Calcium regulation of neural fold formation: visualization of the actin cytoskeleton in living chick embryos

The involvement of calcium ions during chick neurulation was studied by treating neural plate stage embryos with agonists and antagonists of calcium transport and with inhibitors of calmodulin activity. Organotypic shape changes were examined by light and scanning electron microscopy. Changes in size of cell apices were quantitated by computer image analysis of actin filaments labeled with fluorescent phallicidin in time lapse recordings of living embryos. Both ionomycin and A23187 caused precocious fold elevation around the median hinge point and convergence by bending at the lateral furrows only when Ca2+ was in the external medium. As judged by decreased perimeters of 100 fluorescent apical polygons, cell apices constricted medial to the lateral furrows but did not change significantly within the median hinge point. Pretreatment with dihydrocytochalasin B or cytochalasin D prevented precocious folding and apical constriction. Papaverine and verapamil prevented folding but could be reversed by subsequent ionophore treatment. Calmidazolium and trifluoperazine irreversibly blocked folding. The demonstration in living embryos of constriction of lateral cell apices in a calcium-dependent manner is consistent with a contractile process operating during the formation of neural folds.