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.
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