Coupling gene regulatory and lineage analysis of the cardiac neural crest

心脏神经嵴的耦合基因调控和谱系分析

• 批准号: 9764473
• 负责人: Marianne Bronner
• 金额: $ 62.36万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9764473
• 关键词: Ablation; Behavior; Birds; Candidate Disease Gene; Cardiac; Cardiac ablation; Cardiovascular system; Cell Lineage; Cell Separation; Cells; Chick Embryo; Color; Congenital Abnormality; Congenital Heart Defects; Coupled; Coupling; Data; Data Set; Defect; Development; EGR2 gene; ETS1 gene; Elements; Embryo; Enhancers; Failure; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Order; Genes; Genetic Transcription; Genomics; Genus Alpharetrovirus; Goals; Hand; Heart; Heart Abnormalities; Human; Individual; Label; Mediating; Molecular; Mutation; Neural Crest; Neural Crest Cell; Pathogenesis; Peripheral Nervous System; Persistent Truncus Arteriosus; Population; Positioning Attribute; Prevention strategy; Process; Proteins; Recombinants; Regulator Genes; Resolution; Role; Signal Transduction; Signaling Molecule; Skeleton; Stream; Syndrome; Techniques; Testing; Time; Tissues; base; craniofacial; design; experimental study; expression cloning; fluorophore; gain of function; gene function; genome-wide analysis; insight; knock-down; loss of function; melanocyte; neurodevelopment; novel; novel strategies; prevent; public health relevance; relating to nervous system; retroviral-mediated; septal defect; single molecule; single-cell RNA sequencing; stem-like cell; transcription factor; transcriptome

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.

最独特的神经嵴群体之一是“心脏神经嵴”，它有助于 鸟胚胎中流出道和流出隔膜的消融会导致心脏缺陷。 在初步实验中，我们发现了人类先天缺陷——持续性动脉干的迹象。 对早期迁移的心脏神经嵴细胞进行转录组分析，这些细胞通过基于增强子的分离 细胞分选结果揭示了转录因子（例如 MafB、Krox20、Lhx1、Id1、Sall3）以及信号传导。 与早期迁移心脏神经嵴中选择性富集的分子和其他因子相比 在这里，我们建议探索我们的筛选中确定的因素的作用。 基因调控网络赋予心脏神经嵴独特的特性。 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 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。 将实现以下具体目标： 目标 1：测试早期迁移心脏神经嵴中表达的基因的调控联系 我们对心脏神经嵴细胞的活性转录组进行了初步的全基因组分析。 另一方面，我们将进行功能丧失实验来扰乱基因功能并建立基因的顺序 从 MafB 开始，我们将干扰转录因子的功能。 并分析对已知神经嵴基因以及我们发现的新基因表达的影响 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。 目标 2：使用单细胞 RNA-seq 对单个心脏神经嵴细胞进行转录分析 多重单分子荧光原位杂交 (smFISH) 以获得全面的视图。 为了了解单个心嵴细胞的基因表达谱，我们将对多个心嵴细胞进行单细胞 RNA-seq 每个时间点从心嵴分选一百个细胞以进行类似的分析。 为了提供空间信息，我们设计了 smFISH 的改编版，称为空间基因组分析 (SGA) 将在精心分期的胚胎的组织切片上进行，从而实现同步分析 从我们的转录组数据集中确定的心嵴基因中选出的 35 个探针的表达。 目标 3：逆转录病毒介导的克隆分析结合空间基因组分析 (SGA) 进行检查 个体鸡心脏神经嵴的细胞谱系和命运以确定发育。 个体心脏神经嵴细胞对心血管系统做出贡献的潜力，我们将进行 使用混合物对鸡胚胎心脏神经嵴区域进行多色克隆分析 重组复制无能禽逆转录病毒（RIA）编码不同的荧光蛋白进行标记 通过将克隆分析与 SGA 结合起来，我们将确定单个细胞的各个克隆。 分辨率哪些细胞共表达我们筛选中识别的转录因子和信号分子。 这些具体目标旨在定义心脏潜在的分子和细胞机制 最终目标是为隔膜的发病机制提供重要的见解。 缺陷将导致开发预防神经嵴相关心脏缺陷的新策略。