Pu.1 is required for the RBI- but not VDA-derived macrophage development. (A-B) WISH of mfap4 expression in 30-hpf siblings (n = 31/31) and pu.1^∆839 mutants (n = 12/12). (C-D) Neutral red staining of microglia in 3-dpf siblings and pu.1^∆839 mutants. (E-F) WISH of lyz expression in 30-hpf siblings (n = 35/35) and pu.1^∆839 mutants (n = 13/13). (G-H) WISH of cebpa expression in 20-hpf siblings (n = 42/42) and pu.1^∆839 mutants (n = 13/13). (I-J) Fluorescence imaging of RBI- and VDA-labeled pu.1^∆839;Tg(mpeg1:Loxp-Dsred-loxp-GFP) embryos at 8 dpf. Embryos are laterally orientated to view macrophages in the trunk region. (K) Quantification of RBI- or VDA-labeled macrophages (GFP⁺) in the total recovered macrophages (mpeg1⁺) in 8 dpf pu.1^∆839 mutants compared with control embryos without heat shock. ***P < .001 (Student t test, GFP⁺/mpeg1⁺_control (mean/standard error [SE]/n) = 0.09/0.01/9), GFP⁺/mpeg1⁺_RBI (mean/SE/n) = 0.08/0.02/4, GFP⁺/mpeg1⁺_VDA (mean/SE/n) = 0.66/0.04/7). Error bars represent SE. (L-M) Sudan black B (SB) staining of neutrophils in 4-dpf siblings and pu.1^∆839 mutants.