A role for RhoA in the two-phase migratory pattern of post-otic neural crest cells

Neural crest (NC) cells have been elegantly traced to follow stereotypical migratory pathways throughout the vertebrate embryo, yet we still lack complete information on individual cell migratory behaviors and how molecular mechanisms direct NC cell guidance. Here, we analyze the spatio-temporal migratory pattern of post-otic NC and the in vivo role of the small Rho GTPase, RhoA, using fluorescent cell labeling, molecular perturbation, and intravital 4D (3D+ time) confocal imaging in the intact chick embryo. We find that the post-otic NC cell migratory pattern is established in two phases with distinct cell migratory behaviors. An initial wide front of lateral-directed NC cells, led by NC from rhombomere 7 (r7), move as a distinct subpopulation. This is followed in time by fewer NC cells that migrate collectively from r7 to r8 in a follow-the-leader manner with extensive cellular extensions between cells. We show that post-otic migratory NC cells express RhoA, using RT-PCR on isolated, flow cytometry sorted NC cells and in neural tube culture explants. When RhoA function is altered by expression of a dominant negative or constitutively active form, or injection of C3, there are two major consequences. RhoA constitutively active expressing NC cells are less directional, slower and form fewer follow-the-leader chain assemblies. NC cells expressing RhoA-DN are less affective in retracting filopodia, migrate slower and also form fewer follow-the-leader chain assemblies. Together, these alterations to NC cell intrinsic signaling and cell-cell contact disrupt the precise spatio-temporal post-otic NC cell migratory pattern.