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以及初级和替换牙齿形成（RTF）的分子靶点，这些分子靶点可以用于修复人类颅面骨骼和牙齿缺陷。虽然重大的研究工作已经导致了某些基因突变负责人颅面骨骼缺陷的鉴定，功能表征已受到阻碍有限的可用性的人体组织和合适的动物模型。类似地，目前还不存在临床相关的生物疗法来再生牙齿，并且牙齿再生策略的研究也受到类似限制的阻碍。针对性的，基于分子的治疗，以有效地和永久地纠正矿化颅面和牙齿缺损将是一个显着的进步，目前的手术修复方法。斑马鱼颅面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，这一事实为定义和功能性表征指导矿化颅面骨骼和替换牙齿发育的分子信号级联提供了一个独特的机会。斑马鱼和人类之间的基因身份，基因组组织和基因功能的广泛保守性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被操纵来修复颅面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进行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，我们将首先进行正向遗传ENU诱变筛选，结合矿化组织形成的敏感体内测定，以确定新的颅面骨骼和RTF突变体。接下来，我们将确认每个鉴定的突变体的分子身份，并进行功能表征。我们预计，拟议的研究将揭示分子信号级联，可能有助于实施临床相关的基因治疗，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.