Gene Regulatory Network Controlling Premigratory Cranial vs Trunk Neural Crest

描述（由申请人提供）：神经嵴是一种多能胚胎细胞群，可产生多种衍生物，包括外周神经节、面部软骨和骨以及黑素细胞。我们已经提出并测试了一个多步骤的基因调控网络（GRN），由一系列不同的调控步骤组成，这些步骤在协调一致的作用下使颅神经嵴具有其定义的特征。然而， 在发育潜力和迁移途径的不同神经嵴人口产生在不同的轴向水平。在这里，我们建议探讨GRN的差异沿着神经轴，集中在迁移前的神经嵴细胞从两个不同的地区：颅与躯干。我们的初步转录组分析揭示了许多转录因子和信号分子特定于颅，但不是躯干神经嵴，反之亦然。我们的目标是确定这些基因在颅骨和躯干GRNs中的位置。这种系统水平的策略将提供理解为什么神经嵴GRNs产生一个特定的调节状态，用于预编程这些细胞到一个不同的状态。目的1：在颅和躯干水平的GRN连接的多重扰动分析。 随着全基因组的代表性的活动转录组的premigration颅和躯干神经嵴在手，我们将进行功能丧失的实验，以扰乱基因功能，并定量随后的全球转录变化，在假定的靶基因在单个胚胎中使用Nanostring分析。 目的2：神经嵴增强子的系统基因组学和功能分析/解剖。我们将确定顺式调节元件，介导的关键GRN因子在颅与躯干神经嵴人口的表达。我们将进行多维建模，将转录组数据和活性增强子的结果与功能扰动结果结合到神经嵴GRNs的代表性模型中。 目的3：重建躯干神经嵴程序，以测试成骨潜力。 使用GRN信息，我们将挑战的命运，躯干NC通过重新设计他们的监管电路和观察，如果错误表达/删除的关键GRN子电路影响他们的身份和能力，以促进软骨。

DESCRIPTION (provided by applicant): The neural crest is a multipotent embryonic cell population that contributes to diverse derivatives, including peripheral ganglia, cartilage and bone of the face, and melanocytes. We have proposed and tested a multistep gene regulatory network (GRN), comprised of a logical series of distinct regulatory steps that act in concert to imbue the cranial neural crest with its defining traits. However, there are significant differences in developmental potential and migratory pathways of different neural crest populations arising at different axial levels. Here, we propose to explore GRN differences along the neural axis, focusing on premigratory neural crest cells from two distinct regions: cranial versus trunk. Our preliminary transcriptome analysis reveals many transcription factors and signaling molecules specific to the cranial but not trunk neural crest or vice versa. Our goal is to determine the position of these genes in the cranial versus trunk GRNs. This systems level strategy will provide understanding of why neural crest GRNs produces a particular regulatory state for use in preprogramming these cells to a different state. The aims are: Aim 1: Multiplex perturbation analysis of GRN connections at cranial and trunk levels. With the genome-wide representation of the active transcriptome of premigratory cranial and trunk neural crest in hand, we will perform loss-of-function experiments to perturb gene function and quantitate subsequent global transcriptional changes in putative target genes in single embryos using Nanostring analysis. Aim 2: Phylogenomic and functional analysis/dissection of neural crest enhancers. We will identify cis-regulatory elements that mediate expression of key GRN factors in cranial versus trunk neural crest populations. We will perform multidimensional modeling that incorporates results of transcriptome data and active enhancers with functional perturbation results into representational models of neural crest GRNs. Aim 3: Reengineering of the trunk neural crest program to test skeletogenic potential. Using GRN information, we will challenge the fate of trunk NC by reengineering their regulatory circuits and observing if misexpression/deletion of key GRN subcircuits affects their identity and ability to contribute to cartilage.