Molecular genetics of tooth development and disease

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。成牙细胞分泌牙本质外基质，其中含有几种胶原蛋白和非胶原蛋白，形成牙本质前质。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，研究了成釉素、成釉素、DSPP和tgf - β 1基因在牙齿发育中的作用。我们的假设是，这些基因中的每一个在牙齿发育中都起着独特的作用，它们的相互作用协调了牙齿矿化过程。我们使用了各种分子方法，如常规和条件基因靶向，基因组和蛋白质组学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，我们还利用特殊技术，如扫描和透射电子显微镜来分析牙齿的成分和纳米压痕，以确定结合和抗拉强度。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。

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, which contains several collagenous and noncollagenous proteins and forms 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, ameloblastin, 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 crosstalk 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 amelogenesis imperfecta 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 the 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. Amelogenin proteins are products of RNA splicings and we evaluated some of these products for their precise function by breeding the null mice with transgenic mice overexpressing the shortest amelogenin peptide, LRAP, 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 dissociiates to form cell aggregates (epithelial rests of Malassez) that are located between the alveolar bone and the root sheath. 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. Interestingly, our recent studies discovered that two amelogenin splice variants, M180 and LRAP, are predominantly expressed in mouse tooth roots. Additionally also, we have identified LRAP expression by RT PCR from brain, eye, and calvaria tissue. 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. The aim of our ongoing study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament (CM/PDL) cells. Cocultures of wild-type (WT) osteoclast progenitor (OP) and amelogenin-null (KO) CM/PDL cells displayed an increased number of tartrate-resistant acid phosphatase (TRAP)-positive cells as compared to the cocultures of WT-OP + WT-CM/PDL cells. The addition of LRAP to both the cocultures significantly reduced the number of TRAP-positive cells. RANKL expression in the CM/PDL cell cultures was decreased by the addition of LRAP but not P172, a porcine homolog of mouse M180. Proliferation and migration rates of the KO-CM/PDL cells were lower as compared to WT cells and increased with the addition of either LRAP or P172. Our data suggest LRAP inhibits osteoclastogenesis and that both P172 and LRAP promote periodontal cell proliferation and migration of CM/PDL cells. The structural tooth defects observed in the DSPP-/- mice were enlarged pulp chambers, increased width of predentin zone, hypomineralization, pulp exposure, an 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. Their precise role is currently being analyzed using double knockout mice for DSPP/decorin, and DSPP/biglycan. We have begun to analyze specific roles of DSPP cleavage products, DSP and DPP. TGF-beta1 is a key regulator of many cellular processes, including cell adhesion, immune response and synthesis of extracellular matrix proteins. We characterized the enamel defects in a transgenic mouse model overexpressing TGF-beta1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice developed distinct dentin defects similar to those seen in human tooth disorders. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. Our ongoing studies are focused on evaluating precise roles of TGF-beta isoforms and their signaling pathways in development and disease. We are continuing our efforts to characterize craniofacial and skeletal structures in mouse models for Fabry and Mucolipidosis-IV diseases.