Mohammed K Badri1, Honghao Zhang2, Yoshio Ohyama3, Sundharamani Venkitapathi3, Ahmed Alamoudi3, Nobuhiro Kamiya4, Haruko Takeda5, Manas Ray6, Greg Scott6, Takehito Tsuji7, Tetsuo Kunieda7, Yuji Mishina8, Yoshiyuki Mochida9. 1. Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, 700 Albany Street, Boston, MA 02118, USA; Department of Pediatric Dentistry and Orthodontics, College of Dentistry, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia. 2. Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, 1011 N. University Ave., Ann Arbor, MI 48109-1078, USA. 3. Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, 700 Albany Street, Boston, MA 02118, USA. 4. Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, 1011 N. University Ave., Ann Arbor, MI 48109-1078, USA; Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, 111 TW Alexander Drive, Research Triangle Park, NC 27009, USA. 5. Unit of Animal Genomics, GIGA-R & Faculty of Veterinary Medicine, University of Liège, 1 Avenue de l'Hôpital, 4000-Liège, Belgium. 6. Knock Out Core, National Institute of Environmental Health Sciences, 111 TW Alexander Drive, Research Triangle Park, NC 27009, USA. 7. Graduate School of Environmental and Life Science, Okayama University, Okayama City, Japan. 8. Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, 1011 N. University Ave., Ann Arbor, MI 48109-1078, USA; Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, 111 TW Alexander Drive, Research Triangle Park, NC 27009, USA; Knock Out Core, National Institute of Environmental Health Sciences, 111 TW Alexander Drive, Research Triangle Park, NC 27009, USA. 9. Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, 700 Albany Street, Boston, MA 02118, USA. Electronic address: mochida@bu.edu.
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.
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.
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