Molecular Genetics Of Tooth Development

牙齿的发育依赖于口腔上皮和间质之间的相互作用。间充质来源的成牙细胞分泌多种胶原蛋白和非胶原蛋白，形成独特的细胞外基质。在非胶原蛋白中，牙本质唾液蛋白（Dsp）和牙本质磷蛋白（Dpp）具有高度的牙齿特异性，被认为在牙本质向矿化牙本质的转化过程中起着至关重要的作用。这两种蛋白来源于940个氨基酸的多肽牙本质唾液磷蛋白（Dspp）的裂解。Dspp基因由5个长16kb的外显子组成，转录为4.4 kb的mRNA。Dspp的信使RNA主要在成牙细胞中表达，并在前成釉细胞中短暂表达。然而，最近在耳朵和骨头中观察到低水平的Dspp。Dsp是Dspp的氨基末端，是一种富含唾液酸的糖基化蛋白，与骨唾液蛋白、牙本质基质蛋白-1、骨桥蛋白等其他唾液蛋白有相似之处。Dpp是一种高度磷酸化的蛋白，具有天冬氨酸和磷酸丝氨酸的重复，被认为在牙本质钙化过程中羟基磷灰石形成的成核中起关键作用。由于Dspp的表达受限，以及它在人类牙本质发育不全ii （DGI-II）染色体4q位点的物理定位，Dspp被认为是该疾病的潜在候选基因。其他与生物矿化相关的重要基因包括牙本质基质蛋白-1、骨唾液蛋白、DSPP和骨桥蛋白。牙本质发育不全（DGI）是一种主要影响牙本质生物矿化的牙齿常染色体显性遗传病，根据其临床特征按严重程度的顺序可分为三种亚型，其中i型最轻，iii型最严重。dgi - 1与成骨不全有关，而更严重的形式（DGI-II和DGI-III）仅限于牙本质。乳白色牙本质伴牙髓腔消失是DGI-II的特征。DGI-III患者的牙齿被称为壳牙，在牙本质形成后不会发生牙本质矿化。在x线摄影上，这些牙齿的牙髓腔表现为扩大的牙髓腔，牙髓暴露的发生率很高。最近，在患有DGI-II疾病的家庭中发现了Dspp基因的几个突变。这些突变包括位于第3外显子末端的C-T转变（Gln45stop）导致Dspp蛋白过早终止，位于内含子3（剪接供体位点）的G-A转变突变导致外显子跳变，以及Pro17Thr和Val18Phe转变。牙本质发育不良- ii （DD-II）是另一种人类牙本质矿化疾病，与DGI-II有相似之处，由dspp基因疏水核心序列Tyr6Asp蛋白转变突变引起。这种突变使Dspp无法进入内质网。所有这些突变都表明Dspp在牙齿矿化中的潜在功能。为了描述正常牙齿发育和疾病过程中控制牙本质矿化的分子事件，我们在胚胎干细胞（ES）中删除了整个Dspp编码区，并产生了Dspp -/-小鼠。这些空白小鼠显示牙髓腔增大，牙本质前区变宽，牙本质宽度减小，牙髓暴露发生率高，与DGI-III相似。此外，这些小鼠在牙本质中扩大的前牙本质和扇形（空隙）区域内显示出高聚糖和decorin的积累增加，这与矿化缺陷密切相关。成釉原蛋白是形成釉质的主要蛋白质成分，主要参与釉质的形成。此外，淀粉原蛋白与骨质的形成和维持有关。然而，它们的精确表达谱和在胶凝发生中的分子作用尚不清楚。我们首次在野生型小鼠牙根的牙周区发现了淀粉原蛋白mRNA的剪接变体的表达，而在淀粉原蛋白缺失的小鼠中则没有。在无淀粉原的小鼠中，进行性骨质缺损与骨水泥母细胞/牙周韧带细胞中破骨细胞生成的必需分子RANKL表达增加有关。这些发现表明，源自剪接变异体的淀粉原蛋白在调节RANKL通路中发挥了新的作用，通过破骨细胞生成影响骨质的维持。

Tooth development depends on reciprocal interactions between oral epithelium and mesenchyme. The mesenchymal-derived odontoblasts secrete several collagenous and non-collagenous proteins to form a unique extracellular matrix . Among the non-collagenous proteins, dentin sialoprotein (Dsp) and dentin phosphoproteins (Dpp) are highly tooth-specific and are believed to play a crucial role in converting predentin to form mineralized dentin. These two proteins are derived from the cleavage of a 940 amino acid polypeptide, dentin sialophosphoprotein (Dspp). The Dspp gene consists of 5 exons spanning 16 kb and is transcribed as 4.4 kb mRNA . Messenger RNA for Dspp is expressed predominantly by odontoblasts and transiently by pre-ameloblasts. However, recently low levels of Dspp were observed in the ear and in bone. The Dsp, the amino terminal part of Dspp, is sialic acid rich and glycosylated protein that shares similarities with the other sialoproteins including bone sialoprotein, dentin matrix protein-1, and osteopontin. The Dpp, a highly phosphorylated protein with repeats of aspartic acid and phosphoserine, is thought to play a key role in the nucleation of hydroxyapatite formation during dentin calcification. Due to its restricted expression, and its physical localization on the human chromosome 4q within the dentinogenesis imperfecta-II (DGI-II) locus, Dspp was implicated as a potential candidate gene for this disorder. The other important genes involved in biomineralization mapped to this region of chromosome 4q include dentin matrix protein-1, bone sialoprotein, DSPP and osteopontin. Dentinogenesis imperfecta (DGI), an autosomal dominant disorder of the tooth that primarily affects dentin biomineralization, is classified into three subtypes based on the clinical features in an order of degree severity with type-I being the least severe and type-III the most severe . DGI-I is associated with osteogenesis imperfecta while the more severe forms (DGI-II and DGI-III) are restricted to the dentin. Opalescent dentin with an obliterated pulp chambers are the characteristic features in DGI-II. The teeth of patients with DGI-III are referred to as shell teeth in which the dentin mineralization does not occur after mantle dentin is formed. Radiographically, the pulp cavities in these teeth appear as enlarged pulp chambers along with high incidence of pulp exposures .Recently, several mutations in the Dspp gene have been identified in families with DGI-II disorder. These mutations include a C-T transition at the end of 3rd exon (Gln45stop) resulting in a premature termination of Dspp protein, G-A transition mutation in intron 3 (splice donor site) causing exon skipping, and Pro17Thr and Val18Phe transitions. Dentin dysplasia-II (DD-II), another human disorder of dentin mineralization, that shares similarities with DGI-II, and is attributed to a Tyr6Asp protein transition mutation in the hydrophobic core sequence of dspp gene. This mutation disabled the entry of Dspp into the endoplasmic reticulum . All these mutations indicate a potential function for Dspp in tooth mineralization. In order to characterize the molecular events that control dentin mineralization during normal tooth development and disease, we have deleted the entire Dspp coding region in embryonic stem (ES) cells and generated Dspp -/- mice. These null mice displayed an enlarged pulp cavity, widened predentin zone, decreased dentin width and high incidence of pulp exposures similar to DGI-III. Additionally, these mice showed an increased accumulation of biglycan and decorin within the widened predentin and scalloped (void spaces) regions in the dentin correlating well with defective mineralization. Amelogenins form a major protein component of developing enamel and are predominantly involved in the formation of enamel. Additionally, amelogenins have been implicated in the formation and maintenance of cementum. However, their precise expression profile and molecular role in the cementogenesis are not well understood. We have identified for the first time expression of the splice variants of amelogenin mRNA in the periodental region of tooth roots of the wild-type mice but not in the amelogenin-null mice. Progressive cementum defects in the amelogenin-null mice are associated with the increased expression of RANKL, an essential molecule for osteoclastogenesis, in cementoblast/periodontal ligament cells. These findings indicate a novel role for the amelogenin proteins, derived from the splice variants, in regulating RANKL pathway affecting the maintenance of cementum through osteoclastogenisis.