Effect of Reduced Connexin43 Function on Nasal Bone Morphology and Remodelling in the G60S Mutant Mouse Model of Oculodentodigital Dysplasia

Abstract

Connexin43 (Cx43), a gap-junctional protein, is known to play a role in bone development and remodelling. Our understanding of how Cx43 helps regulate bone remodelling has largely been gained through studies of long bones from a variety of Cx43 knockout mouse models, wherein disrupted osteoblast, osteoclast and osteocyte function cause altered bone morphology. However, clinically, altered Cx43 function manifests as the rare disorder oculodentodigital dysplasia (ODDD), caused by mutations in GJA1, the gene that encodes Cx43. Patients with ODDD display a suite of skeletal defects that are largely localized to craniofacial structures. While the important of Cx43 in long bone remodelling has been established, it is unknown if it plays a similar role in the skull. Thus here, using a mouse model of ODDD heterozygous for a mutation in Gja1 (G60S/+) and with known abnormal craniofacial morphology, we investigate the role of Cx43 in remodelling within the skull. Given that skeletally mature G60S/+ mice present with variable curvature of the nasal bones, we measure and compare phenotype and remodelling in nasal bones between G60S/+ and wildtype (WT) mice using micro-CT imaging, dynamic calcein histology, immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining. Results from micro-CT imaging of G60S/+ and WT littermates over a post-natal developmental timeline from post-natal day 7, 14, 21 and three-months of age have shown that this craniofacial asymmetry is absent in younger mice, appears by post-natal day 21 and gets progressively worse with age. By three-months of age, one third of G60S/+ mice display the curved nasal bone phenotype whereas this phenotype is absent in WT mice, suggesting that the phenotype is caused by altered bone remodelling rather than disruption of initial development. Immunohistochemical staining for sclerostin and TRAP staining is greatly pronounced in G60S/+ nasal bones and corresponds to regions that display a concave curvature. These results suggest that the abnormal nasal bone curvature phenotype displayed in G60S/+ mice could be due to altered osteoclast activity and bone resorption and indicate that Cx43 does indeed help regulate bone remodelling in the skull. Our findings provide novel and exciting insights into the role of Cx43 in bone homeostasis and suggests that this protein serves a similar function in remodelling throughout the skeleton. Additionally, our results indicate that many of the skeletal manifestations of ODDD may be due to disrupted bone remodelling, which could underlie the immense phenotypic variation among patients with ODDD.