MOLECULAR REGULATION OF PERIODONTIUM FORMATION

描述(申请人提供)：了解牙根发育和牙周组织形成的遗传控制是本研究的长期目标。分子生物学和发育生物学的进步将众多基因的功能与器官发育的特定阶段联系在一起。就牙齿发育而言，已有300多个基因被报道在这一过程中发挥一定作用。正如预期的那样，这些基因的突变可能会导致牙齿缺陷，其严重程度取决于突变如何影响蛋白质的功能或特定基因调节的过程。将基因与其功能联系起来的许多知识都来自转基因动物的使用，这使得遗传病的动物模型得以建立。就牙齿形成而言，许多参与牙齿发育和牙冠形成早期阶段的基因已经确定。几年前，我们报道了在小鼠中，转录因子核因子I-C(NFI-C)功能的丧失导致了一种独特的表型，其特征是磨牙根部非常短或根本没有根部。没有发现其他表型，也没有在树冠中检测到缺陷，这表明NFI-C基因产物在根形成过程中是必不可少的。NFI-C是第一个也是最新发现的唯一调控牙根发育和牙周组织形成的基因。根的形成，就像牙冠的形成一样，依赖于上皮-间充质的相互作用，导致相应组织的分化和矿化细胞外基质的产生。因为牙冠和牙根的主要区别是牙冠上皮成分，在牙冠中由外釉上皮和内釉上皮组成，外釉上皮和内釉上皮之间由星状网和中间层隔开。牙根只含有内外釉质上皮，形成赫特威格上皮根鞘(HERS)的结构。基于这一差异和初步数据部分提供的数据，我们建议检验这一中心假设，即NFI-c通过调节HERS的功能来控制牙周组织的发育，HERS进而控制牙根形态发生、成牙本质细胞分化、牙本质和牙骨质的产生以及牙周膜的形成。我们建议通过实现以下特定目标来验证这一假说：确定NFI-C在HER分化和成熟中的作用；确定与HER分化和成熟相关的NFI-C下游基因；确定NFI-C在与牙根发育相关的细胞中的相互作用；确定HERS控制成牙本质细胞分化的机制；确定HERS控制牙周膜和牙骨质形成的机制。这些研究将使用NFI-C基因敲除小鼠以及我们建立的与根形成相关的组织永生细胞系、shRNA和微阵列技术。本研究中产生的数据将提供与正常和异常牙根和牙周组织形成过程相关的急需的信息，以及参与这一过程的调节因素。这些数据对了解导致牙根缺陷、牙根发育不全和/或过早牙齿缺失的基因突变具有重要的健康意义，这对于建立未来用于牙周组织再生的遗传和组织工程疗法至关重要。 公共卫生相关性：本研究的长期目标是从形态、细胞和分子水平了解牙根发育和牙周组织形成的过程。这包括导致健康牙周组织中牙根附着的牙根牙本质、牙骨质和牙周膜形成的机制。这项应用将集中在转录因子NFI-C的作用，它似乎是牙周组织形成所必需的。这项研究中产生的数据对于了解导致牙根发育不全的遗传缺陷和导致过早牙齿脱落的牙周组织缺陷具有很大的健康相关性。此外，了解牙根和牙周组织的发育对于建立用于牙周组织再生的遗传和组织工程疗法是至关重要的。

DESCRIPTION (provided by applicant): It is the long-term objective of this study to understand the genetic control of root development and periodontium formation. Advances in molecular and developmental biology have linked the function of numerous genes to specific stages of organ development. In the case of tooth development, more than 300 hundred genes have been reported as having some role in this process. As expected, mutations in these genes can result in dental defects which vary in severity depending on how the mutation affects the function of that protein or what process that particular gene regulates. Much of the knowledge relating a gene to its function is derived from the use of transgenic animals which has allowed the creation of animal models for genetic diseases. In the case of tooth formation, many of the genes involved in the early stages of tooth development and crown formation have been established. A few years ago we reported that the loss of function of the transcription factor Nuclear Factor I-C (NFI-C) in mice results in a unique phenotype characterized by molars with very short or no roots at all. No other phenotype was found and no defects in the crowns were detected indicating that the NFI-C gene product is essential for the process of root formation. NFI- C is the first and up to date only gene that has been found to regulate root development and therefore periodontium formation. Root formation, like crown formation, is dependent on epithelial-mesenchymal interactions resulting in differentiation of the corresponding tissues and production of the mineralized extracellular matrices. Since the main difference between the crown and root is its epithelial component which in the crown is composed by the outer and inner enamel epithelia separated by the stellate reticulum and the stratum intermedium. The root contains only inner and outer enamel epithelia forming the structure known as Hertwig's Epithelial Root Sheath (HERS). Based on this difference and data presented in the preliminary data section, we propose to test the central hypothesis that Nfi-c controls periodontium development by regulating the function of HERS which in turn control root morphogenesis, odontoblast differentiation, dentin and cementum production and periodontal ligament formation. We propose to test this hypothesis by realizing the following Specific Aims: establish the role of NFI-C on HERS differentiation and maturation; determine NFI-C down-stream genes associated with HERS differentiation and maturation; determine NFI-C interactions in cells associated with root development; determine the mechanism by which HERS control odontoblast differentiation and determine the mechanism by which HERS control periodontal ligament and cementum formation. These studies will use the NFI-C knockout mice as well as our established immortal cell lines for the tissues associated with root formation, shRNA and microarray technology. The data generated in this study will provide much needed information related to the process of normal and abnormal root and periodontium formation and the regulatory factors involved in this process. This data is of great health relevance to understand genetic mutations resulting in root defects, root agenesis and/or premature tooth loss which is critical to establish future genetical and tissue engineering therapies for periodontium regeneration. PUBLIC HEALTH RELEVANCE: It is the long-term objective of this study to understand the process of root development and periodontium formation at the morphological, cellular and molecular levels. This includes the mechanisms underlying root dentin, cementum and periodontal ligament formation which lead to root attachment in the healthy periodontium. This application will focus on the role of the transcription factor NFI-C which appears to be essential for periodontium formation. The data generated in this study is of great health relevance to understand genetic defects resulting in root agenesis and periodontium defects which result in premature tooth loss. Additionally, understanding root and periodontium development is critical to establish genetical and tissue engineering therapies for periodontium regeneration.