title: Delayed osteoblastic differentiation and bone development in Cx43 knockout mice.
abstract: GJA1 gene (Connexin43, also known as Cx43) is the most abundant gap junction protein isoform in animal cells and is associated with bone development in embryos. The objective of the present work was to evaluate in vivo osteal development in GJA1-deficient fetal mice through determination of the histological and molecular alterations induced by partial or total deletion of the GJA1 gene. Heterozygous C57BL/6 mice (HT) harboring a null mutation of the GJA1 gene were mated, and pregnant females were submitted to euthanasia and Caesarean section from 12.5 to 19.5 days post coitum (dpc). HT (GJA1(+/-)) and homozygous (GJA1(-/-               )) knockout (KO) mutants and wild-type (WT) fetuses were identified by polymerase chain reaction (PCR), and development curves were constructed on the basis of fetus weight and crown-rump length. Histopathological, histochemical, and real-time PCR analyses were performed in order to assess the expression of markers associated with bone development, namely, osteocalcin, osteopontin, alkaline phosphatase, RUNX2, GJA1, GJC1 (Cx45), and GJA3 (Cx46). HT and KO fetuses exhibited delays in the differentiation of osteoblasts and, consequently, in bone development in comparison with the WT group. Additionally, less deposition of mineralized and osteoid matrix was observed in GJA1-deficient fetuses. Bone development in KO fetuses was delayed through the moment of birth, but in HT animals the delay only extended until 17.5 dpc, following which development was normalized. The expression of genes coding for osteocalcin, osteopontin, alkaline phosphatise, and RUNX2 were also delayed in GJA1-deficient fetuses. Animals that exhibited a lower expression of GJA1 presented delayed expression of the GJC1 and GJA3 genes and their corresponding protein products in the bone tissue. The results of the present study contribute to our understanding of the function of GJA1 during bone development and suggest that GJC1 could play a role in restoring intercellular communication in GJA1-deficient mice.
authors.affiliation: Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil. chaible@usp.br
authors.forename: Lucas Martins
authors.initials: LM
authors.lastname: Chaible
authors.forename: Daniel Soares
authors.initials: DS
authors.lastname: Sanches
authors.forename: Bruno
authors.initials: B
authors.lastname: Cogliati
authors.forename: Gregory
authors.initials: G
authors.lastname: Mennecier
authors.forename: Maria Lucia Zaidan
authors.initials: ML
authors.lastname: Dagli
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D018031:Connexin 43 / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000096:biosynthesis / Q000235:genetics; D018568:Crown-Rump Length; D005260:Female; D020567:Fetal Weight / Q000235:genetics; D017629:Gap Junctions / Q000235:genetics; D017353:Gene Deletion; D006651:Histocytochemistry; D051379:Mice; D018345:Mice, Knockout; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010012:Osteogenesis / Q000235:genetics / Q000502:physiology*; D011247:Pregnancy; D012272:Ribs / Q000737:chemistry
chemical_list: D015415:Biomarkers; D018031:Connexin 43; D050659:Core Binding Factor Alpha 1 Subunit; C493716:Runx2 protein, mouse