Macrophage ontology, morphology, and behavior in stable mpeg1 transgenic zebrafish. (A) Unconverted Kaede expression (green) in dispersed macrophages in Tg(mpeg1:Gal4-VP16/UAS:Kaede) F1 embryos from 28 to 144 hpf. Images at 28, 50, and 120 hpf are composites assembled from photographs of the same embryo taken in 2 focal planes. (B) Loss of transgene expression occurs in young F1 and F2 Tg(mpeg1:Gal4-VP16/UAS:Kaede) adults (i,iv), demonstrated by the absence of dispersed fluorescent macrophages in the tail fin; compare with macrophages in the tail fins of Tg(mpeg1:mCherry) and Tg(mpeg1:EGFP) F0 animals in Figure 1B. However, the direct offspring of an outcross of the F1 adult (i) still shows strong embryonic transgene expression in dispersed macrophages (ii,iii). (I,iv) Arrowheads indicate autofluorescent iridophores. Bar represents 1 mm. (C) Dendritic morphology (arrowheads) of photoconverted Kaede (red) marked cells in Tg(mpeg1:Gal4-VP16/UAS:Kaede) F1 embryos. Bar represents 20 μm. (D) No overlap of fluorophore expression was observed between photoconverted Kaede and EGFP in F1 Tg(mpeg1:Gal4-VP16/UAS:Kaede/mpx:EGFP) compound transgenic embryos, demonstrating that the mpeg1 promoter drives expression in an entirely separate myeloid cell population to that of the mpx promoter (green represents neutrophils; and red, macrophages). Bar represents 50 μm. (E) Macrophage pinocytosis leads to accumulation of neutral red staining in vacuoles of unconverted Tg(mpeg1:Gal4-VP16/UAS:Kaede) positive cells (green) in the brain (arrowheads) of F1 embryos. Bar represents 50 μm. (F) Phagocytosis of heat-killed Penicillium marneffei spores (calcofluor-labeled, blue, arrowhead) by macrophages (red represents photoconverted Kaede) and neutrophils (green represents EGFP). Note phagocytosis of lower fungal spore by macrophage (bottom filled arrowhead) and migration of neutrophil with intracellular spore (open arrowhead). Stills from supplemental Video 1. Bar represents 50 μm. Time: minutes after infection.