Clonal analysis of the cranial neural crest

发育生物学中的一个主要问题是前体细胞如何产生不同的集合 分化的细胞类型。这项建议解决了多项能力和迁徙的问题。 神经脊细胞的行为，由于其广泛的能力，集中在脑神经脊上 促进多种不同类型的细胞，如神经元和软骨。虽然 经典的移植实验已经比较地阐明了神经脊的衍生物。 关于单个脑神经脊细胞在体内的发育潜力，人们知之甚少。 在这里，我们建议使用复制能力不强的禽流感逆转录病毒编码不同的 荧光标记物标记背神经管，以进行克隆分析。 其目的是检查发育潜力、运动和形态发生。 单个或小群的脑神经脊细胞。我们将进行一些实验 对禽类胚胎的研究具有几个优势。小鸡胚胎很容易接触到 逆转录病毒感染和发育早期的实验扰动，允许 时间和空间控制的操控。鸟类和人类一样是羊膜动物，但与老鼠不同， 在母体外发育。因此，它们在早期阶段更容易获得，而 发育的方式在形态上与人类胚胎几乎相同 各阶段。 目的1：逆转录病毒介导的鸡颅神经脊区克隆分析 鸡胚胎的神经管将被感染复制能力不强的禽流感逆转录病毒 编码四个不同的荧光团。克隆性将通过肉眼观察单个 细胞感染后几个小时。然后我们将关注克隆相关细胞的长期命运，因为 利用抗体标记物检测它们的定位和分化的时间函数 具有不同细胞命运的特点。 目的2：单分子荧光原位杂交联用谱系分析 目的：检测克隆性相关细胞的多重基因表达。我们会将血统结合在一起 用我们最近开发的允许多路传输的smFISH的新改编进行分析 单细胞分辨率下的基因表达分析。空间基因组分析(SGA)使 35个或更多基因在迁徙和移行的组织切片上的同时表达分析 迁徙后阶段。我们将结合克隆分析和SGA来确定基因的协同作用。 由克隆性相关细胞使用不同谱系的标记与神经脊一起表达 和多潜能基因，以表征克隆相关基因的转录图谱。 目标3：分析克隆相关细胞之间的迁移相互作用：我们将检查 克隆相关细胞在整架上的迁移行为，也用于OVO成像 就像在切片组织切片中一样，以可视化姐妹细胞和无关邻居之间的相互作用。 一旦建立了正常的迁移模式和细胞相互作用，我们将检查其影响 在个体克隆中扰乱细胞与细胞的相互作用 环境。

A major question in developmental biology is how precursor cells give rise to diverse sets of differentiated cell types. This proposal tackles the question of multipotency and migratory behavior of neural crest cells, focusing on the cranial neural crest due to its broad ability to contribute to numerous and diverse cell types, as distinct as neurons and cartilage. Although classical grafting experiments have elucidated the derivatives of the neural crest, comparatively little is known about the developmental potential of individual cranial neural crest cells in vivo. Here, we propose to use replication incompetent avian retroviruses encoding different fluorescent fluorophores to label dorsal neural tubes in order to perform clonal analyses. The goal is to examine the developmental potential, movement and morphogenesis of individual or small populations of cranial neural crest cells. Experiments will be performed on avian embryos because of several advantages. Chick embryos are easily accessible to retroviral infection and experimental perturbation at early stages of development, allowing temporally and spatially controlled manipulation. Birds like humans are amniotes but, unlike mice, develop outside the mother. Therefore, they are much more accessible at early stage, while developing in a manner that is morphologically nearly identical to human embryos at comparable stages. Aim 1: Retrovirally mediated clonal analysis of the chick cranial neural crest: The cranial neural tube of chick embryos will be infected with replication incompetent avian retroviruses that encode four different fluorophores. Clonality will be established by visual observation of single cells a few hours after infection. We will then follow the long term fate of clonally related cells as a function of time by examining their localization and differentiation using antibody markers characteristic of various cell fates. Aim 2: Coupling lineage analysis with single molecule Fluorescent In Situ Hybridization to examine multiplex gene expression of clonally related cells. We will couple lineage analysis with a novel adaptation of smFISH that we have recently developed that allows multiplex analysis of gene expression at single cell resolution. Spatial Genomic Analysis (SGA) enables simultaneous analysis of the expression of 35 or more genes on tissue sections at migratory and post-migratory stages. We will combine clonal analysis with SGA to determine the genes co- expressed by clonally related cells using markers of various lineages together with neural crest and pluripotency genes to characterize the transcriptional profile of clonally related genes. Aim 3: Analysis of migratory interactions between clonally related cells: We will examine the migratory behavior of clonally related cells both in whole mount, using in ovo imaging, as well as in slice tissue sections to visualize interactions between sister cells and unrelated neighbors. Once normal migratory patterns and cell interactions are established, we will examine the effects of perturbing cell-cell interactions in individual clones migrating through an otherwise normal environment.