Fig. 2.

Neutrophils express arg2:GFP after wound challenge. (A) Fluorescence confocal timelapse micrographs of arg2:GFP-positive cells migrating towards a tailfin nick wound performed at 3 dpf at early timepoints post injury. Dashed lines indicate the edge of the fin and the asterisks mark the nick wound. mpw, minutes post wound. (B) Fluorescence confocal micrographs of the arg2:GFP line crossed to the lyz:mCherry line showing overlap of arg2:GFP with neutrophils at a tailfin wound performed at 3 dpf (dashed-dotted lines). Arrowheads indicate arg2:GFP-positive neutrophils migrating at the wound. Example timelapse images from two independent experiments with six larvae were imaged. (C) Quantification of arg2:GFP-positive neutrophils from B. (D) Number of neutrophils at the wound site that were arg2:GFP positive at 3 hpw. n=6 larvae combined from two independent experiments. (E) Fluorescence confocal timelapse micrographs of the arg2:GFP line crossed to the mpeg:mCherry line showing no overlap of macrophages with arg2:GFP expression early after injury performed at 3 dpf (dashed-dotted lines indicate the wound). The high exposure of the green channel to detect the earliest signs of arg2:GFP expression means that the autofluorescence of pigment cells is also evident, but these do not colocalise with mpeg:mCherry-positive cells. Sixty larvae in total were screened for macrophage arg2:GFP expression over three independent experiments. (F) Fluorescence widefield micrographs of the arg2:GFP line crossed to the lyz:mCherry line. The upper panels show an uninjured tailfin with few neutrophils (yellow arrowhead) and no immune cell-specific arg2:GFP expression overlap at 5 dpf. The middle panels show an injured tailfin (injury performed at 2 dpf) at 2 dpw (4 dpf) showing overlap between lyz:mCherry and arg2:GFP expression (white arrowheads). The lower panels show an injured tailfin at 3 dpw (5 dpf) showing overlap between lyz:mCherry and arg2:GFP expression (white arrowheads). The dashed-dotted lines indicate the edge of the tailfin fold. (G) Fluorescence confocal micrographs of the arg2:GFP line crossed to the lyz:mCherry line at 2 dpw (upper panels) and 3 dpw (lower panels), showing examples of lyz:mCherry-positive arg2:GFP-expressing cells (arrowheads) in the proximity of the healing tailfin wound performed at 2 dpf. (H) Fluorescence widefield micrographs of the arg2:GFP line crossed to the mpeg:mCherry line. The upper panels show an uninjured tailfin with few macrophages (yellow arrowheads) and no immune cell-specific arg2:GFP expression at 5 dpf. The middle panels show an injured tailfin (injury performed at 2 dpf) at 2 dpw (4 dpf) showing no overlap between mpeg:mCherry and arg2:GFP expression. mpeg:mCherry-negative cells expressing arg2:GFP with an amoeboid immune cell shape are shown by white arrowheads. The lower panels show an injured tailfin at 3 dpw (5 dpf) showing no overlap between mpeg:mCherry and arg2:GFP expression, with mpeg:mCherry-negative cells expressing arg2:GFP with an amoeboid immune cell shape shown by white arrowheads. The dashed-dotted lines indicate the edge of the tailfin fold. (I) Fluorescence confocal micrographs of the arg2:GFP line crossed to the mpeg:mCherry line at 2 dpw (upper panels) and 3 dpw (lower panels), showing examples of mpeg:mCherry-positive arg2:GFP-expressing cells in the proximity of the healing tailfin wound (injury performed at 2 dpf) (arrowheads).