Molecular Genetics Of Tooth Development

牙本质发育不全是一种影响牙本质的主要遗传性疾病，估计发病率为1/6000-8000。DGI的临床特征是乳白色牙本质导致牙齿变色。牙本质形成不良是由于牙本质小管排列不规则，矿化度异常低。在这种疾病中，牙齿通常会迅速磨损，留下短而棕色的残留物。牙本质涎磷蛋白(DSPP)是一种非胶原性蛋白，其突变和/或异常表达与DGI-II疾病有关。为了阐明DSPP的精确功能，我们克隆并鉴定了小鼠牙本质涎磷蛋白基因(DSPP)，以确定与该基因产物相关的结构、调控和功能。DSPP最初被翻译成约4.4kb的940aa多肽，后来由于未知的机制被切割成N端的牙本质唾液蛋白(DSP)和C端的牙本质磷蛋白(DPP)。我们还通过在已建立的小鼠成牙本质细胞系MO6-G3中进行了一系列缺失，鉴定了5‘上游序列中的基本启动子、增强子和抑制元件。将报告基因(β-半乳糖苷酶)控制在5.7kb5？侧翼序列，以确定在启动子内存在模仿内源基因的时间和空间表达模式的所有必要元件。对两个独立的转基因株系进行了DSPP-LacZ表达谱分析。转基因的发育表达模式与内源DSPP基因非常相似。我们进一步利用这个启动子产生了成牙本质细胞特异性Cre转基因小鼠系，以建立牙齿特异的基因敲除小鼠模型。 为了研究dspp介导的牙本质形成的分子机制，我们建立了dspp-/-小鼠。突变小鼠表现出牙本质矿化减少，牙髓室扩大，前牙本质和牙髓暴露增加，与人类DGI-III相似。我们发现二聚糖和核心蛋白在钙球间空隙中的分布增加，这表明这些蛋白可能干扰了钙球的结合过程，形成了相对均匀的矿化前沿。Biglycan和Decorin属于一组小亮氨酸重复蛋白，在牙本质矿化过程中与胶原纤维相互作用。除了矿物质成核外，我们还发现了一种新的对Dspp蛋白的负调控功能，即控制Biglycan和Decorin的水平，这是维持正常前牙本质所必需的。 釉原蛋白主要由成釉细胞表达，并分泌形成釉质基质的主要成分。这些蛋白在釉质形成中起着重要作用，其缺陷与釉质形成不完善有关。我们实验室培育的釉原蛋白缺失的小鼠表现出与釉质形成缺陷相关的牙齿变色和磨损的特征。通过使用抗釉原蛋白抗体的免疫染色显示，牙骨质中存在釉原蛋白，这表明这些蛋白在牙骨质中具有潜在的作用。本研究的目的是分析釉原蛋白缺失小鼠牙骨质中的缺陷，以阐明釉原蛋白在牙骨质形成中的作用。用光学显微镜、电子显微镜和显微照相技术对不同日龄的釉原蛋白缺失小鼠及其仔鼠的牙齿进行了分析。对缺牙的EM分析显示，它们的牙根表面有鹅卵石般的粗糙，而野生型小鼠的表面相对光滑。光镜下观察，缺失组小鼠牙周组织中牙本质数目显著增加(约为缺失组的6倍)。空白组小鼠牙骨质中可见大量陷窝，表明牙骨质和牙本质吸收。牙周韧带纤维常深嵌于这些陷窝中。这些发现表明，釉原蛋白不仅在牙釉质形成中起重要作用，而且可能参与牙骨质的形成。 我们早些时候已经报道，在转基因小鼠的牙齿中靶向过表达转化生长因子-1(转化生长因子-1)导致了一种新的牙齿表型，类似于人类最常见的牙病。这种表型与牙齿因矿化缺陷而变色和磨损有关。我们现在已经发现了一个新的晶体蛋白家族成员在小鼠牙齿发育中的表达，并在这些转基因小鼠中受到转化生长因子-β1的调节。晶体蛋白被认为是与压力相关的蛋白质，它们在牙齿中的表达意味着它们具有类似的作用，因为牙齿经常受到物理摩擦和温度波动的影响。

Dentinogenesis imperfecta (DGI) is a major genetic disorder affecting dentin with an estimated incidence of 1 in 6000-8000. DGI is clinically characterized by an opalescent dentin resulting in discoloration of the teeth. The dentin is poorly formed due to the irregular arrangement of dentinal tubules with abnormally low mineralization. In this disorder, teeth usually wear down rapidly, leaving short and brown stumps. Mutation and/or aberrant expression of dentin sialophosphoprotein (DSPP), a noncollagenous protein, has been implicated in DGI-II disorder. In order to delineate precise function of DSPP, we have cloned and characterized the murine dentin sialophosphoprotein gene (dspp) to establish the structure, regulation and functions associated with the gene products. The DSPP is translated initially as a single 940aa peptide from about 4.4kb mRNA and, later by unknown mechanisms, cleaved into N terminal as dentin sialoprotein (DSP) and the C terminal as dentinphosphoprotein (DPP). We have also characterized the basal promoter, enhancer and suppressor elements within 5' upstream sequences using a series of deletions in the established mouse odontoblast cell line MO6-G3. Transgenic animal model was developed with a reporter gene (?-galactosidase) under the control of 5.7 kb 5? flanking sequences to establish the presence of all the necessary elements within the promoter that would mimic the temporal and spatial expression pattern of the endogenous gene. Two independent transgenic lines harboring the transgenes were analyzed for the DSPP-LacZ expression profiles. Developmental expression patterns of the transgene was found to be very similar to the endogenous DSPP gene. We have further harnessed this promoter to generate odontoblast specific Cre transgenic mouse lines to create tooth specific knockout mouse models. To characterize the molecular mechanisms underlying dentinogenesis mediated by dspp, we have generated dspp -/- mice. The mutant mice showed dentin hypomineralization, enlarged pulp chambers, increased predentin and pulp exposures similar to human DGI-III. We found increased distribution of biglycan and decorin in the inter-calcospherite spaces, suggesting that these proteins may likely to interfere progression of calcospheritee coalescence in forming relatively uniform mineralization front. Biglycan and decorin belong to a group small leucine repeat proteins, that are known to interact with collagen fibrils in the process of dentin mineralization. In addition to the mineral nucleation, we have identified a novel negative regulatory function to Dspp protein in controlling the levels of biglycan and decorin, which is essential in maintaining the normal predentin. Amelogenins are mainly expressed by ameloblasts and secreted to form a major component of the enamel matrix. These proteins play an important role in enamel formation, defects in which are implicated in amelogenesis imperfecta. Amelogenin null mice generated in our laboratory displayed characteristic discoloration and attrition of teeth associated with defective enamel formation. Presence of amelogenins in cementum, as shown by immunostaining using anti-amelogenin antibodies, suggests a potential role for these proteins in cementum. The objective of the present study was to analyze the defects in the cementum in the amelogenin null mice in order to delineate the function of amelogenin during cementogenesis. Teeth of amelogenin null mice and their littermate controls at different ages were analyzed using light microscopy, electron microscopy and micro-radiography. EM analysis of the null teeth revealed cobbled roughness on their root surface whereas wild-type mice have a relatively smooth surface. Light microscopic analysis of tooth sections of the null mice displayed significant increase in the number of cementicles (~6 fold increase in the null mice). A number of lacunaes were noticed in the cementum of the null mice indicating resorption of cementum and dentin. Periodental-ligament fibers were frequently seen deeply embedded into these lacunaes. These findings suggest that amelogenin plays an important role not only in enamel formation but may also participate in cementogenesis. We have earlier reported that targeted over-expression of transforming growth factor-?1 (TGF-1) in the teeth of the transgenic mice (dTGF-?1) results into a novel tooth phenotype phenomimicking most prevalent tooth disorders in humans. This phenotype was associated with discoloration and attrition of teeth due to defective mineralization. We have now identified a novel expression of crystallin-family members in developing mouse teeth and its regulation by TGF-?1 in these transgenic mice. Crystallins are believed to be stress-related proteins, their expression in teeth implicates them in similar role since teeth are constantly subjected to physical friction and temperature fluctuations.