Clonal Analysis of the Cranial Neural Crest using RIA Viruses

项目摘要 神经脊是一群对脊椎动物胚胎至关重要的细胞。 发展。这些细胞以其迁移行为而闻名，并对广泛的 衍生产品，如头面部骨骼，表皮色素细胞，以及许多 周围神经系统。这项建议解决了发展中的一个根本问题。 生物学：体内单个颅神经脊细胞是否具有多能性，并保留能力 分化成无数的命运，或者，如果他们代表了受命运限制的人群 细胞，每个细胞都受到独特的细胞命运的限制。要解决这个问题，复制能力不强 编码不同荧光团的禽类逆转录病毒将被用于标记神经 折叠细胞并进行克隆分析以检查发育潜力、运动 以及活体内单个或小群体的脑神经脊细胞的形态发生。 实验将在鸟类胚胎上进行，因为它有几个优点。雏鸡胚胎 在早期很容易受到逆转录病毒感染和实验扰动 开发，允许在时间和空间上控制操作。鸟类和人类一样 但与老鼠不同的是，羊膜在母体外发育，使它们在早期更容易获得 发育阶段，而发育方式在形态上与人类几乎相同 处于可比阶段的胚胎。 目的1：逆转录病毒介导的鸡颅神经脊区克隆分析 鸡胚胎神经管感染限制稀释度复制能力不强 编码荧光团的禽类逆转录病毒。四种不同的病毒会反映不同的颜色 每个胚胎使用，并在几小时后通过肉眼观察建立克隆 感染。克隆相关细胞的命运将通过抗体染色以及 一种单分子荧光原位杂交的新方法 对单个细胞中多达35个转录本的分析。 目标2：分析克隆相关细胞之间的迁移相互作用：迁移 克隆相关细胞的行为将在整个架子上进行检查，也将用于卵子成像 就像在切片组织切片中一样，可视化姐妹细胞、近亲细胞和无关细胞之间的相互作用 邻里。一旦建立了正常的迁移模式和细胞相互作用， 在单个迁移细胞克隆中干扰细胞-细胞和细胞-底物的相互作用将是 在其他正常的环境中进行检查。

Project Summary The neural crest is a population of cells that are critical for vertebrate embryonic development. These cells are known for their migratory behavior, and contribution to a wide range of derivatives such as the craniofacial skeleton, epidermal pigment cells, and much of the peripheral nervous system. This proposal tackles a fundamental question in developmental biology: whether individual cranial neural crest cells in vivo are multipotent, and retain the ability to differentiate into numerous fates, or alternatively, if they represent a population of fate-restricted cells, each confined to a unique cellular fate. To tackle this question, replication incompetent avian retroviruses encoding different fluorescent fluorophores will be used to label neural fold cells and perform clonal analyses to examine the developmental potential, movement and morphogenesis of individual or small populations of cranial neural crest cells in vivo. 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, making them more accessible at early stage, while developing in a manner that is morphologically nearly identical to that of 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 limiting dilutions of replication incompetent avian retroviruses that encode fluorophores. Four different viruses reflecting different colors will be used per embryo and clonality will be established by visual observation a few hours after infection. The fate of clonally related cells will then be tracked using antibody staining as well as a novel method of single molecule fluorescent in situ hybridization that allows simultaneous analysis of up to 35 transcripts in single cells. Aim 2: Analysis of migratory interactions between clonally related cells: The migratory behavior of clonally related cells will be examined in whole mount, using in ovo imaging, as well as in slice tissue sections to visualize interactions between sister cells, cousin cells and unrelated neighbors. Once normal migratory patterns and cell interactions are established, the effects of perturbing cell-cell and cell-substrate interactions in individual clones of migratory cells will be examined within an otherwise normal environment.