Spi-b partially compensates for Pu.1 function during the development of RBI-born macrophages. (A-B) WISH of mfap4 expression in 30-hpf siblings (35/35) and spi-b^∆232 mutants (n = 14/14). (C-D) Neutral red staining of microglia in 3-dpf siblings and spi-b^∆232 mutants. (E-F) WISH of lyz expression in 30-hpf siblings (n = 38/38) and spi-b^∆232 mutants (n = 15/15). (G-H) WISH of pu.1 expression in 20-hpf siblings (n = 44/44) and spi-b^∆232 mutants (n = 17/17). (I-J) WISH of mfap4 expression in 36-hpf pu.1^Δ371 single- and pu.1^Δ371;spi-b^∆232 double-mutant embryos. (K-L) WISH of lyz expression in 36-hpf pu.1^Δ371 single- and pu.1^Δ371;spi-b^∆232 double-mutant embryos. (M) Quantification of mfap4⁺ macrophages and lyz⁺ neutrophils as shown in panels I-J and K-L, respectively. n.s., not significant, ***P < .001, Student t test; mfap4⁺_pu.1Δ371 (mean/standard error [SE]/n) = 39.0/3.0/16, mfap4⁺_dmut (mean/SE/n) = 22.9/2.2/18, lyz⁺_pu.1Δ371 (mean/SE/n)=117.4/4.4/7, lyz⁺_dmut (mean/SE/n) =121.6/4.2/8). Errors bars represent SE.