Cell lineage and transcriptional analysis of the vertebrate neural plate border

布朗纳，M.E. 在早期胚胎中，神经板边界区域决定了不同的细胞命运，从神经细胞到 脊细胞和外胚层胎盘细胞到中枢神经系统的神经元。尽管对…进行了广泛的研究 神经细胞发育后阶段的神经脊和外胚层安慰剂，令人惊讶的是，人们对它们是如何产生的知之甚少。 在早期的神经板边界内，种群变得彼此不同。根据我们的初步调查 数据，我们假设许多神经板边缘细胞是多能的，这从它们的 几个命运特有的标记的伴随表达。我们将通过以下方式检验这一假设：1) 对新出现的神经板边缘区域进行多重蛋白质和基因的详细分析 结合细胞谱系分析的表达谱分析和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。它的意义 这项提议的目的是，它将首次测试外胚层胎盘前体是如何以及何时被分离的 来自神经板边界的神经脊和神经前体。将实现以下目标： 目的1：高分辨率分析神经板、神经脊、胎盘等的蛋白质表达 神经板边缘的外胚层标记随时间变化。我们将研究共同表达的 与神经脊、胎盘、神经板和外胚层谱系相关的转录因子 鸡原肠胚至神经胚期单细胞分辨率的定量研究 重叠和如果/当它们之间在神经板边界发生离散分离。把这个带到一个 多重水平，然后进行单分子荧光原位杂交分析(SmFISH) 相似的阶段，从已知基因中选择35个或更多的探针，从我们的单个细胞中选择新的候选 RNA-seq数据集。 目的2：调节基因表达和细胞命运的相互作用的分子解剖 神经板边缘的选择。我们将研究扰乱个体转录的后果 影响其他神经板边界基因表达的因素(如Pax7、Sox2、SIX1)在人群和 单细胞水平。为了检查调节神经板边界形成的输入，我们将剖析新的 Pax7、SIX1和其他基因的增强子，以确定直接的调控输入。最后，我们将检查 转录因子水平的平衡如何影响神经板边界区域的其他因子。 目的3：神经板缘细胞的单细胞谱系分析。最终测试是否 神经板边缘的单个细胞具有有限或广阔的发育潜力，我们将进行 溶血罗丹明葡聚糖电泳法分析单细胞谱系 单个神经板边缘细胞。我们还将对Pax7、Sox2或SIX1以及 单个细胞的光转换跟踪神经板边缘细胞的长期命运并研究如何 阻断单个转录因子会影响细胞谱系的分配。 1

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