Fig. 3

Cell proliferation decreases and localizes to wound-proximal cells. a EdU staining of S-phase cells in intact and regenerating sea star larvae (1–7 days post-bisection [dpb]). EdU-positive cells are shown in green. Nuclei were stained with DAPI and shown in gray. Cell proliferation in uncut larvae is throughout the ciliary band epithelium (cb), mouth (mo), stomach (s), and coelomic pouches (cp). Regenerating anterior fragments (top row) and posterior fragments (bottom row) demonstrate similar initial distributions of proliferation, although the number of EdU⁺ cells decreased by 3 dpb. Beginning at 6 dpb, EdU⁺ cells are concentrated near the wound site in both anterior and posterior regenerating fragments in a putative regeneration blastema (bl). b Quantitation of the EdU⁺ cells shows a steady decline in the number of proliferating cells in both anterior and posterior regenerating fragments. The difference of the means (i.e., Δ EdU⁺ Cells) is plotted and significance differences are indicated (Mann-Whitney, p < 0.05, red asterisk). n = total number of bisected animals counted. c The fraction of EdU⁺ cells in each of the wound-proximal, middle, and wound-distal thirds of each regenerating larval fragment from panel B is shown. The number of individuals counted is the same as in (b). The difference of the means (i.e., Δ % EdU⁺ cells) is plotted and significance differences are indicated (Mann-Whitney, p < 0.05, red asterisk). d The experimental regimen of the BrdU/EdU pulse-chase experiments is shown. Regenerating larvae (left) or uncut larvae (right) were labeled with BrdU (magenta) for 6 h after which the BrdU was washed out. Larvae are subsequently labeled with a 6 h EdU pulse (green) at the onset of wound-proximal proliferation or after a similar duration for uncut larvae