The formation of RBI-born macrophages is regulated by a vertical Pu.1−Spi-b axis. (A-B) WISH of pu.1 expression in 20-hpf siblings (n = 39/39) and hypomorphic pu.1^G242D mutants (n = 13/13). (C-D) WISH of spi-b expression in 20-hpf siblings (n = 41/41) and pu.1^G242D mutants (n = 12/12). (E-F) WISH of pu.1 expression in 20-hpf siblings (n = 45/45) and spi-b^∆232 mutants (n = 16/16). All embryos in panels A-F are dorsally orientated with anterior to the left. (G) Quantitative RT-PCR for pu.1 and spi-b expression in wild-type embryos at indicated stages (x-axis). Expression level of target genes was normalized with elf1a expression. Error bars represent standard error. (H) Schematic view of the pu.1 or spi-b expression constructs under the control of a -9.0kb pu.1 promoter. Both Pu.1 and Spi-b are flag-myc double tagged at the N terminus. (I-K) WISH of mfap4 expression in 2-dpf pu.1^∆839 (n = 25/25), pu.1^∆839;Tg(-9.0kbpu.1:flag-myc-pu.1)⁺ (shorted as Tgpu.1⁺) (n = 14/15), and pu.1^∆839;Tg(-9.0kbpu.1:flag-myc-spi-b)⁺ (shorted as Tgspi-b⁺) (n = 11/13). (L-N) Neutral red staining of microglia in 3-dpf pu.1^∆839, pu.1^∆839;Tgpu.1⁺, and pu.1^∆839;Tgspi-b⁺. (O) The working model for the vertical Pu.1−Spi-b genetic network in RBI macrophage development.