Fig. 4. V-ATPase function is required for the upregulation of cell proliferation in the tail regeneration bud. Proliferating cells were labeled with immunohistochemical staining for phosphorylated Histone H3B (H3-P). (A) At 24 hpa, proliferating cells in the G2-M transition are randomly distributed. (B) By 48 hpa, the proliferating cells cluster in the regeneration bud and have largely disappeared from the caudal flank. Red arrows indicate H3P-positive cells. (C) Incubation in concanamycin immediately after amputation reduces proliferation in the bud; sample proliferation data are from D (control) and D' (concanamycin-treated) larva. A,B are oriented anterior to the left; all other panels are oriented anterior upwards. n=8 for each condition. (E-G') KCNK1 expression requires V-ATPase activity. The regeneration bud normally expresses the KCNK1 channel by 24 hpa (E,E'). By contrast, larvae in which the V-ATPase was inhibited by concanamycin (F,F') or anti-c subunit antibody (G,G') exhibit no KCNK1 expression at 24 hpa. n=10 for each condition. White arrows indicate lack of expression; red arrows indicate positive signal (expression of KCNK1).