Genetic Hierarchies and Cellular Behaviors during Zebrafish Palatogenesis

描述(申请人提供)：人类有数百种头面部疾病，其中以腭裂最为常见。这一建议的目的是阐明腭裂发生的信号相互作用和细胞行为。斑马鱼为研究腭部发育提供了一个有用的模型系统。斑马鱼拥有强大的遗传和细胞技术，可用于研究基因功能以及细胞和组织信号相互作用。此外，由比哺乳动物少得多的神经脊腭祖细胞组成的简化的腭部骨骼，以及斑马鱼胚胎的视觉清晰度，使其成为分析腭裂发生过程中细胞行为的理想工具。我建议检验互易信号假说的预测，在该假说中，从神经脊到口腔外胚层的信号，然后从口腔外胚层回到神经脊，诱导腭裂发生，并通过细胞嵌入引起腭部的延长。在特定的目标1，1检查候选基因的作用神经脊源信号和口腔外胚层反应基因，开启在口腔外胚层。我使用功能丧失、基因表达、成像和遗传马赛克分析来测试这个模型，即来自神经脊的FgflO和Bmp4信号启动口腔外胚层中的Pitx2，这反过来又促进腭裂的形成。在特定的目标2中，1分析了从口腔外胚层到神经脊的相互信号。我使用功能丧失、成像和遗传镶嵌分析以及构建可诱导的转基因斑马鱼品系来测试来自口腔外胚层的PDGF和ph/ePhin信号促进腭裂发生的预测。在特定的目标3，1中决定了驱动上颌骨伸长的细胞行为。我使用共聚焦时间推移分析以及新的斑马鱼上颚突变体的克隆和表征来测试细胞嵌入驱动斑马鱼上颚延伸的预测。我在这些研究过程中获得的结果将有助于阐明腭裂的遗传和细胞原因。此外，我建议分析的两个基因--Pitx2和ePhrin-B1，都是已知的人类头面部疾病基因。因此，我对这些基因的分析将提供对人类疾病原因的直接洞察。

DESCRIPTION (provided by applicant): There are hundreds of craniofacial diseases in humans and cleft palate is common among these. The goal of this proposal is to elucidate the signaling interactions and cellular behaviors underlying palatogenesis. The zebrafish provides a useful model system in which to study palatal development. Powerful genetic and cellular techniques are available in the zebrafish for studying gene function as well as cell and tissue signaling interactions. Additionally, the simplified palatal skeleton, consisting of far fewer neural crest palate progenitors than in mammals, and the optic clarity of the zebrafish embryo makes it ideal for analyzing cell behaviors occurring in palatogenesis. I propose to examine predictions of a reciprocal signaling hypothesis, in which signals from neural crest to the oral ectoderm and then back from the oral ectoderm to neural crest induce palatogenesis, and cause elongation of the palate through cell intercalations. In Specific Aim 1,1 examine the role of candidate genes for neural crest-derived signals and oral ectoderm response genes, turned on in the oral ectoderm. I use loss-of-function, gene expression, imaging, and genetic mosaic analyses to test the model that FgflO and Bmp4 signaling from the neural crest turns on pitx2 in the oral ectoderm, which, in turn, promotes palatogenesis. In Specific Aim 2,1 analyze the reciprocal signal, from oral ectoderm to neural crest. I use loss-of-function, imaging, and genetic mosaic analyses as well as construction of inducible transgenic zebrafish lines to test the prediction that Pdgf and ph/ephrin signaling from the oral ectoderm promotes palatogenesis. In Specific Aim 3,1 determine the cell behaviors that drive elongation of the palate. I use confocal time lapse analysis as well as cloning and characterization of novel zebrafish palate mutants to test the prediction that cell intercalations drive the extension of the zebrafish palate. The results I obtain during the course of these studies will shed light on the genetic and cellular causes of cleft palate. Additionally, two genes I propose to analyze, pitx2 and ephrin-B1, are known to be human craniofacial disease genes. Therefore, my analyses of these genes will provide direct insight into the cause of human disease.