Alk8 Regulation of Replacement Tooth Formation

Alk8 替换牙齿形成的调节

• 批准号: 7741060
• 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
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-20 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7741060
• 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

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，长期目标是确定斑马鱼中调节替代牙齿形成（RTF）的分子靶标，可以操纵以治疗人类的牙齿发生。斑马鱼是RTF当前唯一可用的可拖动发展模型，因此提供了一个独特的机会来识别和研究调节此过程的信号传导途径。具体而言，我们将检验该假设，并得到该实验室的大量数据支持的假设，即ALK8信号通路介导了RTF。 ALK8基因编码在该实验室中首次鉴定出的新型I型TGF-beta受体家族成员，在斑马鱼原发性和RTF开发中是必需的。在拟议的研究中，我们将使用先前鉴定的LAF/ALK8突变体对ALK8特异性RT信号伴侣的功能表征进行识别和执行，这些LAF/ALK8突变体显示出牙齿发育型表型。 LAF/ALK8突变体中的RT救援将使用高敏感的体内测定法监测和定量，以在活体斑马鱼中以及对固定样本的体外分析中进行矿物化的牙齿形成。首先，我们将对LAF/ALK8突变体进行详细的分子/遗传分析，以定义时间和组织特异性，分子和细胞缺陷，从而导致在这些突变体中观察到的牙齿性发育型表型，包括突变体和WT咽齿组织的微阵列分析。接下来，我们将生成网关转基因，热震诱导的显性负阴性和活性ALK8转基因LAF/ALK8突变型线，以操纵和救援 - 在LAF/ALK8和WT兄弟姐妹中的牙齿性牙齿，并确定这种方法作为一种无来自体内基因递送治疗的方法，以促进牙齿的牙科疗法。最后，我们将使用Zebrafish和Transgenic Mouse模型在RTF中定义Runx2，Renx2，视网膜母细胞瘤（RB）和ALK8信号的分子相互作用，这是基于成骨和runx2在成骨细胞中的RB和Runx2的相互作用，以及我们的初步数据，以及我们的预定数据，这些数据证明了超级牙齿形成rb null null null null null null null null null null miese。拟议研究的重要性包括：1）使用当前可用的，可拖延的，发育模型，斑马鱼来定义RTF信号通路； 2）建立一个体内基因递送模型，以挽救牙齿发育不全； 3）第一次定义RB中RB，Runx2和Alk8的相互作用。拟议的研究的成功完成将通过显着扩大我们当前对RTF的分子信号的了解，为牙齿脱落的治疗治疗提供重要的入门，从而提供了最终建立临床相关疗法以挽救人类牙齿发生的方法。 公共卫生相关性：拟议研究的相关性是开发基于分子的基因递送方法的生物替代牙齿（RT）疗法的潜力。拟议的研究的成功完成将为人类牙齿脱落的治疗治疗提供基础，并通过显着扩展我们当前对调节RTF的分子信号的了解，从而提供了最终建立临床相关疗法以挽救人类牙齿发育不全的方法。