Oral and Craniofacial Development and Disease

牙齿形态发生是口腔上皮和外胚间充质相互作用的结果，最终形成矿化组织、牙釉质和牙本质。小鼠牙齿发育的最早形态发生事件发生在口腔外胚层内陷到下面的神经脊来源的间充质中时。这种内陷的继续导致上皮性牙芽的形成。牙蕾周围的间充质细胞形成牙乳头，随后发育成分泌牙本质的成牙本质细胞和牙髓。蕾期后，牙胚进入帽状期和钟状期，上皮细胞分化为分泌釉质的成釉细胞。在这个口腔和颅面项目中，我们的目标是发现和表征以前未知的基因，以帮助了解牙齿和颅面组织是如何发育的，并确定这些组织异常或口腔癌潜在的分子缺陷。 利用牙胚基因芯片的差异杂交技术，我们已经鉴定出在牙齿中高表达的表普洛林。Epiproin是Sp6的同系物，Sp6是Sp和Kruppel-like-Factor(KLF)家族的成员，包含三个特征的C2H2型锌指基序。在发育中的牙齿、毛囊和四肢等外胚层器官中，均有Epiproin mRNA的表达。在细胞培养中，将表位蛋白表达载体导入牙齿上皮可促进分化为成釉细胞表型。为了确定表皮蛋白在外胚层器官发育中的作用，我们建立了表皮蛋白基因敲除小鼠，发现突变小鼠牙齿数量过多(牙齿肥大)、釉质发育不良和严重的毛发形成缺陷。我们的结果表明，在适当数量的牙齿形成和牙齿上皮细胞分化过程中，表普洛林是一个必不可少的因素。 我们还从小鼠牙胚cDNA文库中鉴定了一个新的胞外蛋白TM14。TM14在中心含有3个EGF模块，C-末端结构域与fibulin模块同源，N-末端有一个独特的Sushi结构域。TM14在发育过程中的成牙本质细胞和成牙本质细胞中均有表达。免疫组织化学染色显示TM14定位于前成牙本质细胞的根尖周区。当牙本质基质完全形成并发生牙本质矿化时，TM14存在于牙本质前基质和牙本质小管上。我们发现重组的TM14蛋白与肝素、纤维连接蛋白、纤维蛋白-1和牙本质涎磷蛋白相互作用。我们还发现，TM14优先结合牙齿间充质细胞和成牙本质细胞，而不结合牙齿上皮细胞或非牙齿细胞，如HeLa，Cos7，或NIH3T3细胞。肝素、EDTA和抗整合素Beta1抗体抑制TM14与牙间充质细胞的结合，提示含有硫酸肝素的细胞表面受体和整合素都参与了TM14细胞的结合。我们的研究结果表明，TM14在成牙本质细胞的分化和维持以及牙本质形成过程中都发挥着重要的作用。根据其蛋白质特性，TM14可归类为纤蛋白-7，属于纤蛋白家族的新成员。

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme, culminating in the formation of mineralized tissues, enamel, and dentin. The earliest morphogenetic event of mouse tooth development occurs when the oral ectoderm invaginates into the underlying neural crest-derived mesenchyme. Continuation of this invagination results in the formation of epithelial tooth buds. Mesenchymal cells surrounding the bud form the dental papillae, which later develop into dentin-secreting odontoblasts and the tooth pulp. After the bud stage, the tooth germ progresses to the cap and bell stages, and the epithelium differentiates into enamel-secreting ameloblasts. In this oral and craniofacial project, our goal is to discover and characterize previously unknown genes to help understand how tooth and craniofacial tissues develop and to define the molecular defects underlying anomalies of these tissues or oral cancer. Using differential hybridization to tooth germ cDNA microchips, we have previously identified epiprofin, which is highly expressed in teeth. Epiprofin, a homologue of Sp6, a member of the Sp and Kruppel-Like-Factor (KLF) family containing three characteristic C2H2-type zinc-finger motifs. Epiprofin mRNA is expressed in certain ectodermal organs such as developing tooth, hair follicle and limb. In cell culture, transfection of the epiprofin expression vector into dental epithelium promotes differentiation into ameloblast phenotypes. To determine the role of epiprofin in ectodermal organ development, we created gene knockout mice for epiprofin and found that mutant mice develop an excess number of teeth (hyperdontia), enamel hypoplasia, and severe defects in hair formation. Our results suggest that epiprofin is an essential factor for the formation of teeth at the proper number and for dental epithelial cell differentiation. We have also identified a new extracellular protein, TM14, from a mouse tooth germ cDNA library. TM14 contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N-terminus. TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, Cos7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin beta1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as fibulin-7, a new member of the fibulin family.