Pu.1 and Spi-b function in parallel during VDA-derived macrophage development. (A) Schematic view of a zebrafish embryo. Purple box indicates the CHT. (B-D) Costaining of spi-b mRNA with Tg(-9.0kbpu.1:GFP) and Lcp1 antibody in the CHT region of 4-dpf wild-type embryos. Panels B/C and B/C/D are merged images from the corresponding panels (B-D). Arrows in each panel indicate cells coexpressing spi-b, pu.1-GFP, and Lcp1. (E-F) WISH of spi-b expression in 4-dpf siblings and pu.1^∆839 mutants. (G-H) Fluorescence images of the pu.1-GFP⁺ myeloid cells in the CHT of 4-dpf Tg(-9.0kbpu.1:GFP) siblings and Tg(-9.0kbpu.1:GFP);spi-b^∆232 mutants. (I) Quantitative RT-PCR for spi-b expression in VDA-derived macrophages isolated from pu.1 MO injected wild-type Tg(mpeg1:LRLG) and pu.1^∆839;Tg(mpeg1:LRLG) at 8 dpf. (J) Quantitative RT-PCR for pu.1 expression in the VDA-derived macrophages isolated from pu.1 MO-injected wild-type Tg(mpeg1:LRLG) and spi-b^∆232;Tg(mpeg1:LRLG) at 8 dpf. (K) Quantitative RT-PCR for pu.1 and spi-b expression in VDA-derived macrophages isolated from pu.1 MO-injected Tg(mpeg1:LRLG) at 8 dpf. Expression level of target genes in panels I-K was normalized with elf1a expression. Error bars represent standard error. (L) The parallel Pu.1−Spi-b genetic network in the development of VDA-derived macrophages.