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: 10213819
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10213819
• 关键词: Ablation; Behavior; Birds; Candidate Disease Gene; Cardiac; Cardiac ablation; Cardiovascular system; Cell Lineage; Cell Separation; Cells; Chick; 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rest人群之一是“心脏神经rest”，有助于 出口和出口隔膜。鸟类胚胎中心脏波峰的消融会导致心脏缺陷 让人联想到人类的先天缺陷，持续的truncus动脉。在初步实验中，我们有 对早期迁移的心脏神经rest细胞进行了转录组分析，该细胞通过增强子基于增强剂分离 细胞分类。结果揭示了转录因子（例如MAFB，KROX20，LHX1，ID1，SALL3）以及信号传导 比较在早期迁移的心脏神经rest中有选择性地富集的分子和其他因素 到其他细胞群体。在这里，我们建议探索在屏幕中确定的因素的作用 基因调节网络将心脏神经rest赋予其独特的识别。损失和收益 功能实验将用于在功能上测试这些因素的作用及其在心脏中的位置 波峰特异性基因调节模块。此外，我们将使用逆转录病毒进行细胞谱系分析 编码的荧光团跟随细胞命运和克隆相关心脏神经rest细胞的基因表达。这 将执行以下特定目标： AIM 1：测试在早期迁移心脏神经CREST中表达的基因的调节连接 细胞。通过我们对心脏神经rest细胞活性转录组的初步基因组分析 手，我们将执行功能丧失实验以驱动基因功能并建立基因的顺序 心脏神经rest的活性。从MAFB开始，我们将扰动转录因子的功能 并分析对我们在我们的中发现的已知神经rest基因表达以及新基因的影响 屏幕。通过这种方式，我们可以在早期迁徙心脏神经rest中组装功能性基因电池。 AIM 2：使用单细胞RNA-Seq和 多重单分子荧光原位杂交（Smfish）。获得全面的看法 单个心脏波峰细胞的基因表达谱，我们将在几个细胞上执行单细胞RNA-seq 每个时间点的数百个细胞从心脏波峰上排序。进行类似的分析优势 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的改编 （SGA）将在精心分期的胚胎的组织部分进行，从而同时进行分析 在我们的转录组数据集中鉴定出的从心脏波峰基因中选择的35个项目的表达。 AIM 3：逆转录病毒介导的克隆分析与空间基因组分析（SGA）相结合以检查 单个雏鸡心脏神经元顶的细胞谱系和命运。确定发展 单个心脏神经元细胞的潜力有助于心血管系统，我们将执行 使用一种混合物的混合 重组复制无能力的禽逆转录病毒（RIA）编码不同的荧光蛋白以标记 具有不同颜色的单个克隆。通过将克隆分析与SGA耦合，我们将在单个单元格处确定 分辨率在我们屏幕上鉴定出的细胞共表达转录因子和信号分子。 这些特定目的旨在定义心脏的分子和细胞机制 神经rest的发展。最终目标是为中间的发病机理提供重要的见解 缺陷将导致发展与神经Crest相关心脏缺陷的新型策略的发展。