Notch Signaling Regulates Development of Stratum Intermedium in Tooth Germs

“Objective:” The stratum intermedium develops as flattened cell layers on the proximal side neighboring the ameloblast layer during the bell stage of tooth development. Stratum intermedium cells strongly express alkaline phosphatase (ALP) activity and have been considered to play a complementary role in the enamel mineralization; however, little information is available regarding the origin and development of these cells. We examined the development of stratum intermedium cells in continuously growing mice incisors and the regulatory mechanisms. “Methods:” We analyzed the cell-lineage of stratum intermedium by using DiI tracers in the incisors organ culture and histological serial section of lower mice incisors (postnatal one day). The expression of Notch signaling associated proteins was examined by immunohistochemistry and in situ hybridization. Further, we examined the effect of the recombinant Jagged1 protein, the neutralization antibody of Jagged1, and the overexpresssion of Notch1 internal domain (active form) during the differentiation of HAT-7 (ameloblast lineage cell line). “Results:” The results indicated that some stratum intermedium cells originated from the IEE. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells expressed the Notch1 and Hes1 mRNA, while the IEE and ameloblasts expressed their ligand Jagged1. Recombinant Jagged1 protein enhanced the appearance of the stratum intermedium cells in HAT-7 and neutralization antibody of Jagged1 inhibited the effects. Additionally, overexpression of Notch1 internal domain also increased the number of the stratum intermedium. “Conclusion:” The development of the stratum intermedium from the ameloblast lineage is regulated by Notch signaling.