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、毛发和四肢都是这些类型的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，牙齿在胚胎11.5天开始发育，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。 牙釉质是体内最坚硬的矿化组织，位于牙冠表面，在咀嚼食物时保护牙齿不受损害，并使其免受温度和化学物质的影响。与再生骨骼和牙本质不同，一旦牙釉质破裂，受损的牙釉质就不可能自我修复。这是因为活的成釉细胞，一种负责通过分泌釉质基质形成釉质的细胞类型，在牙齿萌出时永远失去了。在与福本聪博士的合作下，我们首次通过将iPS细胞与成釉细胞作为饲养细胞共同培养，成功地将iPS细胞分化为能分泌釉质基质的成釉细胞。这些发现表明，局部环境对iPS向成釉细胞分化是必不可少的，并可能为牙齿生物工程提供一种新的途径。 我们先前发现，表普洛芬(Epproin，Epfn)是Sp锌指转录因子家族的成员，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。在Epfn基因敲除小鼠(Epfn-/-)中，牙齿的发育被推迟。然而，在后期，突变的门牙和磨牙长出过多(肥大)，并表现出釉质缺乏和牙本质结构异常。然而，这些缺陷的机制仍不清楚。紧密和黏附的连接对于成釉细胞和成牙本质细胞的分化是必不可少的，因为牢固的连接复合体的建立是这些细胞极化、发挥功能并最终分别分泌釉质和牙本质组织的基础。我们研究了在磨牙和切牙发育过程中，Epiproin/Sp6在紧密和粘连连接复合体形成中的作用。在Epiproin缺失小鼠钟状期发育的切牙和磨牙中，我们发现紧密连接和粘连连接蛋白明显减少，这些连接的丢失与成釉细胞分化失败有关。相反，Epiproin在牙髓MDPC-23细胞中的过度表达导致Wnt-&-catenin信号的激活，导致-catenin蛋白的细胞积累和核重新分布增加，但不包括钙粘素蛋白。这些结果表明，Epiproin增加了间充质细胞的Wnt-&-catenin信号，从而影响了细胞黏附和成釉细胞和成牙本质细胞的分化。

Ectodermal organ development is initiated by inductive tissue interactions. Developing teeth, epidermis, hair, and limbs are classic examples of these types of inductive processes. Tooth development can be divided into the initiation, bud, cap, and bell stages. In mice, tooth development begins at embryonic day (E) 11.5 with the 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 the 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 the inner dental epithelial cells differentiate into enamel matrix-secreting ameloblasts. The goal of this project is to discover novel and previously uncharacterized genes in order to understand how tooth and craniofacial tissues develop, and to define molecular defects underlying anomalies of these tissues. Tooth enamel is the hardest mineralized tissue in the body, and is located on the surface of the crown that protects the tooth from damage when chewing food and insulates it from temperature and chemicals. Unlike regenerative bones and dentin, self-repair of the damaged enamel is impossible once enamel is broken. This is because the live ameloblast, a cell type responsible for forming enamel by secreting enamel matrix, is lost forever when teeth erupt. In collaboration with Dr. Satoshi Fukumoto, we succeeded for the first time in differentiating iPS cells to enamel matrix-secreting ameloblasts by co-culturing them with ameloblasts as feeder cells. These findings suggest that the local environment is essential for iPS differentiation to ameloblasts and may provide a novel approach to tooth bioengineering. We previously identified epiprofin (Epfn) as a member of the Sp zinc-finger transcription factor family that is expressed in certain developing ectodermal tissues such as teeth, hair follicles, skin, and limbs. In Epfn knockout mice (Epfn-/-), development of teeth is delayed. However, at later stages, mutant incisors and molars erupt in excess (hyperdontia) and show enamel deficiency and abnormal dentin structure. However, the mechanism of the defects remains unknown. Tight and adherens junctions are indispensable for ameloblast and odontoblast cell differentiation, since establishment of a firm junctional complex is fundamental for these cells to polarize, become functional, and ultimately secrete enamel and dentin tissue, respectively. We studied the role of Epiprofin / Sp6 in the formation of tight and adherens junction complexes during molar and incisor development. In bell-stage developing incisor and molar teeth of Epiprofin-null mice, we discovered a clear decrease in tight junction and adherens junction proteins, with loss of these junctions correlating with failure of ameloblast differentiation. Conversely, overexpression of Epiprofin in dental pulp MDPC-23 cells results in activation of Wnt-β-catenin signalling, resulting in an increased cellular accumulation and nuclear redistribution of β-catenin protein but not cadherin protein. These results suggest that Epiprofin increases Wnt-β-catenin signaling in mesenchymal cells, which subsequently affects cell adhesion and ameloblast and odontoblast differentiation.