Alk8 Regulation of Replacement Tooth Formation

Alk8 替换牙齿形成的调节

基本信息

批准号：
7911867
负责人：
PAMELA C YELICK
金额：
$ 73.24万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-20 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7911867
关键词：
Activins Amino Acid Sequence Animal Model Autologous Binding Biological Biological Assay Cell Maintenance Data Defect Dental Dentistry Development Dominant-Negative Mutation Exhibits Family member Foundations Gene Delivery Genes Genetic Models Genetic Techniques Genomics Heat-Shock Response Human In Vitro Incisor Knockout Mice Knowledge Laboratories Life Maps Mediating Microarray Analysis Modeling Molecular Molecular Genetics Molecular Target Monitor Mus Mutant Strains Mice Natural regeneration Nucleotides Outcome Study Phenotype Phosphotransferases Point Mutation Principal Investigator Process Property Publishing Regulation Reporting Retinoblastoma Rodent Role Siblings Signal Pathway Signal Transduction Signal Transduction Pathway Specimen Staging Stem cells Supernumerary Tooth Synteny TGF-beta type I receptor Testing Therapeutic Time Tissues Tooth Loss Tooth Tissue Tooth structure Transgenic Mice Transgenic Organisms Zebrafish base clinically relevant design genetic analysis improved in vivo insight mouse model mutant mutant mouse model novel osteoblast differentiation programs public health relevance receptor

项目摘要

DESCRIPTION (provided by applicant): The broad, long-term objective of this study is to identify molecular targets regulating replacement tooth formation (RTF) in zebrafish that can be manipulated to treat tooth agenesis in humans. Zebrafish are the only currently available, tractable developmental model for RTF, and therefore provide a unique opportunity to identify and study signaling pathways regulating this process. Specifically, we will test the hypothesis, supported by much data from this laboratory, that alk8 signaling pathways mediate RTF. The alk8 gene, which encodes a novel type I TGF-beta receptor family member first identified in this laboratory, is expressed during, and is required for, zebrafish primary and RTF development. In the proposed studies, we will identify and perform functional characterization of Alk8 specific RT signaling partners, using previously identified laf/alk8 mutants, which display a tooth agenesis phenotype. RT rescue in laf/alk8 mutants will be monitored and quantified using highly sensitive in vivo assay for mineralized tooth formation in living zebrafish, and by in vitro analyses of fixed specimens. First, we will perform detailed molecular/genetic analyses of laf/alk8 mutants to define the temporal and tissue-specific, molecular and cellular defects leading to the tooth agenesis phenotype observed in these mutants, including microarray analysis of mutant and wt pharyngeal tooth tissues. Next, we will generate Gateway transgenic, heat-shock inducible dominant negative and constitutively active Alk8 transgenic laf/alk8 mutant lines to manipulate - exacerbate and rescue - tooth agenesis in laf/alk8 and wt siblings, establishing this approach as an in vivo gene delivery therapy for rescue of tooth agenesis. Finally, we will define the molecular interactions of Runx2, retinoblastoma (Rb), and Alk8 signaling in RTF, using both zebrafish and transgenic mouse models, based on published interactions of Rb and Runx2 in osteoblast differentiation, and on our preliminary data demonstrating supernumerary tooth formation in Rb null mice. The significance of the proposed studies includes the ability to: 1) define RTF signaling pathways using the only currently available, tractable, developmental model, the zebrafish; 2) establish an in vivo gene delivery model for rescue of tooth agenesis; and 3) define, for the first time, the interactions of Rb, Runx2 and Alk8 in RTF. The successful completion of the proposed studies will provide an important entry for therapeutic treatment of tooth loss, by significantly expanding our current knowledge of molecular signals regulating RTF, providing the means to eventually establish clinically relevant therapies to rescue tooth agenesis in humans. PUBLIC HEALTH RELEVANCE: The relevance of the proposed studies is the potential to develop molecular based, gene delivery approaches for biological replacement tooth (RT) therapies in humans. The successful completion of the proposed studies will provide the foundation for therapeutic treatment of tooth loss in humans, by significantly expanding our current knowledge of molecular signals regulating RTF, providing the means to eventually establish clinically relevant therapies to rescue tooth agenesis in humans.

描述（由申请人提供）：本研究的广泛、长期目标是确定调节斑马鱼替换牙形成（RTF）的分子靶标，可通过操纵这些靶标来治疗人类牙齿发育不全。斑马鱼是目前唯一可用的、易于处理的 RTF 发育模型，因此为识别和研究调节这一过程的信号通路提供了独特的机会。具体来说，我们将测试 alk8 信号通路介导 RTF 的假设，该假设得到了该实验室的大量数据的支持。 alk8 基因编码本实验室首次鉴定的新型 I 型 TGF-β 受体家族成员，在斑马鱼初级和 RTF 发育过程中表达，并且是斑马鱼初级发育和 RTF 发育所必需的。在拟议的研究中，我们将使用先前鉴定的 laf/alk8 突变体（显示牙齿发育不全表型）来鉴定并执行 Alk8 特异性 RT 信号传导伴侣的功能表征。 laf/alk8 突变体中的 RT 救援将通过活体斑马鱼矿化牙齿形成的高灵敏度体内测定以及固定样本的体外分析进行监测和量化。首先，我们将对 laf/alk8 突变体进行详细的分子/遗传分析，以确定导致这些突变体中观察到的牙齿发育不全表型的时间和组织特异性、分子和细胞缺陷，包括突变体和野生型咽齿组织的微阵列分析。接下来，我们将生成 Gateway 转基因、热休克诱导显性失活和组成型活性 Alk8 转基因 laf/alk8 突变体系，以操纵（加剧和拯救）laf/alk8 和 wt 兄弟姐妹中的牙齿发育不全，建立这种方法作为体内基因传递挽救牙齿发育不全的疗法。最后，我们将使用斑马鱼和转基因小鼠模型，基于已发表的 Rb 和 Runx2 在成骨细胞分化中的相互作用以及我们证明多生牙的初步数据，定义 RTF 中 Runx2、视网膜母细胞瘤 (Rb) 和 Alk8 信号传导的分子相互作用Rb 缺失小鼠中的形成。拟议研究的意义包括能够：1）使用当前唯一可用的、易于处理的发育模型斑马鱼来定义 RTF 信号通路； 2）建立挽救牙齿发育不全的体内基因传递模型； 3)首次在RTF中定义了Rb、Runx2和Alk8的相互作用。拟议研究的成功完成将为牙齿缺失的治疗提供重要的切入点，通过显着扩展我们目前对调节 RTF 的分子信号的了解，为最终建立临床相关疗法来拯救人类牙齿发育不全提供手段。公共健康相关性：拟议研究的相关性在于开发基于分子的基因传递方法用于人类生物替代牙齿（RT）疗法的潜力。拟议研究的成功完成将为人类牙齿缺失的治疗奠定基础，通过显着扩展我们目前对调节 RTF 的分子信号的了解，提供最终建立临床相关疗法以挽救人类牙齿发育不全的方法。