Genetic Hierarchies and Cellular Behaviors during Zebrafish Palatogenesis

描述（申请人提供）：人类有数百种颅面疾病，腭裂是其中常见的一种。本研究的目的是阐明腭裂发生背后的信号相互作用和细胞行为。斑马鱼为研究腭发育提供了一个有用的模型系统。强大的遗传和细胞技术可用于研究斑马鱼的基因功能以及细胞和组织信号相互作用。此外，与哺乳动物相比，斑马鱼的腭骨骼结构简化，神经嵴腭祖细胞的数量少得多，而且斑马鱼胚胎的光学清晰度使其成为分析腭发育过程中发生的细胞行为的理想选择。我建议检验一个相互信号传导假说的预测，其中信号从神经嵴到口腔外胚层，然后从口腔外胚层返回到神经嵴诱导腭发育，并通过细胞嵌入引起腭伸长。在Specific Aim 1中，我研究了在口腔外胚层中开启的神经嵴衍生信号和口腔外胚层反应基因的候选基因的作用。我使用功能丧失，基因表达，成像和遗传马赛克分析来测试来自神经嵴的FgflO和Bmp4信号打开口腔外胚层的pitx2的模型，这反过来促进腭发育。在专项目标2中，我分析了从口腔外胚层到神经嵴的互反信号。我使用功能丧失、成像和遗传马赛克分析以及可诱导的转基因斑马鱼系的构建来测试来自口腔外胚层的Pdgf和ph/ephrin信号传导促进腭发育的预测。在特异性目标3中，我确定了驱动腭伸长的细胞行为。我使用共聚焦时移分析以及克隆和表征新的斑马鱼腭突变体来测试细胞插层驱动斑马鱼腭延伸的预测。我在这些研究过程中获得的结果将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是已知的人类颅面疾病基因。因此，我对这些基因的分析将为人类疾病的起因提供直接的见解。

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.