Identification of Chemoattractants in Neural Crest Migration (pilot)

神经嵴迁移中化学引诱剂的鉴定（试点）

• 批准号: 7131852
• 负责人: Maria Elena de Bellard
• 金额: $ 5万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7131852
• 关键词: cell migration; cell proliferation; chemoattractants; chemotaxis; chick embryo; electroporation; method development; minority institution research support; neural crest; neurotrophic factors; small interfering RNA; tissue /cell culture

The neural crest is a migratory population of cells that gives rise to a wide range of cell types in the peripheral nervous system of vertebrate embryos. It has been shown that neural crest cells migrate along very specific pathways throughout the embryo. The reason for such specificity is not fully known. During the last years, some known axon pathfinding repellants (ephrinB2, Semallla, Slit2, etc) have been shown to repel neural crest cells during their migration throughout the embryo. However, we know very little about the migratory clues that guide the neural crest for the rest of their path. It is the goal of this study to find which other molecules are capable of guiding the neural crest along their migratory routes. For this purpose I had set out to screen a group of neurotrophic factors that are expressed at the same time that the crest is migrating through the embryo and which have been shown to be important in neural crest migration by analyzing the corresponding knockout mice. I had taken an in vitro and in vivo approach using a battery of commercially available and cell lines as well that secrete NGF, GDNF, NTS and Neuregulins. In the first case, I modified the already classical collagen gel assay, thus culturing early neural tubes in close proximity to cells that secrete neurotrophic factors. I have also tested neurotrophins' effect on neural crest cells in chemotaxis chambers. The prelimminary results suggest that neural crest cells are attracted to GDNF and Neuregulin. I also tested the effect that these factors would have on live developing chicken embryos. I found that NTS and GDNF were capable of disrupting the migration of trunk neural crest cells. These preliminary data suggests that neural crest cells use a variety of neurotrophic factors as guiding clues during their extensive migration in the embryo. The methods I will use in this proposal will be a) in vitro: isolating neural crest cells and expose them to neurotrophins in chemotaxis chambers, focal points of neurotrophins (as done for growth cone guidance) and in the media. B) in vivo: injecting the celsl secreting neurotrophins, electroporating dominant negative forms and siRNA of their receptors and injecting beads coated with neurotrophins along their regular pathways and also on areas that neural crest cells will not populate to test their potency in attracting them to these novel sites. The relevance that this research will have to public health comes from showing which molecules can attract cells during migration, this knowledge can be tranlated into future therapies with stem cells, especially by helping these cells reach the desired targets for proper regeneration. In addition, the success of this project will demonstrate for the first time that neural crest cells are guided by chemoattractants as well as chemorepellant in the formation of the peripheral nervous system.

神经嵴是细胞的迁移群体，在神经系统中产生多种细胞类型 脊椎动物胚胎的周围神经系统。研究表明，神经嵴细胞沿着 整个胚胎的非常特定的途径。这种特殊性的原因尚不完全清楚。期间 去年，一些已知的轴突寻路驱虫剂（ephrinB2、Semallla、Slit2 等）已被证明可以 在神经嵴细胞在整个胚胎中迁移期间排斥它们。然而，我们对此知之甚少 引导神经嵴完成剩余路径的迁移线索。本研究的目的是找出哪些 其他分子能够引导神经嵴沿着其迁移路线。 为此，我开始筛选一组同时表达的神经营养因子 嵴正在胚胎中迁移，这已被证明对神经嵴很重要 通过分析相应的基因敲除小鼠的迁移。我采用了体外和体内方法 市售细胞系以及分泌 NGF、GDNF、NTS 和神经调节蛋白的电池组。在 第一个案例，我修改了已经经典的胶原蛋白凝胶测定，从而近距离培养早期神经管 接近分泌神经营养因子的细胞。我还测试了神经营养素对神经嵴的影响 趋化室中的细胞。初步结果表明，神经嵴细胞被吸引 GDNF 和神经调节蛋白。我还测试了这些因素对活鸡发育的影响 胚胎。我发现 NTS 和 GDNF 能够破坏躯干神经嵴细胞的迁移。 这些初步数据表明神经嵴细胞使用多种神经营养因子作为指导线索 在它们在胚胎中广泛迁移的过程中。我将在本提案中使用的方法是 a) 体外： 分离神经嵴细胞并将其暴露于趋化室中的神经营养素，趋化室是神经嵴细胞的焦点 神经营养素（如生长锥引导）和媒体中。 B) 体内：注射分泌细胞 神经营养素、电穿孔显性失活形式及其受体的 siRNA 和注射珠 沿着其常规途径以及神经嵴细胞不会覆盖的区域涂有神经营养素 填充以测试他们吸引他们到这些新奇网站的能力。 这项研究与公共健康的相关性在于显示哪些分子可以吸引 细胞在迁移过程中，这些知识可以转化为未来的干细胞疗法，特别是通过 帮助这些细胞达到适当再生的预期目标。此外，该项目的成功 将首次证明神经嵴细胞受到化学引诱剂以及 周围神经系统形成中的化学排斥剂。