Characterization of neural crest gene expression using a new transgenic Xenopus m

描述（申请人提供）：颅面形态发生是一个复杂的发育过程，受多种基因控制。在脊椎动物中，大多数颅面结构来源于颅神经嵴（CNC）， CNC发育异常通常导致颅面疾病。这类疾病在人类中很常见，发生率为每700名新生儿中有1人，迄今为止报告了数百种不同的综合征。这项拟议研究的长期目标是了解CNC发展和颅面疾病的分子基础。为了实现这一目标，在神经嵴谱系中表达荧光报告基因的转基因热带爪蟾株系（snail2-egfp株系）已被用于CNC发育的实时成像。本次申请的目的是测试该转基因系在检测CNC缺陷和纯化CNC细胞用于分子研究中的应用。该应用程序有两个特定的目标。在第一个目标中，snail2-egfp系将进一步表征和评估其在实时检测中的使用

DESCRIPTION (provided by applicant): Craniofacial morphogenesis is a complicated developmental process that is controlled by many genes. In vertebrates, the majority of craniofacial structures are derived from the cranial neural crest (CNC), and abnormalities in CNC development often lead to craniofacial disorders. Such disorders are common in humans, with an occurrence rate of one per 700 newborns and hundreds of different syndromes reported to date. The long-term objective of this proposed research is to understand the molecular bases of CNC development and craniofacial disorders. Toward this objective a transgenic Xenopus tropicalis line that expresses fluorescent reporter in the neural crest lineage (the snail2-egfp line) has been generated for live imaging of CNC development. The goal of the current application is to test the application of this transgenic line in detecting CNC defects and purifyig CNC cells for molecular studies. The application has two specific aims. In the first aim, the snail2-egfp line will be further characterized and assessed for its usage in real-time detection of defects in early and late CNC development. The integration site of the transgene insertion will be mapped, and experiments will be carried out to determine if the transgene has any effect on CNC development or expression of nearby genes. To test if the transgenic line is suitable for real-time detection of aberrant CNC development, deficiencies in the CNC will be induced at different stages and monitored by using high- resolution fluorescence imaging. In the second aim, the snail2-egfp line will be used to isolate high-purity CNC cells for transcriptome analyses. Several methods, including fluorescence activated cell sorting, will be evaluated for their effectiveness in purifying EGFP-labeled CNC cells. Purified CNC cells collected at different developmental stages will be used for cDNA microarray studies to identify genes that are dynamically regulated in premigratory CNC cells. Genes of interest will be selected and their expression patterns will be validated by other molecular techniques; some of these genes will be further tested for their potential functions in CNC development in future studies. Thus the expected outcomes of the proposed research project include identification of new genes that are potentially important for CNC development, as well as establishment of a new methodology for direct detection of CNC defects in live embryos. The successful achievement of these outcomes will provide us new tools and knowledge in craniofacial research. PUBLIC HEALTH RELEVANCE: Head and facial birth defects, such as cleft lip and cleft palate, are very common among newborn babies. Our research aims to understand the development of a special type of cells (the "neural crest") that gives rise to vertebrate head and facial structurs, so that new methods can be developed to prevent or treat the related human birth defects including cleft lip/palate. We have generated transgenic frogs that display fluorescence specifically in the neural crest, so that the development of these cells can be traced in real time In this project we will test the feasibility of using these transgenic frogs to detect any perturbations of neural crest development that may lead to head and facial defects, and to find new genes that are potentially important for the development of head and facial structures.