Alk8 Regulation of Replacement Tooth Formation

描述(申请人提供)：这项研究的广泛、长期目标是确定调控斑马鱼替代牙齿形成(RTF)的分子靶点，这些分子靶点可以用于治疗人类牙齿发育不全。斑马鱼是目前唯一可用的、可驯化的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。具体地说，我们将检验这一假说，该假说得到了本实验室大量数据的支持，即AL8信号通路介导RTF。ALK8基因编码一个新的I型转化生长因子-β受体家族成员，在斑马鱼的初级和RTF发育过程中表达，并且是发育所必需的。在拟议的研究中，我们将使用先前发现的表现牙齿发育不全表型的LAF/Alk8突变体来鉴定并进行Alk8特异性RT信号伙伴的功能鉴定。Laf/alk8突变体中的RT救援将通过对活体斑马鱼矿化牙齿形成的高灵敏度体内检测和固定标本的体外分析来监测和量化。首先，我们将对laf/alk8突变体进行详细的分子/遗传学分析，以确定导致在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，包括突变和wt咽牙齿组织的微阵列分析。接下来，我们将构建Gateway转基因、热休克可诱导的显性显性阴性和结构性活性的Alk8转基因LAF/Alk8突变株，以操纵-加剧和挽救LAF/Alk8和wt兄弟姐妹的牙齿发育不全，建立这种方法作为一种体内基因传递疗法来挽救牙齿发育不全。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，以及我们的初步数据，证明Rb缺失小鼠的多余牙齿形成，定义Runx2、视网膜母细胞瘤(Rb)和Alk8信号在RTF中的分子相互作用。这些研究的意义包括：1)使用目前唯一可用的、易处理的发育模型斑马鱼来确定RTF信号通路；2)建立拯救牙齿发育不全的体内基因传递模型；3)首次确定Rb、Runx2和Alk8在RTF中的相互作用。这项拟议研究的成功完成将为牙齿缺失的治疗提供一个重要的入口，通过显著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的治疗方法来挽救人类牙齿发育不全提供手段。 公共卫生相关性：拟议研究的相关性是为人类的生物替代牙齿(RT)治疗开发基于分子的基因传递方法的潜力。这些研究的成功完成将极大地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的治疗方法来挽救人类牙齿发育不全提供手段，从而为人类牙齿缺失的治疗提供基础。

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.