Expression of Evc2 in craniofacial tissues and craniofacial bone defects in Evc2 knockout mouse

Abstract

Objective Our objectives were to determine the expression of EVC2 in craniofacial tissues and investigate the effect of Evc2 deficiency on craniofacial bones using Evc2 knockout (KO) mouse model. Design Evc2 KO mice were generated by introducing a premature stop codon followed by the Internal Ribosomal Entry Site fused to β-galactosidase (LacZ). Samples from wild-type (WT), heterozygous (Het) and homozygous Evc2 KO mice were prepared. LacZ staining and immunohistochemistry (IHC) with anti-β-galactosidase, anti-EVC2 and anti-SOX9 antibodies were performed. The craniofacial bones were stained with alcian blue and alizarin red. Results The LacZ activity in KO was mainly observed in the anterior parts of viscerocranium. The Evc2-expressing cells were identified in many cartilageous regions by IHC with anti-β-galactosidase antibody in KO and Het embryos. The endogenous EVC2 protein was observed in these areas in WT embryos. Double labeling with anti-SOX9 antibody showed that these cells were mainly chondrocytes. At adult stages, the expression of EVC2 was found in chondrocytes of nasal bones and spheno-occipital synchondrosis, and osteocytes and endothelial-like cells of the premaxilla and mandible. The skeletal double staining demonstrated that craniofacial bones, where the expression of EVC2 was observed, in KO had the morphological defects as compared to WT. Conclusion To our knowledge, our study was the first to identify the types of Evc2-expressing cells in craniofacial tissues. Consistent with the expression pattern, abnormal craniofacial bone morphology was found in the Evc2 KO mice, suggesting that EVC2 may be important during craniofacial growth and development.

A Ciliary Protein EVC2/LIMBIN Plays a Critical Role in the Skull Base for Mid-Facial Development

“…We specifically found decreases in growth axes of the upper face (E-Iu), viscerocranial length (E-Bu), erupted upper incisor length (Pr-Iu), erupted lower incisor length (Id-Ii), distance between the first molar and incisor in mandible (Ml-Ii), length of anterior cranial base (So-E), neurocranial length (Po-E), the distance between the first molar to the most posterior point (Po-Mu), and total skull length (Po-A), respectively (Figure 2A and Table 2 ). These phenotypic observations recapitulate many of the mid-facial defects of the Evc2 global mutants that we previously reported ( Badri et al, 2016a ). Additionally, we found a shallow mid-facial region in the Evc2 P0 mutants, as evidenced by decreased angle measures in nasal bone to cranial base (ANL/SoEL), maxilla-premaxilla to cranial base (MuBuL/SoEL), maxilla-premaxilla to cranial vault (MuBuL/PoEL), upper incisors to cranial base (BuEL/SoEL), and angle of cranial vault (ABrL/PoBrL), respectively (Figure 2B and Table 2 ).…”

“…Generating our own Evc2 mutant mice enabled us to characterize the pathological mechanisms leading to abnormal appendicular bone and hypomorphic enamel development ( Zhang et al, 2015 , 2016a , b ). Our subsequent studies further demonstrated the expression of Evc2 in the mid-facial regions ( Badri et al, 2016a ) and mid-facial defects in Evc2 global mutant mice ( Badri et al, 2016b ). We most recently employed a conditional, Cre -mediated approach toward deleting Evc2 in a neural crest-specific manner.…”

The Role of Ellis‐Van Creveld 2(EVC2) in Mice During Cranial Bone Development

“…These observations are consistent with previous reports using different methods to study abnormal craniofacial development (Badri et al, 2016b) and abnormal tooth development (Zhang et al, 2015(Zhang et al, , 2017. Our previous studies demonstrated that Evc2 is expressed in nearly all tissues in the skull (Badri et al, 2016a). Because the nasal and frontal bones are developmentally derived from neural crest cells, the reasonable speculation is that Evc2 function in the neural crest cells leads to shortened nasal and frontal bones.…”

Whole exome sequencing identified sixty-five coding mutations in four neuroblastoma tumors

“…These include EVC2 on chromosome 4, CDK5RAP2 on chromosome 9, CRY2 on chromosome 11, ATXN2 on chromosome 12, and NUP62 on chromosome 19. EVC2 (EvC ciliary complex subunit 2) contributes to growth and development of bone and skeleton, and regulates Sonic Hedgehog pathway signaling, a pathway described as essential to NB progression 34 – 41 . Mutations in the EVC2 gene have been related to Ellis-van Creveld syndrome and Weyers acrofacial dysostosis 42 .…”