Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

In this study, by embedding a P0-Cre/CAG-CAT-EGFP embryo at E9.5 in collagen gel inside a culture glass slide, we were able to keep the embryo developing ex vivo for more than 24 hours; this development was with enough NCC fluorescent signal intensity to enable single-cell resolution analysis, with the accompanying NCC migration potential intact and with the appropriate NCC response to the extracellular signal maintained. By implantation of beads with absorbed platelet-derived growth factor-AA (PDGF-AA), we demonstrated that PDGF-AA acts as an NCC-attractant in embryos.We also performed assays with NCCs isolated from P0-Cre/CAG-CAT-EGFP embryos on culture plates. The neuromediator 5-hydroxytryptamine (5-HT) has been known to regulate NCC migration. We newly demonstrated that dopamine, in addition to 5-HT, stimulated NCC migration in vitro. Two NCC populations, with different axial levels of origins, showed unique distribution patterns regarding migration velocity and different dose-response patterns to both 5-HT and dopamine.Although avian species predominated over the other species in the NCC study, our novel system should enable us to use mice to assay many different aspects of NCCs in embryos or on culture plates, such as migration, division, differentiation, and apoptosis.The neural crest, a pluripotent cell population, produces a variety of cell types, including neurons, glial cells, sympatho-adrenal cells, melanocytes, and mesenchymal cells. Mesenchymal cells in turn form cartilage, bone, and connective tissue. NCCs undergo an epithelial-mesenchymal transition and migrate away from the neural epithelium in streams to different regions of the embryo, where they contribute to the formation of a variety of structures [1]. The processes of NCC induction and migration have been studied extensively [2-4]. Since one of the most striking characteristics of NCCs is the mechanism involving their long-range and precision-guided migration, many studies have focused on thi