(A) Schematic of the procedure for transient induction of V12RAS/v-Src in embryos that also have fluorescently-tagged neutrophils, with an example of a V12RAS⁺ melanoblast clone (red) in a 3dpf larva also expressing eGFP (green) in neutrophils. (B) (i) A 5dpf MPO:GFP larva with a V12RAS⁺ clone (red). (ii) High magnification view of the inset in (B) (i), which is a single image from a time-lapse movie (Video S1A) of GFP-tagged neutrophils actively interacting with a V12RAS⁺ clone; note that most of the red fluorescent signal is quenched by melanocyte pigment. (C) (i) A single image from a time-lapse movie showing LysC:DsRed⁺ cells recruited v-Src⁺ (green) cells in a 3dpf larva (Video S2B) (ii) An equivalent image from a time-lapse movie showing no recruitment of LysC:DsRed⁺ cells to GAP43-eGFP expressing cells in a control larva (Video S2A). (D) (i) Low magnification, two-channel, lateral view of a control Tg (kita:GalTA4; UAS:eGFP; LysC:DsRed) larva at 4dpf. (ii) Single channel view to highlight only the DsRed-tagged leukocytes. The box highlights these cells located within the caudal hematopoietic tissue. (E) As for D but of a Tg (kita:GalTA4; UAS:V12RASeGFP; LysC:DsRed) larva at 4 dpf. The boxed zone in E (ii) indicates how the LysC:DsRed⁺ cells have largely dispersed from the caudal hematopoietic tissue into the flank skin. (F) A confocal Z-stack projection of the flank of a control larva in the trunk region; (G) Equivalent image to E but of a V12RAS+ larva; both are stained with the anti-L-plastin antibody (magenta). (H) A high magnification view of a larva similar to that in (F), illustrating the association of L-plastin⁺ cells (magenta) with V12RAS⁺ cells (green). (I) A similar larvae to that in H, but with v-Src expressing cells (green) (J) Anti-BrdU (red) immunostaining of control mucus secreting cells (green). (K) Anti-BrdU (red) immunostaining of V12RAS⁺ mucus secreting cells (green). (L) Quantification of numbers of LysC:DsRed⁺ cells present in the skin of the trunk in the region indicated by boxes in (D) and (E). (M) Quantification of the number of L-plastin⁺ cells present in the trunk epidermis in regions indicated in (F) and (G). *** p<0.001; Larval images in (D) and (E) have been “tiled” together from several micrographs and the tiling borders are indicated with white dotted lines. Scale bars: (A) = 48 μm; (B) = 24 μm; (C) = 20 μm; (D, E, F, G) = 150 μm; (I, J, K) = 16 μm.

https://doi.org/10.1371/journal.pbio.1002377.g001

(A) A confocal image of a LysC:DsRed⁺ (red) cell and a macrophage (pale green with dotted line outline) interacting with a V12RAS⁺ cell (green) (Video S8). (B, C) Images showing LysC:DsRed⁺ leukocytes establishing tethers with V12RAS⁺ cells, as observed in Tg(Fli:GFP; LysC:DsRed; kita:GalTA4; UAS:V12RASeGFP) larvae: (B) shows a tether entirely composed of V12RAS cytoplasm (green) (Video S9); (C) shows a chimeric tether composed of both V12RAS (green) and LysC:DsRed⁺ (red) cytoplasm. (D) A single image from a 3D movie (Video S10), of a neutrophil as it glides over and engulfs pieces of a V12RAS⁺ cell; (E) 3D reconstruction of a V12RAS⁺ cell clump (green cells) showing two macrophages (L-plastin⁺, magenta staining–yellow arrowheads) as they deform to engulf individual V12RAS⁺ cells. Small white arrows and dotted outline indicates lamellipodial protrusions extending over another pair of transformed cells. (F) A single focal plane (corresponding to that indicated in [E]) of the same pair of macrophages, showing V12RAS⁺ cell shaped phagosomes (asterisks), within them. One macrophage has partially enveloped another V12RAS⁺ cell with a thin lamellipodial extension (indicated by small white arrows). (G) 3D reconstruction of an anti-L-plastin stained mitfa:V12RAS-mitfa:mCherry injected Tg(BACMPO:eGFP) ⁱ¹¹⁴ larval flank region, showing the presence of mCherry⁺ (red) debris within L-plastin⁺MPO^- cells. (H) Image as in (G) but with single channel to highlight the mCherry⁺ V12RAS⁺ debris; green arrows indicate co-localization within L-plastin⁺ MPO⁺ neutrophils; blue arrowheads indicate co-localization within L-plastin⁺ MPO^- macrophages. Note that the larger clumps of debris all reside within macrophages. The grey tonal bars/domains at the bottom of (D) and (E) are the “floor” effect offered by Velocity software when generating a 3D reconstruction from a “Z-stack” of optical sections. Scale bars = 8 μm.

https://doi.org/10.1371/journal.pbio.1002377.g002