Tooth Root Formation: An Emerging Signaling Pathway

描述(申请人提供)：尽管在识别牙胚和牙冠形态发生中的信号通路方面取得了进展，但导致牙根形成的确切分子事件在很大程度上是未知的。由于牙本质构成了牙根的主体，这些知识对于探索牙髓细胞如何通过形成修复性牙本质基质来应对损伤以及开发可预测的牙本质-牙髓复合体修复方法至关重要。目前认为牙根发育是从HERS(Hertwig上皮根鞘)开始的，但成釉发育不全的患者没有明显的牙根缺陷。我们最近的研究结果表明，E14.5位的角蛋白5 Cre诱导的BMPR1A靶向缺失导致了严重的釉质缺陷，但没有表现出明显的根部牙本质表型，提示来源于间充质细胞的分子在牙根形成过程中可能起着关键作用。牙根牙本质形成模型为从分子水平研究成牙本质细胞分化的调控机制提供了一种实用的方法。在这项研究中，我们将进行一系列实验来验证 BMPR1A信号来源于牙髓/成牙本质细胞，通过一条新的NFIC-OSX-β-Catenin-DSPP信号通路控制牙根的形成。我们提出了三个目标。在特定目标1中，我们将检验OSX是BMPR1A的关键下游分子的假设。为了验证这一假设，我们将使用3.2 COL1ERTM-CRE(由注射他莫昔芬诱导)来确定Osx1的重新表达在多大程度上可以挽救BMPR1ACKO的异常根表型。在具体目标2中，我们将验证OSX通过(1)抑制细胞增殖和(2)通过抑制β-连环蛋白和上调DSPP促进CLL分化来控制牙根牙本质形成的假说。在具体目标3中，我们将 NFIC是根牙本质发生的重要转录因子，它是BMP的KE下游分子，通过OSX控制根牙本质的发生。为了验证这一假说，我们将在BMPR1ACKO成骨细胞中重新表达NFIC，以确定NFIC在多大程度上可以挽救异常的根表型。我们还将在NFICKO成牙本质细胞中重新表达OSX，以确定OSX的重新表达在多大程度上可以挽救异常的牙根表型。在一个未被研究的地区完成这个项目后，我们将提供新的机制洞察这些来自间充质细胞的信号分子在根形成过程中的作用。这些有价值的信息不仅将挑战目前的教条(牙根信号的诱导来自她)，而且还将应用于新的生物活性修复疗法的开发，旨在恢复损伤后牙本质-牙髓复合体的完整性。

DESCRIPTION (provided by applicant): Despite the advances made in identifying the signaling pathways in tooth germ and crown morphogenesis, the precise molecular events leading to root formation are largely unknown. Since dentin forms the bulk of the root, such knowledge is critical for exploring how pulpal cells respond to injury by forming a reparative dentin matrix and for developing predictable therapies for dentin-pulp complex repair. The current belief is that root development is initiated from HERS (Hertwig's Epithelial Root Sheath), but amelogenesis imperfecta patients exhibit no apparent root defects. Our recent data showed that the target deletion of Bmpr1a in epithelium by keratin 5 Cre induced at E14.5 leads to severe enamel defects but showed no apparent root dentin phenotype, suggesting that molecules originating from mesenchymal cells may play critical roles during root formation. The root dentinogenesis model offers a practical means of studying how odontoblast differentiation is regulated at the molecular level. In this study, we will perform a series of experiments to test the hypothesis that BMPR1A signaling originating from pulp/odontoblast cells controls root formation via a novel NFIC-OSX-¿-catenin-DSPP signaling pathway. We have proposed three Aims. In specific Aim 1 we will test the hypothesis that OSX is a key downstream molecule of BMPR1A. To test this hypothesis, we will use the 3.2 Col 1ERTM-Cre (induced by injection of Tamoxifen) to determine the degree to which re-expression of Osx1 can rescue the abnormal root phenotype in Bmpr1a cKO. In Specific Aim 2 we will test the hypothesis that OSX controls root dentinogenesis through (i) the inhibition of cell proliferation and (ii) the acceleration of cll differentiation via the inhibition of ?-catenin and upregulation of DSPP. In Specific Aim 3 we will test the hypothesis that NFIC, an essential transcriptional factor for root dentinogenesis, is a ke downstream molecule of BMP, which controls root dentinogenesis via OSX. To test this hypothesis, we will re-express Nfic in the Bmpr1a cKO osteoblasts to determine the degree to which Nfic can rescue the abnormal root phenotype. We will also re-express Osx in the Nfic KO odontoblasts to determine the degree to which re-expression of Osx can rescue the abnormal root phenotype. Upon completion of this project in an understudied area, we will provide new mechanistic insights into the role of these signaling molecules from mesenchymal cells during root formation. This valuable information will not only challenge the current dogma (induction of root signal is from HERS) but will also be applied to the development of new bioactive restorative therapies aimed at restoring the integrity of the dentin-pulp complex after injury.