Figure 6

Re-initiation of regeneration following secondary bisection. (A) Schematic of experiments. (B-E) Nuclei of proliferating cells (green) labeled with the thymidine analog EdU, and all nuclei counterstained with Hoechst (blue). (F-I) Nuclei of proliferating cells labeled with EdU (green). In all experiments, polyps were incubated with EdU for 30 minutes immediately prior to fixation. (B, F) Region of the wound site in a control polyp maintained in 1/3 seawater until 24 hpa. (C, G) Polyp incubated with 0.1 μM nocodazole from 18 hpa to 24 hpa and fixed. (D, H) Polyp maintained in 1/3x seawater until 24 hpa, re-bisected, and maintained in 1/3x seawater until 24 hours after secondary amputation (hpa2). (E, I) Polyp incubated with 0.1 μM nocodazole from 18 hpa to 24 hpa, re-bisected, and maintained in 1/3x seawater until 24 hpa2. (J-M) Staining of F-actin stained with BODIPY FL phallacidin. (J) Regeneration of oral structures in a control polyp maintained in 1/3 seawater until 96 hpa. (K) Polyp incubated with 0.1 μM nocodazole from 18 hpa to 24 hpa, washed, and maintained until 96 hpa. (L) Polyp maintained in 1/3x seawater until 24 hpa, re-bisected, and maintained in 1/3x seawater until 96 hpa2. (M) Polyp incubated with 0.1 μM nocodazole from 18 hpa to 24 hpa, re-bisected, and maintained in 1/3x seawater until 96 hpa2. (N) Chart of average tentacle lengths 96 hours after initial or secondary amputation. Values are the mean of measurements for at least 4 polyps at each time point. Error bars: standard error. Asterisk: p value < 0.05; Student’s t-test of treatment versus control. Scale bars = 50 μm.