Oral and Craniofacial Development and Disease

外胚层器官的发育是由诱导性组织相互作用启动的。发育中的牙齿、表皮、毛发和四肢都是这种诱导过程的典型例子。牙齿发育可分为萌动期、蕾状期、帽状期和钟状期。在小鼠中，牙齿的发育在E11.5开始，牙齿上皮变厚。牙板经历进一步的增殖，随后发育成牙芽和牙胚。牙芽是由胎盘的内陷和靠近牙蕾的间充质细胞凝聚而成的。在帽状期(E14.5)，牙齿上皮细胞分化为几种细胞类型，如内牙上皮和釉质结节细胞。牙釉质结节内的细胞死亡是磨牙牙尖形成的关键。钟状期(E17.5)，牙本质间充质细胞分化为分泌牙本质基质的成牙本质细胞，内牙本质上皮细胞分化为分泌釉质基质的成釉细胞。该项目的目标是发现新的和以前未描述的基因，以了解牙齿和颅面组织是如何发育的，并确定这些组织异常背后的分子缺陷。 成釉蛋白(Amen)是釉质基质蛋白中的一个新成员。我们发现AMBN由成釉细胞分泌，定位于细胞的顶端，是成釉细胞的黏附分子。当成牙本质细胞从牙间充质分化出来时，分隔牙间充质和上皮组织的基底膜被前牙本质所取代。然而，负责成牙本质细胞与前牙本质附着的基质分子尚不清楚。我们发现的新的牙本质基质分子TM14可能起到这种作用。我们利用牙胚基因芯片进行差异杂交，初步确定TM14是一个在牙齿发育过程中优先表达的基因。TM14蛋白具有EGF模块、纤维蛋白基序和一个独特的N-末端结构域，可归类为纤维蛋白家族的一个新成员：纤维蛋白-7。TM14在成牙本质细胞和成牙本质细胞中均有表达。TM14优先与牙间充质细胞和成牙本质细胞结合。我们证明该蛋白还与肝素、纤维连接蛋白、纤维蛋白-1和牙本质涎磷蛋白相互作用。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时，TM14定位于前牙本质小管和牙本质小管。这些结果提示TM14在牙本质发育和牙本质小管结构中起作用。 我们先前发现EPFn/Sp6是一种锌指转录因子，在某些发育中的外胚层组织中表达，如牙齿、毛囊、皮肤和四肢。我们建立了Epfn基因敲除小鼠(Epfn-/-)，以确定其在体内的功能。Epfn-/-小鼠的牙齿有缺陷，皮肤变厚，无毛，并进行数字融合。突变的门牙和磨牙爆裂，数量过多，并有严重的釉质缺陷。突变小鼠牙尖和牙根形成存在缺陷，牙本质结构异常。在Epfn-/-小鼠中，在最初阶段形成了一个单一的牙齿胎盘和牙蕾。然而，后来，牙齿上皮的多个分支插入到牙齿间充质中。在Epfn-/-牙中，内牙上皮的快速增殖和分化受到抑制，并且保持了祖细胞的表型。异位形成多个早熟的非增殖性釉质结节结构。我们发现，Epfn载体的转染促进了牙上皮细胞的分化，并激活了成釉蛋白基因的启动子活性，表明Epfn促进了成釉细胞的分化。我们的结果表明，在Epfn缺陷的牙齿中，异位的非增殖区可能从自我更新的牙齿上皮中萌发出来，形成多个分支，最终发育成多生牙齿。因此，Epfn通过调节细胞的增殖和分化，阻止牙齿的持续萌发和生成，具有决定牙齿上皮细胞命运的多种功能。

Ectodermal organ development is initiated by inductive tissue interactions. Developing teeth, epidermis, hair, and limbs are classic examples of such inductive processes. Tooth development can be divided into the initiation, bud, cap, and bell stages. In mice, tooth development begins at E11.5 by thickening of the dental epithelium. The dental lamina undergoes further proliferation and subsequently develops into the tooth bud and germ. The tooth bud is formed by the invagination of the placode and the condensation of mesenchyme cells adjacent to the bud. At the cap stage (E14.5), dental epithelial cells differentiate into several cell types, such as the inner dental epithelium and enamel knot cells. Cell death by apoptosis within the enamel knot is critical for cusp formation in molars. At the bell stage (E17.5), the dental mesenchyme differentiates into dentin matrix-secreting odontoblasts, and then the inner dental epithelial cells differentiate into enamel matrix-secreting ameloblasts. The goal of the project is to discover novel and previously uncharacterized genes to understand how tooth and craniofacial tissues develop and to define molecular defects underlying anomalies of these tissues. We previously identified ameloblastin (Ambn) as a new member of the enamel matrix proteins. We found that Ambn was secreted by ameloblasts, localized at the apical region of the cells, and served as the adhesion molecule for ameloblasts. When odontoblasts differentiate from the dental mesenchyme, the basement membrane separating the dental mesenchyme and epithelium is replaced by predentin. However, the matrix molecule responsible for odontoblast attachment to predentin is unknown. The new dentin matrix molecule we discovered, TM14, may serve this role. We initially identified TM14 as a gene preferentially expressed in developing teeth by differential hybridization using tooth germ cDNA microchips. The TM14 protein has EGF modules, a fibulin motif, and a unique N-terminal domain and can be classified as a new member of the fibulin family: fibulin-7. TM14 mRNA was expressed in preodontoblasts and odontoblasts. TM14 bound preferentially to dental mesenchyme cells and odontoblasts. We demonstrated that the protein also interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed, TM14 was localized in predentin and dentinal tubules. These results suggest that TM14 plays roles in odontogenesis and dentinal tubule structures. We previously identified epiprofin (Epfn)/Sp6 as a zinc-finger transcription factor that is expressed in certain developing ectodermal tissues such as teeth, hair follicles, skin, and limbs. We created Epfn knockout mice (Epfn-/-) to identify its in vivo function. Epfn-/- mice had defects in tooth and developed a thick skin, hairlessness, and digital fusions. Mutant incisors and molars erupted with excess numbers and had severe enamel defects. Mutant mice have defects in cusp and root formation and abnormal dentin structure. In Epfn-/- mice, a single dental placode and bud formed at the initial stage. However, later, multiple branches of the dental epithelium had invaginated into the dental mesenchyme. In Epfn-/- teeth, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Multiple premature non-proliferating enamel knot-like structures were formed ectopically. We found that transfection of an Epfn vector promoted dental epithelial cell differentiation and activated promoter activity of the ameloblastin gene, suggesting that Epfn promotes ameloblast differentiation. Our results suggest that in Epfn-deficient teeth, ectopic, non-proliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.