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

项目摘要 牙齿和颅面畸形是美国最常见的先天性疾病， 3-4%的成年人出现牙齿畸形。对于几种牙齿和颅面疾病， 已经鉴定了调控基因。例如，PITX 2和EDA中的突变都与 缺失或畸形的乳牙和成年牙。了解这些基因是如何调节和功能， 因此，正常和疾病背景对于更好地理解牙齿如何形成和再生是重要的。然而，在这方面， 因为发育中的牙齿是动态的、异质性的组织， 信号通常是细胞类型特异性的，我们目前缺乏对Pitx 2和Eda在空间上如何表达的清晰理解。 在牙齿形成过程中受到时间调控，以及它们影响哪些基因网络以促进牙齿形成 基板增殖因此，这项工作的总体目标是确定细胞类型和组织特异性 基因调控和信号网络的基础牙基板启动和形态发生。这一目标将 可以通过完成几个目标的三刺鱼，一个听话的鱼模型， 对于发育生物学，像人类但不像老鼠，有能力取代牙齿。第一，基因组 将鉴定和分析调节发育中牙齿中Pitx 2和Eda表达的增强子， 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，Pitx 2和 EDA突变等位基因对早期牙胚细胞增殖和形态学的影响将被定量， 2、牙齿的更换（具体目标2）。最后，研究了Pitx 2和Eda突变体的下游转录效应。 将使用几种互补技术，特别是原位杂交和细胞杂交， 增殖测定（特定目标2）。这项工作将直接测试Pitx 2和Eda调节的假设， 牙齿上皮细胞增殖的邻近细胞通过不同的下游目标。除了 提高我们对导致人类牙齿的两个重要主调节基因的功能理解 这项工作将提供传统和尖端发育生物学技术的培训， 充满活力和支持性的科学环境。获得这些技能，与当地的 通过研究所的课程进行培训，将有助于提高科学界的能力， 申请人必须对牙齿形成的遗传和发育基础进行严格的独立研究。

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.