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

Pitx2 和 Eda 表达在牙齿萌生和替换过程中的细胞特异性调控和下游效应

• 批准号: 10640204
• 负责人: Sophie L Archambeault
• 金额: $ 7.51万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10640204
• 关键词: Address; Adult; Affect; Alleles; Anhidrotic Ectodermal Dysplasia; Axenfeld-Rieger syndrome; Binding Sites; Bioinformatics; Biological Assay; Biology; Cell Proliferation; Cells; Cellular Morphology; Chromatin; Communities; Conserved Sequence; Craniofacial Abnormalities; DNA Sequence; Dental; Development; Developmental Biology; Disease; Enhancers; Environment; Epithelial Cell Proliferation; Epithelium; Exhibits; Eye Abnormalities; Fishes; Gasterosteidae; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Enhancer Element; Genetic Research; Genetic Transcription; Genomics; Goals; Hair; Heart Abnormalities; Histologic; Human; Hypodontia; In Situ Hybridization; Individual; Knowledge; Laboratories; Logic; Measures; Mediating; Mesenchyme; Methods; Modeling; Molecular; Morphogenesis; Mus; Mutation; Natural regeneration; Organogenesis; Outcome; Pathway interactions; Proliferating; Regulation; Regulator Genes; Research Institute; Signal Transduction; Signaling Molecule; Sweat Glands; Syndrome; System; Techniques; Testing; Time; Tissues; Tooth Abnormalities; Tooth Diseases; Tooth Germ; Tooth structure; Training; United States; Vertebrates; Work; Zebrafish; animal facility; cell behavior; cell type; craniofacial disorder; gain of function; gene network; genetic manipulation; genome-wide; improved; loss of function; loss of function mutation; mouse model; mutant; novel; pleiotropism; response; skill acquisition; transcription factor; transcriptomics

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.

项目摘要 牙齿和颅面异常是美国最常见的先天性条件， 3-4％的成年人发生牙齿异常。对于几种牙科和颅面疾病，突变 调节基因已被鉴定出来。例如，Pitx2和EDA中的突变都与 缺失或畸形的初级和成人牙齿。了解这些基因的调节方式并在 因此，正常和疾病环境对于更好地了解牙齿的形成和再生很重要。然而， 因为发育牙齿是动态的，异质的组织，并且对分子的启动和反应 信号通常是细胞类型特异性的，我们目前对PITX2和EDA的空间方式缺乏清晰的了解 并在牙齿启动期间受到时间调节，以及它们影响哪个基因网络以促进牙齿 位置增殖。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是牙齿位置启动和形态发生的基础。这个目标将 可以通过完成三个目标（一种可拖延鱼模型）中的几个目标来实现 对于像人类一样，与小鼠不同的发育生物学，具有替代牙齿的能力。首先，基因组 将确定并分析调节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（特定目标1）。接下来，pitx2和 在初始和 替换牙齿（特定目标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技术进行量化，特别是在原位杂交和细胞中 扩散测定（特定目标2）。这项工作将直接检验PITX2和EDA调节的假设 通过不同的下游靶标，邻近细胞的牙齿上皮细胞增殖。此外 提高我们对两个引起人牙的两个重要主调节基因的功能理解 疾病，这项工作将提供传统和尖端发育生物学技术的培训 动态和支持的科学环境。这些技能的获取，当地的互动 科学界和通过研究所的课程培训将有助于促进 申请人对牙齿形成的遗传和发育基础进行严格的独立研究。