Molecular Genetics Of Tooth Development

牙本质发育不全（DGI）是一种影响牙本质的主要遗传疾病，估计发病率为6000-8000分之一。DGI的临床特征是乳白色牙本质导致牙齿变色。由于牙本质小管排列不规则且矿化异常低，牙本质形成不良。在这种疾病中，牙齿通常磨损得很快，留下短而棕色的残肢。牙本质唾液磷酸蛋白（DSPP）是一种非胶原蛋白，其突变和/或异常表达与DGI-II疾病有关。为了准确描述DSPP的功能，我们克隆并鉴定了小鼠牙本质唾液磷蛋白基因（DSPP），以确定该基因产物的结构、调控和功能。DSPP最初从约4.4kb mRNA中翻译为一个940aa肽，随后通过未知机制分裂为N端作为牙本质唾液蛋白（DSP）和C端作为牙本质磷蛋白（DPP）。我们还在已建立的小鼠成牙细胞系MO6-G3中使用一系列缺失来表征5'上游序列中的基础启动子、增强子和抑制元件。用报告基因(？-半乳糖苷酶)在5.7 KB 5？侧翼序列以确定启动子内所有必要元素的存在，这些元素将模仿内源基因的时间和空间表达模式。对2个独立的转基因系进行了DSPP-LacZ表达谱分析。该基因的发育表达模式与内源DSPP基因非常相似。我们进一步利用该启动子生成成牙细胞特异性Cre转基因小鼠系，以创建牙齿特异性敲除小鼠模型。

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.