Septal chondrocyte hypertrophy contributes to midface deformity in a mouse model of Apert syndrome

Abstract

Midface hypoplasia is a major manifestation of Apert syndrome. However, the tissue component responsible for midface hypoplasia has not been elucidated. We studied mice with a chondrocyte-specific Fgfr2^S252W mutation (Col2a1-cre; Fgfr2^S252W/+) to investigate the effect of cartilaginous components in midface hypoplasia of Apert syndrome. In Col2a1-cre; Fgfr2^S252W/+ mice, skull shape was normal at birth, but hypoplastic phenotypes became evident with age. General dimensional changes of mutant mice were comparable with those of mice with mutations in EIIa-cre; Fgfr2^S252W/+, a classic model of Apert syndrome in mice. Col2a1-cre; Fgfr2^S252W/+ mice showed some unique facial phenotypes, such as elevated nasion, abnormal fusion of the suture between the premaxilla and the vomer, and decreased perpendicular plate of the ethmoid bone volume, which are related to the development of the nasal septal cartilage. Morphological and histological examination revealed that the presence of increased septal chondrocyte hypertrophy and abnormal thickening of nasal septum is causally related to midface deformities in nasal septum-associated structures. Our results suggest that careful examination and surgical correction of the nasal septal cartilage may improve the prognosis in the surgical treatment of midface hypoplasia and respiratory problems in patients with Apert syndrome.

Figure 1

Chondrocyte-specific Fgfr2^S252W mutation shows progressive midface hypoplasia in the absence of premature craniofacial suture closure. (a) Micro-computed tomographic (CT) images of the skulls of the mouse model of Apert syndrome on postnatal days 0, 7, and 21 (P0, P7, and P21). The white and red arrows in the images of EIIa-SW mice indicate the fusion of nasofrontal and coronal sutures, respectively. The red asterisk indicates the fusion of the premaxillo–maxillary suture at P0 and P7. The yellow line indicates the curvature of the nasion. Abbreviations: pm, premaxilla; m, maxilla. Scale bar: 2 mm. (b) Micro-CT images of the skulls of Col2-SW mice at P0, P7, and P21. The yellow line indicates the curvature of the nasion. Scale bar: 2 mm. (c) Schematic of linear measurements of the craniofacial bones. (d) Skull lengths in each group at P0, P7, and P21. (n = 5, each stage) (e) Linear measurements of the cranium, the cranial base, and the face at P21 (n = 5). (f) The facial width and the height, and the cranial width in relation to their length at P21 (n = 5). (g) The facial shapes of the two groups were compared in discriminant function analysis (DFA). The first column indicates the analyzed facial region in the lateral and inferior views. The mean facial shape is displayed by wireframe images. The p values for permutation tests (1000 permutations) between the two groups are listed below of the wireframe images. (n = 5) (h) The variance in the facial shape was analyzed by principal components analysis (PCA). The percentage of total variance for each principal component is displayed on the axis. The shape changes are represented with wireframe images by the axis along the negative principal component axis (gray line) or positive principal component axis (black line) (n = 5). Values are presented as means ± standard deviations. *p ≤ 0.0332, **p ≤ 0.0021, ***p ≤ 0.0002, ****p ≤ 0.0001.

Figure 2

Facial deformities in Col2-SW mice are mainly developed in nasal septum-associated structure. (a) Micro-CT images of mouse skulls in the midsagittal plane at P7 and P21. The red arrows indicate the closure between the premaxilla and the vomer. The red arrowheads indicate palate perforation. Scale bar: 1 mm. (b) Hematoxylin, eosin, and Alcian blue staining of skulls of P7 mice in the midsagittal plane. The dotted line indicates the edge of each bone. Abbreviations: n, nasal bone; f, frontal bone; pm, premaxilla bone; v, vomer; et, ethmoid bone; m, maxilla; ns, nasal septum. Scale bar: 1 mm. (c) Superior view of the skull. The white arrows on the mutant mice indicate the deviation of the nasal bone. Scale bar: 2 mm. (d) Micro-CT images of the perpendicular plate of the ethmoid bone at P21. (e) Measurement of perpendicular plate of ethmoid bone volume. Scale bar: 1 mm. Values are presented as means ± standard deviations. *p ≤ 0.0332, **p ≤ 0.0021 (n = 5).

Figure 3

Thickening of septal cartilage altered the paraseptal structures in mutant mice. (a) Hematoxylin, eosin, and Alcian blue staining of the septal cartilage (ns) and vomer (v) at P21. The dotted line indicates the vomer. Scale bar: 200 µm. (b) Schematic image of the vomer for linear measurements. The measurements were conducted in the coronal plane of 3D reconstructed images, at the highest and the lowest points on the vomer are seen (red dotted line). The distance between the two vomer wings (c) (distance (1) in Fig. 3b) and the length of the vomer wings (d) (length (2) in Fig. 3b) were measured. The lengths of the two wings were averaged (n = 5). (e) Micro-CT images of the vomer and premaxilla bone in the coronal plane. The red arrows indicate the fusion between the vomer (v) and the premaxilla bone (pm). Scale bar: 0.5 mm. Values are presented as means ± standard deviations. *p ≤ 0.0332, **p ≤ 0.0021, ***p ≤ 0.0002, ****p ≤ 0.0001.

Figure 4

Thickening and deviation of nasal septal cartilage caused crooked snout in mutant mice. (a) Midsagittal micro-CT image of the nasal cavity of mice at P21, which were stained with potassium triiodide, the contrast agent (Lugol solution), for 48 h. The dotted red line indicates the nasal septum. The white line indicates the coronal and transverse planes for further analyses. Abbreviations: n, nasal bone; ns, nasal septum; pm, premaxilla; v, vomer; et, the perpendicular plate of the ethmoid bone. (b) Coronal view (upper images) and transverse view (lower images) of the nasal cavity. The dotted red line indicates the septal cartilage. Scale bar: 1 mm. (c,d) The measurement of septal deviation in the coronal view of the nasal cavity stained with the contrast agent. The measured plane was matched between groups based on the structural features of incisors, premaxilla, eyeballs and vomeronasal organ. The angle of septal deviation between the most deviated point of the septum and the midline (crossing two points on the nasal bone and the caudal most point of the septum) was measured (n ≥ 3). (e) Measurement of nasal deviation. The angle between the two dotted black lines, parallel to the interfrontal suture and the nasal bone, was measured (n ≥ 3). (f) Correlation between the degree of deviation of the snout and the septum in the mutant mice. The triangle indicates the same deviating direction of the nasal septal cartilage and nose, and the circle indicates the direction opposite of deviation. (g) 3D reconstructed images of the nasal septal cartilage at P21 in lateral and inferior views (magnified in the white box). Scale bar: 1 mm. (h) Mean septal cartilage shape of EIIa-SW and Col2-SW was compared with that of WT mice by DFA with the wireframe images. The inferior view of wireframe image is displayed in the box (n ≥ 3). The p values for 1000 permutation tests between two groups are shown with the wireframe images. Values are presented as means ± standard deviations. *p ≤ 0.0332.

Figure 5

Increased chondrocyte hypertrophy and cellular size, which induced thickening of the nasal septal cartilage. (a, b) Immunohistochemistry of SOX9 and SOX9-positive cell ratio in the septal cartilage at P0. Scale bar: 50 µm. Three mice per genotype were used. (c,d) Immunohistochemistry of RUNX2 and RUNX2-positive cell ratio in the septal cartilage of mice at P21. Scale bar: 100 µm. (e,f). Immunohistochemistry of type X collagen (COLX) and COLX-positive area ratio in the septal cartilage of mice at P21. Positive cell and area ratio from randomly selected three slides from each mouse was averaged. The black arrowheads indicate the expression of COL X at the edge of the septal cartilage of Col2-SW. Scale bar: 100 µm. For RUNX2 and COLX positive cell or area measurements, three WT mice and four Col2-SW mice were used. Three slides were randomly selected from each mouse. Gene expression of markers of chondrocyte hypertrophy: Col10a1 (g), Osteopontin (h), and matrix metalloproteinase-13 (Mmp13) (i) in the septal cartilage at P0 and P7. The expression level was normalized to Gapdh. RNA from P0 mice (n = 4 per genotype) and P7 mice (n = 3 per genotype) were used. (j) Septal chondrocyte size distribution in the septal cartilage at P21. (k) The extracellular matrix (ECM) (gray) and cellular (white) area in the septal cartilage at P21. The difference of ECM area between two groups was not significant. (l) The cell density (cell number/area) was measured in the septal cartilage at P21. For histological assessments (j–l), three WT mice and four Col2-SW mice were used. Four slides were randomly selected from each mouse. (m) Gene expression of Ki-67 in the septal cartilage at P0 and P7. Values are presented as means ± standard deviations. For Student’s t test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. n.s., not significant.