The skull is a complex structure formed by the craniofacial bones’ elaborate organization. The growth pattern in each craniofacial bone of the postnatal skull has been presented in wild-type mice. However, the skull’s growth pattern, determined by the craniofacial bones’ coordinated growth, is unfamiliar. This study aimed to examine the overall morphological change in the mid-sagittal plane of the postnatal mice’s skulls and interaction between the craniofacial bones.
Geometric morphometric principal component analysis was performed in the mid-sagittal plane of 31 wild-type mice’s skulls from postnatal days 28 to 98. The relationship between the cranial base and cranial vault was investigated by comparing skulls with early fusion and non-fusion of intersphenoid synchondrosis (ISS).
The cranial vault flattening and sphenoid bone length increased with age. The cranial vault curvature and sphenoid base length showed a positive correlation that was confirmed by comparing the skulls with early fusion and non-fusion of ISS. The sphenoid bone length and cranial vault angle significantly decreased in the skulls with early fusion of ISS compared to non-fusion skulls.
It is suggested that the cranial vault flattening is sphenoid bone length-induced but cranial vault length-independent during postnatal mice skull development.
The craniofacial skeleton is formed by 2 mechanisms of bone formation; intramembranous bone formation and endochondral bone formation.[
The cranial base contains multiple growth centers called synchondroses, which are made up of mirror-image growth plates.[
The skull is a complex structure formed by the elaborate organization of many bones. Therefore, it was not easy to fully understand the growth of the craniofacial bone. Accurate analysis of craniofacial structures in various mouse models largely depends on an in-depth understanding of the normal craniofacial development of mice. Two recent studies have well-documented the 3-dimensional (3D) metrics of the normal craniofacial development in postnatal skulls in male and female C57BL/6 mice which is one of the most commonly used inbred mouse strains.[
In this study, we performed geometric morphometric analysis of postnatal growth pattern in the mid-sagittal skulls of C57BL/6N mice from postnatal day (P) 28 to P98, and found 2 distinct geometric morphometric changes in the angle of the cranial vault and the length of sphenoid bone. We also found a close relationship between the cranial vault and the sphenoid bone, and tried to verify the relationship.
All animal experiments were performed under approved protocols of the Intramural Animal Use and Care Committee of the College of Dentistry, Yonsei University. C57BL/6N mice were euthanized utilizing CO2 exposure at selected postnatal ages (P28 mice N=6, P42 mice N=4, P63 mice N=4, P70 mice N=4, P98 non-fusion mice N=8, P98 early fusion mice N=5). The number of mice per stage was determined based on previous C57BL/6 mouse studies, all of which used 4 mice per stage.[
Micro-CT images of the fixed mouse skulls were obtained using a micro-CT scanner (Skyscan1173; Bruker-CT, Kontich, Belgium) at 130 kV and 60 μA and were reconstructed using NRecon (Version 1.6; Bruker-CT) with consistent parameters (
The reconstructions were converted to 3D volumes using the software 3D Slicer (Version 4.1;
The 14 landmarks (
The correlations were calculated as the coefficient of determination (R2) value by Excel (Microsoft Corp., Redmond, WA, USA). The difference in bone length between groups in each parameter was tested statistically with Mann–Whitney U test using SPSS (version 26.0; IBM Corp., Armonk, NY, USA). All
To investigate the postnatal growth pattern in the mid-sagittal skulls of C57BL/6N mice, a 3D geometric morphometric analysis was performed in skulls at P28, P42, P63, P70, and P98 (
Furthermore, we investigated the correlation between the curvature of the cranial vault and the length of sphenoid bone. To quantify the curvature of the cranial vault, we measured and summed the angles at bregma and lambda, the 2 points with the most prominent positional change in PC analysis. We found that the cranial vault curvature and sphenoid base length had a positive correlation (R2=0.7662) (
To confirm the positive correlation between cranial vault curvature and sphenoid base length, a 3D geometric morphometric PC analysis was performed in the mid-sagittal skulls showing early fusion of ISS. Five skulls showing early fusion of ISS were obtained at P98 mice, but we could not find skulls with early fusion of SOS at P98 mice. The skulls with the early fusion of ISS show a domed cranial vault compared to non-fusion skulls (
In PC analysis, the early fusion skulls were clustered on the left along the PC1 value on a PC plot, and the non-fusion skulls were clustered on the right (
To investigate the postnatal growth pattern in the midline of C57BL/6 mouse skulls, the geometric morphometric analysis was performed in the mid-sagittal skulls at P28, P42, P63, P70, and P98. We excluded skulls between p7 and P21, which have been reported to show a rapid change in all dimensions of the skull.[
We found that the cranial vault flatness and the sphenoid bone length in the mid-sagittal skulls increased with age and that the cranial vault curvature was positively correlated with the sphenoid bone length but not with the cranial vault length. These results are consistent with the result of previous reports.[
To confirm the correlation between cranial vault curvature and sphenoid base length, we compared skulls with early fusion and non-fusion of ISS. The fusion time of ISS and SOS was reported in previous studies. The ISS completely fused sometime after P120 [
The data from C57BL/6 mice in the present study are unlikely to be directly applicable to humans because there are striking morphological differences between human and mouse skull bases. First, the overall shape of the sphenoid bone is completely different between humans and mice. In the lateral view, the body of the sphenoid bone is curved in humans and the sphenoid bone in mice is flat. Due to these differences, the angle at the base of the skull is highly flexed at an acute angle in humans. On the other hand, the skull base angle is retroflexed (>180° when measured ventrally) in mice, just as it is flat or obtuse in most mammals.[
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT). (No.2020R1A2C2005790).
All animals were treated in accordance with the Guide for the Intramural Animal Use and Care Committee of the College of Dentistry, Yonsei University.
No potential conflict of interest relevant to this article was reported.
Lateral view of C57BL/6 mouse skulls and landmarks in the mid-sagittal plane of the skull. (A) Three-dimensional reconstructed skulls at postnatal day (P) 28, P42, P63, P70, and P98. (B–E) The skull with landmarks is viewed from various directions; frontal (B), lateral (C), dorsal (D), and ventral view (E). The 14 landmarks, which are placed in the mid-sagittal plane of the skull, corresponding to landmarks in
Geometric morphometric changes in the mid-sagittal skull of the postnatal mice. (A) Principal component (PC) analysis based on procrustes coordinates of 14 cranial mid-sagittal landmarks is presented as scatter plots of individual scores along the PC1 and PC2. The PC 1 explains 49.46% of total shape variance. Skulls from the same age group are well clustered. The postnatal day 28 skulls are located on the left, and the skulls with age gradually moved further to the right along the PC1 axis. The gray wireframe shows the spatial relationships of the landmarks represented at all PC values of 0. Skull with negative PC1 scores (blue wireframe) exhibit a cranial vault that is more curved than average, whereas skulls with positive PC1 scores (red wireframe) exhibit the flatter cranial vault. In addition, the basisphenoid bone of the skull gradually becomes longer as the PC1 score increase. Therefore, a positive score for PC1 corresponds to a polygon composed of a flatter cranial vault and a longer sphenoid bone length. (B) The angles at the bregma and lambda landmarks were summed and regarded as the cranial vault angle. The sphenoid bone length shows a positive correlation with the cranial vault angle (R2=0.7662). (C) There is no direct relationship between cranial vault bone length (total of the frontal, parietal, and interparietal bone length) and the cranial vault angle (R2=0.4887).
Mouse skulls with early fusion and non-fusion in intersphenoid synchondrosis (ISS). (A, B) Lateral view of 3-dimensional ISS reconstructed skulls at postnatal day 98. The skull with the early fusion of ISS shows a domed cranial vault compared to the non-fusion skull. (C, D) The dorsal view of early fusion and non-fusion skull. The skull with the early fusion of ISS shows a complete fusion between presphenoid bone and basisphenoid bone. (E, F) In the longitudinal section of the cranial base, early fusion of ISS with poor demarcation in the ISS compared to the non-fusion skull.
Geometric morphometric changes in the mid-sagittal skulls with an early fusion of intersphenoid synchondrosis (ISS) and non-fusion skulls. (A) Skulls from the same age group are well clustered. The principal component (PC) 1 explains 66.98% of total shape variance. The early fusion skulls with negative scores of PC1 (blue wireframe), which corresponds to a polygon composed of a domed cranial vault and a shorter sphenoid bone length. The gray wireframe shows the spatial relationships of the landmarks represented at all PC values of 0. (B, C) The ISS early fusion skulls show a significant decrease in the sphenoid bone length and the cranial vault angle summed with the angles at bregma and lambda compared to those of non-fusion skulls in the box plots (P < 0.005).
Landmark placed in the mid-sagittal plane of mouse skull
No. of landmark | Landmark definition |
---|---|
1 | Anterior nasal spine |
2 | Rhinion |
3 | Nasion |
4 | Bregma |
5 | The intersection of parietal bones with anterior aspect of interparietal bone, midline |
6 | The intersection of interparietal bone with squamous portion of occipital bone, midline |
7 | Opisthion |
8 | Basion |
9 | The central point of spheno-occipital synchondrosis |
10 | The central point of intersphenoidal synchondrosis |
11 | Rostral end of the curvature of the palatine bone (lamina horizontalis ossis palatine) |
12 | The intersection of the maxillo-palatine suture in the midline |
13 | Posterior end of the wing of vomer |
14 | The central point on the premaxilla between the incisors |