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

基本信息

批准号：
10331009
负责人：
Marianne Bronner
金额：
$ 39.38万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10331009
关键词：
Affect CHARGE syndrome Cartilage Cell Lineage Cell physiology Cells Cephalic Characteristics Chick 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 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 factor protein expression public health relevance relating to nervous system repaired rhodamine dextran single molecule single-cell RNA sequencing stem cells transcription factor

项目摘要

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

布朗纳，M.E. 在早期胚胎中，神经板边界区域有助于不同的细胞命运，从神经嵴细胞和外胚层基板细胞连接到中枢神经系统的神经元。尽管进行了广泛的研究神经嵴和外胚层基板在神经胚后阶段，令人惊讶的是，我们对这些如何发生知之甚少。在早期神经板边界内，种群变得彼此不同。根据我们的初步根据数据，我们假设许多神经板边缘细胞是多能的，正如它们的证据所证明的那样几种命运特征标记的同时表达。我们将通过以下方式检验这个假设：1) 通过多重蛋白质和基因对新兴神经板边界区域进行详细分析表达谱与细胞谱系分析相结合，2) 检查转录因子的扰动水平影响单个神经板边缘细胞的表达谱和谱系分配。意义该提案的主要内容是它将成为第一个测试外胚层基板前体如何以及何时分离的项目来自神经板边界的神经嵴和神经前体。将实现以下目标：目标 1：神经板、神经嵴、基板等蛋白表达的高分辨率分析神经板边界的外胚层标记作为时间的函数。我们将检查共表达与神经嵴、基板、神经板和外胚层谱系相关的转录因子在雏鸡原肠胚到神经胚阶段进行定量和单细胞分辨率测定，以确定它们的程度重叠以及如果/当它们在神经板边界中发生离散分离时。为了把这个带到一个多重水平，然后我们将进行单分子荧光原位杂交分析（smFISH）类似的阶段，具有从已知基因中选择的 35 个或更多探针以及来自我们单细胞的新候选基因 RNA-seq 数据集。目标 2：介导基因表达和细胞命运的调控相互作用的分子剖析神经板边界的选择。我们将检查扰乱个体转录的后果影响群体中其他神经板边缘基因表达的因素（例如 Pax7、Sox2、Six1）以及单细胞水平。为了检查调节神经板边界形成的输入，我们将剖析新颖的 Pax7、Six1 和其他基因的增强子以确定直接调控输入。最后，我们将检查转录因子的平衡水平如何影响神经板边缘区域的其他因素。目标 3：神经板边缘细胞的单细胞谱系分析。为了最终测试是否神经板边缘的单个细胞具有有限或广泛的发育潜力，我们将进行通过离子电渗法将赖氨酸罗丹明葡聚糖注射到单细胞谱系分析中单个神经板边缘细胞。我们还将使用 Pax7、Sox2 或 Six1 以及单个细胞的光转换以跟踪神经板边缘细胞的长期命运并检查如何阻断单个转录因子会影响细胞谱系分配。 1