Clonal Analysis of the Cranial Neural Crest using RIA Viruses

项目摘要 神经嵴是脊椎动物胚胎发育的关键细胞群 发展这些细胞以其迁移行为而闻名，并对广泛的 的衍生物，如颅面骨骼，表皮色素细胞，和大部分的 外周神经系统。这一建议解决了发展中的一个根本问题。 生物学：是否个别颅神经嵴细胞在体内是多能的，并保留能力， 分化成众多的命运，或者，如果他们代表了一个人口的命运限制， 每个细胞都有自己独特的命运为了解决这个问题，复制能力不足 编码不同荧光团的禽类逆转录病毒将用于标记神经细胞， 折叠细胞并进行克隆分析，以检查发育潜力、运动 和在体内的单个或小群体的颅神经嵴细胞的形态发生。 实验将在鸟类胚胎上进行，因为有几个优点。鸡胚 很容易受到逆转录病毒感染和实验干扰的早期阶段， 发展，允许时间和空间控制操纵。鸟类和人类一样， 但是，与小鼠不同的是，它们在母体外发育， 阶段，同时以形态上与人类几乎相同的方式发育 胚胎在相似的阶段。 目的1：鸡颅神经嵴的逆转录病毒介导的克隆分析： 鸡胚的神经管将感染有限稀释的复制缺陷 编码荧光团的鸟类逆转录病毒。四种不同的病毒反射不同的颜色， 每个胚胎使用，并在接种后几小时通过目视观察建立克隆性。 感染克隆相关细胞的命运将使用抗体染色以及 一种新的单分子荧光原位杂交方法， 分析单细胞中多达35种转录物。 目的2：分析克隆相关细胞之间的迁移相互作用： 克隆相关细胞的行为将在整个安装中进行检查，也使用卵内成像 如在切片组织切片中，以可视化姐妹细胞、表亲细胞和无关细胞之间的相互作用， 邻居一旦建立了正常的迁移模式和细胞相互作用， 在迁移细胞的单个克隆中干扰细胞-细胞和细胞-基质相互作用将是 在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.