Cell proliferation during mouth development in Priapulus caudatus. (A) Schematic summary of the experimental setup to identify proliferative cells (by means of EdU incorporation) after 3.5, 4.5, and 5.5 days of development, and their position 6, 12, and 24 hours after the EdU pulse. Drawings are not to scale. (B-J) z projections of confocal stacks of embryos labeled for EdU-positive cells (green) and nuclei (magenta). (B-D) After gastrulation, cell proliferation is mostly concentrated in the animal hemisphere (white arrowheads, encircled by the dashed line), where the introvert and mouth (inset in D, white arrowhead) forms. (E-G) With the appearance of the ventral invagination that forms the mouth at 4.5 days of development, proliferation in the introvert becomes asymmetric (white arrowheads), on the side of mouth development, as observed by the co-localization of proliferative cells and cells expressing the mouth marker foxA (inset in F, white arrowhead; dashed line outlines the embryo). (H-J) Beyond 5.5 days of development, after mouth migration and the formation of the digestive tract, proliferative cells appear scattered throughout the introvert and trunk of the embryo (white arrowheads). In B-D, F, H-J, and inset in E, lateral view; and in E, G, inset in D and F, top view. In D-J, ventral to the left. Scale bars, 50 μm. hpt, hours post-treatment.