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

描述（由申请人提供）：颅面形态发生是一个复杂的发育过程，由许多基因控制。在脊椎动物中，大多数颅面结构来自颅神经嵴（CNC），CNC发育异常通常会导致颅面疾病。这种疾病在人类中很常见，发生率为每700名新生儿中有一名，迄今为止报道了数百种不同的综合征。这项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，一个转基因热带爪蟾线，表达荧光报告的神经嵴谱系（蜗牛2-EGFP线）已产生的CNC发展的实时成像。本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。该申请有两个具体目标。在第一个目标中，将进一步表征和评估snail 2-egfp系在实时检测 早期和晚期CNC发展的缺陷。将绘制转基因插入的整合位点，并进行实验以确定转基因是否对CNC发育或附近基因的表达有任何影响。为了测试转基因系是否适合于实时检测异常CNC发育，将在不同阶段诱导CNC缺陷并通过使用高分辨率荧光成像进行监测。在第二个目标中，snail 2-egfp系将用于分离用于转录组分析的高纯度CNC细胞。 几种方法，包括荧光激活细胞分选，将评估其在纯化EGFP标记的CNC细胞的有效性。在不同发育阶段收集的纯化CNC细胞将用于cDNA微阵列研究，以鉴定在迁移前CNC细胞中动态调节的基因。将选择感兴趣的基因，并通过其他分子技术验证其表达模式;其中一些基因将在未来的研究中进一步测试其在CNC开发中的潜在功能。因此，拟议研究项目的预期成果包括鉴定对CNC发育具有潜在重要性的新基因，以及建立直接检测活胚胎中CNC缺陷的新方法。这些成果的成功实现将为我们提供新的工具和知识的颅面研究。

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.