CELLULAR PATHWAYS INVOLVED IN EPITHELIAL-TO-MESENCHYMAL TRANSITIONS IN NEURAL CREST CELLS

Abstract

Neural crest cells are a population of multi-potent progenitor cells in the developing vertebrate embryo that undergo an epithelial-to-mesenchymal transition (EMT) and migrate extensively to generate diverse derivatives. As such, abnormal development of neural crest cells can lead to human congenital and hereditary malformations, diseases and cancers. Both internal molecular signals and external mechanical factors play essential roles in facilitating neural crest cell EMT. How cells modulate their adhesion machinery and dynamically reorganize their actin cytoskeleton to respond to the mechanical features of their external environment during EMT is not well understood. To evaluate the role of the actomyosin cytoskeleton during neural crest cell EMT and migration, midbrain neural folds that contain premigratory neural crest cells were dissected out from chick embryos, explanted into chamber slides, and incubated to allow for the formation of migratory neural crest cells. Time-lapse imaging technique was used to record cell behaviors. To elucidate cellular pathways controlling EMT and migration, chemical inhibitors (blebbistatin, Y-27632, latrunculin-A, and nocodazole) that perturb molecular cascades regulating cellular structures were employed. Effects of these perturbations on neural crest cell EMT and migration were quantified in terms of the spreading rate of the explants, and vorticity of collectively moving cell groups. We observed that blebbistatin treatment reduced the overall velocity of migratory neural crest cells to negligible levels. Moreover, migratory neural cells developed rounder cell bodies, and lamellipodia were transformed into filopodia at the periphery of the extract. Y-27632 treatment led to more neural crest cells coming out from these explants within a shorter time period compared to control. Nocodazole treatment blocked neural crest cell EMT and the resumption was dose-dependent. Latrunculin-A caused cell death at a very low concentration. These results implicate roles for non-muscle myosin II, the target of blebbistatin, and ROCK, the target of Y-27632, as well as microtubules and actin filaments, in chick midbrain neural crest cell EMT and migration. Actin crosslinkers such as &#945;-actinin and actin-associated proteins like palladin also participate in pathways affected by these cytoskeletal inhibitors through their regulation of focal adhesion formation and cytoskeletal organization, thereby modulating cell stiffness and migration. We are also documented the distribution of &alpha;-actinin and palladin in migratory neural crest cells <italic>in vivo</italic>. Collectively, our studies have provided insight into specific cellular pathways regulating neural crest cell EMT and migration and the impact on various biophysical parameters upon perturbation of these pathways.