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。该提案涉及多能性和迁移性问题， 神经嵴细胞的行为，由于其广泛的能力， 这些细胞与神经元和软骨一样，都是多种多样的细胞类型。虽然 经典的嫁接实验已经阐明了神经嵴的衍生物， 关于个体颅神经嵴细胞在体内的发育潜力知之甚少。 在这里，我们建议使用无法复制的鸟类逆转录病毒编码不同的 荧光荧光团标记背神经管，以便进行克隆分析。 其目的是检查发育潜力，运动和形态发生， 单个或小群体的颅神经嵴细胞。将进行实验 因为有几个优点。鸡胚很容易被 逆转录病毒感染和实验干扰在早期阶段的发展， 时间和空间控制操纵。和人类一样，鸟类也是食草动物，但与老鼠不同， 在母亲之外发展。因此，他们在早期阶段更容易获得，而 以与人类胚胎在形态上几乎相同的方式发育， 阶段 目的1：鸡颅神经嵴的逆转录病毒介导的克隆分析： 鸡胚的神经管将被复制缺陷型禽逆转录病毒感染， 编码四种不同的荧光团。将通过目视观察单个 感染后几个小时。然后，我们将跟踪克隆相关细胞的长期命运， 通过使用抗体标记物检查它们的定位和分化， 具有不同细胞命运的特征。 目的2：将谱系分析与单分子荧光原位杂交相结合 以检测克隆相关细胞的多重基因表达。我们将结合血统 我们最近开发的一种新的适应smFISH的分析， 以单细胞分辨率分析基因表达。空间基因组分析（SGA） 同时分析迁移和迁移时组织切片上35个或更多个基因的表达， 后迁移阶段。我们将联合收割机克隆分析与SGA相结合，以确定基因的共同作用。 由克隆相关细胞使用各种谱系的标记物与神经嵴一起表达 和多能性基因以表征克隆相关基因的转录谱。 目的3：分析克隆相关细胞之间的迁移相互作用：我们将研究 克隆相关细胞的迁移行为，无论是在整个安装，使用卵成像，以及 如在切片组织切片中，以可视化姐妹细胞和不相关的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， 干扰单个克隆中细胞间的相互作用， 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.