Identification of Chemoattractants in Neural Crest Migration (pilot)

神经嵴是细胞的迁移群体，其在神经细胞中产生广泛的细胞类型。 脊椎动物胚胎的周围神经系统。研究表明，神经嵴细胞沿着 在整个胚胎中有非常特殊的路径。这种特殊性的原因尚不完全清楚。期间 近年来，一些已知的轴突寻路排斥剂（ephrinB2、Semallla、Slit2等）已被证明 排斥神经嵴细胞在整个胚胎中迁移。然而，我们对它知之甚少。 迁移的线索，指导神经嵴的其余部分的路径。这是这项研究的目标，以找到 其它分子能够引导神经嵴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， 嵴在胚胎中迁移，这在神经嵴中很重要。 通过分析相应的基因敲除小鼠迁移。我采用了体外和体内的方法， 一组可商购获得的细胞系以及分泌NGF、GDNF、NTS和神经调节蛋白的细胞系。在 在第一种情况下，我修改了已经经典的胶原凝胶试验，从而在封闭的环境中培养早期神经管。 接近分泌神经营养因子的细胞。我还测试了神经营养因子对神经嵴的影响 细胞在趋化室中。初步结果表明，神经嵴细胞被吸引到 GDNF和神经调节蛋白。我还测试了这些因素对活的发育中的鸡的影响 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，神经嵴细胞利用多种神经营养因子作为指导线索 在胚胎中的大规模迁移过程中我将在本提案中使用的方法将是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因子， 神经营养因子（如用于生长锥引导）和培养基中。B）体内：注射分泌 神经营养素、电穿孔显性负性形式和它们的受体的siRNA以及注射珠 在它们的正常通路上以及在神经嵴细胞不会 来测试它们吸引它们到这些新地点的能力。 这项研究与公共卫生的相关性来自于显示哪些分子可以吸引 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 帮助这些细胞达到所需的目标，以进行适当的再生。此外，该项目的成功 将首次证明神经嵴细胞受化学引诱物的引导， 化学排斥剂在周围神经系统的形成。

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.