Molecular Genetics Of Tooth Development

牙齿发育的分子遗传学

• 批准号: 6966505
• 负责人: Ashok B. KULKARNI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6966505
• 关键词: Fabry' s disease; dental development; dental disorder; extracellular matrix; gene targeting; genetically modified animals; growth factor; laboratory mouse; molecular genetics; polymerase chain reaction; toluene; tooth

Mammalian tooth development is regulated by dynamic interactions among many molecules encoded either by globally expressed genes or tooth-specific genes regulated in a spatiotemporal manner. The tooth is a unique organ that develops through a number of morphological and cytological changes leading to four structurally and functionally distinct components: enamel, dentin, cementum, and dental pulp. Dentin, enamel, and cementum are highly mineralized and provide strength to fully formed teeth, whereas dental pulp provides constant metabolic support. Ameloblasts secrete several proteins to form the enamel extracellular matrix (ECM). Amelogenins constitute more than 90% of secreted enamel ECM proteins and are believed to play an important role in the biomineralization of enamel. Odontoblasts secrete dentin ECM that contains several collagenous and noncollagenous proteins to form predentin. The predentin mineralizes to form mature dentin. Dentin sialophosphoprotein (DSPP), one of the major noncollagenous proteins, has been considered to be a key regulator in dentin formation. Genetic mutations in amelogenin and DSPP genes are implicated in the most common genetic disorders of enamel and dentin. TGF-beta1 is expressed throughout tooth development and regulates the synthesis of ECM proteins and adhesion molecules. TGF-beta also modulates immune responses and wound healing. We have used the powerful research tool of mouse molecular genetics to study the developmental roles of the amelogenin, DSPP and TGF-beta1 genes in the tooth. Our broad hypothesis is that each of these genes plays a unique role in tooth development, and their ?crosstalk? orchestrates the tooth mineralization process. We have used a variety of molecular approaches such as conventional and conditional gene targeting, genomic and proteomic analysis, and microarray screening to investigate the cross-talk amongst these genes. With the help of expert collaborators, we have also utilized special techniques such as scanning and transmission electron microscopy to analyze the composition and nanoindentation of teeth to determine bonding and tensile strength. Our present studies will not only contribute to a greater understanding of the molecular roles of these candidate genes in tooth development, but should also aid future efforts toward the development of more effective treatments for tooth disorders. Enamel matrix is secreted by ameloblasts and mineralizes to form enamel. Amelogenins are major constituents of the enamel matrix and are believed to play an important role in enamel mineralization. Mutations in the human amelogenin gene have been reported in AI patients. We generated amelogenin-null mice, which displayed a typical X-linked amelogenesis imperfecta phenotype characterized by chalky white teeth, enamel hypoplasia, a lack of prismatic crystals, and cuspal attrition. Elemental analysis indicated that the enamel contained normal hydroxyapatite crystals, confirming continuation of mineralization in the absence of the amelogenins. These results establish that amelogenins are essential for the organization of the crystal pattern and enamel development but are not required for initiation of mineral crystal formation. We crossed these null mice with transgenic mice overexpressing bovine leucine-rich amelogenin peptide (LRAP), one of the alternately spliced amelogenins, to assess its effects on the amelogenin-null phenotype. These double-transgenic mice failed to rescue the tooth defects seen in the amelogenin-null mice, indicating the importance of functional differences in amelogenin splice variants. In addition to their enamel-specific roles, amelogenins are also implicated in the formation of root cementum. During cementogenesis, Hertwig?s epithelial root sheath dissociates to form cell aggregates (epithelial rests of Malassez) that are located between the alveolar bone and the root shheath. The mesenchyme-derived cementoblasts secrete cementum matrix onto the root surface to form cementum. The presence of amelogenins was reported earlier on the root surface close to the site of extracellular cementum and in the epithelial remnants of the root sheath. However, these reports were based on immunostaining studies using polyclonal antibodies and therefore could not identify specific amelogenins. We have analyzed the expression of various spliced variants of amelogenin in tooth roots using the reverse-transcribed polymerase chain reaction (RT-PCR) technique. The amplified products were cloned and sequenced to confirm their sequence identity. Interestingly, these studies discovered that two amelogenin splice variants, M180 and LRAP, are predominantly expressed in mouse tooth roots. Thus, our studies clearly demonstrate that the amelogenin splice variants are expressed in a nonenamel component of the tooth, namely tooth roots, thereby implying additional roles. In order to determine the precise role of amelogenins in tooth roots, we carefully analyzed tooth roots of aging amelogenin-null mice. This analysis unexpectedly revealed progressive cementum defects in the null mice. The cementum of the null mice displayed resorptive lacunae at sites where periodontal ligaments attach to the cementum surface. Multiple intrusive attachments of periodontal ligament cells extended through the cementum into the root dentin of the null mice. Thus, our studies have clearly established that amelogenins play critical roles in enamel formation and also in the development and maintenance of tooth roots and periodontium. To gain insights into the molecular roles of DSPP in dentinogenesis, we pursued a gene targeting strategy to generate DSPP knockout mice. The structural tooth defects observed in these mice were enlarged pulp chambers, increased width of predentin zone, hypomineralization, pulp exposure, irregular mineralization front, and a lack of uniform coalescence of calcospherites in the dentin. The levels of the proteoglycans biglycan and decorin were increased in the widened predentin zone and in the void spaces among the calcospherites in the null dentin. These enhanced levels correlated well with the regions defective in mineralization and further indicated that these molecules may adversely affect the dentin mineralization process by interfering with the coalescence of calcospherites. However, type I collagen levels were unaffected in the null teeth. Therefore, we speculate that the increased levels of biglycan and decorin in the DSPP knockout mice interact with collagen fibrils and promote maturation, but they fail to dissociate from mature collagen, which is required for subsequent dentin mineralization. We propose that DSPP or its cleaved peptides, in addition to their suggested role in nucleation of mineralization, may play a pivotal role in the regulation of biglycan and decorin levels during dentinogenesis and together may form the basis for the dentin defects seen in the DSPP-null mice. TGF-beta1 is a member of a superfamily of multifunctional growth factors involved in key processes, such as cell proliferation, differentiation, embryonic development, carcinogenesis, immune dysfunction, inflammation, and wound healing. Three highly homologous isoforms of TGF-beta (1, 2, and 3) have been identified in mammals, and they share a common signaling pathway. Of these three isoforms, TGF-beta1 is expressed throughout tooth development, but its specific role in tooth biology is far from clear. We are continuing to analyze its role by overexpressing it in dentin and also by substituting one isofrom for another. We have also extended our studies to analyze tooth defects in rare heridetary disorders of bones and salivary glands, Fabry disease and mucolipidosis-IV disorder.

哺乳动物的牙齿发育受全局表达基因或牙齿特异性基因编码的许多分子之间的动态相互作用调控，这些分子在时空上受到调控。牙齿是一种独特的器官，经过一系列形态学和细胞学的变化，形成了四种结构和功能上不同的组成部分：牙釉质、牙本质、牙骨质和牙髓。牙本质、牙釉质和牙骨质是高度矿化的，为完全成形的牙齿提供强度，而牙髓则提供持续的代谢支持。成釉细胞分泌多种蛋白质形成釉质细胞外基质（ECM）。成釉原蛋白占分泌的牙釉质ECM蛋白的90%以上，被认为在牙釉质的生物矿化中起重要作用。成牙本质细胞分泌含有多种胶原蛋白和非胶原蛋白的牙本质外基质，形成牙本质前期。前牙本质矿化形成成熟牙本质。牙本质唾液磷蛋白（DSPP）是主要的非胶原蛋白之一，被认为是牙本质形成的关键调节因子。淀粉原蛋白和DSPP基因的基因突变与牙釉质和牙本质最常见的遗传性疾病有关。tgf - β在整个牙齿发育过程中表达，并调节ECM蛋白和粘附分子的合成。tgf - β也调节免疫反应和伤口愈合。我们利用小鼠分子遗传学这一强大的研究工具，研究了amelogenin、DSPP和TGF-beta1基因在牙齿发育中的作用。我们的广泛假设是，这些基因中的每一个在牙齿发育中都起着独特的作用，它们之间的“串扰”？协调牙齿矿化过程。