Studies of the Roles of Twist1 and E12 in Tooth Morphogenesis

描述（由申请人提供）：发育中的牙齿是了解器官发生过程中形态发生和终末分化方式的遗传和分子基础的有价值的范例。牙齿形态发生涉及由转录因子和生长因子以及细胞表面和细胞外基质（ECM）分子介导的牙齿上皮和间充质之间的相互信号传导相互作用。成纤维细胞生长因子（fibroblastgrowthfactors，FGF）作为促有丝分裂因子，在牙上皮细胞和间充质细胞的生长过程中起着重要的作用，上皮细胞FGF主要调控牙间充质细胞的生长，间充质FGF主要调控上皮细胞的生长。Twist 1和Twist 2属于进化上保守的Twist家族的碱性螺旋-环-螺旋（bHLH）转录因子，并且在牙齿形态发生期间在牙齿间充质中高度表达。它们在牙齿间充质中的表达可以由上皮FGF诱导，我们实验室的研究表明Twist 1及其异二聚体结合伴侣E12调节FGF受体2（Fgfr 2）和Fgf 10启动子活性。这些观察结果表明，上皮FGF和间充质FGF可能通过Twist 1和Twist 2在牙齿间充质中偶联。本研究的长期目标是了解牙齿形态发生和成牙本质细胞终末分化的分子机制。拟开展的研究将检验Twist 1与E12在牙齿间充质中形成异源二聚体，并通过调节Fgfr 2和Fgf 10的表达来调节牙齿形态发生的假设。为了检验这一假设，提出了两个具体目标。目的1将确定Twist 1，沿着其异源二聚体结合伴侣E12，是否在转录水平上使用体外方法包括定量实时PCR，染色质免疫沉淀（ChIP）测定和Fgfr 2和Fgf 10启动子的缺失分析来调节Fgfr 2和Fgf 10的表达。目的2将确定Twist 1和Twist 2表达的改变是否影响体内牙齿形态发生和成牙本质细胞分化。这一目的将通过产生和分析Twist 1和Twist 2复合突变体胚胎来实现，以检查牙间充质中Fgfr 2和Fgf 10的表达是否存在Twist剂量依赖性变化以及突变体牙齿中的表型改变。成功完成拟议的研究将表明，Twist 1和Twist 2在介导由上皮FGF触发的FGF信号传导和在推进牙齿形态发生（芽到早期钟状阶段）期间在牙齿间充质中产生递归间充质FGF中发挥关键作用。

DESCRIPTION (provided by applicant): The developing tooth is a valuable paradigm for understanding the genetic and molecular basis for the way in which morphogenesis and terminal differentiation are achieved during organogenesis. Tooth morphogenesis involves reciprocal signaling interactions between the dental epithelium and mesenchyme mediated by transcription factors and growth factors, as well as cell surface and extracellular matrix (ECM) molecules. As mitogenic factors, fibroblast growth factors (FGFs) play essential roles in coupling dental epithelial and mesenchymal growth, since epithelial FGFs mainly regulate dental mesenchymal growth and mesenchymal FGFs regulate epithelial growth. Twist1 and Twist2 belong to the evolutionarily conserved Twist family of basic helix-loop-helix (bHLH) transcription factors and are highly expressed in the dental mesenchyme during tooth morphogenesis. Their expression in the dental mesenchyme can be induced by epithelial FGFs, and studies in our laboratory have shown that Twist1, together with its heterodimeric binding partner, E12, regulates FGF receptor 2 (Fgfr2) and Fgf10 promoter activities. These observations suggest that epithelial FGFs and mesenchymal FGFs might be coupled through Twist1 and Twist2 in the dental mesenchyme. The long-term goal of this research project is to understand the molecular mechanisms governing tooth morphogenesis and terminal differentiation of odontoblasts. The proposed studies will test the hypothesis that Twist1 forms heterodimers with E12 in the dental mesenchyme, and modulates tooth morphogenesis by regulating the expression of Fgfr2 and Fgf10. To test this hypothesis, two specific aims are proposed. Aim 1 will determine whether Twist1, along with its heterodimeric binding partner E12, regulates the expression of Fgfr2 and Fgf10 at the transcriptional level using in vitro approaches including quantitative real-time PCR, chromatin immunoprecipitation (ChIP) assay, and deletion analyses of the Fgfr2 and Fgf10 promoters. Aim 2 will determine if alterations in the expression of Twist1 and Twist2 affect tooth morphogenesis and odontoblast differentiation in vivo. This aim will be achieved by generating and analyzing the Twist1 and Twist2 compound mutant embryos to examine whether there are Twist dosage-dependent changes in the expression of Fgfr2 and Fgf10 in the dental mesenchyme as well as phenotypic alterations in the mutant teeth. Successful completion of the proposed studies will show that Twist1 and Twist2 play key roles in mediating FGF signaling triggered by epithelial FGFs and in generating recursive mesenchymal FGFs in the dental mesenchyme during advancing tooth morphogenesis (bud to early bell stages).