A novel Ambn-IRESCre mouse line allows ameloblast-specific Smad4 silencing

For more than two decades, enamel researchers have primarily used the keratin-14-Cre (Krt14-Cre) driver line to study ameloblast-specific molecular activities. Keratin-14 is expressed in multiple tissues apart from ameloblasts; thus, the use of Krt14-Cre to study post-birth events of amelogenesis has limitations. Therefore, to specifically study various gene functions during amelogenesis, we developed a novel Ambn-IRESCre mouse line that expresses Cre-recombinase only in ameloblastin-expressing cells.

Cre RNA expression visualized by in situ hybridization closely matched Ambn RNA localization in Ambn-IRESCre⁺ incisors and molars. Using two reporter lines, namely, mTmG and LacZ, we confirmed robust Cre-mediated recombination in ameloblast. We used the Ambn-IRESCre⁺ mice as a platform to study conditional Smad4 gene silencing mirroring the Ambn gene expression profile. Smad4 is a transcription factor expressed in all cell types and inhibits epithelial cell proliferation, but its role in post-birth amelogenesis has not been studied due to neonatal lethal phenotype in K14Cre-Smad4 conditional knockout models. We examined the Ambn-IRESCre⁺/Smad4^fl/fl mice at 8 weeks and found ameloblast dysmorphology, enamel hypomineralization, lack of rod-interrod structure, and enamel loss from the surface of the incisor in otherwise healthy and viable mice.

Overall, the Ambn-IRESCre mouse model has been created to study amelogenesis and offers advantages over previously used Cre-recombinase models. Data collected from Ambn-IRESCre^+/-/Smad4^fl/fl mice demonstrate efficient targeting of Smad4 in ameloblast and support the utility of this model for studying gene function during enamel formation.