Cell lineage and transcriptional analysis of the vertebrate neural plate border

脊椎动物神经板边界的细胞谱系和转录分析

• 批准号: 10178170
• 负责人: Marianne Bronner
• 金额: $ 7.35万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10178170
• 关键词: Affect; CHARGE syndrome; Cartilage; Cell Lineage; Cell physiology; Cells; Cephalic; Characteristics; Chick Embryo; Competence; Congenital Abnormality; Cornea; Coupled; Data; Data Set; Defect; Development; DiGeorge Syndrome; Diagnosis; Disease; Dissection; Ear; Ectoderm; Embryo; Endocrine Gland Neoplasms; Enhancers; Equilibrium; Expression Profiling; Eye; Face; Fluorescent in Situ Hybridization; Ganglia; Gastrula; Gene Expression; Gene Expression Profiling; Genes; Genetic Transcription; Head; Human; Image; Individual; Injections; Lateral; Maintenance; Malignant Neoplasms; Medial; Mediating; Molecular; Natural regeneration; Neural Crest; Neural Crest Cell; Neural tube; Neuraxis; Neuroblastoma; Neurons; Neurula; Nose; Organ; Peripheral Nervous System; Pigmentation physiologic function; Population; Protein Analysis; Reporter; Resolution; Sensory; Signal Transduction; Structure; Techniques; Testing; Textbooks; Therapeutic Intervention; Time; Transcript; base; bone; cell type; epithelial to mesenchymal transition; experimental study; infancy; lens; melanoma; migration; multipotent cell; nerve stem cell; neural plate; novel; pluripotency; protein expression; public health relevance; relating to nervous system; repaired; rhodamine dextran; single molecule; single-cell RNA sequencing; stem cells; transcription factor

Bronner, M.E. In the early embryo, the neural plate border region contributes to diverse cell fates, ranging from neural crest cells and ectodermal placode cells to neurons of the central nervous system. Despite extensive studies of the neural crest and ectodermal placodes at post-neurula stages, surprisingly little is known about how these populations become distinct from one another within the early neural plate border. Based on our preliminary data, we hypothesize that many neural plate border cells are multipotent as evidenced by their concomitant expression of markers characteristic of several fates. We will test this hypothesis by: 1) conducting a detailed analysis of the emerging neural plate border region by multiplex protein and gene expression profiling coupled with cell lineage analysis and 2) examining how perturbation of transcription factor levels affects expression profiles and lineage allocations of individual neural plate border cells. The significance of this proposal is that it will be the first to test how and when ectodermal placode precursors are segregated from neural crest and neural precursors at the neural plate border. The following aims will be performed: Aim 1: High resolution analysis of protein expression of neural plate, neural crest, placode and other ectodermal markers in the neural plate border as a function of time. We will examine co-expression of transcription factors associated with neural crest, placode, neural plate and ectodermal lineages quantitatively and at single cell resolution in chick gastrula to neurula stages to determine their degree of overlap and if/when a discrete separation occurs between them in the neural plate border. To take this to a multiplex level, we will then perform single molecule fluorescent in situ hybridization analysis (smFISH) at similar stages with 35 or more probes selected from known genes and new candidates from our single cell RNA-seq dataset. Aim 2: Molecular dissection of regulatory interactions that mediate gene expression and cell fate choice at the neural plate border. We will examine the consequence of perturbing individual transcription factors (e.g. Pax7, Sox2, Six1) on expression of others neural plate border genes at the population and single cell level. To examine inputs that regulate neural plate border formation, we will dissect novel enhancers for Pax7, Six1 and other genes to determine direct regulatory inputs. Finally, we will examine how balancing levels of transcription factors may influence other factors in the neural plate border region. Aim 3: Single cell lineage analysis of cells at the neural plate border. To definitively test whether individual cells at the neural plate border have restricted or broad developmental potential, we will carry out single cell lineage analysis by performing iontophoretic injection of lysinated rhodamine dextran into individual neural plate border cells. We also will use enhancers for Pax7, Sox2, or Six1 as well as photoconversion of individual cells to follow the long term fate of neural plate border cells and examine how blocking individual transcription factors affects cell lineage allocation. 1

Bronner，M.E. 在早期胚胎中，神经板边缘区域有助于不同的细胞命运，从神经细胞到神经细胞， 嵴细胞和外胚层基板细胞到中枢神经系统的神经元。尽管广泛的研究表明， 在后神经胚阶段的神经嵴和外胚层基板，令人惊讶的是，很少有人知道这些 种群在早期神经板边界内变得彼此不同。根据我们初步的 数据，我们假设，许多神经板边缘细胞是多能的，证明了他们的 伴随表达几种命运特征性标志物。我们将通过以下方式检验这一假设：1） 通过多重蛋白和基因对新生神经板边缘区进行详细分析， 表达谱分析结合细胞谱系分析和2）检测转录因子的扰动如何影响细胞的表达， 水平影响单个神经板边缘细胞的表达谱和谱系分配。意义 这一建议的一个重要方面是，它将是第一个测试外胚层基板前体是如何以及何时被分离的。 从神经嵴和神经板边缘的神经前体细胞中分离。将实现以下目标： 目的1：高分辨分析神经板、神经嵴、基板等组织的蛋白表达 神经板边缘的外胚层标记物作为时间的函数。我们将研究 与神经嵴、基板、神经板和外胚层谱系相关的转录因子 定量和单细胞分辨率在鸡原肠胚神经胚阶段，以确定其程度， 重叠，以及如果/当它们之间在神经板边界中发生离散分离时。把这个带到 多重水平，然后我们将进行单分子荧光原位杂交分析（smFISH）， 类似的阶段，从已知基因和来自我们的单细胞的新候选物中选择35个或更多个探针 RNA-seq数据集。 目的2：对基因表达和细胞命运调控相互作用的分子解剖 选择在神经板边界。我们将研究扰乱个体转录的后果 因子（例如Pax 7、Sox 2、Six 1）对群体中其他神经板边缘基因表达的影响， 单细胞水平。为了检查调节神经板边界形成的输入，我们将解剖新的 Pax 7，Six 1和其他基因的增强子，以确定直接的调控输入。最后，我们将研究 转录因子的平衡水平如何影响神经板边缘区域的其他因子。 目的3：神经板边缘细胞的单细胞谱系分析。为了明确地测试 神经板边缘的单个细胞具有有限或广泛的发育潜力，我们将进行 单细胞谱系分析通过将赖氨酸化罗丹明葡聚糖进行离子电渗注射到 单个神经板边缘细胞。我们还将使用Pax 7、Sox 2或Six 1的增强子， 单个细胞的光转换，以跟踪神经板边缘细胞的长期命运，并研究如何 阻断单个转录因子影响细胞谱系分配。 1