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

通过采用已获得巨大成功的方法来测试海胆发育基因 ，我们建议对指定脊椎动物所涉及的基因相互作用进行详细分析 神经脊细胞。我们的目标是通过揭示大多数/全部，在系统水平上了解这一过程的基因组控制 系统的输入，并有条不紊地干扰它们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脊基因调控网络(NC-GRN)。 单一脊椎动物。新的高速技术的出现将极大地促进这些努力，这些技术将 显著提高数据采集和解释的速度，以及使用新的生物信息学工具进行讯问 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 是将这些应用于脊椎动物系统的中等到高吞吐量。这代表着两个领域的巨大飞跃 可以测试的范围和深度。最近获得的雏鸡基因组为发现 基因和调控区域。此外，作为羊膜动物，雏鸟的发育与人类相似，与哺乳动物不同， 因为胚胎是在母亲之外发育的，所以在早期阶段就可以进行成像。我们将测试雏鸟体内的联系 神经冠基因调控网络，识别调控元件并测试直接相互作用。 目的1：检验已知的神经板边缘和神经脊指示符的功能丧失的影响。 通过将吗啡反义寡核苷酸引入预期的神经板边缘或关闭神经 管子。对潜在下游靶点的影响将通过原位杂交和定量聚合酶链式反应来检测。 目的2：检测新发现的转录因子在NC-GRN中的功能 我们将测试额外的转录因子在网络中的作用和位置，并将继续尝试 识别送入NC-GRN的转录因子。 目的3：分离神经脊特异体和下游靶标的调控区域。 我们将分离神经脊指定子基因的假定调节区，首先是Sox 10，然后是其他 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以 确定神经脊调节元件。 目的4：通过鉴定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。 我们将询问神经脊增强子元件的调节区，以寻找负责 神经板边界指定子基因和/或神经脊指定子基因的结合。我们将化验直接结合 使用染色质免疫沉淀分析、电泳迁移率改变分析和 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.