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

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; Crouzon syndrome; Saethre-Chotzen syndrome; coronal suture; craniosynostosis; geometric morphometrics; inter-sphenoid synchondrosis; midfacial hypoplasia.

FIGURE 1

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).

FIGURE 2

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).

FIGURE 3

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

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

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.

FIGURE 6

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.