Geometric Morphometry Of The Craniomaxillofacial Skeleton In An IGSF3 Knockout Mouse Model Based On EDMA

Objective Current studies have shown that IGSF3 gene is related to the development of human nasolacrimal duct,and IGSF3 gene mutation may lead to congenital nasolacrimal duct obstruction.At the same time,based on the previous research of our group,we found that IGSF3 gene point mutation may affect the morphology of human craniofacial bones.Therefore,to explore whether IGSF3 gene function affects the changes of craniofacial morphology in humans,we measured the craniofacial morphology of IGSF3 knockout mice based on EDMA to explore the effect of IGSF3 gene on the craniofacial morphology of mice.This study lays a theoretical foundation for further study on the specific mechanism of IGSF3 gene affecting craniomaxillofacial skeletal morphology.Current studies have shown that IGSF3 gene is related to the development of human nasolacrimal duct,and IGSF3 gene mutation may lead to congenital nasolacrimal duct obstruction.At the same time,based on the previous research of our group,we found that IGSF3 gene point mutation may affect the morphology of human craniofacial bones.Therefore,to explore whether IGSF3 gene function affects the changes of craniofacial morphology in humans,we measured the craniofacial morphology of IGSF3 knockout mice based on EDMA to explore the effect of IGSF3 gene on the craniofacial morphology of mice.This study lays a theoretical foundation for further study on the specific mechanism of IGSF3 gene affecting craniomaxillofacial skeletal morphology.Methods The obtained F0 generation was crossed with wild-type（WT）mice to obtain heterozygous F1 generation mice,and the mice carrying the target gene deletion were screened by gene identification and sequencing.IGSF3-/-mice,IGSF3+/-mice and IGSF3^+/+ mice were obtained from heterozygous F1 mice.Eight weeks old IGSF3-/-mice were used as experimental group,8 weeks old IGSF3-/+ mice were used as control group,and 8 weeks old IGSF3^+/+ mice were used as control group.The genotypes of the mice were verified by polymerase chain reaction（PCR）and sanger sequencing.Then the mice were euthanized,and the skulls of the isolated mice were scanned by SCANCO Micro-CT（model μCT100）to obtain DICOM image data.Then VG Studio3.2MAX software was used to reconstruct the three-dimensional images of the mouse skull,and the reconstructed images of the mouse skull were divided into three large regions and seven sub-regions according to the morphological and anatomical characteristics of the mouse skull and maxillofacial region.The three large regions were the skull region,the zygomatic ring region,the mandibular region,and the mandibular region.The seven sub-regions were nasal bone region,frontoparietal bone region,left zygomatic ring region,right zygomatic ring region,left mandibular region,right mandibular region,and intermandibular region.Euclidean distance matrix analysis（EDMA）was used to compare the IGSF3-/-,IGSF3+/-and IGSF3^+/+ mice in different groups.The first group of IGSF3-/-mice was compared with IGSF3+/-mice,the second group of IGSF3-/-mice was compared with IGSF3^+/+ mice,and the third group of IGSF3+/-mice was compared with IGSF3^+/+ mice.For the form matrix（FM）regions formed by the cranium region（without duplication）and the intermandibular region,a ttest was used for the former and a one-way ANOVA was used for the latter.Results1.Fourteen IGSF3 gene chimeric mice were successfully constructed,and three F0 generation IGSF3 gene chimeric mice met the experimental conditions,and the mice were three males.The results of gene sequencing showed that there was a deletion of 1816 bp between the third and fourth exons and a random insertion of four bases,respectively,which were CCAT.2.5 IGSF3-/-mice,6 IGSF3+/-mice and 5 IGSF3^+/+ mice were obtained in F2 generation.3.The skull region was divided into nasal bone region,frontoparietal bone region and cranium（without overlap）region.The nasal bone morphology of IGSF3-/-mice was reduced,mainly manifested as the length and width of the nasal bone and the overall shape of the pyriform foramen,and the right side of the pyriform foramen was more obvious than the left side.Comparison of the Euclidean distance matrix of the nasal bone region of the three types of mice,the first group T=1.484,P=0.001,the number of line segments with FDM ratio difference accounted for 50%（14/28）of the total,the second group T=1.267,P=0.332,the number of line segments with FDM ratio difference accounted for 35.7%（10/28）of the total.The third group T=1.349,P=0.203,the number of line segments with FDM ratio differences accounted for 21.42%（6/28）of the total.There was no significant difference in skeletal morphology between IGSF3-/-mice and the other two types of mice.Comparison of the Euclidean distance matrix of the frontoparietal bone region of the three mouse types,the first group T=1.094,P=0.142,the number of line segments with FDM ratio difference accounted for 7.14%（2/28）of the total;In the second group,T=1.051,P=0.586,the number of line segments with FDM ratio difference accounted for 0 of the total;The third group T=1.075,P=0.227,the number of line segments with FDM ratio difference accounted for 0 of the total.There was no significant difference in the width of the skull between IGSF3-/-mice and the other two types of mice.The distance from nasal process to occipital bone was reduced.The number of line segments with FDM ratio difference in the first group accounted for 19.04%（4/21）of the total.The number of line segments with FDM ratio difference in the second group accounted for 4.76%（1/21）of the total.The number of line segments with FDM ratio differences in the third group accounted for 0 of the total.maxna,l-paro（P=0.0003）,maxna,r-pari（P=0.0014）,nsl-paro（P=0.0033）.nsl-pto,r by t test P=0.3750.4.The zygomatic ring region and mandibular region were divided into the left zygomatic ring region,the right zygomatic ring region,the left mandibular region,the right mandibular region,and the intermandibular region.There was no significant difference in skeletal morphology between the left and right zygomatic ring regions in IGSF3-/-mice.The comparison results of the bone morphology of the left zygomatic ring showed that the first group T=1.175,P=0.058,the second group T=1.067,P=0.503,and the third group T=1.121,P=0.178.The comparison results of the bone morphology of the right zygomatic ring region showed that the first group T=1.072,P=0.394,the second group T=1.065,P=0.451,and the third group T=1.085,P=0.148.The left and right mandibular regions of IGSF3-/-mice were reduced,and the right mandibular region was more reduced than the left.The comparison of the left mandibular bone morphology showed that the first group T=1.360,P=0.004,and the number of line segments with FDM ratio difference accounted for 42.30%（33/78）of the total.The second group T=1.249,P=0.035,the number of line segments with FDM ratio difference accounted for 5.12%（4/78）of the total;The third group T=1.194,P=0.067,the number of line segments with FDM ratio difference accounted for 17.95%（14/78）of the total.The comparison of the bone morphology of the right mandibular region showed that the first group T=1.368,P=0.028,and the number of line segments with FDM ratio difference accounted for 46.15%（36/78）of the total.The second group T=1.304,P=0.048,the number of line segments with FDM ratio difference accounted for 17.94%（14/78）of the total;The third group T=1.194,P=0.150,the number of line segments with FDM ratio difference accounted for 23.07%（18/78）of the total.The bone morphology of IGSF3-/-mice was significantly smaller than that of IGSF3^+/+ mice,but less so than that of IGSF3+/-mice.In the intermandibular region,the comparison results of the three types of mice showed that the inter-group comparison results were P < 0.001,and the intra-group comparison results were P < 0.001.The number of line segments with FDM ratio difference accounted for 46.15%（78/169）of the total.In the second group,P > 0.05,the number of line segments with FDM ratio difference accounted for 5.91%（10/169）.The third group P < 0.001 the number of line segments with FDM ratio differences accounted for 25.44%（43/169）of the total.5.In asymmetry comparison,it mainly includes two regions,the left and right zygomatic ring region and the left and right mandibular region.There was no significant difference in skeletal asymmetry in the zygomatic ring region among the three mouse types,and the IGSF3-/-mice had a higher variation in the asymmetry than the other two mouse types.IGSF3-/-mice: T=1.129,P=0.185;IGSF3+/-mice T=1.145,P=0.070 and IGSF3^+/+ mice T=1.064,P=0.296.IGSF3-/-mice > IGSF3+/-mice > IGSF3^+/+ mice.There was no significant difference in the asymmetry of bone morphology in the mandibular region among the three mouse types.IGSF3-/-mice: T=1.216,P=0.120;IGSF3+/-mice T=1.119,P=0.330 and IGSF3^+/+ mice T=1.149,P=0.386.Conclusions1.The IGSF3 gene knockout mouse model was successfully constructed.2.Nasal bone area: The length and width of the nasal bone and pyriform foramen are reduced overall.3.Cranial region（no overlap）: The distance from the nasal process to the interoccipital bone is shortened.4.Mandibular region: IGSF3-/-mice shortened the length and width of the mandible,and the distance between the mandible decreased.5.In terms of asymmetry,there was no significant difference in the asymmetry of thethree mouse types,but from the perspective of the number of FDM ratio line segments,the asymmetry of IGSF3-/-mice was higher than that of IGSF3-/+ mice and IGSF3^+/+ mice.6.The IGSF3 gene has an important influence on the craniofacial morphological development of mice.