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.

哺乳动物牙齿的发育受到许多分子之间动态相互作用的调节，这些分子由全局表达的基因或以时空方式调节的牙齿特异性基因编码。牙齿是一种独特的器官，通过许多形态和细胞学变化而发育，形成四种结构和功能不同的成分：牙釉质、牙本质、牙骨质和牙髓。牙本质、牙釉质和牙骨质高度矿化，为完全形成的牙齿提供强度，而牙髓提供持续的代谢支持。成釉细胞分泌多种蛋白质来形成牙釉质细胞外基质（ECM）。釉原蛋白占分泌型牙釉质 ECM 蛋白的 90% 以上，被认为在牙釉质生物矿化中发挥重要作用。成牙本质细胞分泌牙本质 ECM，其中含有多种胶原蛋白和非胶原蛋白，形成前牙本质。前牙本质矿化形成成熟的牙本质。牙本质唾液酸磷蛋白（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。牙釉质和 DSPP 基因的基因突变与最常见的牙釉质和牙本质遗传性疾病有关。 TGF-β1 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 还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来研究牙釉蛋白、DSPP 和 TGF-β1 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发挥着独特的作用，以及它们的“串扰”。协调牙齿矿化过程。 我们使用了多种分子方法，例如常规和条件基因靶向、基因组和蛋白质组分析以及微阵列筛选来研究这些基因之间的串扰。在专家合作者的帮助下，我们还利用扫描和透射电子显微镜等特殊技术来分析牙齿的成分和纳米压痕，以确定粘合和拉伸强度。我们目前的研究不仅有助于更好地了解这些候选基因在牙齿发育中的分子作用，而且还有助于未来开发更有效的牙齿疾病治疗方法。 牙釉质基质由成釉细胞分泌并矿化形成牙釉质。釉原蛋白是牙釉质基质的主要成分，被认为在牙釉质矿化中发挥重要作用。据报道，人工智能患者的人类釉原蛋白基因发生突变。我们培育了无牙釉蛋白的小鼠，其表现出典型的 X 连锁牙釉质生成不全表型，其特征是白垩牙齿、牙釉质发育不全、缺乏棱柱状晶体和牙尖磨损。元素分析表明牙釉质含有正常的羟基磷灰石晶体，证实了在没有牙釉蛋白的情况下矿化的继续。这些结果表明，牙釉蛋白对于晶体图案的组织和牙釉质发育至关重要，但对于矿物晶体形成的起始不是必需的。我们将这些无效小鼠与过度表达牛富含亮氨酸的牙釉蛋白肽（LRAP）（交替剪接的牙釉蛋白之一）的转基因小鼠进行杂交，以评估其对牙釉蛋白无效表型的影响。这些双转基因小鼠未能挽救在牙釉蛋白无效小鼠中观察到的牙齿缺陷，这表明牙釉蛋白剪接变体中功能差异的重要性。除了牙釉质特有的作用外，牙釉蛋白还参与牙骨质的形成。在牙骨质形成过程中，赫特维希的上皮根鞘分离形成位于牙槽骨和根鞘之间的细胞聚集体（马拉塞斯的上皮残余物）。间充质来源的成牙骨质细胞将牙骨质基质分泌到牙根表面以形成牙骨质。早期报道在靠近细胞外牙骨质部位的根表面和根鞘的上皮残余物中存在牙釉蛋白。然而，这些报告基于使用多克隆抗体的免疫染色研究，因此无法识别特定的牙釉蛋白。我们使用逆转录聚合酶链反应（RT-PCR）技术分析了牙根中牙釉蛋白各种剪接变体的表达。对扩增产物进行克隆和测序以确认其序列同一性。有趣的是，这些研究发现两种牙釉蛋白剪接变体 M180 和 LRAP 主要在小鼠牙根中表达。因此，我们的研究清楚地表明，牙釉质剪接变体在牙齿的非釉质成分（即牙根）中表达，从而意味着额外的作用。为了确定牙釉蛋白在牙根中的精确作用，我们仔细分析了衰老的牙釉蛋白缺失小鼠的牙根。该分析出乎意料地揭示了无效小鼠的进行性牙骨质缺陷。无效小鼠的牙骨质在牙周膜附着于牙骨质表面的位置处显示出再吸收陷窝。牙周膜细胞的多个侵入性附着通过牙骨质延伸到无效小鼠的牙根牙本质。因此，我们的研究清楚地表明，釉原蛋白在牙釉质形成以及牙根和牙周组织的发育和维护中发挥着关键作用。 为了深入了解 DSPP 在牙本质发生中的分子作用，我们采用基因靶向策略来产生 DSPP 敲除小鼠。在这些小鼠中观察到的结构性牙齿缺陷包括牙髓腔扩大、牙本质前区宽度增加、矿化不足、牙髓暴露、矿化前沿不规则以及牙本质中钙质球体缺乏均匀聚结。在加宽的前牙本质区和无效牙本质中的钙质球体之间的空隙中，蛋白多糖双聚糖和核心蛋白聚糖的水平增加。这些增强的水平与矿化缺陷区域密切相关，并进一步表明这些分子可能通过干扰钙质球粒的聚结而对牙本质矿化过程产生不利影响。然而，空牙中的 I 型胶原蛋白水平不受影响。因此，我们推测 DSPP 敲除小鼠中双糖链蛋白聚糖和核心蛋白聚糖水平的增加与胶原纤维相互作用并促进成熟，但它们无法与成熟胶原解离，而成熟胶原是随后牙本质矿化所必需的。我们认为，DSPP 或其裂解肽除了在矿化成核中发挥的作用外，还可能在牙本质形成过程中双糖链蛋白聚糖和核心蛋白聚糖水平的调节中发挥关键作用，并且共同可能构成 DSPP 中所见的牙本质缺陷的基础。无效小鼠。 TGF-β1 是多功能生长因子超家族的成员，参与细胞增殖、分化、胚胎发育、癌变、免疫功能障碍、炎症和伤口愈合等关键过程。已在哺乳动物中鉴定出三种高度同源的 TGF-β 异构体（1、2 和 3），并且它们具有共同的信号传导途径。在这三种亚型中，TGF-β1 在整个牙齿发育过程中表达，但其在牙齿生物学中的具体作用尚不清楚。我们正在通过在牙本质中过度表达它以及用一种异构体替换另一种异构体来继续分析其作用。我们还扩展了研究范围，分析了罕见的遗传性骨骼和唾液腺疾病、法布里病和粘脂沉积症 IV 性疾病中的牙齿缺陷。