Aberrantly activated Wnt/ß-catenin pathway co-receptors LRP5 and LRP6 regulate osteoblast differentiation in the developing coronal sutures of an Apert syndrome (Fgfr2S252W/+ ) mouse model.

BACKGROUND: Apert syndrome is an autosomal, dominant inherited disorder characterized by craniosynostosis and syndactyly caused by gain-of-function mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. Wnt/ß-catenin signaling plays critical roles in regulating the skeletal development. Here, we analyzed the role of this pathway in the developing coronal sutures (CS) of a murine Apert syndrome model (Fgfr2S252W/+ ). RESULTS: We observed aberrantly increased mRNA expression of Lrp5 and Lrp6 in CS of Fgfr2S252W/+ mice, whereas both wild type (WT) and Fgfr2S252W/+ mice showed similar expression of other Wnt/ß-catenin-related genes, such as Wnt3, Wnt3a, Fzd4, Fzd6, Axin2, and Dkk1 as evidenced by in situ hybridization. Significantly increased Lrp5 and Lrp6 mRNA expression was observed by quantitative PCR analysis of cultured cells isolated from CS of Fgfr2S252W/+ mice. Phospho-LRP5, phospho-LRP6, and non-phospho-ß-catenin were upregulated in Fgfr2S252W/+ CS compared with that in WT CS. Short-interfering RNA targeting Lrp5 and Lrp6 significantly reduced runt-related transcription factor 2, collagen type 1 alpha 1, and osteocalcin mRNA expression, and alkaline phosphatase activity in cultured cells. CONCLUSIONS: The Wnt/ß-catenin pathway was activated in the CS of Fgfr2S252W/+ mice during craniofacial development, suggesting the involvement of the Wnt/ß-catenin pathway in the pathogenesis of CS synostosis in Fgfr2S252W/+ mice.

Soluble form of FGFR2 with S252W partially prevents craniosynostosis of the apert mouse model.

BACKGROUND: Apert syndrome (AS) is characterized by craniosynostosis, midfacial hypoplasia, and bony syndactyly. It is an autosomal dominantly inherited disease caused by point mutations (S252W or P253R) in fibroblast growth factor receptor (FGFR) 2. These mutations cause activation of FGFR2 depending on ligand binding. Recently, an AS mouse model, Fgfr2(+/) (S252W) , showed phenotypes similar to those of AS patients. We previously reported that the soluble form of FGFR2(S252W) (sFGFR2IIIc(S252W) ) efficiently inhibits enhanced osteoblastic differentiation caused by FGFR2 activation in AS in vitro, presumably because FGFs binding to FGFRs is interrupted. In this study, we developed Fgfr2(+/) (S252W) (Ap) mice expressing the sFGFR2IIIc(S252W) protein, and we investigated the effects of sFGFR2IIIc(S252W) on AS-like phenotypes. RESULTS: In Ap mice, the coronal suture (CS) was fused prematurely at P1. In addition, the mice exhibited a widened interfrontal suture (IFS) with ectopic bone and thickened cartilage formation. In Fgfr2(+/) (S252W) sFGFR2IIIc(S252W) (Ap/Sol) mice, the CS was similar to that of wild-type mice. Ap/Sol mice did not show any ectopic bone or cartilage formation in the IFS, but showed a wider IFS than that of the wild-type mice. CONCLUSIONS: sFGFR2IIIc(S252W) may partially prevent craniosynostosis in the Apert mouse model by affecting the CS and IFS in vivo.

Craniofacial divergence by distinct prenatal growth patterns in Fgfr2 mutant mice.

BACKGROUND: Differences in cranial morphology arise due to changes in fundamental cell processes like migration, proliferation, differentiation and cell death driven by genetic programs. Signaling between fibroblast growth factors (FGFs) and their receptors (FGFRs) affect these processes during head development and mutations in FGFRs result in congenital diseases including FGFR-related craniosynostosis syndromes. Current research in model organisms focuses primarily on how these mutations change cell function local to sutures under the hypothesis that prematurely closing cranial sutures contribute to skull dysmorphogenesis. Though these studies have provided fundamentally important information contributing to the understanding of craniosynostosis conditions, knowledge of changes in cell function local to the sutures leave change in overall three-dimensional cranial morphology largely unexplained. Here we investigate growth of the skull in two inbred mouse models each carrying one of two gain-of-function mutations in FGFR2 on neighboring amino acids (S252W and P253R) that in humans cause Apert syndrome, one of the most severe FGFR-related craniosynostosis syndromes. We examine late embryonic skull development and suture patency in Fgfr2 Apert syndrome mice between embryonic day 17.5 and birth and quantify the effects of these mutations on 3D skull morphology, suture patency and growth. RESULTS: We show in mice what studies in humans can only infer: specific cranial growth deviations occur prenatally and worsen with time in organisms carrying these FGFR2 mutations. We demonstrate that: 1) distinct skull morphologies of each mutation group are established by E17.5; 2) cranial suture patency patterns differ between mice carrying these mutations and their unaffected littermates; 3) the prenatal skull grows differently in each mutation group; and 4) unique Fgfr2-related cranial morphologies are exacerbated by late embryonic growth patterns. CONCLUSIONS: Our analysis of mutation-driven changes in cranial growth provides a previously missing piece of knowledge necessary for explaining variation in emergent cranial morphologies and may ultimately be helpful in managing human cases carrying these same mutations. This information is critical to the understanding of craniofacial development, disease and evolution and may contribute to the evaluation of incipient therapeutic strategies.

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).

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.

The developing mouse coronal suture at single-cell resolution.

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.

Early onset of craniosynostosis in an Apert mouse model reveals critical features of this pathology.

Activating mutations of FGFRs1-3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model. We find that the critical event of CS is the early loss of basal sutural mesenchyme as the osteogenic fronts, expressing activated Fgfr2, unite to form a contiguous skeletogenic membrane. A mild increase in osteoprogenitor proliferation precedes but does not accompany this event, and apoptosis is insignificant. On the other hand, the more apical coronal suture initially forms appropriately but then undergoes fusion, albeit at a slower rate, accompanied by a significant decrease in osteoprogenitor proliferation, and increased osteoblast maturation. Apoptosis now accompanies fusion, but is restricted to bone fronts in contact with one another. We correlated these in vivo observations with the intrinsic effects of the activated Fgfr2 S252W mutation in primary osteoblasts in culture, which show an increased capacity for both proliferation and differentiation. Our studies suggest that the major determinant of Fgfr2-induced craniosynostosis is the failure to respond to signals that would halt the recruitment or the advancement of osteoprogenitor cells at the sites where sutures should normally form.

Increased expression of protein kinase Calpha, interleukin-1alpha, and RhoA guanosine 5'-triphosphatase in osteoblasts expressing the Ser252Trp fibroblast growth factor 2 receptor Apert mutation: identification by analysis of complementary DNA microarray.

Apert (Ap) syndrome is a craniofacial malformation characterized by premature fusion of cranial sutures (craniosynostosis). We previously showed that the Ser252Trp fibroblast growth factor receptor 2 (FGFR-2) mutation in Ap syndrome increases osteoblast differentiation and subperiosteal bone matrix formation, leading to premature calvaria ossification. In this study, we used the emerging technology of complementary DNA (cDNA) microarray to identify genes that are involved in osteoblast abnormalities induced by the Ser252Trp FGFR-2 mutation. To identify the signaling pathways involved in this syndrome, we used radioactively labeled cDNAs derived from two sources of cellular messenger RNAs (mRNAs) for hybridization: control (Co) and mutant Ap immortalized osteoblastic cells. Among genes that were differentially expressed, protein kinase Ca (PKC-alpha), interleukin-1alpha (IL-1alpha), and the small guanosine-5'-triphosphatase (GTPase) RhoA were increased in FGFR-2 mutant Ap cells compared with Co cells. The validity of the hybridization array was confirmed by Northern blot analysis using mRNAs derived from different cultures. Furthermore, immunochemical and Western blot analyses showed that mutant Ap cells displayed increased PKC-alpha, IL-1alpha, and RhoA protein levels compared with Co cells. Treatment of Co and Ap cells with the PKC inhibitor calphostin C decreased IL-1alpha and RhoA mRNA and protein levels in Ap cells, indicating that PKC is upstream of IL-1alpha and RhoA. Moreover, SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), and PD-98059, a specific inhibitor of MAPK kinase (MEKK), also reduced IL-1alpha and RhoA expression in Ap cells. These data show that the Ser252Trp FGFR-2 mutation in Ap syndrome induces constitutive overexpression of PKC-alpha, IL-1alpha, and small GTPase RhoA, suggesting a role for these effectors in osteoblast alterations induced by the mutation. The cDNA microarray technology appears to be a useful tool to gain information on abnormal gene expression and molecular pathways induced by genetic mutations in bone cells.

Negative autoregulation of fibroblast growth factor receptor 2 expression characterizing cranial development in cases of Apert (P253R mutation) and Pfeiffer (C278F mutation) syndromes and suggesting a basis for differences in their cranial phenotypes.

OBJECT: Heterogeneous mutations in the fibroblast growth factor receptor 2 gene (FGFR2) cause a range of craniosynostosis syndromes. The specificity of the Apert syndrome-affected cranial phenotype reflects its narrow mutational range: 98% of cases of Apert syndrome result from an Ser252Trp or Pro253Arg mutation in the immunoglobulin-like (Ig)IIIa extracellular subdomain of FGFR2. In contrast, a broad range of mutations throughout the extracellular domain of FGFR2 causes the overlapping cranial phenotypes of Pfeiffer and Crouzon syndromes and related craniofacial dysostoses. METHODS: In this paper the expression of FGFR1, the IgIIIa/c and IgIIIa/b isoforms of FGFR2, and FGFR3 is investigated in Apert syndrome (P253R mutation)- and Pfeiffer syndrome (C278F mutation)-affected fetal cranial tissue and is contrasted with healthy human control tissues. Both FGFR1 and FGFR3 are normally expressed in the differentiated osteoblasts of the periosteum and osteoid, in domains overlapped by that of FGFR2, which widely include preosseous cranial mesenchyme. Expression of FGFR2, however, is restricted to domains of advanced osseous differentiation in both Apert syndrome- and Pfeiffer syndrome-affected cranial skeletogenesis in the presence of fibroblast growth factor (FGF)2, but not in the presence of FGF4 or FGF7. Whereas expression of the FGFR2-IgIIIa/b (KGFR) isoform is restricted in normal human cranial osteogenesis, there is preliminary evidence that KGFR is ectopically expressed in Pfeiffer syndrome-affected cranial osteogenesis. CONCLUSIONS: Contraction of the FGFR2-IgIIIa/c (BEK) expression domain in cases of Apert syndrome- and Pfeiffer syndrome-affected fetal cranial ossification suggests that the mutant activation of this receptor, by ligand-dependent or ligand-independent means, results in negative autoregulation. This phenomenon, resulting from different mechanisms in the two syndromes, offers a model by which to explain differences in their cranial phenotypes.

Clinical variability in patients with Apert's syndrome.

OBJECT: Apert's syndrome is characterized by faciocraniosynostosis and severe bony and cutaneous syndactyly of all four limbs. The molecular basis for this syndrome appears remarkably specific: two adjacent amino acid substitutions (either S252W or P253R) occurring in the linking region between the second and third immunoglobulin domains of the fibroblast growth factor receptor (FGFR)2 gene. The goal of this study was to examine the phenotype/genotype correlations in patients with Apert's syndrome. METHODS: In the present study, 36 patients with Apert's syndrome were screened for genetic mutations. Mutations were detected in all cases. In one of the patients there was a rare mutation consisting of a double-base pair substitution in the same codon (S252F). A phenotypical survey of our cases was performed and showed the clinical variability of this syndrome. In two patients there was no clinical or radiological evidence of craniosynostosis. In two other patients with atypical forms of syndactyly and cranial abnormalities, the detection of a specific mutation was helpful in making the diagnosis. CONCLUSIONS: The P253R mutation appears to be associated with the more severe forms, with regard to the forms of syndactyly and to mental outcome. The fact that mutations found in patients with Apert' s syndrome are usually confined to a specific region of the FGFR2 exon IIIa may be useful in making the diagnosis and allowing genetic counseling in difficult cases.

The role of bone centers in the pathogenesis of craniosynostosis: an embryologic approach using CT measurements in isolated craniosynostosis and Apert and Crouzon syndromes.

This paper describes the role of the displacement of bone centers, i.e., the tubers, in the pathogenesis of craniosynostosis. This displacement was studied in 54 patients with isolated or syndromic craniosynostosis in the form of CT scans as well as in two dry neonate skulls with Apert syndrome. For comparison, 49 fetal and 8 normal infant dry skulls were studied. Our investigation was restricted to the coronal and metopic sutures. The results showed a significantly more occipital localization of the frontal bone center and a more frontal localization of the parietal bone center at the side of a synostotic coronal suture in the isolated form as well as in Apert syndrome. In contrast, this was not the case in Crouzon syndrome, thus showing that these two syndromes have a different pathogenesis. For trigonocephaly, a more anteromedial localization of the frontal bone centers was found.

Metopic suture in fetuses with Apert syndrome at 22-27 weeks of gestation.

OBJECTIVES: To examine the possible association of skull deformity and the development of the cranial sutures in fetuses with Apert syndrome. METHODS: Three-dimensional (3D) ultrasound was used to examine the metopic and coronal sutures in seven fetuses with Apert syndrome at 22-27 weeks of gestation. The gap between the frontal bones in the transverse plane of the head at the level of the cavum septi pellucidi was measured and compared to findings in 120 anatomically normal fetuses undergoing routine ultrasound examination at 16-32 weeks. RESULTS: In the normal group, the gap between the frontal bones in the metopic suture at the level of the cavum septi pellucidi, decreased significantly with gestation from a mean of 2.2 mm (5th and 95th centiles: 1.5 mm and 2.9 mm) at 16 weeks to 0.9 mm (5th and 95th centiles: 0.3 mm and 1.6 mm) at 32 weeks. In the seven cases with Apert syndrome, two-dimensional ultrasound examination demonstrated the characteristic features of frontal bossing, depressed nasal bridge and bilateral syndactyly. On 3D examination there was complete closure of the coronal suture and a wide gap in the metopic suture (15-23 mm). CONCLUSION: In normal fetuses, cranial bones are believed to grow in response to the centrifugal pressure from the expanding brain and proximity of the dura to the suture is critical in maintaining its patency. In Apert syndrome, the frontal bossing may be a mere consequence of a genetically predetermined premature closure of the coronal suture. Alternatively, there is a genetically predetermined deformation of the brain, which in turn, through differential stretch of the dura in the temporal and frontal regions, causes premature closure of the coronal suture and impaired closure of the metopic suture.

[Fetal ventilation and craniomaxillary development]. / Ventilation foetale et développement cranio-maxillaire.

Data acquired by means of color Doppler ultrasound very explicitly suggest what the role of the fetal ventilation and nasal capsules in the morphogenesis of the maxillary prognathism, turbinates, nasal valves and nasopharynx could be. Furthermore, the dysmorphologies observed in Apert or Crouzon craniosynostosis, achondroplasia or unilateral cleft lip would also testify that the influence of the fetal ventilatory dynamics goes beyond the limits of the face and extends to the cranial base and the cranium. The wealth of raised hypothesis thanks to the contribution of this imaging system could question the validity of some conceptions of the fetal craniomaxillary morphogenesis.

Apert's syndrome: cephalometric evaluation and considerations on pathogenesis.

Apert's syndrome is a malformation characterized by abnormalities in the cranial vault, midfacial malformations, and syndactylia. The present study analyzes the lateral and frontal projections of the teleradiograms from five patients. Data were taken on the skeletal features and an attempt was made to interpret them in terms of functional matrices. We have used cephalometry as a descriptive device although the intent was also to use it to provide a perspective on the pathogenetic data derived from the literature. The hypothesis analyzed is that from the outset of craniofacial pathogenesis there may be a primitive alteration of the cartilaginous template from which are derived endochondral bones. The progressive involvement of the synchondrosis of the cranial base is subsequently transmitted to the membranous structures of the vault and face through the coronal ring and lambdoid suture systems. Although the data gained do not actually confirm this hypothesis, they do provide further support for it.

The variable expressivity and incomplete penetrance of the twist-null heterozygous mouse phenotype resemble those of human Saethre-Chotzen syndrome.

Most targeted gene mutations are recessive and analyses of gene function often focus on homozygous mutant phenotypes. Here we describe parts of the expression pattern of M-twist in the head of developing wild-type mice and present our analysis of the phenotype of heterozygous twist- null animals at around birth and in adults. A number of twist -null heterozygous mice present skull and limb defects and, in addition, we observed other malformations, such as defects in middle ear formation and the xyphoïd process. Our study is of interest to understand bone formation and the role of M-twist during this process, as within the same animal growth of some bones can be accelerated while for others it can be delayed. Moreover, we show here that expressivity of the mouse mutant heterozygous phenotype is dependent on the genetic background. This information might also be helpful for clinicians, since molecular defects affecting one allele of the human H-twist ( TWIST ) gene were identified in patients affected with Saethre-Chotzen syndrome (SCS). Expressivity of this syndrome is variable, although most patients present craniofacial and limb malformations resembling those seen in mutant mice. Thus the mutant mouse twist -null strain might be a useful animal model for SCS. The twist -null mutant mouse model, combined with other mutant mouse strains, might also help in an understanding of the etiology of morphological abnormalities that appear in human patients affected by other syndromes.

Prenatal diagnosis of facial malformations.

In a prospective study, 5407 pregnant women were screened by ultrasound to detect malformations of the fetal face. Of a total of 11 facial anomalies, eight were detected by prenatal ultrasound (72 per cent). Three pregnancies were terminated because of associated developmental abnormalities or aneuploidy. In all of them, the facial malformations were correctly diagnosed. When associated with other developmental abnormalities, facial malformations were picked up at a rate of 100 per cent. Isolated facial malformations, by contrast, were detected in no more than 50 per cent of cases. Eight cases with suspected facial dysmorphism ended with the delivery of normal babies (specificity 99.8 per cent). None of them prompted karyotyping or any other invasive testing. Only two correctly detected facial malformations (bilateral cleft lips/palate) had a minor influence on obstetrical management. There would not have been disadvantages for the newborns in any of the cases if the malformations had been missed.