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，其中常见的是腭裂。本研究的目的是阐明腭发育过程中的信号传导和细胞行为。斑马鱼为腭发育的研究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，简化的腭骨骨架，由比哺乳动物少得多的神经嵴腭祖细胞组成，以及斑马鱼胚胎的光学清晰度，使其成为分析腭发生中细胞行为的理想工具。我建议检查一个相互信号假说的预测，其中信号从神经嵴到口腔外胚层，然后从口腔外胚层返回神经嵴诱导腭发生，并通过细胞插入导致腭伸长。在具体目标1中，1检查了在口腔外胚层中开启的神经嵴源性信号和口腔外胚层应答基因的候选基因的作用。我使用功能丧失、基因表达、成像和遗传镶嵌分析来测试来自神经嵴的FgflO和Bmp 4信号传导在口腔外胚层中开启pitx 2的模型，这反过来促进腭发育。在具体目标2中，分析了从口腔外胚层到神经嵴的相互信号。我使用功能丧失，成像和遗传镶嵌分析，以及诱导型转基因斑马鱼系的建设，以测试的预测，Pdgf和ph/ephrin信号从口腔外胚层促进腭发育。在具体目标3中，1确定驱动腭伸长的细胞行为。我使用共聚焦时间推移分析，以及克隆和表征新的斑马鱼腭突变体，以测试预测，细胞嵌入驱动的斑马鱼腭的延伸。我在这些研究过程中获得的结果将揭示腭裂的遗传和细胞原因。此外，我建议分析的两个基因，pitx 2和ephrin-B1，已知是人类颅面疾病基因。因此，我对这些基因的分析将为人类疾病的原因提供直接的见解。

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.