Methods: Twist1 and Twist2 double heterozygous mice were generated by breeding Twist1 floxed mice with Twist2-Cre Knock-in mice. Combinations of plain X-radiography, micro-CT, alizarin red/alcian blue staining, immunohistochemistry, and real-time PCR were used to characterize the tooth and skeletal phenotypes of the double heterozygous mice. Promoter-luciferase assay was used to determine the effects of Twist1, Twist2 and E12 on a 4.9 kb Fgfr2 promoter fragment in C3H10T1/2, MC3T3-E1, and MDPC-23 cells. Co-immunoprecipitation and Western-blot analyses were used to analyze the protein-protein interaction of Twist1 and E12.
Results: Plain X-radiography, micro-CT, and Alizarin red/alcian blue staining showed delayed development of the third molars and reduced bone formation in the double heterozygous mice. Immunohistochemistry revealed that osteoblast differentiation was affected in the double heterozygous mice; In addition, osteoblast progenitors in the periosteal tissues were also reduced. Real-time PCR demonstrated a decrease in Fgfr2 expression while in vitro promoter studies confirmed that both Twist1 and Twist2 were able to activate a 4.9 kb Fgfr2 promoter fragment synergistically with E12 in C3H10T1/2, MC3T3-E1, and MDPC-23 cells. Consistently, co-immunoprecipitation and Western-blot analyses showed that only intact Twist1 was able to interact with E12.
Conclusion: These findings suggest that Twist1 and Twist2 might have redundant function in regulating tooth and bone development through modulating FGF signaling.
Keywords: Bone, Odontoblasts, Osteoblasts/osteoclasts and Twist1, Twist2