Method: Twist1 floxed mice (Twist1f/f) were bred with Twist2-Cre knock-in mice (Twist2Cre/+) to generate Twist1 and Twist2 double heterozygous mice (Twist1f/+;Twist2Cre/+), which were crossed again with the (Twist1f/f) mice to obtain mice with the deletion of both Twist1 alleles and one Twist2 allele (Twist1f/f;Twist2Cre/+). Alizarin red/alcian blue staining, H&E staining, BrdU immunohistochemistry, in situ hybridization, real-time PCR were employed to investigate the dental and skeletal defects and the molecular changes in the developing teeth of Twist1f/f;Twist2Cre/+ embryos. Promoter luciferase assay and chromatin immunoprecipitation (ChIP) analysis were conducted to analyze the regulatory roles of Twist1 on Fgf10.
Result: Twist1f/f;Twist2Cre/+ embryos died before birth due to a severe hernia and brain defect. These embryos developed hypoplastic craniofacial bones as revealed by alizarin red/alcian blue staining and smaller tooth germs with abnormal cusps by E18.5 as shown by histological analyses. Compared with the control embryos, the tooth germs of the E14.5 Twist1f/f;Twist2Cre/+ embryos had low levels of Shh transcripts in the enamel knot as shown by in situ hybridization, decreased number of proliferative cells as demonstrated by BrdU immunohistochemistry, and reduced levels of Fgf3, Fgf10, Fgfr1 and Fgfr2 transcripts as shown by quantitative real-time PCR. In vitro promoter luciferase assay indicated that Twist1 and E12 synergistically stimulated the activities of Fgf10 promoter. ChIP assay confirmed that Twist1 protein interacted with Fgf10 promoter in a region between -1.8 kb to -1.2 kb.
Conclusion: Twist1 and Twist2 are essential for normal tooth morphogenesis through upregulating FGF signaling.