Quantification of macrophage and neutrophil numbers and their basal migratory capability in the 3 dpf tlr2 and myd88 mutants and wild sibling controls larvae. (A) Experimental scheme. At 3 dpf, numbers and basal migratory capability of GFP-labeled neutrophils and mCherry-labeled macrophages in tail region were quantified using Leica TCS SP8 confocal laser scanning microscopy (CLSM). Red boxes show the area in which cells were counted or tracked. (B–E) The quantification of neutrophil and macrophage numbers in tail region by using tlr2 and myd88 zebrafish larvae. Data (mean ± SD) are combined from three pools of zebrafish larvae. No significant differences (ns) in the number of neutrophils (B,D) and macrophages (C,E) was detected with an unpaired, two-tailed t-test. Each point represents one larva and different colors represent different pools. Sample size (n): 28, 32 (B,C); 24, 24 (D,E). (F,G,J,K) Quantification of basal migratory capability in 3 dpf tlr2 zebrafish. The total displacement and mean speed of individual neutrophils (F,J) and macrophages (G,K) were quantified by using a manual tracking plugin. Data (mean ± SD) are combined from 5 larvae of tlr2^+/+ Tg (mpeg1:mCherry-F);TgBAC (mpx: EGFP) and tlr2^{– /–} Tg (mpeg1:mCherry-F);TgBAC (mpx: EGFP) larvae, respectively. Each color indicates a different larva. No significant differences (ns) in the total displacement and mean speed of neutrophils (F,J) and macrophages (G,K) were detected with an unpaired, two-tailed t-test. Sample size (n): 28, 28 (F,J); 40, 39 (G,K). Cell tracking movies are shown in Supplementary Movies S1–S4) (H,I,L,M) Quantification of basal migratory capability in 3 dpf myd88 zebrafish. The total displacement and mean speed of individual neutrophils (H,L) and macrophages (I,M) were quantified by using a manual tracking plugin. Data (mean ± SD) are combined from 5 larvae of myd88^+/+ Tg (mpeg1:mCherry-F);TgBAC (mpx: EGFP) and myd88^{– /–} Tg (mpeg1:mCherry-F);TgBAC (mpx: EGFP) larvae, respectively. Each color indicates a different larva. No significant differences (ns) in the total displacement and mean speed of neutrophils (H,L) and macrophages (I,M) were detected with an unpaired, two-tailed t-test. Sample size (n): 34, 33 (H,L); 47, 55 (I,M). Cell tracking movies are shown in Supplementary Movies S5–S8).

The number of neutrophils recruited to the wounded area in the tlr2 and myd88 mutants and wild type sibling controls larvae. (A) Experimental scheme. Tlr2 and myd88 homozygous mutants and sibling control larvae were wounded at 3 dpf. Their tails were wounded to the tip of the notochord. The red dashed line shows the site of wounding. Recruited neutrophils at the wound were imaged at 1, 2, 4, and 6 hpw by using CLSM. For recruited cell counting analysis, cells localized within an area of 200 μm from the wounding edge toward the body trunk were counted as recruited cells. The red dashed box shows the area where neutrophils were counted as recruited neutrophils. (B,D) Representative images of 3 days dpf larvae at 1, 2, 4, and 6 h post-wounding (hpw). Scale bar: 50 μm. (C) Quantification of recruited neutrophil numbers to the wounded area at 1, 2, 4, and 6 hpw in 3 dpf tlr2^+/+ and tlr2^{– /–} larvae. Each point represents a different larva. Sample size (n): 45, 46, 82, 72, 74, 68, 50, 50. (E) Quantification of recruited neutrophil numbers to the wounded area at 1, 2, 4, and 6 hpw in 3 dpf myd88^+/+ and myd88^{– /–} larvae. Each point represents a different larva. Sample size (n): 29, 28, 37, 38, 45, 39, 51, 45. In all cases, statistical analyses were done from three independent experiments. An unpaired, two-tailed t-test was used to assess significance (ns, no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001) and data are shown as mean ± SD.

The number of macrophages recruited to the wounded area in the tlr2 and myd88 mutants and wild type sibling controls larvae. (A) Experimental scheme. Tlr2 and myd88 homozygous mutants and sibling control larvae were wounded at 3 dpf. Their tails were wounded to the tip of the notochord. The red dashed line shows the site of wounding. Recruited macrophages at the wound were imaged at 1, 2, 4, and 6 hpw by using CLSM. For recruited cell counting analysis, cells localized within an area of 200 μm from the wounding edge toward the body trunk were counted as recruited cells. The red dashed box shows the area where macrophages were counted as recruited macrophages. (B,D) Representative images of 3 dpf larvae at 1, 2, 4, and 6 hpw. Scale bar: 50 μm. (C) The quantification of recruited macrophage numbers to the wounded area at 1, 2, 4, and 6 hpw in 3 dpf tlr2^+/+ and tlr2^{– /–} larvae. Each point represents a different larva. Sample size (n): 45, 45, 82, 71, 69, 68, 51, 50. (E) The quantification of recruited macrophage numbers to the wounded area at 1, 2, 4, and 6 hpw in 3 dpf myd88^+/+ and myd88^{– /–} larvae. Each point represents a different larva. Sample size (n): 35, 34, 40, 43, 56, 42, 60, 58. In all cases, statistical analyses were done with data of three independent experiments. An unpaired, two-tailed t-test was used to assess significance (ns, no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001) and data are shown as mean ± SD.

Calculated track measures. (A) Depiction of distance to the wound. It measured for each frame as the shortest distance from the cell’s current position to the entire line of the wound, i.e., the orthogonal projection to the wound. (B) Depiction of V_AP: velocity in anteroposterior axis direction. The visible part of the spine is taken as the y-axis. (C) Depiction of the net displacement, total displacement, meandering index, and mean speed: the net displacement is the distance of the cell between the first and final time frame. Total displacement is the sum of the net displacement between two successive frames. Meandering index corresponds to the net displacement divided by the total displacement. Mean speed is the total displacement divided by traveled time. Formulas show in Table 1 (Eqs 1–4). (D) Depiction of the construction of the mean squared displacement: the displacement between the first time frame and time frame t from all cells is squared and averaged (see Table 1, Eq. 5).

Quantification of distant neutrophils behavior in wounded tlr2 mutant and sibling control larvae. (A) Experimental scheme. Tlr2^+/+ and tlr2^{– /–} larvae were wounded at 3 dpf. The red dashed line shows the site of wounding. Neutrophils of wounded zebrafish larvae were tracked for 2 h and images were taken every 1 min by using CLSM. For cell tracking analysis, cells localized outside an area of 200 μm from the wounding edge toward the body trunk were counted as distant cells. Blue dashed box shows the area where distant neutrophils were tracked. (B) Representative images of distant neutrophil tracks in the wounded tail fin of 3 dpf tlr2^+/+ or tlr2^{– /–} larvae at frame 1, frame 60 and frame 120. Time interval between two successive frames is 1 min. Each color track represents an individual neutrophil. Cell tracking movies are shown in Supplementary Movies S9, S10). Scale bar: 50 μm. (C) Distance to the wound. Black dash line represents average distance to the wound. Each color line represents one cell. (D–I) Quantification of distant neutrophil tracks. In (D–F,H), each color indicates a different larva. There was no significant difference between the groups in terms of mean speed (D), net displacement (E), and MSD (green) and fitted MSD (black) (G). However, meandering index (F) and mean V_AP(H) of neutrophils at the wound in tlr2^+/+ is greater than in tlr2^{– /–} larvae. The fitted MSD (G, black) was fitted for dt < 80 min. The shaded regions in MSD (G) and mean V_AP over time (I) indicate standard error of the mean. Statistical analyses were done with 7 and 8 fish, respectively, for each group. An unpaired, two-tailed t-test was used to assess significance (ns, non-significance, *P < 0.05) and data are shown as mean ± SD. Sample size (n): 25, 22 (D–F,H).

Quantification of distant neutrophils behavior in wounded myd88 mutant and sibling control larvae. (A) Experimental scheme. Myd88^+/+ and myd88^{– /–} larvae were wounded at 3 dpf. The red dashed line shows the site of wounding. Neutrophils of wounded myd88 zebrafish larvae were tracked for 2 h and images were taken every 1 min by using CLSM. For cell tracking analysis, cells localized outside an area of 200 μm from the wounding edge toward the body trunk were counted as distant cells. Blue dashed box shows the area where distant neutrophils were tracked. (B) Representative images of distant neutrophil tracks in the wounded tail fin of 3 dpf myd88^+/+ or myd88^{– /–} larvae at frame 1, frame 60, and frame 120. Time interval between two successive frames is 1 min. Each color track represents an individual neutrophil. Cell tracking movies are shown in Supplementary Movies S11, S12). Scale bar: 50 μm. (C) Distance to the wound. Black dash line represents average distance to the wound. Each color line represents one cell. (D–I) Quantification of distant neutrophil tracks. In (D–F,H), each color indicates a different larva. There was no significant difference between the groups in terms of mean speed (D). However, the net displacement (E), meandering index (F), MSD (green) and fitted MSD (black) (G), and mean V_AP(H) of neutrophils at the wound in myd88^+/+ is greater than in myd88^{– /–} larvae. The shaded regions MSD (G) and in mean V_AP over time (I) indicate standard error of the mean. The fitted MSD (G, black) was fitted for dt < 80 min. Statistical analyses were done with 8 and 7 fish, respectively, for each group. An unpaired, two-tailed t-test was used to assess significance (ns, non-significance, *P < 0.05, **P < 0.01) and data are shown as mean ± SD. Sample size (n): 30, 22 (D–F,H).

Quantification of distant macrophage behavior in wounded tlr2 mutant and sibling control larvae. (A) Experimental scheme. Tlr2^+/+ and tlr2^{– /–} larvae were wounded at 3 dpf. The red dashed line shows the site of wounding. Macrophages of wounded tlr2 zebrafish larvae were tracked for 2 h and images were taken every 1 min by using CLSM. For cell tracking analysis, cells localized outside an area of 200 μm from the wounding edge toward the body trunk were counted as distant cells. Blue dashed box shows the area where distant macrophages were tracked. (B) Representative images of distant macrophage tracks in the wounded tail fin of 3 dpf tlr2^+/+ or tlr2^{– /–} larvae at frame 1, frame 60, and frame 120. Time interval between two successive frames is 1 min. Each color track represents an individual macrophage. Cell tracking movies are shown in Supplementary Movies S13, S14). Scale bar: 50 μm. (C) Distance to the wound. Black dash line represents average distance to the wound. Each color line represents one cell. (D–I) Quantification of distant macrophage tracks. In (D–F,H), each color indicates a different larva. There was a significant difference between the groups in terms of mean speed (D), net displacement (E), meandering index (F), MSD (red) and fitted MSD (black) (G), and mean V_AP(H) of macrophages. The shaded regions in MSD (G) and mean V_AP over time (I) indicate standard error of the mean. Statistical analyses were done with 6 and 8 fish, respectively, for each group. An unpaired, two-tailed t-test was used to assess significance (ns, non-significance, *P < 0.05, **P < 0.01, ****P < 0.0001) and data are shown as mean ± SD. Sample size (n): 23, 22 (D–F,H).

Quantification of distant macrophages behavior in wounded myd88 mutant and sibling control larvae. (A) Experimental scheme. Myd88^+/+ and myd88^{– /–} larvae were wounded at 3 dpf. The red dashed line shows the site of wounding. Macrophages of wounded zebrafish larvae were tracked for 2 h and images were taken every 1 min by using CLSM. For cell tracking analysis, cells localized outside an area of 200 μm from the wounding edge toward the body trunk were counted as distant cells. Blue dashed box shows the area where distant macrophages were tracked. (B) Representative images of distant macrophage tracks in the wounded tail fin of 3 dpf myd88^+/+ or myd88^{– /–} larvae at frame 1, frame 60 and frame 120. Time interval between two successive frames is 1 min. Each color track represents an individual macrophage. Cell tracking movies are shown in Supplementary Movies S15, S16). Scale bar: 50 μm. (C) Distance to the wound. Black dash line represents average distance to the wound. Each color line represents one cell. (D–I) Quantification of distant macrophage tracks. In (D,F,H), each color indicates a different larva. There was a significant difference between the groups in terms of mean speed (D), net displacement (E), meandering index (F), MSD (red) and fitted MSD (black) (G) and mean V_AP(H) of macrophages. Statistical analyses were done with 9 and 8 fish, respectively, for each group. The shaded regions in MSD (G) and mean V_AP over time (I) indicate standard error of the mean. An unpaired, two-tailed t-test was used to assess significance (ns, non-significance, ^∗∗P < 0.01, ^∗∗∗P < 0.001, ^****P < 0.0001) and data are shown as mean ± SD. Sample size (n): 50, 44 (D–F,H).

Graphic summary of the data of cell migration behavior in the tlr2 and myd88 mutants and wild type siblings. (A) Cell migration behavior in the wild type siblings. (B) Cell migration behavior in the tlr2 mutant. (C) Cell migration behavior in the myd88 mutant. In all cases, the green and red tracks are representative for the medians of the measured total displacements and net displacements in the anteroposterior axis of distant neutrophils and macrophages, respectively. The number of drawn leukocytes at the wound are only representing estimates of the relative numbers in the different genotypes. For the wild type sibling the tlr2^+/+ sibling was used as an example (A).