Fig 1

(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.