Craniofacial Morphogenesis in Zebrafish

描述（由申请人提供）：为了与NIDCR改善口腔、牙齿和颅面健康的使命保持一致，本提案侧重于面部骨骼模式的发育遗传基础。它利用斑马鱼作为模型系统。该模型在研究胚胎细胞如何构建和形成一组相互连接的骨骼元素的详细性质以及了解指导这些细胞活动的基因方面具有许多积极的属性。斑马鱼与包括人类在内的其他脊椎动物在颌骨和颅骨形成的基本模式机制上大致相同。此外，有越来越多的例子表明，人类、老鼠和斑马鱼的相同基因突变会导致头部骨骼畸形，这些畸形在细节上是相似的。因此，斑马鱼可以告诉我们很多关于遗传性颅面疾病（如腭裂）缺陷的本质。提出了三个项目来测试解释早期骨骼模式关键方面的假设的具体预测，并发现新的颅面调节基因。第一项研究是检查胚胎咽弓中由上游信号分子内皮素1激活的遗传通路的功能，内皮素1对斑马鱼和小鼠的下颌模式至关重要。即使这条通道受到轻微破坏，上颚和下颚之间的关节也会消失，取而代之的是软骨，这种方式似乎与严重骨关节炎的早期阶段相似。实验检查了内皮素1靶基因的特定作用，内皮素1靶基因通常调节关节处软骨发育的抑制。到目前为止，所有对斑马鱼颅面的研究都局限于骨骼发育的最初几天，第二和第三个项目将扩大我们的知识，包括更多的日子，到少年-成年阶段。第二个项目利用共聚焦延时记录在活的、完整的鱼幼虫中荧光标记的骨和骨形成细胞的发育。它将探索动态细胞重组的本质，似乎是新骨形态形成的基础。第三项研究是在少年-成年阶段使用骨骼突变筛选来发现新的颅面基因。最重要的是找到能够在胚胎形成的早期骨骼支架上扩展的基因。对这个屏幕上恢复的突变体的分析将揭示这些基因是如何工作的。

DESCRIPTION (provided by applicant): In keeping with the mission of NIDCR to improve oral, dental and craniofacial health, this proposal focuses on the developmental genetic basis of patterning of the facial skeleton. It utilizes zebrafish as a model system. The model has many positive attributes for study of the detailed nature of how embryonic cells build and shape a set of interconnected skeletal elements, and for learning about the genes that guide these cellular activities. Zebrafish broadly share basic patterning mechanisms of jaw and skull formation with other vertebrates, including humans. Furthermore, there are increasing numbers of examples known where mutations in the very same genes of humans, mouse, and zebrafish produce head skeletal malformations that are similar in detail. Hence, zebrafish can teach us much about the nature of the defects in inherited craniofacial disorders such as cleft palate. Three projects are proposed to test specific predictions of hypotheses explaining critical aspects of early skeletal patterning, and to discover new craniofacial regulatory genes. The first investigation is to examine function of a genetic pathway that is activated in the embryonic pharyngeal arches by an upstream signaling molecule, Endothelin1, critical for jaw patterning in zebrafish and mouse alike. With even mild disruption of this pathway the joint between the upper and lower jaw is lost, replaced with cartilage in a way that seems to mimic early stages of severe osteoarthritis. The experiments examine specific roles of Endothelin1 target genes that normally regulate repression of cartilage development where the joint arises. All of the zebrafish craniofacial studies carried out so far have been limited to the first few days of skeletal development, and the second and third projects will expand our knowledge to include many more days, to the juvenile-adult stage. The second project utilizes confocal time-lapse recording of development of fluorescently labeled bone and bone-forming cells in living, intact fish larvae. It will explore the nature of the dynamic cellular reorganizations that appear to underlie formation of new bone morphologies. The third investigation is to use a skeletal mutant screen at the juvenile-adult stage to discover new craniofacial genes. The highest priority is to find genes that function to expand upon the early skeletal scaffold that forms in the embryo. Analyses of the mutants recovered in this screen will reveal how the genes work. PUBLIC HEALTH RELEVANCE: Our project will provide basic understanding of genetic and cellular mechanisms regulating morphogenesis of the craniofacial skeleton. We will investigate cellular behaviors that shape dermal bones, and we will identify and characterize new craniofacial genes, informing us of the potential disturbances underlying inherited human disorders such as in DeGeorge and Frasier syndromes.