Clonal analysis of the cranial neural crest

颅神经嵴的克隆分析

基本信息

批准号：
9929202
负责人：
Marianne Bronner
金额：
$ 4.47万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9929202
关键词：
Affect Antibodies Back Behavior Biological Models Birds CHARGE syndrome Cartilage Cell Communication Cell Differentiation process Cell Lineage Cell physiology Cells Cephalic Characteristics Chick Embryo Chimera organism Clonality Congenital Abnormality Coupling DNA Defect Development Developmental Biology DiGeorge Syndrome Diagnosis Disease Dorsal Embryo Embryonic Development Endocrine Gland Neoplasms Enteral Environment Face Fluorescent in Situ Hybridization Ganglia Gene Expression Gene Expression Profiling Genes Genetic Transcription Genomics Genus Alpharetrovirus Glial Fibrillary Acidic Protein Goals Head Hour Human Image Individual Infection Knowledge Label Maintenance Malignant Neoplasms Molecular Morphogenesis Morphology Mothers Movement Mus Natural regeneration Neural Crest Neural Crest Cell Neural Fold Neural Tube Closure Neural tube Neuraxis Neuroblastoma Neuroglia Neurons Pattern Peripheral Peripheral Nervous System Pheochromocytoma Pigmentation physiologic function Pigments Population Quail Resolution Route Schwann Cells Sensory Sister Skeleton Slice Smooth Muscle Actin Staining Method Stains Stem cells Therapeutic Intervention Time Tissues Transcript Visual blastomere structure bone cell motility cell type comparative experimental study expression cloning fluorophore in vivo infancy melanoma migration neuroepithelium neurofilament novel pluripotency precursor cell repaired retroviral-mediated single molecule stem-like cell

项目摘要

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.

发育生物学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。该提案解决了多责任和迁移的问题神经rest细胞的行为，重点是颅神经rest，因为它的广泛能力有助于多种细胞类型，与神经元和软骨一样不同。虽然经典的嫁接实验已经阐明了神经rest的衍生物，相对关于体内个体颅神经rest细胞的发育潜力知之甚少。在这里，我们建议使用编码不同的复制能力的禽逆转录病毒荧光荧光团以标记背部神经管以进行克隆分析。目的是检查发育潜力，运动和形态发生颅神经rest细胞的个体或小种群。实验将进行在禽类胚胎上，因为有几个优势。小鸡胚胎很容易进入发育早期阶段的逆转录病毒感染和实验性扰动时间和空间控制的操作。像人类这样的鸟是羊水，但与老鼠不同，在母亲外面发展。因此，它们在早期更容易获得在形态学上以与人类胚胎相同的方式发展阶段。 AIM 1：雏鸡颅神经rest的逆转录病毒介导的克隆分析：颅骨雏鸡胚胎的神经管将被复制无能力的逆转录病毒感染编码四个不同的荧光团。克隆性将通过视觉观察到单个感染几个小时后的细胞。然后，我们将遵循克隆相关细胞的长期命运通过使用抗体标记来检查其定位和分化，来的函数各种细胞命运的特征。 AIM 2：与单分子荧光原位杂交耦合谱系分析检查克隆相关细胞的多重基因表达。我们将伴随血统我们最近开发的Smfish的新型Smfish进行了分析分析单细胞分辨率下的基因表达。空间基因组分析（SGA）启用同时分析35个或更多基因在迁移和组织切片上的表达移民后阶段。我们将将克隆分析与SGA结合在一起，以确定共同的基因由克隆相关的细胞使用各种谱系的标记与神经rest表示和多能性基因以表征克隆相关基因的转录谱。目标3：分析克隆相关细胞之间的迁移相互作用：我们将检查在OVO成像中也使用克隆相关细胞的迁移行为，既有就像在切片组织中一样，可视化姐妹细胞与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，我们将检查效果单个克隆中的细胞细胞相互作用的扰动，迁移到原本正常环境。