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、毛发和四肢是这些类型的诱导过程的典型例子。牙齿的发育可分为起始期、蕾状期、帽状期和钟状期。在小鼠中，牙齿发育开始于胚胎第11.5天，牙齿上皮增厚。牙板经历进一步增殖，随后发育成牙芽和牙胚。牙芽是由基板内陷和芽旁间充质细胞凝聚而成。在帽状期（E14.5），牙上皮细胞分化成几种细胞类型，如内牙上皮和釉结细胞。细胞凋亡导致的釉结内细胞死亡对磨牙牙尖的形成至关重要。钟状期（E17.5），牙间充质分化为分泌牙本质基质的成牙本质细胞，内牙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表征的基因，以了解牙齿和颅面组织如何发育，并确定这些组织异常的分子缺陷。 牙釉质是身体中最坚硬的矿化组织，位于牙冠表面，在咀嚼食物时保护牙齿免受损害，并将其与温度和化学品隔离。与再生骨骼和牙本质不同，一旦牙釉质破裂，受损牙釉质的自我修复是不可能的。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，在牙齿萌出时永远丢失。在与Satoshi Fukumoto博士的合作中，我们首次成功地将iPS细胞分化为分泌釉基质的成釉细胞，将其与成釉细胞作为饲养细胞共培养。这些发现表明，局部环境是必不可少的iPS分化成釉细胞，并可能提供一种新的方法，牙齿生物工程。 我们以前确定epiprofin（Epfn）作为Sp锌指转录因子家族的成员，在某些发育中的外胚层组织，如牙齿，毛囊，皮肤和四肢中表达。在Epfn敲除小鼠（Epfn-/-）中，牙齿发育延迟。然而，在后期阶段，突变的门牙和磨牙过度萌出（多齿），并显示出釉质缺乏和牙本质结构异常。然而，缺陷的机制仍然未知。紧密连接和粘附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对于这些细胞粘附、变得有功能并最终分别分泌釉质和牙本质组织是基本的。我们研究了Epiprofin /Sp 6在磨牙和切牙发育过程中形成紧密和粘附连接复合物的作用。在钟形阶段发展门齿和臼齿Epiprofin无效的小鼠，我们发现了一个明确的减少紧密连接和粘附连接蛋白，这些连接的损失与成釉细胞分化失败。相反，在牙髓MDPC-23细胞中表普罗芬的过度表达导致Wnt-β-连环蛋白信号的激活，导致β-连环蛋白而不是钙粘蛋白的细胞积累和核再分布增加。这些结果表明，Epiprofin增加间充质细胞中的Wnt-β-catenin信号传导，其随后影响细胞粘附以及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.