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，受多个基因控制。在脊椎动物中，大多数颅面结构起源于颅神经脊，而颅神经脊发育的异常往往会导致头面部疾病。这种疾病在人类中很常见，到目前为止，每700名新生儿中就有一名发生，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数控发育和颅面部疾病的分子基础。为了达到这一目的，我们已经建立了一个在神经脊谱系中表达荧光报告的转基因热带非洲爪哇品系(Snail2-EGFP品系)，用于计算机数控系统发育的实时成像。目前应用的目标是测试该转基因系在检测数控缺陷和提纯用于分子研究的数控细胞方面的应用。该应用程序有两个具体目标。在第一个目标中，将进一步表征和评估Snail2-EGFP系在实时检测 在早期和后期的数控开发缺陷。转基因插入的整合部位将被绘制出来，并将进行实验，以确定转基因是否对计算机数控系统的发育或附近基因的表达产生任何影响。为了测试转基因系是否适合于实时检测数控系统的异常发育，将在不同的阶段诱导出数控系统的缺陷，并使用高分辨率荧光成像进行监测。在第二个目标中，Snail2-EGFP系将被用于分离高纯度的NC细胞用于转录组分析。 包括荧光激活细胞分选在内的几种方法将评估它们在纯化EGFP标记的NC细胞方面的有效性。在不同发育阶段收集的纯化的数控细胞将用于基因芯片研究，以确定在迁移前的数控细胞中动态调节的基因。将选择感兴趣的基因，并将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数控系统开发中的潜在功能。因此，拟议研究项目的预期结果包括识别对数控系统发展具有潜在重要性的新基因，以及建立一种直接检测活体胚胎中数控系统缺陷的新方法。这些成果的成功实现将为我们在颅面研究中提供新的工具和知识。 与公共卫生相关：头部和面部出生缺陷，如唇裂和腭裂，在新生儿中非常常见。我们的研究旨在了解一种特殊类型的细胞(神经脊)的发育过程，这种细胞可以形成脊椎动物的头部和面部结构，以便开发新的方法来预防或治疗相关的人类出生缺陷，包括唇腭裂。我们已经培育出专门在神经脊显示荧光的转基因青蛙，这样就可以实时追踪这些细胞的发育。在这个项目中，我们将测试使用这些转基因青蛙来检测可能导致头部和面部缺陷的神经脊发育的任何扰动的可行性，并寻找对头部和面部结构的发育具有潜在重要性的新基因。

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.