JCS009159 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Macrophage immunolabeling in wild-type and hi2217 embryos. (A-C) Wild-type embryos immunolabeled with an antibody to zebrafish L-plastin at various stages of development, arrows indicate individual cells. (A) 26 hpf, composite image, (B) 48 hpf, composite image, (C) 72 hpf, tailfin after 2.5 hours of response to wound (indicated with *). Note elongated morphology of L-plastin immunolabeled cells responding to wound. (D-G′) hi2217 embryos at 26 hpf; D-G are wild-type siblings and D′-G′ are homozygous mutants. (D-E′) Views of yolk and yolk-sac extension in hi2217 embryos at 26 hpf; (D,D′) Oblique coherent contrast, (E,E′) L-plastin immunolabeling; note the presence of macrophages at the intersection between the yolk sac and yolk sac extension in mutants (D′,E′). (F-G′) hi2217 embryos injected with either a standard control MO (F,G) or a MO to pu.1 (F′,G′) and immunolabeled for L-plastin. Note the same mutant phenotype in the absence of macrophages (G′, compare with E′). Embryos are oriented anterior to the left, dorsal side up.

• Movie 1 -

  Movie S1. Neutrophil migration in hi2217;MPO:GFP embryo tailfin at 2 dpf. Time-lapse movie of the distal tailfin of a homozygous mutant hi2217;MPO:GFP embryo, with fluorescence overlaid onto DIC images; migratory GFP-positive cells are neutrophils. Frames were collected at 30 seconds per frame over 135 minutes, and displayed at 15 frames per second. Note that images from this movie were used for Fig. 7A-C,F,H.

• Movie 2 -

  Movie S2. Pseudopod extension during neutrophil migration in hi2217;MPO:GFP embryo tailfin at 2 dpf. Time-lapse movie (fluorescence only) of a section of the tailfin of a homozygous mutant hi2217;MPO:GFP embryo, focusing on a single migrating neutrophil that extends multiple pseudopodia. Frames were collected at 30 seconds per frame over 14 minutes, and displayed at 7 frames per second.

• Movie 3 -

  Movie S3. NS-398 treatment causes neutrophil arrest in hi2217;MPO:GFP embryos. Time-lapse movie (fluorescence only) of the tailfin of a mutant hi2217;MPO:GFP embryo pre-treated for 1 hour and maintained in 250 μM NS-398. This is followed by a movie of the same embryo 1 hour after washing out the drug (‘Wash’, as indicated in the lower left corner). For each part (NS-398 treatment and Wash) frames were collected at 30 seconds per frame over 22 minutes, and displayed at 6 frames per second. Note that cell tracks from this movie are shown in Fig. 9.