Fig. 2

Tregs are required for blastemal proliferation during caudal fin regeneration. (A) Experimental scheme for Mtz application in foxp3a:NTR fish to achieve Treg cell-specific ablation and study caudal fin regeneration. Three continuous days of overnight treatment of Mtz were performed before the initiation of caudal fin amputation at day 0. (B) Brightfield microscopic images of caudal fins show the rate of fin regeneration after 5 and 10 dpa in wild-type and Treg cell ablated fish. (C) Rate of fin regeneration length was quantified in the wild-type fish against the Treg ablated fish. The average length of wild-type 10 dpa was considered as 100% length of fin regeneration (mean ± SEM, n = 7, Student’s T-test). (D, E) Confocal images of fin blastema at 4 dpa after EdU labeling indicates the foxp3a:RFP+ cells are spatially localized in close proximity of EdU+ blastemal cells (D) and sometimes they are also directly in contact with the EdU+ blastemal cells (E). EdU was injected intraperitoneally 30 mins before the collection of fin tissue. (F) The wholemount preparation of 4 dpa fin with EdU and H3P immunostaining in wild-type and after Treg cell ablation. (G) Quantification of H3P+ cells in the 4 dpa fin blastema of wild type and Treg ablated fish (mean ± SEM, n = 12, Mann–Whitney U test). *P < 0.01; **P < 0.001; Mtz, metronidazole; NTR, nitroreductase; Scale Bars, 50 mm.