BACKGROUND

Ameloblasts are epithelially derived cells responsible for enamel formation through a process known as amelogenesis. Amongst the several transcription factors that are expressed during amelogenesis, both and transcription factors play important role. and mouse mutants, exhibit similar amelogenesis defects, namely enamel hypoplasia, while humans with amelogenesis imperfecta (AI) carry mutations in the human homologues of MSX2 or SP6 genes. These across species similarities in function indicate that these two transcription factors may reside in the same developmental pathway. In this paper, we test whether they work in a coordinated manner to exert their effect during amelogenesis.

METHODS

Two different dental epithelial cell lines, the mouse LS8 and the rat G5 were used for either overexpression or silencing of or or both. mutant mouse embryos or pups were used for studies. hybridization, semi-quantitative and quantitative real time PCR were employed to study gene expression pattern. was used to identify several potential putative binding sites upstream of the murine promoter region. Chromatin Immunoprecipitation (chIP) was used to confirm the binding of to promoter at the putative sites.

RESULTS

Using the above methods we identified that (i) and exhibit overlapping expression in secretory ameloblasts, (ii) expression is reduced in the mouse mutant secretoty ameloblasts, and (iii) that , like inhibits expression. Specifically, our function studies by silencing and/or in mouse dental epithelial (LS8) cells showed significant downregulation of but upregulation of expression. Transient transfection of overexpression plasmid, up-regulated and downregulated expression. Additionally, using , we identified several potential putative binding sites, 3.5 kb upstream of the murine promoter region. By chIP, we confirmed the binding of to promoter at these sites, thus suggesting that is a direct target of .

CONCLUSION

Collectively, these results show that and work in a concerted manner to form part of a network of transcription factors that operate during later stages of tooth development controlling ameloblast life cycle and amelogenesis.