- Title
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Zebrafish Regulatory T Cells Mediate Organ-Specific Regenerative Programs
- Authors
- Hui, S.P., Sheng, D.Z., Sugimoto, K., Gonzalez-Rajal, A., Nakagawa, S., Hesselson, D., Kikuchi, K.
- Source
- Full text @ Dev. Cell
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Inducible ablation of zTreg cells, Related to Figure 4. (A) foxp3a:NTR fish were treated overnight on 3 consecutive days with vehicle (Veh) or metronidazole (Mtz), and total fish samples were used for qRT-PCR analysis of lck (T cells), pax5 (B cells), spi1b (myeloid cells), mpx (neutrophils), and mpeg1.1 and coronin1a (macrophages) (mean ± SEM, n = 5). Exposure of foxp3a:NTR fish to Mtz reduced endogenous foxp3a expression by approximately 85%, without altering the expression of other immune cell markers. Gene expression is shown relative to the levels in vehicle controls. (B) foxp3a:NTR fish were injured at the spinal cord and treated overnight with Veh (left) or Mtz (middle and right) at 1, 2, 3 and 5 dpi. The spinal cords were analyzed via immunofluorescence against TagCFP at 7 dpi (left and middle) and 14 dpi (right; after an 8-day interval without Mtz treatment). Asterisk, injury epicenter. (C) foxp3a:NTR fish were injured at the cardiac ventricle and treated overnight with Veh (left) or Mtz (middle and right) at 4, 5, and 6 dpi. The hearts were analyzed at 7 dpi (left and middle) and 10 dpi (right; after a 3-day interval without Mtz). Dotted line, wound border. (D) foxp3a:NTR fish were injured at the retina and treated overnight with Veh (left) or Mtz (middle and right) at 1, 2, and 3 dpi. The retinas were analyzed at 4 dpi (left and middle) and 14 dpi (right; after a 11-day interval without Mtz treatment). Asterisk, injury epicenter. (E) zTreg cell ablation protocol used throughout this study. After 7 days, Mtz or Veh treatments were performed every second day for the duration of the experiment. (F–H) Clutch mate WT and foxp3a:NTR fish were subjected to injury and drug treatments as described in (E), and foxp3a expression in the spinal cord (F), heart (G), and retina (H) was measured by qRT-PCR (mean ± SEM, n = 5). (I) qRT-PCR analysis of inflammatory cytokine genes in foxp3a:NTR fish after a 30- day treatment regimen as described in E (mean ± SEM, n = 5). dpt, days post treatment. Single confocal sections are shown in B-D. DAPI: 4',6-diamidino-2- phenylindole. *P < 0.05, **P < 0.01, ***P < 0.001, Mann–Whitney U test. NS, not significant. Scale bars, 50 μm. |
Survival gene expression and apoptosis in injured zTreg cell depleted tissues, Related to Figure 5. (A–C) qRT-PCR analysis of survival factors in injured spinal cords (A), hearts (B), and retinas (C) from control and zTreg cell-depleted fish (mean ± SEM, n = 6). (D–F) Immunofluorescence for activated Caspase-3. Arrows indicates apoptotic neurons (D; HuC/D+Caspase- 3+), cardiomyocytes (E; MHC+Caspase-3+), and Müller glia (F; gfap:GFP+Caspase-3+). Images show the rostral stump of a 7-dpi spinal cord. cc, central canal; epi, injury epicenter. Dotted line, wound border plane. (G–I) Quantification of D–F (mean ± SEM, n = 5). Mann–Whitney U test. NS, not significant. Scale bars, 100 μm. |
Regeneration factor administration and receptor gene expression in regenerating parenchymal cells, Related to Figure 6. (A) NTF3 treatment regimen. NTF3 or PBS was injected intraperitoneally (i.p.) from 6 dpi. (B–E) Proliferation of neural progenitor cells (B; Sox2+PCNA+), immature neurons (C; HuC/D+EdU+), cardiomyocytes (D; Mef2+PCNA+) and Müller glia (E; gfap:GFP+PCNA+). Insets show single-channel confocal slices (B, C, E) or epifluorescent images (D) of the demarcated regions. Dotted line, wound border plane. (F) Quantified proliferating immature neurons (HuC/D+EdU+) in C (mean ± SEM, n = 5). (G–I) Semi-qRT-PCR analysis of purified gfap:GFP+ cells from spinal cords (G; ependymo-radial glial cells), cmlc2:GFP+ cells from hearts (H; cardiomyocytes), and gfap:GFP+ cells from retinas (I; Müller glia). Confocal projections of z-stacks are shown, except for the epifluorescent images shown in D. *P < 0.05, **P < 0.01, Mann–Whitney U test. NS, not significant. Scale bars, 50 μm. |
zTreg cell-mediated immunomodulation during regeneration, Related to Figure 6. (A–C) qRT-PCR analysis of inflammatory gene expression in injured spinal cords (A), hearts (B), and retinas (C) from control and zTreg celldepleted fish (mean ± SEM, n = 6). (D–F) Expression levels of the same genes analyzed in A–C were unchanged in the remainder of the fish after removing the injured spinal cord (D), heart (E), or retina (F) (mean ± SEM, n = 5). (G–I) The expression of M1 macrophage marker nos2b and M2 macrophage marker marco was measured in injured spinal cord (G), heart (H), and retina (I) samples obtained from Mtz-treated WT and foxp3a:NTR fish (mean ± SEM, n = 6). (J–L) Sections of spinal cords (J), hearts (K), and retinas (L) from lck:RFP (left) or lck:RFP; foxp3a:NTR fish (right) treated with Mtz (mean ± SEM, n = 6). Asterisks, injury epicenter. (M–O) Quantification of J (M), K (N), and L (O). zTreg (+) and (−) indicate zTreg cell ablated or non-ablated samples, respectively (mean ± SEM, n = 6). (P–R) qRT-PCR analysis of the expression of effector T cell marker genes in T cells (lck:RFP+ cells) purified from damaged spinal cords (P), hearts (Q), retinas (R) (mean ± SD; *P < 0.05). AcTub, Acetylated Tubulin; HT, heart; marco, macrophage receptor with collagenous domain; MHC, myosin heavy chain; nos2b, inducible nitric oxide synthase 2b; RT, retina; SC, spinal cord. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant, Mann–Whitney U test (A–I, M–O), Student’s t-test (P–R). Scale bars, 100 μm. |
Regeneration phenotype of il10–/– zebrafish, Related to Figure 6. (A) il10 expression in injured spinal cords (left), hearts (middle), and retinas (right) from control and zTreg cell-depleted fish (mean ± SEM, n = 6). (B) il10 expression in injured spinal cords (left), hearts (middle), and retinas (right) from clutch mate WT (il10+/+) and il10–/– fish (mean ± SEM, n = 5). (C–E) qRT-PCR analysis of inflammatory cytokine (left) and M1 macrophage marker (middle) and M2 macrophage marker (right) gene expression in injured spinal cords (C), hearts (D), and retinas (E) from il10+/+ and il10–/– fish (mean ± SEM, n = 5). Gene expression is normalized to the levels in WT fish. (F–H) Proliferation of immature neurons (F; HuC/D+EdU+), cardiomyocytes (G; Mef2+PCNA+) and Müller glia (H; gfap:GFP+PCNA+) in injured spinal cords (F), hearts (G), and retinas (H) from il10+/+ and il10–/– fish. Insets show single-channel confocal slices (F, H) or epifluorescent images (G) of the demarcated regions. The rostral spinal cord stumps are shown in F. Arrows indicate co-labeled cells, and dotted lines indicate the wound border plane. (I–K) Quantification of F (I), G (J), and H (K). (mean ± SEM, n = 5). (L–N) qRT-PCR analysis of regeneration factor genes in injured spinal cords (L), hearts (M), and retinas (N) from il10+/+ and il10–/– fish (mean ± SEM, n = 5). cc, central canal; epi, injury epicenter. *P < 0.05, **P < 0.01, ***P < 0.001, Mann–Whitney U test. Scale bars, 100 μm. |
Regeneration phenotype of foxp3a–/– zebrafish, Related to Figure 7. (A) Sections of 30 dpi spinal cords from clutch mate WT (foxp3a+/+; left) and foxp3a–/– fish (right). Immunofluorescence for acetylated Tubulin (AcTub) marks nerves. Asterisks, injury epicenter. (B) Picro-Mallory staining of sections from 45 dpi hearts from WT (left) and foxp3a–/– fish (right). (C) Luxol fast blue staining of sections from 30 dpi retinas from WT (left) and foxp3a–/– fish (right). Asterisks, injury epicenter. (D–F) Quantification of regeneration in A–C (n = 6–8). (G–I) Quantification of zTreg cells in Figure 7B (mean ± SEM, n = 5). (J–L) il10 expression in injured spinal cords (J), hearts (K), and retinas (L) from WT and foxp3a–/– fish (mean ± SEM, n = 5). (M–O) The expression of inflammatory cytokine genes in injured spinal cords (M), hearts (N), and retinas (O) from WT and foxp3a–/– fish (mean ± SEM, n = 5). Gene expression is normalized to the levels in WT fish. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001, Fisher’s exact test (D–F), Mann–Whitney U test (G–O). Scale bars, 50 μm. PHENOTYPE:
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Reprinted from Developmental Cell, 43, Hui, S.P., Sheng, D.Z., Sugimoto, K., Gonzalez-Rajal, A., Nakagawa, S., Hesselson, D., Kikuchi, K., Zebrafish Regulatory T Cells Mediate Organ-Specific Regenerative Programs, 659-672.e5, Copyright (2017) with permission from Elsevier. Full text @ Dev. Cell