Cell-specific regulation and downstream effects of Pitx2 and Eda expression in tooth initiation and replacement

项目总结 牙齿和颅面畸形是美国最常见的先天性疾病之一， 3%-4%的成年人会出现牙齿畸形。对于几种牙科和颅面疾病，基因突变 调控基因已经被识别出来。例如，PITX2和EDA的突变都与 乳牙和成人牙齿缺失或畸形。了解这些基因是如何调控和发挥作用的 因此，正常和疾病的背景对于更好地了解牙齿形成和再生是很重要的。然而， 因为发育中的牙齿是动态的、异质的组织，分子的启动和反应 信号通常是特定于细胞类型的，我们目前缺乏对Pitx2和EDA在空间上的清楚了解 和在牙齿起始过程中的时间调节，以及它们影响哪个基因网络(S)促进牙齿 胎盘增殖。因此，这项工作的总体目标是确定细胞类型和组织特异性 构成牙齿胎盘启动和形态发生的基因调控和信号网络。这一目标将 通过完成三刺鱼的几个目标来实现，这是一种易驯养的鱼模型 对于发育生物学来说，它和人类一样，但不像老鼠，有能力取代牙齿。第一，基因组 将确定和分析在发育中的牙齿中调节Pitx2和EDA表达的增强剂 提高我们从基因组序列预测增强子的能力(具体目标1)。接下来，Pitx2和Pitx2的影响 EDA突变等位基因对早期牙胚细胞增殖和形态的影响将在初始和 替换牙齿(具体目标2)。最后，Pitx2和edA突变体的下游转录效应 等位基因将使用几种互补的技术进行量化，特别是原位杂交和细胞 增殖实验(特异靶2)。这项工作将直接检验Pitx2和Eda调控的假设 牙科上皮细胞通过不同的下游靶点促进邻近细胞的增殖。除了……之外 提高我们对导致人类牙齿的两个重要主控基因的功能理解 这项工作将提供传统和尖端发育生物学技术方面的培训 充满活力和支持性的科学环境。这些技能的获得，当地内部的互动 科学界，通过研究所的课程进行培训将有助于提高 申请人对牙齿形成的遗传和发育基础进行严格、独立的研究。

PROJECT SUMMARY Dental and craniofacial abnormalities are some of the most common congenital conditions in the United States, and tooth abnormalities occur in 3-4% of adults. For several dental and craniofacial disorders, mutations in regulatory genes have been identified. For example, mutations in PITX2 and EDA are both associated with missing or misshapen primary and adult teeth. Understanding how these genes are regulated and function in normal and disease contexts is thus important to better understand how teeth form and regenerate. However, because developing teeth are dynamic, heterogeneous tissues, and the initiation of and response to molecular signals is often cell-type-specific, we currently lack a clear understanding of how Pitx2 and Eda are spatially and temporally regulated during tooth initiation, and which gene network(s) they affect to promote tooth placode proliferation. Therefore, the overall goal of this work is to determine the cell-type and tissue-specific gene regulation and signaling networks that underlie tooth placode initiation and morphogenesis. This goal will be achieved through the completion of several objectives in the threespine stickleback, a tractable fish model for developmental biology that, like humans but unlike mice, has the ability to replace teeth. First, genomic enhancers that regulate the expression of Pitx2 and Eda in developing teeth will be identified and analyzed to improve our ability to predict enhancers from genomic sequence (Specific Aim 1). Next, the effects of Pitx2 and Eda mutant alleles on early tooth germ cell proliferation and morphology will be quantified, both in initial and replacement teeth (Specific Aim 2). Finally, the downstream transcriptional effects of Pitx2 and Eda mutant alleles will be quantified using several complementary techniques, specifically in situ hybridization and cell proliferation assays (Specific Aim 2). This work will directly test the hypothesis that Pitx2 and Eda regulate dental epithelial cell proliferation of neighboring cells through distinct downstream targets. In addition to improving our functional understanding of two important master regulator genes that cause human tooth disorders, this work will provide training in traditional and cutting-edge developmental biology techniques in a dynamic and supportive scientific environment. The acquisition of these skills, interactions within the local scientific community, and training through courses at the research institute will facilitate the ability of the applicant to conduct rigorous, independent research of the genetic and developmental basis of tooth formation.