Identification of Chemoattractants in Neural Crest Migration (pilot)

神经脊是一种迁移的细胞群体，它产生了广泛的细胞类型。 脊椎动物胚胎的周围神经系统。已有研究表明，神经脊细胞沿 在整个胚胎中都有非常特殊的途径。这种特殊性的原因尚不完全清楚。在.期间 在过去的几年里，一些已知的轴突寻路驱避剂(ehorinB2，Semallla，Slit2，等等)已经被证明 在整个胚胎的迁移过程中排斥神经脊细胞。然而，我们对此知之甚少 迁移的线索，引导神经峰的其余部分的路径。这项研究的目的是找出 其他分子能够引导神经脊沿着它们的迁移路线。 为此，我开始筛选一组同时表达的神经营养因子 脊线是通过胚胎迁移的，并且已经被证明在神经线纹线中很重要 通过分析相应的基因敲除小鼠进行迁移。我采取了体外和体内的方法，使用了 商业上可用的电池和细胞系以及分泌NGF、GDNF、NTS和NeuRegins的细胞系。在……里面 第一例，我改进了已经很经典的胶原胶试验，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峰的影响 趋化室中的细胞。初步结果表明，神经脊细胞被吸引到 GDNF和Neuregin。我还测试了这些因素对活体发育鸡的影响。 胚胎。我发现NTS和GDNF能够干扰干神经脊细胞的迁移。 这些初步数据表明，神经脊细胞使用各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。我将在这项建议中使用的方法将是a)体外： 分离神经脊细胞并将它们暴露于趋化小室中的神经营养素，焦点是 神经营养因子(用于生长锥体引导)和介质中。B)体内：注射体内分泌的CESL 神经营养因子、电穿孔显性阴性形式及其受体和注射微球的siRNA 沿着正常的路径和神经脊细胞不会被覆盖的区域 以测试它们在将它们吸引到这些新奇网站方面的效力。 这项研究与公众健康的相关性来自于展示了哪些分子可以吸引 在细胞迁移过程中，这一知识可以转化为未来的干细胞疗法，特别是通过 帮助这些细胞达到预期的目标，以进行适当的再生。此外，这个项目的成功 将首次证明神经脊细胞是由化学诱导剂以及 化学排斥剂在外周神经系统的形成中。

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.