Two phases of N. vectensis aPKC (NvaPKC) and Par-6 (NvPar-6) localization. Immunostaining against NvaPKC (A) and NvPar-6 (B) during different stages of N. vectensis development. These proteins do not localize asymmetrically in unfertilized eggs and in the cells of cleavage stages; however, they do localize asymmetrically in the epithelial cells of blastula and gastrula stage embryos. Similar to some bilaterians, NvaPKC and NvPar-6 localize at the apical zone of the cell (insets in blastula and gastrula). Both proteins distribute throughout the microtubule cytoskeleton (labeled with tubulin antibody staining), and labeling was not observed in the endoderm/gastrodermis of gastrula and polyps. White arrows indicate astral/centrosomal and (pro)nuclear structures. White arrowheads indicate the endodermal layer in gastrula and polyp stages. Actin cytoskeleton is shown in gray. Animal/vegetal axis (A/V) and apical/basal axis are indicated. Insets shown in blastula, gastrula, and polyp stages are higher magnification of the zone indicated with the dashed lines. Images ranging from unfertilized to gastrula and insets in polyp stages correspond to a single slice from z-stack series. Image of the whole polyps corresponds to a 3D reconstruction from z-stack series.