Experimental solution of the GRN underlying development of the chick neural crest

雏鸡神经嵴发育的GRN基础实验解决方案

• 批准号: 8092701
• 负责人: Marianne Bronner
• 金额: $ 31.83万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8092701
• 关键词: Antisense Oligonucleotides; Binding; Binding Sites; Bioinformatics; Biological Assay; Cartilage; Cells; Chick Embryo; Data; Development; Developmental Gene; Developmental Process; Ectoderm; Electrophoretic Mobility Shift Assay; Embryo; Enhancers; Face; Family; Fibroblast Growth Factor; Genes; Genetic Enhancer Element; Genome; Genomics; Goals; Human; Image; In Situ Hybridization; Mammals; Methodology; Mothers; Mus; Mutation; Nature; Neural Crest; Neural Crest Cell; Neural tube; Neurons; Nucleic Acid Regulatory Sequences; Pathway interactions; Phase; Population; Positioning Attribute; Process; Property; Regulator Genes; Regulatory Element; Role; Sea Urchins; Sensory; Sequence Analysis; Signal Transduction; Site; Snails; Solutions; Specific qualifier value; Speed; Staging; System; Technology; Testing; Up-Regulation; Xenopus; Zebrafish; cell type; chromatin immunoprecipitation; comparative; data acquisition; feeding; gene discovery; gene interaction; loss of function; melanocyte; neural plate; novel; progenitor; prospective; relating to nervous system; slug; success; tool; transcription factor

By adapting approaches that have been applied with great success to testing the sea urchin developmental gene regulatory network, we propose to perform a detailed analysis of the gene interactions involved in specifying vertebrate neural crest cells. Our aim is to understand the genomic control of this process at a systems level by revealing most/all of the inputs into the system and methodically functionally perturbing them to examine interactions amongst players. In other words, we propose to test a putative neural crest gene regulatory network (NC-GRN) at a systems levels in a single vertebrate. These efforts will be greatly facilitated by the advent of new, high speed technologies that will significantly increase the rate of data acquisition and interpretation as well as novel bioinformatics tools to interrogate genomic information. We will draw heavily on methodologies and concepts developed in the Davidson lab. The goal is to apply these to a vertebrate system at moderate to high throughput. This represents a huge leap forward in both the scale and depth of what can be tested. The recent availability of the chick genome affords a rich tool for discovery of genes and regulatory regions. In addition as an amniote, chick development is similar to humans and, unlike mammals, is accessible to imaging at early stages since the embryo develops outside the mother. We will test linkages in the chick neural crest gene regulatory network, identify regulatory elements and test direct interactions. Aim 1: Examine effects of loss-of-function of known neural plate border and neural crest specifiers. By introducing morpholino antisense oligonucleotides into the prospective neural plate border or closing neural tube. Effects on potential downstream targets will be examined by in situ hybridization and QPCR. Aim 2: Test the function of newly identified transcription factors in the NC-GRN We will test the role and position additional transcription factors in the network and we will continue to attempt to identify transcription factors that feed into the NC-GRN. Aim 3: Isolate regulatory regions of neural crest specifer and downstream targets. We will isolate putative regulatory regions of neural crest specifier genes, initially for Sox 10 and then other specifiers, via comparative sequence analysis. Candidate regions will be electroporated into early chick embryos to identify neural crest regulatory elements. Aim 4: Establish direct relationships within the network by identification of transcription factor binding sites within regulatory regions of downstream genes. We will interrogate the regulatory regions of neural crest enhancer elements for critical sequences responsible for binding of neural plate border specifiers genes and/or neural crest specifier genes. We will assay for direct binding interactions within the network using chromatin immunoprecipitation assay, electrophoretic mobility shift assays and mutational analysis.

通过适用于测试海胆发育基因的适应方法 调节网络，我们建议对指定脊椎动物涉及的基因相互作用进行详细分析 神经rest细胞。我们的目的是通过揭示大多数/所有 在系统中的输入，并在功能上有条不紊地扰动它们以检查玩家之间的相互作用。 换句话说，我们建议在系统级别的系统级别测试假定的神经克雷斯特基因调节网络（NC-GRN） 单脊椎动物。新的高速技术的出现将极大地促进这些努力 显着提高数据获取和解释的速度以及新颖的生物信息学工具来询问 基因组信息。我们将大力利用戴维森实验室中开发的方法和概念。目标 是将其应用于中等至高通量的脊椎动物系统。这代表了这两个方面的巨大飞跃 可以测试的规模和深度。 Chick Genome最近的可用性为发现的丰富工具提供了 基因和监管区域。此外，作为一种羊水，雏鸡的发育类似于人类，与哺乳动物不同， 由于胚胎在母亲之外发展，因此可以在早期阶段进行成像。我们将在小鸡中测试联系 神经rest基因调节网络，确定调节元件并测试直接相互作用。 AIM 1：检查已知神经板界和神经冠指定功能丧失的影响。 通过将Morpholino反义寡核苷酸引入前瞻性神经板边界或关闭神经 管子。将通过原位杂交和QPCR检查对潜在下游目标的影响。 AIM 2：测试NC-GRN中新鉴定的转录因子的功能 我们将测试网络中的角色和位置其他转录因素，我们将继续尝试 确定输入NC-GRN的转录因子。 AIM 3：神经Crest规范和下游靶标的分离的调节区域。 我们将隔离神经Crest指定基因的假定调节区域，最初是Sox 10，然后是其他 通过比较序列分析说明器。候选区域将被电穿孔到早期的小鸡胚胎到 确定神经rest调节元素。 目标4：通过识别转录因子在网络中建立直接关系 下游基因的调节区域内的结合位点。 我们将询问有关负责的关键序列的神经rest增强子元素的调节区域 神经板边框指定基因和/或神经rest指定基因的结合。我们将分析直接绑定 使用染色质免疫沉淀测定，电泳迁移率转移测定法和网络之间的相互作用 突变分析。