NOVEL SCREEN FOR MINERALIZED CRANIOFACIAL AND TOOTH MUTANTS IN ZEBRAFISH

描述(申请人提供)：这项研究的广泛目标是确定调控斑马鱼头面部骨骼形成以及原牙和替代牙齿形成(RTF)的分子靶标，这些靶标可以用于修复人类颅面骨骼和牙齿缺陷。虽然大量的研究工作导致了导致人类头面部骨骼缺陷的某些基因突变，但由于人类组织和合适的动物模型的有限，功能表征一直受到阻碍。同样，目前还没有临床上相关的生物疗法来再生牙齿，牙齿再生策略的研究也受到类似限制的阻碍。与目前的手术修复方法相比，靶向的、基于分子的治疗有效并永久地纠正矿化的颅面和牙齿缺陷将是一个重大的进步。斑马鱼头面部骨骼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，提供了一个独特的机会来定义和功能表征引导矿化头面部骨骼和替换牙齿发育的分子信号级联。斑马鱼和人类之间的基因同一性、基因组组织和基因功能的广泛保守使得对斑马鱼的研究能够与人类发育直接相关。我们的长期目标是确定可以被操纵来修复头面部骨骼缺陷并启动人类RTF的遗传途径。本研究的目的是利用斑马鱼来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。在这项建议中，我们将首先进行正向遗传ENU突变筛选，结合体内矿化组织形成的灵敏测试，以鉴定新的头面部骨骼和RTF突变体。接下来，我们将确认每个已识别突变体的分子身份，并进行功能鉴定。我们预计，拟议的研究将揭示分子信号级联，这可能有助于临床相关基因治疗的实施，以纠正人类矿化的头面部骨骼和牙齿缺陷。

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.