Wnt signaling in the induction of new tooth formation

描述（由申请人提供）：尽管对牙齿形态发生和分化的了解有所进展，但对诱导牙齿形成的分子基础知之甚少。我最近的研究表明，在上皮细胞缺失Ape或激活Wnt/(3-catenin)信号后，成人口腔组织，特别是青年成人组织仍保留成牙潜能，仍能形成新牙，这表明Ape和Wnt/p-catenin是牙齿诱导的关键调控基因。此外，我发现猿缺乏症通过激活Wnt/p-catenin信号来诱导多牙。一旦开始，这些新牙齿的发育就会自主地进入细胞分化和根形成的阶段。我的长期目标是阐明调节牙齿形成起始的分子机制，从而为体外牙齿工程铺平道路。本课题的目的是利用mRNA和miRNA微阵列技术鉴定Wnt/p-catenin信号传导的下游靶基因，进一步分析其作用，并利用其体外诱导牙齿。在指导阶段（Aim 1），我将使用激光捕获显微镜获得牙齿样本，并通过比较野生型小鼠与Ape上皮缺失或Pcatenin组成性激活小鼠的基因表达谱来确定Wnt/p-catenin信号传导的下游靶点。这种方法的基本原理是，在这两个转基因小鼠中表达的基因子集应该是相似的。因此，只选择在两种转基因小鼠中突出的基因进行进一步分析。这次培训将扩展我在颅面生物学方面的专业知识，并且在系统生物信息学和干细胞生物学方面获得的知识将有助于我向一个成功的独立科学家过渡。在独立阶段，Aim 2将通过体外和体内方法分析候选调节基因在牙齿形成中的作用。在Aim 3中，我将建立体外培养体系来概括Wnt信号的非细胞自主作用，并进一步操纵关键调控基因来体外诱导牙齿。这些研究如果成功，将为牙齿和其他器官的发育提供基本的见解，也可能有助于临床的牙齿工程。公共卫生相关性：牙齿脱落会导致严重的生理和心理损伤。我将阐明诱导牙齿形成的分子机制，从而为体外牙齿工程铺平道路。

DESCRIPTION (provided by applicant): Despite advances in knowledge about tooth morphogenesis and differentiation, relatively little is known about the molecular basis of the induction of tooth formation. My recent studies show that upon epithelial deletion of Ape or activation of Wnt/(3-catenin signaling, adult oral tissues, especially young adult tissues, retain odontogenic potential and can still form new teeth, indicating that Ape and Wnt/p-catenin are key regulatory genes in tooth induction. Moreover, I have found that the induction of supernumerary teeth by Ape deficiency occurs through activation of Wnt/p-catenin signaling. Once initiated, the development of these new teeth proceeds autonomously to stages of cell differentiation and root formation. My long-term goal is to elucidate the molecular mechanisms that regulate the initiation of tooth formation, thus paving the way for in vitro tooth engineering. The objective of this proposal is to use mRNA and miRNA microarrays to identify the downstream target genes of Wnt/p-catenin signaling, and further analyze their roles and utilize them to induce teeth in vitro. In the Mentored phase (Aim 1), I will use laser capture microscopy to obtain tooth samples and to identify the downstream targets of Wnt/p-catenin signaling by comparing gene expression profiles of wild type mice with mice with epithelial loss of Ape or with constitutive activation of Pcatenin. The rationale for this approach is that a subset of genes expressed in these two transgenic mice should be similar. Therefore, only genes highlighted in both transgenic mice will be selected for further analysis. This training will expand my expertise in craniofacial biology, and the acquired knowledge in system bioinformatics and stem cell biology will facilitate my transition into a successful independent scientist. In the Independent phase, Aim 2 will focus on analyzing the role of candidate regulatory genes in tooth initiation using in vitro and in vivo approaches. In Aim 3, I will establish an in vitro culture system to recapitulate the non-cell autonomous effect of Wnt signaling, and will further manipulate the key regulatory genes to induce teeth in vitro. These studies, if successful, will provide fundamental insights into the development of teeth and other organs, and may also assist in tooth engineering in the clinic. Public Health Relevance: Loss of teeth leads to great physiological and psychological impairments. I will elucidate the molecular mechanisms underlying the induction of tooth formation, thus paving the way for in vitro tooth engineering.