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

通过采用已经成功应用于测试海胆发育基因的方法， 调控网络，我们建议进行详细的分析基因的相互作用，参与指定脊椎动物 神经嵴细胞我们的目标是通过揭示大多数/所有的基因组控制，在系统水平上了解这一过程 系统的输入，并有条不紊地对其进行功能扰动，以检查玩家之间的交互。 换句话说，我们建议在系统水平上测试一个假定的神经嵴基因调控网络（NC-GRN）， 单一脊椎动物。新的高速技术的出现将大大促进这些努力， 显着提高数据采集和解释的速度，以及新的生物信息学工具，以询问 基因组信息我们将大量借鉴戴维森实验室开发的方法和概念。目标 是将这些应用于脊椎动物系统中的中到高通量。这代表了一个巨大的飞跃， 可以测试的范围和深度。最近鸡基因组的可用性为发现 基因和调控区。此外，作为一种哺乳动物，小鸡的发育与人类相似，与哺乳动物不同， 由于胚胎在母体外发育，因此在早期阶段可以进行成像。我们将在幼鸟体内测试 神经嵴基因调控网络，确定调控元件和测试直接相互作用。 目的1：检查已知神经板边界和神经嵴指定符的功能丧失的影响。 通过将吗啉代反义寡核苷酸引入到预期的神经板边缘或闭合神经板， 管材.将通过原位杂交和QPCR检查对潜在下游靶标的影响。 目的2：检测新发现的转录因子在NC-GRN中的功能 我们将测试网络中其他转录因子的作用和位置，我们将继续尝试 鉴定进入NC-GRN的转录因子。 目的3：分离神经嵴特异性蛋白调控区及其下游靶点。 我们将分离神经嵴特异性基因的假定调控区域，首先是Sox 10，然后是其他基因。 说明符，通过比较序列分析。候选区域将被电穿孔到早期鸡胚中， 鉴定神经嵴调节元件。 目的4：通过识别转录因子在网络中建立直接关系 下游基因调控区域内的结合位点。 我们将询问神经嵴增强子元件的调控区，寻找负责 神经板边界指定基因和/或神经嵴指定基因的结合。我们将检测直接结合 使用染色质免疫沉淀分析、电泳迁移率变动分析和 突变分析

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.