Regulation of Wnt signaling in tooth development and regeneration

描述（由申请人提供）：在美国，因蛀牙或意外事故导致的牙齿脱落是一个主要的健康问题，遗传性少齿症发生在近1%的人口中。目前的治疗方法依赖于外科植入物，但在骨质流失的情况下缺乏敏感性、机械故障和有限的效用。牙科生物工程的一个长期目标是利用患者自身的细胞在体外再生牙芽，使其在植入后发育成功能正常的牙齿。最近在这方面取得的进展包括成功地从游离的胚胎牙细胞或胚胎牙上皮结合成体间充质干细胞再生啮齿动物的牙芽，并将这些重建体植入产生神经支配和血管化的牙齿结构。然而，这些重建结构的大小和形状是可变的，这表明需要确定控制这些特性的内源性因素，并可以应用于生物工程策略。人类患者中Wnt/ β -连环蛋白通路组分的突变与牙齿缺陷有关。特别是，WNT10A突变存在于超过50%的先天性非综合征性下颌畸形病例中，以及外胚层发育不良综合征的一个子集中。小鼠基因功能缺失实验揭示了Wnt/ β -连环蛋白信号在牙齿发育早期阶段的需求，以及尖和根结构的形成。相反，在小鼠胚胎中，口腔上皮β -连环蛋白的强制激活激活了一系列下游的牙齿调节因子，刺激牙齿的持续发育，并在成人中导致门牙上皮形成胚胎样的牙芽。由胚胎或成人激活的口腔上皮β -连环蛋白产生的结构分别是单尖状，或矿化但不爆发。因此，Wnt信号必须严格控制正常的牙齿形态发生。Wnt/ β -连环蛋白活性可由分泌拮抗剂调节，也可通过Wnt配体、受体和辅助因子的特定组合激活β -连环蛋白独立通路。根据已发表的数据和我们的初步研究，我们假设Wnt10a及其相关基因Wnt10b通过激活β -catenin信号传导促进牙齿发育和磨牙尖形成，并与分泌的Sostdc1、Wif1和Dkk家族抑制剂竞争，影响牙齿和牙尖的发育。我们进一步假设Wnt受体Fzd2介导Wnt5a的非规范信号传导，其拮抗β -catenin通路，在牙齿生长和尖形成中起关键作用。我们将使用基因功能丧失、抢救实验和体外方法来验证这些假设。我们将进一步利用空间和时间控制的递送系统来确定重组WNT10A和Wnt10B蛋白是否可以促进胚胎牙胚和三维培养牙齿重建，以及SOSTDC1、WIF1和DKK蛋白是否可以抑制胚胎牙胚的生长和尖端形成。这些实验的结果将为确定大小和形状的生物工程牙齿以及治疗影响牙齿发育的遗传疾病提供潜在的策略。

DESCRIPTION (provided by applicant): Tooth loss through decay or accident is a major health problem in the USA, and genetic oligodontia occurs in almost 1% of the population. Current therapies rely on surgical implants that suffer from lack of sensitivity, mechanical failur and restricted utility in cases of bone loss. A long term goal of dental bioengineering is to use a patient's own cells for in vitro regeneration of tooth buds that can develop into normally functioning teeth following implantation. Recent progress towards this goal includes successful regeneration of rodent tooth buds from dissociated embryonic dental cells, or embryonic dental epithelia combined with adult mesenchymal stem cells, and implantation of these reconstructs to produce tooth structures that are innervated and vascularized. However, the sizes and shapes of these reconstructed structures are variable, indicating a need to identify endogenous factors that control these properties and could be applied in bioengineering strategies. Mutations of Wnt/beta-catenin pathway components in human patients are associated with dental defects. In particular, mutations in WNT10A are present in more than 50% of congenital non-syndromic hypodontia cases, as well as in a subset of ectodermal dysplasia syndromes. Genetic loss of function experiments in mice reveal requirements for Wnt/beta-catenin signaling at early stages of tooth development, and for formation of both cusp and root structures. Conversely, forced activation of oral epithelial beta-catenin in mouse embryos activates a cascade of downstream dental regulators, stimulating continuous tooth development, and in adults causes formation of embryonic-like tooth buds from incisor epithelia. The structures resulting from embryonic or adult activation of oral epithelial beta-catenin are, respectively, unicuspid, or ca mineralize but do not erupt. Thus, Wnt signaling must be tightly controlled for normal tooth morphogenesis. Wnt/beta-catenin activity is modulated by secreted antagonists, and by competition with alternate, beta-catenin independent pathways that are activated by specific combinations of Wnt ligands, receptors and co-factors. Based on published data and our preliminary studies we hypothesize that Wnt10a and its relative Wnt10b promote tooth development and molar cusp formation by activating beta-catenin signaling, and compete with secreted Sostdc1, Wif1 and Dkk family inhibitors to pattern tooth and cusp development. We further hypothesize that the Wnt receptor Fzd2 mediates non- canonical signaling by Wnt5a, which antagonizes the beta-catenin pathway and plays a critical role in tooth growth and cusp formation. We will test these hypotheses using genetic loss of function and rescue experiments and in vitro approaches. We will further utilize spatially and temporally-controlled delivery systems to determine whether recombinant WNT10A and Wnt10B proteins can be used to promote, and SOSTDC1, WIF1 and DKK proteins to inhibit, growth and cusp formation in embryonic tooth germs and tooth reconstructs in 3-D culture. Results from these experiments will suggest potential strategies for bioengineering teeth of defined size and shape, and for treating inherited conditions that affect tooth development.