Coupling gene regulatory and lineage analysis of the cardiac neural crest

最独特的神经嵴群之一是“心脏神经嵴”，它有助于心脏的运动。 流出道和流出隔。鸟类胚胎心脏嵴的消融导致心脏缺陷 让人联想到人类的先天缺陷，永存动脉干。在初步实验中，我们有 对早期迁移的心脏神经嵴细胞进行了转录组分析， 细胞分选结果揭示了转录因子（例如MafB、Krox 20、Lhx 1、Id 1、Sall 3）以及信号传导 分子和其他因子选择性地富集在早期迁移的心脏神经嵴中， 到其他细胞群。在这里，我们建议探索在我们的屏幕中识别的因素在 基因调控网络，赋予心脏神经嵴独特的身份。损失和收益 功能实验将用于功能测试这些因素的作用及其在心脏中的位置。 冠特异性基因调控模块。此外，我们将使用逆转录病毒进行细胞谱系分析， 编码荧光团，以跟踪克隆相关心脏神经嵴细胞的细胞命运和基因表达。的 将实现以下具体目标： 目的1：检测早期迁移心脏神经嵴中基因表达的调控联系 细胞通过我们对心脏神经嵴细胞活性转录组的初步全基因组分析， 另一方面，我们将进行功能丧失实验，以扰乱基因功能，并建立基因的顺序， 心脏神经嵴的活动从MafB开始，我们将扰乱转录因子的功能 并分析对已知神经嵴基因表达的影响，以及在我们的研究中发现的新基因， 屏幕通过这种方式，我们可以在早期迁移的心脏神经嵴中组装功能基因电池。 目的2：使用单细胞RNA-seq和 多重单分子荧光原位杂交（smFISH）。全面了解 单个心脏嵴细胞的基因表达谱，我们将对几个细胞进行单细胞RNA-seq 每个时间点从心脏嵴分选出100个细胞。进行类似的分析， 为了提供空间信息，我们设计了一种称为空间基因组分析的smFISH适应方法 (SGA)这将在精心分阶段的胚胎组织切片上进行， 从我们的转录组数据集中鉴定的心脏嵴基因中选择的35个探针的表达。 目的3：逆转录病毒介导的克隆分析与空间基因组分析（SGA）相结合， 单个鸡心脏神经嵴的细胞谱系和命运。为了确定发展 我们将研究单个心脏神经嵴细胞对心血管系统的贡献潜力， 使用混合物对鸡胚心脏神经嵴区域进行多色克隆分析 重组复制缺陷型禽逆转录病毒（RIA）编码不同的荧光蛋白标记 具有不同颜色的个体克隆。通过将克隆分析与SGA相结合，我们将确定单个细胞 在我们的筛选中鉴定的细胞共表达转录因子和信号分子的分辨率。 这些特定的目的是为了确定心脏病的分子和细胞机制， 神经嵴发育其最终目的是提供重要的见解，间隔的发病机制， 缺陷，这将导致开发新的战略，以预防神经嵴相关的心脏缺陷。

One of the most unique neural crest populations is the “cardiac neural crest” that contributes to the outflow tract and outflow septum. Ablation of the cardiac crest in bird embryos causes a heart defect reminiscent of the human birth defect, persistent truncus arteriosus. In preliminary experiments, we have performed a transcriptome analysis of early migrating cardiac neural crest cells, isolated by enhancer-based cell sorting. The results reveal transcription factors (e.g. MafB, Krox20, Lhx1, Id1, Sall3) as well as signaling molecules and other factors that are selectively enriched in the early migrating cardiac neural crest compared to other cell populations. Here, we propose to explore the role of factors identified in our screen in the gene regulatory network that imbues the cardiac neural crest with its unique identify. Loss- and gain-of- function experiments will be used to functionally test the role of these factors and their position in a cardiac crest-specific gene regulatory module. In addition, we will perform cell lineage analysis using retrovirally encoded fluorophores to follow cell fate and gene expression of clonally related cardiac neural crest cells. The following specific aims will be performed: Aim 1: Testing regulatory connections of genes expressed in early migrating cardiac neural crest cells. With our preliminary genome-wide analysis of the active transcriptome of cardiac neural crest cells in hand, we will perform loss-of-function experiments to perturb gene function and establish the order of gene activity in the cardiac neural crest. Starting with MafB, we will perturb function of the transcription factors and analyze effects on expression of known neural crest genes as well as new genes uncovered in our screen. In this way, we can assemble a functional gene battery in the early migratory cardiac neural crest. Aim 2: Transcriptional profiling of individual cardiac neural crest cells using single cell RNA-seq and multiplex single molecule fluorescent in situ hybridization (smFISH). To gain a comprehensive view of the gene expression profile of individual cardiac crest cells, we will perform single cell RNA-seq on several hundred cells per time point sorted from the cardiac crest. To perform a similar analysis with the advantage of providing spatial information, we have devised an adaptation of smFISH called Spatial Genomic Analysis (SGA) that will be performed on tissue sections of carefully staged embryos, enabling simultaneous analysis of the expression of 35 probes selected from cardiac crest genes identified in our transcriptome dataset. Aim 3: Retrovirally mediated clonal analysis coupled with Spatial Genomic Analysis (SGA) to examine the cell lineage and fate of individual chick cardiac neural crest. To determine the developmental potential of individual cardiac neural crest cells to contribute to the cardiovascular system, we will perform multi-color clonal analysis of the cardiac neural crest region of chick embryos using a mixture of recombinant replication incompetent avian retroviruses (RIA) encoding different fluorescent proteins to label individual clones with distinct colors. By coupling clonal analysis with SGA, we will determine at single cell resolution which cells co-express transcription factors and signaling molecules identified in our screen. These specific aims are designed to define the molecular and cellular mechanisms underlying cardiac neural crest development. The ultimate goal is to provide important insights into the pathogenesis of septal defects that will lead to development of novel strategies for the prevention of neural crest-related heart defects.