Reciprocal epithelial-mesenchymal interactions are a key step in early tooth formation and defects in these interactions are likely to lead to tooth agenesis. BMP-4 a crucial signal molecule in development of many tissues is very likely required at early stages of tooth formation. Its spatial and temporal expression correlates highly with the epithelial-mesenchymal interactions during the initiation phase of odontogenesis. Local application of BMP-4 to tooth mesenchyme can mimic the action of the dental epithelium during early stages of odontogenesis. However, there is no direct in vivo evidence to show the importance of BMP-4 in odontogenesis, since the BMP-4 knock out die at an early stage of development prior to tooth formation. Recently BMP-4 has been shown to be strongly linked to Msx-1, a critical transcription factor, for tooth development. Msx-1 knock out show an arrest in the bud stage of tooth formation which is associated with substantial reduction of BMP-4 expression in the dental mesenchyme . Addition of recombinant BMP-4 to arrested tooth from Msx-1 knock out in vitro can rescue the tooth development into cap stage. Previously the applicant have cloned the mouse BMP-4 genes and identified the potential Msx-1 binding sites in the BMP-4 promoter. Using in vitro transfection studies they have shown that Msx-1 expressing plasmid can upregulate BMP-4 promoter activity. They have also been able to identify the tooth-specific elements of the BMP-4 promoter region mapped to region between -1144 to -260 bp. Using transgenic mice carrying LacZ reporter the tooth specific expression of this fragment has been confirmed. Seven transgenic animal expressing different levels of human BMP-4 have been generated.
The aim of this proposal is to examine the role of BMP-4 and its regulation by Msx-1 in normal and abnormal tooth formation in vivo. The underlying hypothesis for the project is that Msx-1 act upstream of the BMP-4 at the transcription levels and BMP-4 is required for tooth formation. This will be tested by using transgenic mice, BMP-4 knock in and Msx-1 knock out mice.
Four specific aims have been proposed.
Specific Aim 1 will examine the changes in the expression of reporter gene driven by intact or specific BMP-4 fragments in the presence and absence of Msx-1 to determine the regulatory role of Msx-1 on BMP-4 transcription in vitro.
Specific Aim 2 will examine if over expression of BMP-4 driven by specific Msx-1 promoter or the BMP-4 promoter can rescue the tooth phenotype of Msx-1 mutant in vivo.
Specific Aim 3 will extend these animal studies to examine the potential mutation of the Msx-1 gene in autosomal dominant form of familial tooth agenesis. Research into this relatively unexplored area will contribute to our understanding of basic Msx-1-BMP-4 signal pathway mechanisms of tooth formation and possible cause of autosomal dominant forms of familial tooth agenesis.
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