Migration capacity, tissue deployment and lifespan of RBI-derived and CHT-derived neutrophils during homeostasis.

A Experimental strategy design. At 34 h post fertilization (hpf) the head or the CHT was photoconverted and at 44 hpf the number of RBI-derived and CHT-derived neutrophils located in or outside the corresponding original region, was quantified. B Quantification of the number of RBI-derived and CHT-derived neutrophils present in and outside the head or CHT, n = 11 embryos for head or CHT photoconvertion per experiment. C Experimental design. At 34 hpf and 44 hpf the first and second photoconversion were performed either in the head or CHT according to the experimental needs. D, E Representative images and quantification of CHT-derived neutrophils at 72 h post second photoconversion (hpp) present in the head, CHT, dorsum, tail, and intestine. n = 15 larvae for head or CHT photoconvertion per experiment. F, G Representative images and quantification of RBI-neutrophils at 72 hpp present in the head, CHT, dorsum, tail, and intestine, n = 15 larvae for head or CHT photoconvertion. H Quantification of photoconverted RBI and CHT-derived neutrophils present in the whole body compared to their initial number (24 hpp), n = 14 larvae for head or CHT photoconvertion per experiment. I Loss rate of RBI-derived (red line) and CHT-derived neutrophils (green line) during 72 h. n = 14 larvae for head or CHT photoconvertion per experiment. Data are represented as mean ± SD. Statistical analyses were performed using a two-sided Mann-Whitney U test. Experiments were performed independently at least 3 times. Scale bar: 100 μm.

Recruitment of RBI-derived and CHT-derived neutrophils after tail transection.

A Experimental design. At 34 h post fertilization (hpf) and 44 hpf the first and second photoconversions were performed in the head to label RBI-derived neutrophils red. At 54 hpf, caudal fin transections were made, and time lapse analysis was conducted during the 3 h post damage (hpd). B Representative images of RBI-derived/red and CHT-derived/green neutrophils at the wound site (dotted white line) at 1, 2, and 3 hpd. C Quantification of the number of RBI-derived and CHT-derived neutrophils present at the wound at 0, 1, 2, and 3 hpd. n = 16 larvae per experiment. D Representative images of RBI-derived/red and CHT-derived/green neutrophils at the wound (dotted white line) at 1, 2, and 3 hpd treated or not with diphenyliodonium (DPI). E Quantification of the number of RBI-derived and CHT-derived neutrophils that reach the wound at 1, 2, and 3 hpd treated or not with DPI. n = 10 larvae per experiment. In all the cases data are represented as mean ± SD. Statistical analysis was done with a two-sided Mann-Whitney U test. Experiments were performed independently at least 3 times. Scale bar: 100 μm.

Migration dynamic of RBI-derived and CHT-derived neutrophils during inflammation.

A Representative images of the tail at 3 h post damage (hpd) showing RBI-derived and CHT-derived neutrophil tracks to reach the wound. The blue line represents the circulatory loop. B Quantification of the migration route (bloodstream or extracellular matrix) utilized by RBI-derived and CHT-derived neutrophils to reach the wound at 1, 2, and 3 hpd. Analysis of the (C) Euclidian distance, (D) maximal velocity, (E) cumulative distance and (F) directionally of RBI-derived and CHT-derived neutrophils recruited to the wound. n = 13 larvae per experiment Statistical analyses were done with a two-sided Mann-Whitney U test. Data are represented as mean ± SD (G) Dispersion. Rose diagram showing the deviation angles in each segment of the route followed by neutrophils with respect to the direct route (0°) to the damage area. White numbers inside the green or red bars represent the percentage of neutrophils that follow a segment with this angle. The p-values were obtained after applying a Rayleigh test to angles distribution in each subpopulation. n = 10 larvae per experiment. Experiments were performed independently at least 3 times.

Single-cell transcriptomic reveal neutrophil and macrophage cell populations.

A UMAP plot displaying the 3 main cell type clusters: neutrophils (3732 cells), macrophages (1830 cells), and an unknown cell type (1466 cells). B Heatmap of top 15 marker genes per cluster (complete list available in Supplementary Data 1, filtered on a minimum detection fraction threshold = 0.1, log fold-change threshold = 0.25 and p-value < 0.05 (not adjusted), negative binomial test, out of the 2000 most variable genes, only genes with positive fold change were selected). C Expression plots of selected markers for neutrophils: mpx, liz, mmp13a and coro1a. D Expression plots of selected markers for macrophages: mfap4, mpeg1.1, marco, irf8.

Neutrophil and macrophage populations both contain cells from all 4 conditions.

A UMAP plot showing the distribution of cells from the four experimental conditions within the entire dataset. B, C Pie charts showing the number of cells of each condition present in macrophages (B) and neutrophils (C). D Stacked bar plot showing the number of neutrophils and macrophages present in each condition.

RBI- and CHT-derived neutrophils have different responses to resection.

A Heatmap of the top 5 markers in primitive and definitive neutrophils at steady state (complete list available in Supplementary Data 2, filtered on a minimum detection fraction threshold = 0.1, log fold-change threshold = 0.25 and p-value < 0.05 (not adjusted), negative binomial test, out of the 2000 most variable genes, only genes with positive fold change were selected). B, C Heatmaps of the top 15 upregulated and downregulated genes upon tail resection among the RBI- derived (B) and CHT-derived (C) neutrophil populations (complete list available in Supplementary Data 3 and 4, filtered on a minimum detection fraction threshold = 0.1, log fold-change threshold = 0.25 and p-value < 0.05 (not adjusted), negative binomial test, out of the 2000 most variable genes, only genes with positive fold change were selected). D Venn diagram showing the number of overlapping upregulated and downregulated genes between RBI-derived and CHT-derived neutrophils. E Network of the pathways associated with upregulated and downregulated genes among RBI-derived and CHT-derived neutrophils upon tail resection. Each gene associated with a given pathway is displayed. Pie charts indicated the relative contribution of each type of neutrophil to that term/gene (see Methods). Note that csf3b appears both as associated with chemotaxis upon resection and as a specific marker of the RBI-derived neutrophils when compared with CHT-derived neutrophils. Heatmaps show genes with p-value < 0.05, based on a negative binomial distribution, out of the 2000 most variable genes.

Csf3b regulates RBI-derived neutrophil egress from hematopoietic tissue at steady state.

A Experimental strategy. At 34 hpf the yolk or CHT of control and morphant embryos was photoconverted and at 44 hpf the amount of RBI-derived and CHT-derived neutrophils was quantified. B Quantification of RBI-derived and CHT-derived neutrophils in and outside the corresponding hematopoietic region in control and morphant embryos. n = 13 embryos for yolk or CHT photoconvertion per experiment. C Experimental strategy. At 34 hpf and 44 hpf the head of control and morphant embryos was photoconverted, at 54 hpf caudal fin transection was performed and the number of RBI-derived and CHT-derived neutrophils present at the wound was quantified during the first three hours post damage. n = 18 larvae per condition per experiment. D Quantification of the number of RBI-derived and CHT-derived neutrophils present at the wound at 0, 1, 2 and 3 hpd in control and morphant embryos. Statistical analysis was done using a two-sided Kruskal-Wallis Test. Data are represented as mean ± SD Experiments were performed independently at least 3 times.