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的遗传和分子基础的有价值的范例。牙齿的形态发生涉及到转录因子、生长因子、细胞表面和细胞外基质（ECM）分子介导的牙上皮和间质之间的相互信号作用。作为有丝分裂因子，成纤维细胞生长因子（fibroblast growth factors, FGFs）在牙上皮细胞和间充质细胞的生长耦合过程中起着至关重要的作用，因为上皮细胞的FGFs主要调控牙间充质细胞的生长，而间充质细胞的FGFs则调控上皮细胞的生长。Twist1和Twist2属于基本螺旋-环-螺旋（bHLH）转录因子Twist家族，在牙齿形态发生过程中在牙间质中高度表达。它们在牙间质中的表达可被上皮性FGFs诱导，我们实验室的研究表明，Twist1及其异二聚体结合伙伴E12调节FGF受体2 （Fgfr2）和Fgf10启动子活性。这些观察结果表明，上皮性FGFs和间充质FGFs可能通过牙间质中的Twist1和Twist2偶联。本研究的长期目标是了解成牙细胞牙齿形态发生和终末分化的分子机制。本研究将验证Twist1在牙间质中与E12形成异源二聚体，并通过调节Fgfr2和Fgf10的表达来调节牙齿形态发生的假设。为了验证这一假设，提出了两个具体目标。目的1将确定Twist1及其异二聚体结合伙伴E12是否在转录水平上调节Fgfr2和Fgf10的表达，使用体外方法，包括定量实时PCR、染色质免疫沉淀（ChIP）测定和Fgfr2和Fgf10启动子的缺失分析。目的2将确定Twist1和Twist2表达的改变是否影响体内牙齿的形态发生和成牙细胞分化。这一目标将通过生成和分析Twist1和Twist2复合突变胚胎来实现，以检测突变牙间质中Fgfr2和Fgf10的表达是否存在Twist剂量依赖性变化以及表型改变。上述研究的成功完成将表明，Twist1和Twist2在介导上皮细胞FGFs触发的FGF信号传导以及在牙齿形态发生（萌芽期至早期钟形期）的牙间质中产生递归间充质FGFs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). PUBLIC HEALTH RELEVANCE: Successful completion of the proposed studies will provide fundamental information on the molecular mechanisms that regulate tooth morphogenesis and will contribute new insights about how teeth are formed. This information is essential for understanding the pathogeneses of congenital tooth defects, establishing scientifically based treatment modalities, and for developing methods leading to tooth regeneration.