NOVEL SCREEN FOR MINERALIZED CRANIOFACIAL AND TOOTH MUTANTS IN ZEBRAFISH

描述（由申请人提供）：本研究的主要目的是确定调节斑马鱼颅面骨骼发生和初级和替代牙齿形成（RTF）的分子靶点，这些靶点可以被操纵来修复人类颅面骨骼和牙齿缺陷。尽管重要的研究工作已经导致了对人类颅面骨骼缺陷的某些基因突变的鉴定，但由于人类组织和合适的动物模型的有限可用性，功能表征受到阻碍。同样，临床相关的牙齿再生生物疗法目前还不存在，牙齿再生策略的研究也受到类似限制。有针对性的、基于分子的治疗方法可以有效和永久地纠正矿化的颅面和牙齿缺陷，这将是目前外科修复方法的重大进步。事实上，斑马鱼颅面骨骼的发育与哺乳动物非常相似，再加上斑马鱼一生中不断再生牙齿的事实，为定义和功能表征指导矿化颅面骨骼和替代牙齿发育的分子信号级联提供了独特的机会。斑马鱼和人类之间基因特性、基因组组织和基因功能的广泛保守使得斑马鱼的研究与人类发育直接相关。我们的长期目标是确定可以修复颅面骨骼缺陷和启动人类RTF的遗传途径。本研究的目的是利用斑马鱼进行调节颅面矿化骨骼和牙齿形成的分子机制的功能研究。在这个建议中，我们将首先进行前向遗传ENU突变筛选，结合敏感的体内矿化组织形成试验，以鉴定新的颅面骨骼和RTF突变体。接下来，我们将确认分子身份，并执行功能表征，每个已确定的突变体。我们期望所提出的研究将揭示分子信号级联，这可能促进临床相关基因治疗的实施，以纠正人类矿化颅面骨骼和牙齿缺陷。

DESCRIPTION (provided by applicant): The broad objective of this study is to identify molecular targets regulating zebrafish craniofacial skeletogenesis and primary and replacement tooth formation (RTF), that can be manipulated to repair human craniofacial skeletal and tooth defects in humans. Although significant research efforts have resulted in the identificaton of certain gene mutations responsible for human craniofacial skeletal defects, functional characterizations have been hampered by limited availability of human tissues and suitable animal models. Similarly, clinically relevant biological therapies to regenerate teeth do not currently exist, and research on tooth regeneration strategies is hampered by similar limitations. Targeted, molecular based therapies to effectively and permanently correct mineralized craniofacial and tooth defects would be a significant advancement over current surgical repair methods. The fact that zebrafish craniofacial skeletal development closely resembles that of mammals, combined with the fact that zebrafish continuously regenerate teeth throughout their lives, provides a unique opportunity to define and funcitionally characterize molecular signaling cascades directing mineralized craniofacial skeletal and replacement tooth development. The extensive conservation of gene identity, genomic organization and gene function between zebrafish and humans allows for studies in zebrafish to be directly related to human development. Our long term goal is to identify genetic pathways that can be manipulated to repair craniofacial skeletal defects and initiate RTF in humans. The objective of this study is to exploit the zebrafish to perform functional studies of molecular mechanisms regulating craniofacial mineralized skeletal and tooth formation. In this proposal we will first perform a forward genetic ENU mutagenesis screen, combined with a sensitive in vivo assay for mineralized tissue formation, to identify novel craniofacial skeletal and RTF mutants. Next, we will confirm the molecular identity, and perform functional characterization, of each identified mutant. We anticipate that the proposed studies will reveal molecular signaling cascades that may facilitate the implementation of clinically relevant gene based therapies to correct mineralized craniofacial skeletal and tooth defects in humans.