Analysis of trunk neural crest cell migration using a modified Zigmond chamber assay.

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands. However, not until recently have any chemoattractants of trunk NCCs been identified. Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

Effect of NRG1, GDNF, EGF and NGF in the migration of a Schwann cell precursor line.

The Schwann cells are the myelinating glia of the peripheral nervous system that originated during development from the highly motile neural crest. However, we do not know what the guidance signals for the Schwann cell precursors are. Therefore, we set to test some of the known neurotrophins that are expressed early in developing embryos and have been shown to be critical for the survival and patterning of developing glia and neurons. The goal of this study was to determine more specifically if GDNF, NRG1 and NGF are chemoattractants and/or chemokinetic molecules for a Schwann cell precursor line, the Spl201. We performed live chemoattraction assays, with imaging and also presented these molecules as part of their growing substrate. Our results show for the first time that GDNF and NRG1 are potent chemoattractive and chemokinetic molecules for these cells while NGF is a chemokinetic molecule stimulating their motility.