Functional Redundancy of Twist1 and Twist2 in Mouse Development

Objective: Twist1 and Twist2 are basic helix-loop-helix transcription factors that have a high degree of sequence similarity. They exhibit extensive overlapping expression patterns during embryonic development of tooth and bone, however, their mutations in humans result in quite different phenotypic changes, suggesting that they may have a redundant function in regulating the development of tooth and bone. The goal of this study was to determine if Twist1 and Twist2 play a redundant function in tooth and bone development using both in vitro and in vivo approaches.

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.