Gene Regulatory Network Controlling Premigratory Cranial vs Trunk Neural Crest

描述(申请人提供)：神经脊是一个多能的胚胎细胞群体，有助于各种衍生品，包括外周神经节、面部软骨和骨骼，以及黑素细胞。我们已经提出并测试了一个多步骤基因调控网络(GRN)，它由一系列不同的调控步骤组成，这些步骤协同行动，使脑神经脊部具有其定义特征。然而，它们之间存在着显著的差异 在不同轴向水平出现的不同神经脊种群的发育潜力和迁移路径中。在这里，我们建议探索GRN沿神经轴的差异，重点关注来自两个不同区域的迁移前神经脊细胞：颅骨和躯干。我们的初步转录组分析揭示了许多转录因子和信号分子，这些转录因子和信号分子只针对颅骨，而不是躯干神经脊，反之亦然。我们的目标是确定这些基因在颅骨和躯干GRN中的位置。这一系统水平的策略将提供对为什么神经脊GRN产生特定的调节状态以用于将这些单元预编程到不同状态的理解。目标1：头颅和躯干GRN联系的多重摄动分析。随着迁移前颅骨和躯干神经脊活性转录组的全基因组表达，我们将进行功能丧失实验来扰乱基因功能，并使用纳米串分析来定量单个胚胎中可能的靶基因随后的整体转录变化。目的2：神经脊增强剂的系统基因组和功能分析/解剖。我们将确定在脑神经脊群和躯干神经脊群中调节关键GRN因子表达的顺式调控元件。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的结果合并到神经脊GRN的代表性模型中。目的3：重建躯干神经脊程序以测试其成骨潜能。利用GRN信息，我们将通过重组躯干NC的调节电路并观察关键GRN亚电路的错误表达/缺失是否会影响它们的身份和对软骨的贡献来挑战它们的命运。

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.