression in the cranial neural crest (CNC)‐derived dental mesenchyme. Nfic is crucial for root development, and loss of Nfic results in a CNC‐derived dentin defect similar to the one of K14‐Cre;Smad4^fl/fl mice. Significantly, we show that ectopic Shh induces Nfic expression in dental mesenchyme and partially rescues root development in K14‐Cre;Smad4^fl/fl mice. Taken together, our study has revealed an important signaling mechanism in which TGF‐β/BMP signaling relies on a Smad‐dependent mechanism in regulating Nfic expression via Shh signaling to control root development. The interaction between HERS and the CNC‐derived dental mesenchyme may guide the size, shape, and number of tooth roots. © 2010 American Society for Bone and Mineral Research     

Expression of high-affinity receptors for TGF-beta during rat cranial suture fusion.

The etiology of craniosynostosis is unknown. The elucidation of the biological pathways responsible for this disorder has been hampered by an inability to evaluate cranial sutures before, during, and after cranial suture fusion. The programmed fusion of the rat posterofrontal (PF) suture postnatally provides an excellent model to study the molecular events that occur during cranial suture fusion. Previous experiments have implicated transforming growth factor beta (TGF-beta) growth factors in the regulation of PF suture fusion. The purpose of these experiments was to localize the expression of high-affinity receptors for these growth factors during cranial suture fusion. Four rats were sacrificed on postnatal days 8, 12, 17, and 40 (N = 16). The PF and sagittal sutures were harvested and prepared for immunohistochemical localization of TGF-beta receptor 1 and receptor 2 (Tbeta-RI, Tbeta-RII) protein. Results indicate that immunostaining for Tbeta-RI and Tbeta-RII is markedly increased in the dura mater and osteoblasts of the sutural margin of the PF suture during active suture fusion (on postnatal days 12, 17, and 40) compared with the osteoblasts and dura mater underlying the patent sagittal suture. These results, in combination with the authors' previous findings as well as studies supporting a role for TGF-beta molecules in the regulation of osteogenesis, implicate TGF-beta signaling in the regulation of suture fusion. The possible mechanisms of ligand-receptor interaction are discussed.     

Enzyme replacement for craniofacial skeletal defects and craniosynostosis in murine hypophosphatasia

Hypophosphatasia (HPP) is an inborn-error-of-metabolism disorder characterized by deficient bone and tooth mineralization due to loss-of function mutations in the gene (Alpl) encoding tissue-nonspecific alkaline phosphatase (TNAP). Alpl^−/− mice exhibit many characteristics seen in infantile HPP including long bone and tooth defects, vitamin B6 responsive seizures and craniosynostosis. Previous reports demonstrated that a mineral-targeted form of TNAP rescues long bone, vertebral and tooth mineralization defects in Alpl^−/− mice. Here we report that enzyme replacement with mineral-targeted TNAP (asfotase-alfa) also prevents craniosynostosis (the premature fusion of cranial bones) and additional craniofacial skeletal abnormalities in Alpl^−/− mice. Craniosynostosis, cranial bone volume and density, and craniofacial shape abnormalities were assessed by microscopy, histology, digital caliper measurements and micro CT. We found that craniofacial shape defects, cranial bone mineralization and craniosynostosis were corrected in Alpl^−/− mice injected daily subcutaneously starting at birth with recombinant enzyme. Analysis of Alpl^−/− calvarial cells indicates that TNAP deficiency leads to aberrant osteoblastic gene expression and diminished proliferation. Some but not all of these cellular abnormalities were rescued by treatment with inorganic phosphate. These results confirm an essential role for TNAP in craniofacial skeletal development and demonstrate the efficacy of early postnatal mineral-targeted enzyme replacement for preventing craniofacial abnormalities including craniosynostosis in murine infantile HPP.     

Smad6 is essential to limit BMP signaling during cartilage development

Bone morphogenetic protein (BMP) signaling pathways regulate multiple aspects of endochondral bone formation. The importance of extracellular antagonists as regulators of BMP signaling has been defined. In vitro studies reveal that the intracellular regulators, inhibitory Smads 6 and 7, can regulate BMP‐mediated effects on chondrocytes. Although in vivo studies in which inhibitory Smads were overexpressed in cartilage have shown that inhibitory Smads have the potential to limit BMP signaling in vivo, the physiological relevance of inhibitory Smad activity in skeletal tissues is unknown. In this study, we have determined the role of Smad6 in endochondral bone formation. Loss of Smad6 in mice leads to defects in both axial and appendicular skeletal development. Specifically, Smad6?/? mice exhibit a posterior transformation of the seventh cervical vertebra, bilateral ossification centers in lumbar vertebrae, and bifid sternebrae due to incomplete sternal band fusion. Histological analysis of appendicular bones revealed delayed onset of hypertrophic differentiation and mineralization at midgestation in Smad6?/? mice. By late gestation, however, an expanded hypertrophic zone, associated with an increased pool of proliferating cells undergoing hypertrophy, was evident in Smad6 mutant growth plates. The mutant phenotype is attributed, at least in part, to increased BMP responsiveness in Smad6‐deficient chondrocytes. Overall, our results show that Smad6 is required to limit BMP signaling during endochondral bone formation. © 2011 American Society for Bone and Mineral Research     

A comprehensive screen of genes implicated in craniosynostosis

Recent advances in human molecular genetics have identified mutations in the TWIST, FGFR-1, FGFR-2 and FGFR-3 genes to be important causes of craniosynostosis. Despite this, however, mutations cannot be identified in the majority of patients. This study reports the first comprehensive screen of mutations in TWIST, FGFR-1, FGFR-2 and FGFR-3 genes in a cohort of patients with craniosynostosis. This has led to the identification of Saethre-Chotzen syndrome to be a new microdeletion disorder and reports the first example of a gene-environment interaction leading to craniosynostosis. In addition, investigation of the expression patterns of the Fgfr and Twist genes in the normal developing mouse coronal suture has identified the TWIST protein to be important in cranial suture initiation and biogenesis. These findings have significant clinical implications and will form the basis of future attempts to develop novel therapies aimed at inhibiting cranial suture fusion.     

A Ser252Trp [corrected] substitution in mouse fibroblast growth factor receptor 2 (Fgfr2) results in craniosynostosis

Apert syndrome (AS) is one of the most severe craniosynostoses and is characterized by premature fusion of craniofacial sutures. Mutations of either Ser252Trp or Pro253Arg in fibroblast growth factor receptor 2 (FGFR2) are responsible for nearly all known cases of AS. Here we show that mutant mice carrying the activation mutation, Ser252Trp [corrected] which corresponds to Ser252Trp in human FGFR2, have malformations mimicking the skull abnormalities found in AS patients. Mutant mice (Fgfr2(250/+)) are smaller in body size with brachycephaly and exhibit distorted skulls with widely spaced eyes. Unexpectedly, the premature closure of the coronal suture is accompanied by decreased, rather than increased, bone formation. We demonstrate that the Fgfr2-Ser252Trp [corrected] mutation does not cause obvious alterations in cell proliferation and differentiation; however, it results in increased Bax expression and apoptosis of osteogenic cells in mutant coronal suture. The accelerated cell death possibly reduces the space between osteogenic fronts of flat bones and results in the physical contact of these bones. Thus, our data reveal that dysregulated apoptosis plays an important role in the pathogenesis of AS related phenotypes.     

Further Analysis of the Crouzon Mouse: Effects of the FGFR2C342Y Mutation Are Cranial Bone–Dependent

Crouzon syndrome is a debilitating congenital disorder involving abnormal craniofacial skeletal development caused by mutations in fibroblast growth factor receptor-2 (FGFR2). Phenotypic expression in humans exhibits an autosomal dominant pattern that commonly involves premature fusion of the coronal suture (craniosynostosis) and severe midface hypoplasia. To further investigate the biologic mechanisms by which the Crouzon syndrome–associated FGFR2^C342Y mutation leads to abnormal craniofacial skeletal development, we created congenic BALB/c FGFR2^C342Y/+ mice. Here, we show that BALB/c FGFR2^C342Y/+ mice have a consistent craniofacial phenotype including partial fusion of the coronal and lambdoid sutures, intersphenoidal synchondrosis, and multiple facial bones, with minimal fusion of other craniofacial sutures. This phenotype is similar to the classic and less severe form of Crouzon syndrome that involves significant midface hypoplasia with limited craniosynostosis. Linear and morphometric analyses demonstrate that FGFR2^C342Y/+ mice on the BALB/c genetic background differ significantly in form and shape from their wild-type littermates and that in this genetic background the FGFR2^C342Y mutation preferentially affects some craniofacial bones and sutures over others. Analysis of cranial bone cells indicates that the FGFR2^C342Y mutation promotes aberrant osteoblast differentiation and increased apoptosis that is more severe in frontal than parietal bone cells. Additionally, FGFR2^C342Y/+ frontal, but not parietal, bones exhibit significantly diminished bone volume and density compared to wild-type mice. These results confirm that FGFR2-associated craniosynostosis occurs in association with diminished cranial bone tissue and may provide a potential biologic explanation for the clinical finding of phenotype consistency that exists between many Crouzon syndrome patients.     

Sustained BMP Signaling in Osteoblasts Stimulates Bone Formation by Promoting Angiogenesis and Osteoblast Differentiation

Angiogenesis and bone formation are tightly coupled during the formation of the skeleton. Bone morphogenetic protein (BMP) signaling is required for both bone development and angiogenesis. We recently identified endosome‐associated FYVE‐domain protein (endofin) as a Smad anchor for BMP receptor activation. Endofin contains a protein‐phosphatase pp1c binding domain, which negatively modulates BMP signals through dephosphorylation of the BMP type I receptor. A single point mutation of endofin (F872A) disrupts interaction between the catalytic subunit pp1c and sensitizes BMP signaling in vitro. To study the functional impact of this mutation in vivo, we targeted expression of an endofin (F872A) transgene to osteoblasts. Mice expressing this mutant transgene had increased levels of phosphorylated Smad1 in osteoblasts and showed increased bone formation. Trabecular bone volume was significantly increased in the transgenic mice compared with the wildtype littermates with corresponding increases in trabecular bone thickness and number. Interestingly, the transgenic mice also had a pronounced increase in the density of the bone vasculature measured using contrast‐enhanced μCT imaging of Microfil‐perfused bones. The vessel surface and volume were both increased in association with elevated levels of vascular endothelial growth factor (VEGF) in osteoblasts. Endothelial sprouting from the endofin (F872A) mutant embryonic metatarsals cultured ex vivo was increased compared with controls and was abolished by an addition of a VEGF neutralizing antibody. In conclusion, osteoblast targeted expression of a mutant endofin protein lacking the pp1c binding activity results in sustained signaling of the BMP type I receptor, which increases bone formation and skeletal angiogenesis.