Inactivation of Pu.1 and Spi-b impairs the VDA-born macrophage development. (A) A schematic view of a zebrafish embryo. The red box represents the trunk region, the purple box represents CHT, and the blue line indicates the imaged cross section of brain. (B-G) Fluorescence imaging indicates macrophages in Tg(mpeg1:LRLG);pu.1^∆839 single and Tg(mpeg1:LRLG);pu.1^∆839;spi-b^Δ232 double mutants at indicated developmental stages. (F′-G′) Magnified images of the boxed regions as indicated in F and G. (H) Quantification of tissue macrophages in the trunk and CHT (as shown in panels B-G) of Tg(mpeg1:LRLG);pu.1^∆839 single and Tg(mpeg1:LRLG);pu.1^∆839;spi-b^Δ232 double mutants at indicated developmental stages. n.s., not significant; ***P < .001, Student t test; mpeg1⁺_pu.1∆839 (3 dpf, mean/standard error [SE]/n) = 0.6/0.4/7, mpeg1⁺_dmut (3 dpf, mean/SE/n) = 0/0/4, mpeg1⁺_pu.1∆839 (8 dpf, mean/SE/n) = 29.7/1.6/6, mpeg1⁺_dmut (8 dpf, mean/SE/n) = 5.0/1.6/6, mpeg1⁺_pu.1Δ839 (15 dpf, mean/SE/n) = 64.0/5.6/6, mpeg1⁺_dmut (8 dpf, mean/SE/n) = 24.8/2.9/6). Error bars represent SE. (I-J) Costaining of TUNEL signals and coro1a-GFP in the CHT of 4 dpf pu.1^∆839 single and pu.1^∆839;spi-b^Δ232 double mutants. (K-L) Co-staining of acridine orange (AO) signals (K′-L′, green) and Tgmpeg1 (K′′- L′′, red) on the trunk of 15-dpf Tg(mpeg1:LRLG);pu.1^Δ839 single and Tg(mpeg1:LRLG);pu.1^Δ839;spi-b^∆232 double mutants. White arrows in panel L indicate mpeg1⁺AO⁺ apoptotic cell bodies of dying macrophages in Tg(mpeg1:LRLG);pu.1^Δ839;spi-b^∆232 double mutants. (M-N) Costaining of Lcp1 antibody and mpeg1-DsRed on the whole-mount brain sections of pu.1^Δ839 mutants and pu.1^Δ839;spi-b^∆232 double mutants at 15 dpf. Dashed circles indicate the brain regions on the sections.