title: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Mohammed K
authors.initials: MK
authors.lastname: Badri
authors.affiliation: Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, 1011 N. University Ave., Ann Arbor, MI 48109-1078, USA.
authors.forename: Honghao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, 700 Albany Street, Boston, MA 02118, USA.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Ohyama
authors.affiliation: Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, 700 Albany Street, Boston, MA 02118, USA.
authors.forename: Sundharamani
authors.initials: S
authors.lastname: Venkitapathi
authors.affiliation: Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, 700 Albany Street, Boston, MA 02118, USA.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Alamoudi
authors.affiliation: 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.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Kamiya
authors.affiliation: Unit of Animal Genomics, GIGA-R & Faculty of Veterinary Medicine, University of Liège, 1 Avenue de l'Hôpital, 4000-Liège, Belgium.
authors.forename: Haruko
authors.initials: H
authors.lastname: Takeda
authors.affiliation: Knock Out Core, National Institute of Environmental Health Sciences, 111 TW Alexander Drive, Research Triangle Park, NC 27009, USA.
authors.forename: Manas
authors.initials: M
authors.lastname: Ray
authors.affiliation: Knock Out Core, National Institute of Environmental Health Sciences, 111 TW Alexander Drive, Research Triangle Park, NC 27009, USA.
authors.forename: Greg
authors.initials: G
authors.lastname: Scott
authors.affiliation: Graduate School of Environmental and Life Science, Okayama University, Okayama City, Japan.
authors.forename: Takehito
authors.initials: T
authors.lastname: Tsuji
authors.affiliation: Graduate School of Environmental and Life Science, Okayama University, Okayama City, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Kunieda
authors.affiliation: 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.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Mishina
authors.affiliation: 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.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Mochida
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000378:metabolism / Q000473:pathology; D019902:Chondrocytes / Q000378:metabolism; D019465:Craniofacial Abnormalities / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004195:Disease Models, Animal; D004613:Ellis-Van Creveld Syndrome / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D000072081:Patched-1 Receptor; D001616:beta-Galactosidase
chemical_list: C541939:EVC2 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000072081:Patched-1 Receptor; C000606240:Ptch1 protein, mouse; D001616:beta-Galactosidase
keywords: Craniofacial bone; Development; EVC2; Ellis-van Creveld syndrome; Expression; Knockout (KO) mouse