Morales et al., 2019 - Peripheral Macrophages Promote Tissue Regeneration in Zebrafish by Fine-Tuning the Inflammatory Response. Frontiers in immunology   10:253 Full text @ Front Immunol

Fig. 1

 Kinetic differences between peripheral tissue-resident and CHT-resident macrophages in steady state and after damage. (A) At 72 hpf, macrophages in the Tg(mpeg1:Dendra2) reporter line were classified as peripheral tissue-resident macrophages (white area) or CHT-resident macrophages (yellow area). (B) Average speed of peripheral and CHT macrophages (Mϕ) in steady state conditions. A tail portion of Tg(mpeg1:Dendra2)larva was imaged every 3 min for 3 h. (C) Time lapse imaging of photoconverted macrophages recruited to the damaged site in Tg(mpeg1:Dendra2) larvae. The photoconversion was performed before damage, and the complete tail region was captured every 5 min for a total of 24 h. Representative images showing a peripheral macrophage (white arrowhead) in the start of the time lapse (upper image), and at the time of recruitment to the damage site (lower image). Scale bar = 50 μm. (D) The average speed and (E) the speed of arrival of individual photoconverted macrophages. A total of 14 peripheral macrophages and 12 CHT macrophages from a pool of photoconverted individuals were used for the analysis. **p < 0.01; ***p < 0.001.

Fig. 2

Panther (csf1ra−/−) larvae have a fewer peripheral macrophage population and a delayed recruitment of macrophages after tail fin amputation. (A) Representative images of Tg(mpeg1:Dendra2) larvae in a wild type (WT) or a panther genetic background. Scale bar = 250 μm. (B) Total number of macrophages ± SD in the tail of panther and WT larvae at 72 hpf. (C) Quantification of peripheral tissue-resident and CHT-resident macrophages in the tail of panther and WT larvae. Means ± SDs for each condition are shown in the graph. (D)Recruitment of macrophages (green cells in the yellow dashed rectangle) in panther and WT individuals after tail fin amputation. Scale bar = 100 μm. (E) Quantification of recruited macrophages ± SEM after tail fin amputation in panther and WT larvae from 0 to 48 hpa. Twenty larvae per condition were used. (F) The previous quantification was normalized by the number of total macrophages in the tail of the respective larva (the sum of peripheral, CHT, and recruited macrophages). ***p < 0.001.

Fig. 3

Partial reduction of macrophages pool does not affect the kinetic of macrophage recruitment after tail fin amputation. (A) Images of 72 hpf Tg(mpeg1:Dendra2) larvae, 18 h after injection of an 1:50 dilution of Lipo-PBS and Lipo-clodronate in the bloodstream, respectively. Scale bar = 250 μm. (B) Total macrophages ± SD in the tail of Lipo-clodronate 1:50 and Lipo-PBS 1:50 larvae at 72 hpf. (C) Mean ± SD of peripheral tissue-resident and CHT-resident macrophages in the tail. (D) Recruited macrophages (green cells in the yellow dashed rectangle) in Lipo-clodronate 1:50 and Lipo-PBS 1:50 individuals after tail fin amputation. Scale bar = 100 μm. (E) Quantification of recruited macrophages ± SEM after tail fin amputation in Lipo-clodronate 1:50 and Lipo-PBS 1:50 larvae from 0 to 48 hpa. A total of 20 larvae per condition were used. (F) Normalized number of recruited macrophages at each time point. **p < 0.01; ***p < 0.001.

Fig. 4

Panther individuals exhibit an impaired tail fin regeneration. The tail fin area of regenerating tail fins was calculated at 3 and 5 dpa. (A) Representative regenerating tail fins in panther and WT larvae. Scale bar = 200 μm. (B) Tail fin area quantification of regenerating tail fins in panther and WT larvae at both timepoints. A total of 27 larvae per group was used for the analysis. (C) Regenerating tail fin images of Lipo-clodronate 1:50 and Lipo-PBS 1:50 treated larvae. (D) Quantification of the tail fin area in Lipo-clodronate 1:50 and Lipo-PBS 1:50 treated individuals. A total of 24 larvae per group was used for the analysis. n.s not significant; **p < 0.01; ***p < 0.001.

Fig. 5

Increased cell death and reduced cell proliferation after tail fin amputation in panther larvae. (A) Cell death was measured at 24 hpa through TUNEL assays. Red dots represent TUNEL+ cells. (B) Quantification of TUNEL+ cells ± SD in the damage site of panther and WT larvae at 24 hpa. Twenty larvae per condition were used. (C) Cell proliferation was assessed by BrdU incorporation from 6 to 24 hpa. Green dots represent BrdU+ cells (D) The number of BrdU+ cells ± SD in panther and WT individuals was obtained from 12 larvae per condition. **p < 0.01; ***p < 0.001.

Fig. 6

Heightened il1b expression and ROS in the damage site of panther larvae after tail fin amputation. (A) The amount of ROS generated in the damage site (yellow dashed circle) was measured through the accumulation of the fluorescent 2′7′-dichlorofluorescein (DCF) sensor. Scale bar = 100 μm. (B) Relative fluorescence in the damage site ± SD of panther and wild type larvae. Twenty larvae per condition were analyzed. (C) Quantitative RT-PCR for il1b, tnfa, tgfb1a, and il10 from tails of panther and WT larvae at 6 and 24 hpa. The expression of ef1a was used as housekeeping for the 2−ΔCt calculation. A pool of ~20 larval tails was collected for RNA isolation, and the graphs show the mean ± SD of three independent experiments per condition. n.s not significant; *p < 0.05; ***p < 0.001.

Acknowledgments:
ZFIN wishes to thank the journal Frontiers in immunology for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Front Immunol