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. 2022 Nov 17;242(3):387–401. doi: 10.1111/joa.13790

Synchondrosis fusion contributes to the progression of postnatal craniofacial dysmorphology in syndromic craniosynostosis

Yukiko Hoshino 1,2, Masaki Takechi 1,3,✉, Mehran Moazen 4, Miranda Steacy 5, Daisuke Koyabu 1,6, Toshiko Furutera 1,3, Youichirou Ninomiya 7, Takashi Nuri 8, Erwin Pauws 5, Sachiko Iseki 1,✉
PMCID: PMC9919486  PMID: 36394990

Abstract

Syndromic craniosynostosis (CS) patients exhibit early, bony fusion of calvarial sutures and cranial synchondroses, resulting in craniofacial dysmorphology. In this study, we chronologically evaluated skull morphology change after abnormal fusion of the sutures and synchondroses in mouse models of syndromic CS for further understanding of the disease. We found fusion of the inter‐sphenoid synchondrosis (ISS) in Apert syndrome model mice (Fgfr2 S252W/+) around 3 weeks old as seen in Crouzon syndrome model mice (Fgfr2c C342Y/+). We then examined ontogenic trajectories of CS mouse models after 3 weeks of age using geometric morphometrics analyses. Antero‐ventral growth of the face was affected in Fgfr2 S252W/+ and Fgfr2c C342Y/+ mice, while Saethre–Chotzen syndrome model mice (Twist1 +/−) did not show the ISS fusion and exhibited a similar growth pattern to that of control littermates. Further analysis revealed that the coronal suture synostosis in the CS mouse models induces only the brachycephalic phenotype as a shared morphological feature. Although previous studies suggest that the fusion of the facial sutures during neonatal period is associated with midface hypoplasia, the present study suggests that the progressive postnatal fusion of the cranial synchondrosis also contributes to craniofacial dysmorphology in mouse models of syndromic CS. These morphological trajectories increase our understanding of the progression of syndromic CS skull growth.

Keywords: Apert syndrome, coronal suture, craniosynostosis, Crouzon syndrome, geometric morphometrics, inter‐sphenoid synchondrosis, midfacial hypoplasia, Saethre–Chotzen syndrome

We investigated ontogenic trajectories of the skull in postnatal CS mouse models using geometric morphometrics analyses. The synchondrosis fusion in the cranial base contributes to the postnatal morphological change of the skull in Apert and Crouzon syndrome model mice.

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1. INTRODUCTION

The skull vault is composed of several bony elements that are connected by the cranial sutures. The suture is a fibrous tissue formed by undifferentiated mesenchymal cells and osteoblast precursors (Beederman et al., 2014; Opperman, 2000). The cranial sutures of mammals allow temporary deformation during parturition and play a crucial role as growth sites of the developing skull (Flaherty et al., 2016). In humans, the majority of the cranial sutures are replaced with bony tissues in adulthood except for the metopic suture between the frontal bones that fuses around 2 years old (Cohen Jr., 2005). Synchondroses in the cranial base are also growth centers of craniofacial skeleton after birth (Krishan & Kanchan, 2013). The inter‐sphenoid synchondrosis (ISS) and the sphenoid‐occipital synchondrosis (SOS) are the main synchondroses present along the midline of the skull. In humans, the ISS begins to fuse before birth and completely ossifies by 2 or 3 years old (Hayashi, 2003; Madeline & Elster, 1995), whereas the SOS remains patent up to the adolescent, which indicates that the SOS contributes to postnatal craniofacial skeletal growth (Cendekiawan et al., 2010).

Craniosynostosis (CS) defined as premature fusion of one or more cranial suture(s) occurs approximately 1 in 2000–2500 newborns (Boulet et al., 2008). CS leads to morphological abnormalities, such as deformation of the cranial vault and facial asymmetry, accompanied by increased intracranial pressure and dysfunction of the brain (Slater et al., 2008). CS is classified into the non‐syndromic and the syndromic, the latter usually demonstrates the severe phenotypes (Flaherty et al., 2016). Mutations in fibroblast growth factor type 2 receptor (FGFR2) are associated with Apert syndrome characterized by CS with cranial, neural, limb, and visceral malformations (OMIM#101200). The majority of Apert syndrome patients carry one of two mutations associated with the change of Serine (Ser252Trp) and Proline (Pro253Arg) in exon IIIa that comprises the linker region between the second and the third extracellular immunoglobulin‐like domains of the ligand binding site (Wilkie et al., 1995). These mutations are gain‐of‐function types and alter the ligand‐binding affinity and specificity (Ibrahimi et al., 2001; Plotnikov et al., 2000), resulting in abnormal cell proliferation, differentiation, and migration (Ornitz & Marie, 2002). Anomalies and dysfunctions in Apert syndrome include midface hypoplasia, hypertelorism, syndactyly of hand and feet, and hearing loss (Lance & Governale, 2015). Crouzon syndrome is also caused by mutations in FGFR2, a missense mutation at Cysteine 342 in exon 9 (Cys342Tyr) is the most common in the disease, resulting in ligand‐independent receptor dimerization (Fenwick et al., 2014). Patients with Crouzon syndrome often exhibit hypertelorism and exophthalmos (OMIM#123500) (Lance & Governale, 2015). Haploinsufficiency of TWIST1 gene induces a syndromic CS, Saethre–Chotzen syndrome (OMIM #101400), with brachydactyly, facial dysmorphism and limb abnormalities (El Ghouzzi et al., 1997; Paznekas et al., 1998). In these syndromes, the fusion of the coronal suture is the main phenotype, and the fusion is initiated prenatally and commonly followed by the fusion of other cranial sutures postnatally (Lance & Governale, 2015). Regarding the cranial base in syndromic CS patients, both Apert and Crouzon syndrome patients exhibit earlier closure of the SOS compared to the matched controls, the closure begins around 2–3 years old (McGrath et al., 2012; Tahiri et al., 2014). Of note, the fusion has not been reported in Saethre–Chotzen syndrome patients.

CS mice are invaluable models to advance our understanding of syndromic CS in humans (Lee et al., 2019). One of the mouse models of Apert syndrome, Fgfr2 S252W/+ mouse, demonstrates premature craniosynostosis and skull malformation as well as abnormalities of internal organs (Figure 1b; Chen et al., 2003; Wang et al., 2005). About 80% of Fgfr2 S252W/+ mice show partial to complete fusion of the coronal suture at postnatal day (P) 0 (Motch Perrine et al., 2014). The Fgfr2c C342Y/+ mouse has been reported as a mouse model of Crouzon syndrome (Figure 1c), showing shortened face, protruding eyes, and premature fusion of the cranial sutures (Eswarakumar et al., 2004). Martínez‐Abadías et al. (2013) noted that over 95% of Fgfr2c C342Y/+ mutant mice at P0 exhibit partial to complete bilateral coronal suture fusion. Postnatal fusion of the ISS is also reported in the mutant with various genetic backgrounds (Liu et al., 2013). Twist1 heterozygous mice (Twist1 +/−) recapitulate the phenotype clinically observed in Saethre–Chotzen syndrome patients (El Ghouzzi et al., 1997). Twist1 +/− mice postnatally demonstrate the fusion of the coronal suture either in bilateral or unilateral (Figure 1d) in about 80% of the skulls by P15. On the other hand, the ISS remains patent up to the adult stage (Carver et al., 2002; Nuri et al., 2022; Parsons et al., 2014) as seen in wild‐type mice.

FIGURE 1.

FIGURE 1

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3D reconstructions of the skull in craniosynostosis mouse models at 3 weeks old and the cranial base in Fgfr2 S252W/+ mice and Fgfr2c C342Y/+ mice at 3 and 5 weeks old. Left lateral (top) and superior (bottom) views of wild type (a), Fgfr2 S252W/+ Apert syndrome mouse model (b), Fgfr2c C342Y/+ Crouzon syndrome mouse model (c), and Twist1 +/− Seathre‐Chotzen syndrome mouse model (d), respectively. Fusion of the coronal suture is apparent in each CS mouse model (white arrows in b–d) whereas the coronal suture of the wild‐type mouse keeps patency (white arrowhead in a). (e–l) 3D reconstructions of μCT images of cranial base at 3 (e, g, i, k) and 5 weeks old (f, h, j, l) of littermate controls of Fgfr2 S252W/+ mice (e, f), Fgfr2 S252W/+ mice (g, h), littermate controls of Fgfr2c C342Y/+ mice (i, j) and Fgfr2c C342Y/+ mice (k, l). White arrows and white arrowheads indicate the inter‐sphenoid synchondrosis (ISS) and the sphenoid‐occipital synchondrosis (SOS), respectively. (m–x) Hematoxylin–Eosin staining of sagittal sections of the cranial base at 5 weeks old in littermate controls of Fgfr2 S252W/+ mice (m, o, q), Fgfr2 S252W/+ mice (n, p, r), littermate controls of Fgfr2c C342Y/+ mice (s, u, w) and Fgfr2c C342Y/+ mice (t, v, x). White arrows and white arrowheads indicate the ISS and the SOS, respectively. High magnification of the ISS (o, p, u, v) and the SOS (q, r, w, x) indicated by hatched line boxes in (m), (n), (s) and (t) are shown. Black arrowheads in (p) and (v) indicate ossification of the ISS. bo, basioccipital bone; bs, basisphenoid bone; pl, palatine bone; ps, presphenoid bone; hz, hypertrophic zone; nc, nasal cavity; pz, proliferating zone; rz, resting zone. Scale bars: 1 mm (e), 200 μm (m), 20 μm (o), 50 μm (q).

Contrary to description of skull morphology by qualitative observation, quantitative analysis such as geometric morphometrics (Adams et al., 2004) provides a more accurate description and evaluation of craniofacial dysmorphogenesis, and these data may contribute to improved clinical diagnosis and subsequent therapeutic strategies. Cranial morphologies of Fgfr2 S252W/+ and Fgfr2 P253R/+ mice, Apert syndrome models, can be distinguished as early as P0 by principal component analysis (PCA) (Martínez‐Abadías et al., 2010). Growth Difference Matrix Analysis (GDMA) has revealed that mutant and non‐mutant mice display statistically different growth trajectories, and the difference becomes larger with age (Motch Perrine et al., 2014). A Crouzon mouse model with Fgfr2c C342Y/+ mutation at P0 shows significant features, expansion of height and width of the posterior region of the cranial vault, and the length of the anterior part of the facial skeleton including nasal bones shows shorter than wild‐type mice (Martínez‐Abadías et al., 2013). Parsons et al. (2014) reported that the skull morphology of Twist1 +/− mice at P15 is induced not simply by compensatory growth disturbances resulting from the coronal suture fusion. Although the fusion of the coronal suture is the main phenotype of the patients and the model mice, these observations suggest that morphological abnormalities are induced not only by the coronal suture fusion but also by other factors. Given that dysmorphology of the skull and the brain increases with age in CS patients (Breakey et al., 2018), ontogenic trajectories of the skull shape may be fundamental information for understanding the nature of the syndromic CS cases caused by a specific gene mutation.

Previous reports on CS mouse models using geometric morphometric analyses mainly revealed morphological features of prenatal or neonatal skull shape, the relation to brain development, and the interaction between craniofacial dysmorphogenesis and coronal suture patency. However, evaluation of skull morphology after abnormal fusion of cranial sutures and synchondroses in CS mouse models has not been reported. As described in the existing literature, the three CS mouse models (Fgfr2 S252W/+, Fgfr2c C342Y/+ and Twist1 +/−) usually exhibit closure of the coronal suture by 3 weeks old (Martínez‐Abadías et al., 2013; Motch Perrine et al., 2014; Nuri et al., 2022). In addition, Fgfr2c C342Y/+ mice have the fusion of the ISS by 4 weeks old (Liu et al., 2013) whereas wild‐type mice maintain patency of the ISS and the SOS up to the adult stage. In this study, we therefore investigated the growth pattern of each CS mouse model using geometric morphometric analysis focusing on the postweaning period, corresponding approximately to 2–3 years old onward in humans based on the skull growth (Libby et al., 2017; Wei et al., 2017).

2. MATERIALS AND METHODS

2.1. Mice

Twist1 +/− mice (Chen & Behringer, 1995) and Fgfr2 S252W/+ mice (Chen et al., 2003; Wang et al., 2005) were maintained on a C57Bl/6J background (Sankyo Labo Service Corporation, Inc.), and Fgfr2c C342Y/+ mice (Eswarakumar et al., 2004) were maintained on a CD‐1 genetic background. The sample sizes of Twist1 +/−, Fgfr2 S252W/+, Fgfr2c C342Y/+ and their littermate controls, which include either EIIa‐Cre or Fgfr2 ploxpneo‐S252W positive mice, for this study are shown in Table S1. In our Fgfr2 S252W/+ colony, only individuals without fusion of the coronal suture survived after 3 weeks old. Three types of mixed anesthetic agent (Kawai et al., 2011) were injected to each mouse before micro‐computed tomography (μCT) scanning. Antagonistic agent (Kawai et al., 2011) was injected at 3, 5, and 7 weeks old for awakening while euthanasia was carried out by cervical dislocation at 9 weeks old for Twist1 +/− and Fgfr2 S252W/+ mice. Fgfr2c C342Y/+ mice were euthanized using carbon dioxide and were kept in formaldehyde until they were μCT scanned. Animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (A2018‐048C, A2019‐060C3, A2021‐198C7) and approved by the UK Home Office and performed as part of a Project License (PP8161503) under the UK Animals (Scientific Procedures) Act 1986.

2.2. μCT scanning

μCT images of heads of Twist1 +/− mice and Fgfr2 S252W/+ mice were acquired using Inspexio SMX‐100CT (Shimadzu) with the X‐ray source at 50–100 tube kV, 30–50 μA tube current. The whole skull was first scanned with voxel size ranging 0.061–0.063 mm, and then the cranial base was additionally rescanned with 0.013–0.015 mm voxel sizes for further detailed observation. μCT images of heads of Fgfr2c C342Y/+ mice were acquired by X‐Tek HMX 160 (XTek Systems Ltd). The images had a voxel size of 0.02 mm. Three dimensional (3D) reconstruction of the skull was conducted using Avizo 6.3 (Visualization Sciences Group, RRID:SCR_014431). 3D surfaces were reconstructed with Isosurface function in Avizo 6.3.

2.3. Sample preparation for histological analysis

The cranial base of Fgfr2 S252W/+ and Fgfr2c C342Y/+ mice were dissected and fixed in 4% PFA, Bouin's solution, Carnoy's solution, or 70% ethanol, then demineralized in either 10% EDTA 2Na solution or 10% citric acid in 22.5% formic acid. The samples were embedded in paraffin after dehydration, and sagittal sections were obtained at 7–8 μm thickness for Hematoxylin–Eosin staining. The stained slides were cleared by xylene and applied to mounting agent for observation.

2.4. Landmark data collection and shape analysis

Landmark‐based geometric morphometrics were conducted to assess the growth pattern of each mouse model. 33 landmarks (Figure S1) were placed by Landmarks function in Avizo 6.3. Morpho J v1.07 (RRID:SCR_016483) was used for statistical analyses of acquired landmarks. All geometric morphometric analyses were applied to each genotype of mice regardless of sex because our preliminary analysis showed no significant sexual difference in the skull shape (data not shown). Generalized Procrustes analysis with superimposition was applied to normalize the size and align size‐free shape information of skull landmarks regardless of size (Klingenberg, 2011). Centroid size was computed for each sample as the square root of the total sums of distances between each landmark coordinate. In order to extract the group differences, canonical variates analysis (CVA), which maximizes the separation of defined groups such as genotypes and ages (Klingenberg, 2011), was applied to landmark configuration of Fgfr2 S252W/+, Fgfr2c C342Y/+, and Twist1 +/− mice, and littermate controls grouped by age and genotype. In order to assess the overall variation of samples and covariation of landmarks, PCA was applied to landmark configuration of all CS mouse models with coronal suture fusion. Allometric effect in skull shape was assessed by linear regressions between centroid sizes and CVA or PCA scores.

2.5. Statistics

Pearson's correlation and statistical analysis were performed by Excel (Microsoft, RRID:SCR_06137) or PRISM ver. 6.01 (GraphPad Software, RRID:SCR_005375). p values of <0.05 were considered significant in Student's t‐test.

3. RESULTS

3.1. Fgfr2 S252W /+ mice showed abnormalities of the synchondroses in the cranial base after weaning

It was previously reported that a Crouzon syndrome mouse model (Fgfr2c C342Y/+) and an Apert syndrome mouse model (Fgfr2 P253R/+) exhibit fusion of the ISS by 4 weeks old and P9, respectively (Liu et al., 2013; Yin et al., 2008). In contrast, the synchondroses appear normal at P18 in another Apert mouse model (Fgfr2 S252W/+) (Chen et al., 2003), therefore, we carefully observed the cranial base of Fgfr2 S252W/+ mice at later stages. The μCT images revealed that the ISS was partially fused in two out of five (2/5) and 5/5 Fgfr2 S252W/+ mice, at 3 and 5 weeks old, respectively (Figure 1g,h), while the littermate controls did not show the fusion (Figure 1e,f, white arrow). All Fgfr2 S252W/+ mice at 9 weeks old showed fusion of the ISS (4/4) while 3/4 littermate controls did not show the fusion (data not shown). In the histological section of the 5‐week‐old Fgfr2 S252W/+, there was bone tissue observed in the ventral part of the ISS (Figure 1p, black arrowheads), and the cartilage was protruding towards the intracranial space (Figure 1n). The SOS remained patent in both Fgfr2 S252W/+ mice and the littermate controls through the investigated period except for one Fgfr2 S252W/+ mouse (1/5) indicating the partial fusion (data not shown). However, the arrangement of chondrocytes in the SOS (the resting zone, the proliferating zone, and the hypertrophic zone), which was clearly recognized in the littermate controls (Figure 1q), seemed to be disturbed in the Fgfr2 S252W/+ (Figure 1r). We then investigated earlier stages, Fgfr2 S252W/+ mice demonstrated relatively normal ISS and SOS at P7 and P14. Although one Fgfr2 S252W/+ mouse (1/4) at P14 indicated minor protrusion and aberrant arrangement of the chondrocytes in the ISS (data not shown), almost all the histology did not show the difference between the mutants and the littermate controls (Figure 2c–f,i–l). All Fgfr2c C342Y/+ mice at 3 (3/3) and 5 weeks old (4/4) indicated the fused ISS (Figure 1k,l) while the littermate controls did not show the fusion (Figure 1i,j, white arrow). In the 5‐week‐old Fgfr2c C342Y/+, the ventral part of the ISS had bone tissue (Figure 1v, black arrowheads), and protrusion towards the brain side was also seen (Figure 1t), while the SOS maintained patency (Figure 1t,x). Histological sections revealed that Fgfr2c C342Y/+ mice at P7 and P14 had protrusion of the ISS (Figure 2n,p,t,v), which was never seen in the littermate controls (Figure 2m,o,s,u). All the Fgfr2c C342Y/+ mice at P7 and P14 indicated normal SOS (Figure 2r,x). We previously reported that Twist1 +/− mice do not demonstrate any fusion of the synchondroses until 8 weeks old (Nuri et al., 2022). Similarly, our present study also indicated no fusion up to 9 weeks old in Twist1 +/− mice (data not shown).

FIGURE 2.

FIGURE 2

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Hematoxylin–eosin staining of the cranial base in Fgfr2 S252W/+ mice and Fgfr2c C342Y/+ mice at P7 and P14. (a, b, g, h, m, n, s, t) hematoxylin–eosin staining of sagittal section of the cranial base at P7 and P14 in littermate controls of Fgfr2 S252W/+ mice (a, g), Fgfr2 S252W/+ mice (b, h), littermate controls of Fgfr2c C342Y/+ mice (m, s), Fgfr2 S252W/+ mice (s, t). High magnification of the ISS (c, d, i, j, o, p, u, v) and SOS (e, f, k, l, q, r, w, x) indicated by hatched line boxes in (a), (b), (g), (h), (m), (n), (s) and (t) are shown. Bo, basioccipital bone; bs, basisphenoid bone; pl, palatine bone; ps, presphenoid bone; hz, hypertrophic zone; nc, nasal cavity. Scale bars: 200 μm (a), 50 μm (c).

3.2. Growth patterns of skull morphology of three CS mouse models after weaning

We next investigated growth pattern of skull morphology of the mouse models by geometric morphometric analysis (Klingenberg, 2010, 2016) using the μCT images of skulls of each mouse model (Fgfr2 S252W/+, Fgfr2c C342Y/+, and Twist1 +/−; Figure 1b–d) from 3 to 6 or 9 weeks old. 33 landmarks were placed on the reconstructed neurocranium of each mouse model in Avizo 6.3 by Isosurface function (Figure S1, Table S2), then growth pattern of skull morphology was assessed by landmark‐based geometric morphometrics (Klingenberg, 2010, 2016). CVA was applied to coordinates of the 33 landmarks of each CS mouse model. Canonical variates provide linear combinations of the original variables that maximally separate a priori classified groups. In the present study, samples were classified according to the genotype and age prior to analyses. The detail of the sample size is shown in Table S1. Table 1 summarizes variance, correlation coefficient (r), and p‐value corresponding to each CS model. CVA showed clear separation between CS model mice and their control littermates along CV1 (Figures 3, 4, 5, scatter plot). CV1 of Fgfr2 S252W/+, Fgfr2c C342Y/+ and Twist1 +/− accounted for 74.3%, 83.5% and 84.3% of the total shape variation, respectively (Table 1). CV2 of Fgfr2 S252W/+, Fgfr2c C342Y/+, and Twist1 +/− explained 14.1%, 10.5% and 9.43% of the total shape variation, respectively (Table 1). In Fgfr2 S252W/+ and Fgfr2c C342Y/+ mice, both CV1 and CV2 expressed the morphological changes with age, while only CV2 expressed the morphological changes in Twist1 +/− mice. The distribution of scores along CV3 of Fgfr2 S252W/+ and Fgfr2c C342Y/+ as well as their littermate controls overlapped regardless of age, they were not clearly separated (Figures S2 and S3). CV1 scores and centroid sizes of individuals were positively correlated (Fgfr2 S252W/+: r = −0.866, p < 0.0001, Fgfr2c C342Y/+: r = 0.903, p < 0.0001, Twist1 +/−: r = −0.772, p < 0.0001), while other CV scores except CV2 of Twist1 +/− (r = 0.3320, p < 0.0001) did not show significant correlations to centroid sizes (Table 1). These results indicate that CV1 in each analysis summarizes size‐correlated skull shape variation. In other words, the clear separation between each mouse model and the littermate controls along CV1 is partly due to their size differences.

TABLE 1.

Results of CVA analysis for each three craniosynostosis mouse model

CVx CV1 CV2 CV3 CV4
Fgfr2 S252W/+
Variance (%) 74.3 14.1 4.91 2.96
Correlation coefficient (r) −0.866 0.355 0.125 −0.109
p‐value <0.0001 0.013 0.396 0.460
Fgfr2c C342Y/+
Variance (%) 83.5 10.5 3.18 1.60
Correlation coefficient (r) 0.903 0.357 0.022 −0.004
p‐value <0.0001 0.062 0.910 0.985
Twist1 +/−
Variance (%) 84.3 9.43 2.66
Correlation coefficient (r) −0.772 −0.481 −0.130
p‐value <0.0001 <0.0001 0.238
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Note: Bold values indicate statistically significant p‐value.

FIGURE 3.

FIGURE 3

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Scatter plot of CVA applied to the skulls of Fgfr2 S252W/+ mice and their littermate controls after the weaning period. Closed circles, opened square, closed triangle, and opened star indicate mice at 3, 5, 7 and 9 weeks old in Fgfr2 S252W/+ mice (magenta) and littermate controls (black), respectively. Red arrows indicate ontogenic trajectories of Fgfr2 S252W/+ mice and littermate controls. The wireframes with blue lines along the CV1 axis show morphological features of the lateral view (top) and the frontal view (bottom) of the positive extreme (right, CV1 = 15) and those of the negative extreme (left, CV1 = −9). The wireframes with blue lines along the CV2 axis show morphological features of the lateral view (right) and the frontal view (left) of the positive extreme (top, CV2 = 10) and those of the negative extreme (bottom, CV2 = −5). The gray dashed lines and landmarks indicate mean shape of all observed samples.

FIGURE 4.

FIGURE 4

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Scatter plot of CVA applied to the skulls of Fgfr2c C342Y/+ mice and their littermate controls after the weaning period. Closed circles, opened square, closed triangle and opened star indicate 3, 4, 5 and 6 weeks old in Fgfr2c C342Y/+ (pale green) and littermate controls (black), respectively. Red arrows indicate ontogenic trajectories of Fgfr2c C342Y/+ and littermate controls. The wireframes with blue lines along the CV1 axis show morphological features of the lateral view (top) and the frontal view (bottom) of the positive extreme (right, CV = 15) and those of the negative extreme (left, CV = −9). The wireframes with blue lines along the CV2 axis show morphological features of the lateral view (right) and the frontal view (left) of the positive extreme (top, CV = 5) and those of the negative extreme (bottom, CV = −10). The gray dashed lines and landmarks indicate mean shape of all observed samples.

FIGURE 5.

FIGURE 5

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Scatter plot of CVA applied to the skulls of Twist1 +/− mice and littermate controls after the weaning period. Closed circles, opened square, closed triangle, and opened star indicate mice at 3, 5, 7 and 9 weeks old in Twist1 +/− (blue) and wild‐type (black) mice, respectively. Red arrows indicate ontogenic trajectories of Twist1 +/− mice and littermate controls. The wireframes with blue lines along the CV1 axis show morphological features of the lateral view (top) and the frontal view (bottom) of the positive extreme (right, CV = 20) and those of the negative extreme (left, CV = −30). The wireframes with blue lines along the CV2 axis show morphological features of the lateral view (right) and the frontal view (left) of the positive extreme (top, CV = 20) and those of the negative extreme (bottom, CV = −10). The gray dashed lines and landmarks indicate mean shape of all observed samples.

3.2.1. Fgfr2 S252W /+ mice

The distribution of scores along CV1 was different between Fgf2 S252W/+ mice and their littermate controls. Fgf2 S252W/+ mice were mainly distributed in the positive area of CV1 and their littermate controls were localized in the negative area of CV1 (Figure 3, scatter plot). The wire frames with blue lines under the scatter plot indicate the morphological feature in negative/positive extreme of CV1, and those on the left side of the scatter plot are associated with CV2 (Figure 3, scatter plot). The morphological feature of Fgf2 S252W/+ mice exhibited taller and wider skull vaults (Figure 3, wireframes along the CV1 axis) relative to that of their littermate controls (Figure 3, wireframes along the CV1 axis). This brachycephalic phenotype was contributed by displacement of the frontal and parietal bones dorsally and bilateral expansion of the skull vault (Figure 3, wireframes along the CV1 axis). Furthermore, the facial area was greatly modified, the zygomatic arch shifted ventrally while the anterior part of the frontal process and the most anterior point of the first molar shifted dorsally (Figure 3, wireframes along the CV1 axis). Their growth trajectory was different from that of their littermate controls; the morphological changes with growth of Fgf2 S252W/+ mice were mainly indicated by CV2 rather than CV1, and the extent of change from 3 to 9 weeks old along CV2 is greater in Fgf2 S252W/+ mice than littermate controls (Figure 3, scatter plot). The posterior region of the calvarium became more flattened along growth (Figure 3, wireframes along the CV2 axis), while the anteroventral extension of the facial area was relatively restricted in Fgf2 S252W/+ mice (Figure 3, wireframes along the CV1 axis).

3.2.2. Fgfr2c C342Y /+ mice

The scores of Fgfr2c C342Y/+ mice were distributed in the positive area of CV1 (Figure 4, scatter plot), they showed brachycephalic phenotypes similar to Fgfr2 S252W/+ mice (Figures 3 and 4, wireframes along the CV1 axis). The dorsal displacement of the parietal and interparietal bones mainly contributed to the morphological feature of Fgfr2c C342Y/+ mice (Figure 4, wireframes along the CV1 axis). Furthermore, the posterior part of the skull vault, especially at the intersection of the temporal bone and the interparietal bone, showed marked expansion in width (Figure 4, wireframes along the CV1 axis). The foramen magnum of Fgfr2c C342Y/+ mice was larger than that of their littermate controls (Figure 4, wireframes along the CV1 axis). In the facial area, they showed shorter zygomatic arch and the anterior displacement of the zygomatic process of the temporal bone (Figure 4, wireframes along the CV1 axis). The anterior part of the palate was shifted dorsally similar to Fgfr2 S252W/+ mice (Figures 3 and 4, wireframes along the CV1 axis). The ontogenic trajectory of Fgfr2c C342Y/+ mice was similar to that of Fgfr2 S252W/+ mice, which is reflected in CV2 (Figures 3 and 4, scatter plot); the anterior region of the calvarium became more flattened along growth (Figure 4, wireframes along the CV2 axis), while the anteroventral extension of the facial area was relatively restricted (Figure 4, wireframes along the CV1 axis).

3.2.3. Twist1 +/− mice

We then investigated Twist1 +/− mice that do not display fusion of the ISS but show coronal suture synostosis around the weaning period. The distribution of scores along CV1 was different between Twist1 +/− mice and their wild‐type littermates while scores of each age group were separated along CV2 (Figure 5, scatter plot). The scores of Twist1 +/− mice were distributed in positive area of CV1 (Figure 5, scatter plot). Twist1 +/− mice showed brachycephalic phenotype as seen in Fgf2 S252W/+ and Fgfr2c C342Y/+ mice (Figures 3, 4, 5, wireframes along the CV1 axis). Twist1 +/− mice were also characterized by the dysmorphogenesis of the zygomatic arch (Figure 5, wireframes along the CV1 axis). The zygomatic process of the temporal bone was shifted anteriorly, which was also observed in Fgfr2c C342Y/+ mice (Figures 4 and 5, wireframes along the CV1 axis). On the other hand, ontogenetic trajectory of Twist1 +/− mice was obviously different from those of Fgf2 S252W/+ and Fgfr2c C342Y/+ mice (Figures 3, 4, 5, scatter plot). Both the littermate controls and Twist1 +/− mice were plotted parallel to the CV2 axis, indicating that CV2 reflects the normal growth pattern of the littermate controls (Figure 5, scatter plot). Taking into account that CV2 scores and centroid sizes of individuals were positively correlated, the differences of the skull shape along CV2 axis are partly due to their sizes (Table 1). The calvarium becomes flattened and the facial area extends anteriorly with age (Figure 5, wireframes along the CV2 axis). While the trajectories along CV2 of Twist1 +/− mice and the littermate controls were comparable, the extent of change is different. In particular, the extent from 3 to 5 weeks old was much greater in the littermate controls than Twist1 +/− mice (Figure 5, scatter plot).

3.3. No obvious shared features were detected among CS mouse models characterized by coronal suture fusion

Since the three CS mouse models share features of coronal suture synostosis in the early stage of their life and consequent skull dysmorphogenesis, we further investigated the extent that the fusion of the coronal suture contributes to the skull shape using geometric morphometric analysis. The sample size of each group and the basic statistics of PCA are shown in Table 2. PC1 scores and centroid sizes of individuals were positively correlated (r = 0.6999, p < 0.0001) while other PC scores, excluding the PC3 score (r = −0.625, p < 0.0001), did not show significant correlations to centroid sizes (Table 2). Despite the three CS mouse models being on different genetic backgrounds (Fgfr2 S252W/+ and Twist1 +/− mice are on C57Bl/6J while Fgfr2c C342Y/+ mice is on CD1), PC scores of the littermate controls of each model overlapped with each other (Figure 6). The PC scores of littermate controls and those of the three CS mutant mice separated along the PC1 axis. Considering PC1 scores and centroid sizes of individuals were positively correlated, the differences of PC scores of three CS mouse models and those of the littermate controls along the PC1 axis are partly due to their size differences (Table 2). Other PC scores of the three CS mouse models did not overlap each other along any PC axis (Figure S4). These results indicate that although the CS mouse models commonly show a brachycephalic phenotype, they do not have shared morphological features associated with coronal suture synostosis.

TABLE 2.

Sample size and result of PCA analysis for three craniosynostosis mouse models

Sample size PCx PC1 PC2 PC3 PC4 PC5 PC6

Fgfr2 S252W/+: 3

Fgfr2 +/+(S252W): 32

Fgfr2c +/C342Y: 12

Fgfr2c +/+(C342Y): 11

Twist1 +/: 52

Twist1 +/+: 33

 Variance (%) 41.1 12.5 8.17 4.42 3.01 2.89
Correlation coefficient (r) 0.699 −0.081 −0.625 0.001 −0.020 −0.069
p‐value <0.0001 0.334 <0.0001 0.991 0.812 0.412
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Note: Bold values indicate statistically significant p‐value.

FIGURE 6.

FIGURE 6

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Scatter plot of PCA applied to the skulls of three CS models with synostosis of the coronal suture and their littermate controls. Closed circles with blue, magenta and pale green represent Twist1 +/−, Fgfr2 S252W/+, and Fgfr2c C342Y/+ mice, respectively. Opened circles represent littermate controls of each mouse model.

4. DISCUSSION

Fgfr2 S252W/+, Fgfr2c C342Y/+, and Twist1 +/− mice have been used for revealing molecular mechanisms of syndromic CS and further understanding of the pathogenesis in CS patients (Chen et al., 2003; El Ghouzzi et al., 1997; Eswarakumar et al., 2004; Wang et al., 2005). In particular, the correlation between suture patency and craniofacial dysmorphology has been investigated by various strategies (Carver et al., 2002; Motch Perrine et al., 2014; Parsons et al., 2014). The growth pattern of the skull during perinatal stage by geometric morphometric analysis has been of long interests for researchers and clinicians working on craniosynostosis (Motch Perrine et al., 2014). However, long‐term data of postnatal skull growth of CS mouse models have not yet been analyzed. To the best of our knowledge, the present study is the first report that investigated ontogenic trajectories of CS mouse models during fusion of the cranial synchondrosis and the coronal suture using a landmark‐based geometric morphometric analysis of the skull. Furthermore, we examined whether coronal suture synostosis causes any shared morphological features among three CS mouse models.

The craniofacial growth pattern of Fgfr2 S252W/+ and Fgfr2c C342Y/+ mice was successfully extracted using CVA. CV1 reflected the anteroventral extension of the facial area and flattening of calvaria while CV2 reflected the overall shape of the calvaria (Figures 3 and 4). The growth pattern of Fgfr2 S252W/+ and Fgfr2c C342Y/+ mice was different from their littermate controls (Figures 3 and 4, scatter plot). The covariation among CV1 suggests that Fgf2 S252W/+ and Fgfr2c C342Y/+ mice have difficulties in extending the facial area anteroventrally while their calvaria become flattened after weaning (Figures 3 and 4, wireframes along the CV1 and CV2 axis).

Considering the anatomical position and the role of craniofacial sutures and the synchondroses, it is reasonable to presume that the patency of facial sutures and the cranial synchondroses are key elements for the growth of the facial area. Purushothaman et al. (2011) reports that neonatal Fgf2 S252W/+ mice with abnormal synostosis of facial sutures, such as the premaxillary‐maxillary suture and the zygomatic arch, exhibit midface hypoplasia without fusion of the cranial synchondroses. On the other hand, Pfeiffer syndrome patients, who carry autosomal dominant mutations in FGFR1 or FGFR2, show strong correlation between midface hypoplasia and premature SOS closure (Paliga et al., 2014). Apert and Crouzon syndrome patients also show earlier fusion of the SOS (McGrath et al., 2012; Tahiri et al., 2014). Although early ossification of the ISS in Fgfr2c C342Y/+ and Fgfr2 P253R/+ mice has been reported (Liu et al., 2013; Moazen et al., 2022; Yin et al., 2008), the detail in Fgfr2 S252W/+ mice has not been sufficiently studied. In this study, we identified fusion of the ISS in Fgfr2 S252W/+ mice occurs later than 3 weeks old (Figures 1 and 2), as seen in Fgfr2c C342Y/+ mice (Eswarakumar et al., 2004; Liu et al., 2013, this study). Of contrast, Twist1 +/− mice maintain patency of the ISS and show the similar facial growth to the littermate controls (Figure 5, wireframes along the CV2 axis).

The fusion process of the cranial synchondroses in the mutants remains to be elucidated. Among the Fgfr2 mRNA isoforms, Fgfr2b and Fgfr2c are transcribed in the perichondria, and the proliferative and the resting zone of the chondrocytes in the cranial base (Rice et al., 2003). A recent study showed that expression of Fgfr2 with an Apert mutation in chondrocytes accelerates maturation and hypertrophy of the chondrocytes, resulting in ossification of the synchondroses in the cranial base (Nagata et al., 2011). The ossification wave seems to break into the SOS from the edges of bone forming region of the sphenoid or the occipital bones (Nagata et al., 2011). In our study, we observed the bone at the ventral side of the ISS bridging the presphenoid and the basisphenoid bones in Fgfr2 S252W/+ and Fgfr2c C342Y/+ mice (Figure 1). These observations are consistent with the finding that the secondary ossification center is absent in the cranial base (Wei et al., 2016). We found that a few Fgfr2 mutant mice at P7 and P14 demonstrated protrusion of the ISS without mineralization. These ISS exhibited slight aberrant arrangement of the chondrocytes (Figure 2p,v). Therefore, it is possible that a primary defect of the chondrocytes in the ISS induced morphological disturbance of the cartilage in the Fgfr2 mutant mice. In addition, the morphology of the chondrocytes may be further exacerbated by the pressure resulting from ossification of the ventral side of the ISS. Another possibility is that the pressure induced by the fusion of the facial sutures in Fgfr2 S252W/+ and Fgfr2c C342Y/+ mice (Purushothaman et al., 2011: Motch Perrine et al., 2014; Martínez‐Abadías et al., 2013) brought the mechanical force resulting in protrusion and disturbed morphology of the chondrocytes in the ISS of Fgfr2 mutant mice.

Fgfr2 S252W/+ mice are previously shown to have the fusion of sutures in the zygomatic‐maxillary, premaxillary‐maxillary, and fronto‐maxillary bones at P0 (Motch Perrine et al., 2014; Purushothaman et al., 2011). Some of Fgfr2c C342Y/+ mice also show fusion of the zygomatic‐maxillary bones (Martínez‐Abadías et al., 2013). Furthermore, in neonatal Fgfr2 S252W/+ mice, the angle of the presphenoid to the horizon plane is larger than that in control mice, and the anterior part of the presphenoid bone in Fgfr2 S252W/+ mice raises up the premaxilla bone (Kim et al., 2020; Martínez‐Abadías et al., 2010), which was presumed to result from an increased intracranial pressure (Connolly et al., 2004; Kreiborg et al., 1993). Those premature fusion of the facial sutures and dysmorphology of the cranial base precedes the fusion of the ISS. Therefore, these factors may also contribute to the characteristics of craniofacial morphology of the mutant mice detected in this study. However, since the length of the cranial base continues to grow until around 8 weeks old in normal condition (Vora et al., 2016), the fusion of the ISS, occurring around 3 weeks of age in the mutants, could contribute to the midface hypoplasia at least after this age, which is indicated by CV1 and CV2 in this study (Figures 3 and 4).

To our surprise, Twist1 +/− mice showed a similar growth pattern to that of littermate controls contrary to the Fgfr2 mutant mice. Our data demonstrated that growth pattern of the skull shape (CV2 in Figure 5) is not largely affected by the synostosis of the coronal suture. Both Twist1 +/− mice and their littermate controls continue to grow into a more flattened calvarium and anteriorly extended the facial area (Figure 5, wireframes along the CV2 axis). Twist1 plays an important role in mesenchymal cells fate, especially in differentiation from mesenchymal stem cells into osteoblasts, chondrocytes, or adipocytes (Miraoui & Marie, 2010) and functions in maintenance of the suture through regulation of FGFR2 and RUNX2 (Connerney et al., 2006; Johnson et al., 2000). Furthermore, Twist1 regulates proliferation, differentiation and cell death of osteoblasts (Miraoui & Marie, 2010). It has been suggested that the growth of the brain affects skull morphology and vice versa through developmentally indispensable signaling (Hill et al., 2013; Richtsmeier & Flaherty, 2013). Yu et al. (2021) reported that regeneration of the coronal suture at P14 leads to a normalized skull shape, intracranial pressure, and cognitive function to Twist1 +/− mice, indicating that the growth of the brain is potentially normal in Twist1 +/− mice. In contrast, Fgfr2 is expressed in developing brain (Wilke et al., 1997), and the skull growth of the Fgfr2 S252W/+ and the Fgfr2c C342Y/+ mice seems to be affected by brain (Motch Perrine et al., 2017). Of note, timing of the morphological change of Twist1 +/− mice was different from that of the littermate controls (Figure 5, scatter plot). The relative skull height of the littermate controls rapidly decreases, which is consistent with the previous report (Wei et al., 2017). In contrast, Twist1 +/− mice exhibit accelerated growth in the skull height until 3 weeks old (also see Nuri et al., 2022), and the skull gradually flattened (Figure 5, scatter plot and wireframes along the CV2 axis), probably due to the synostosis of the coronal suture at early stage. Based on these data, although the growth pattern of the skull shape in Twist1 +/− mice is relatively similar to that in littermate controls, the fusion of the coronal suture seems to delay the change of skull growth pattern in Twist1 +/− mice. In other words, Twist1 has an important role in the early phase of skull development, while abnormalities in the skull of Twist1 +/− mice after weaning is attributed to the premature fusion of the coronal suture, but not decreased function of this gene. Although the expression of Twist1 in mouse embryos and fetuses is detected in many tissues (Barnes & Firulli, 2009; Bourgeois et al., 1998), it is not clear whether this gene is expressed in craniofacial regions including the brain during the postnatal period. These data may give some insights into the role of Twist1 after birth and the treatment plan for Saethre–Chotzen syndrome patients.

In the suture mesenchyme, the TWIST1 homodimer functions as a FGFR2 agonist in cells of the osteogenic front while the TWIST1 heterodimer acts as an antagonist of FGFR2 (Connerney et al., 2006) and downregulates BMP signaling, which are downstream of the FGFR2 signaling pathway (Connerney et al., 2008). However, in the cranial base, the role of Twist1 has been elusive. Twist1 +/− mice showed accelerated tether thickening at the SOS between P25 and P30 compared to the control mice while thickness, but volume and tether number of the SOS was not different from the control (Hermann et al., 2012). In addition, Twist1 +/− mice have no fusion in the ISS and the SOS until 9 weeks old (Nuri et al., 2022, this study). Twist1 +/− mice and Fgfr2 mutant mice decisively differ in the growth pattern of the skull, which is mainly at the facial area, and the difference seems to be partially caused by fusion of the ISS of the cranial base.

We also tried to detect shared morphological features among three CS mouse models based on the coronal suture fusion. The brachycephalic phenotype characterized by higher, wider, and shorter dimensions in the anteroposterior direction of the neurocranium was found by the wireframes, indicating morphological features of those CS mouse models, in common (Figures 3, 4, 5, wireframes along the CV1 axis). This phenotype is consistent with prenatal and postnatal CS models (El Ghouzzi et al., 1997; Eswarakumar et al., 2004; Marghoub et al., 2018; Martínez‐Abadías et al., 2010; Parsons et al., 2014; Wang et al., 2005) and CS patients of Saethre–Chotzen, Apert, and Crouzon syndromes (Flaherty et al., 2016). However, strict overlaps of distribution on CS mouse models were not detected by PCA for all CS models (Figure 6, Figure S4) while the distribution of each littermate control group of the three CS mouse models being on different genetic backgrounds (Fgfr2 S252W/+ and Twist1 +/− mice are on C57Bl/6J while Fgfr2c C342Y/+ mice is on CD1) were overlapped each other (Figure 6). These results obtained by geometric morphometric analysis indicate that although these CS mouse models commonly show a brachycephalic phenotype, they do not have other shared features. These data corroborate the previous reports which suggest that craniofacial dysmorphology found in CS mouse models is not induced simply by early fusion of the coronal suture (Martínez‐Abadías et al., 2010; Nagata et al., 2011; Parsons et al., 2014). Considering that Twist1 and Fgfr2 regulate not only the coronal suture but also patency of other sutures (Connerney et al., 2008; Martínez‐Abadías et al., 2013; Motch Perrine et al., 2014) and various biological phenomenon (Miraoui & Marie, 2010; Ornitz & Itoh, 2015; Peskett et al., 2017), it is reasonable to think that an orchestration of those functions brings craniofacial dysmorphology.

In this study, we analyzed ontogenic trajectories after weaning in three syndromic CS mouse models using geometric morphometrics. Our main finding is that the skull base phenotype, which occur after loss of patency in the coronal and facial sutures, contributes to the craniofacial phenotype progression in Fgfr2 mutant mice. The detailed characterization of morphological changes in a long‐term period can provide the fundamental information for revealing the pathologic progression and improving treatment of syndromic CS patients.

AUTHOR CONTRIBUTIONS

This project was conceived by Sachiko Iseki, Masaki Takechi, Youichirou Ninomiya and Takashi Nuri. The data collection of μCT scanning images of Fgfr2 S252W/+ and Twist1 +/− mice was performed by Yukiko Hoshino, Masaki Takechi and Takashi Nuri. Images of Fgfr2 C342Y/+ mice were collected and scanned by Mehran Moazen and Erwin Pauws. Geomorphometric morphometric analysis was performed by Masaki Takechi, Daisuke Koyabu and Yukiko Hoshino. Sample preparation for histological analysis of Fgfr2 S252W/+ mice was carried out by Yukiko Hoshino and Toshiko Furutera, and that of Fgfr2 C342Y/+ mice was performed by Miranda Steacy and Erwin Pauws. Yukiko Hoshino, Masaki Takechi and Sachiko Iseki drafted the manuscript. All authors contributed to critical revision of the manuscript and approval of the article.

FUNDING INFORMATION

This work was supported by grants‐in‐aid from: Ministry of Education, Culture, Sports, Science, and Technology of Japan [18K06821 to M.T., 18K19605 and 21H03098 to S.I.], Royal Academy of Engineering (10216/119 to MM), Engineering and Physical Science Research Council (EP/W008092/1 to MM), Newlife, the charity for disabled children (award 17‐18/18 to EP) and by the NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.

CONFLICT OF INTEREST

The authors declare no competing or financial interests.

Supporting information

Figure S1

Figure S2

Figure S3

Figure S4

Table S1

Table S2

Click here for additional data file. (828.5KB, pdf)

ACKNOWLEDGMENTS

We appreciate Jin Cheng‐Xue for his technical support. We also thank Michael Fagan and Christian Babbs for assistance with collection and scanning of Fgfr2 C342Y/+ mice.

Hoshino, Y. , Takechi, M. , Moazen, M. , Steacy, M. , Koyabu, D. & Furutera, T. et al. (2023) Synchondrosis fusion contributes to the progression of postnatal craniofacial dysmorphology in syndromic craniosynostosis. Journal of Anatomy, 242, 387–401. Available from: 10.1111/joa.13790

Yukiko Hoshino and Masaki Takechi contributed equally to this work.

Contributor Information

Masaki Takechi, Email: takechi.emb@tmd.ac.jp.

Sachiko Iseki, Email: s.iseki.emb@tmd.ac.jp.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

Figure S2

Figure S3

Figure S4

Table S1

Table S2

Click here for additional data file. (828.5KB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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