Disruption of Spi-1l, the zebrafish ortholog of mammalian SPI-B, does not affect the development of VDA-derived macrophages. (A) Phylogenic tree analysis of mouse SPI genes (PU.1, SPI-B, and SPI-C) with zebrafish counterparts. The phylogenic tree was generated by multiple protein sequence alignment with Clustal Omega online software. (B) Percent identity matrix was created by multiple protein sequence of mammalian SPI-B and zebrafish Pu.1, Spi-b, and Spi-c. (C) Alignment of N-terminal transactivation domain of mouse PU.1 (mPU.1), SPI-B (mSPI-B), and zebrafish Pu.1 (zPu.1) and Spi-1-like (zSpi-1l). Red letters represent the conserved amino acids in acidic motifs (D/E-X-D/E-X-X-X-D/E, blue dashed boxes) and the Q-rich region (purple dashed box), whereas underlined black letters indicate the mutated residues in these position. The black dashed box indicates the P/S/T region in mSPI-B and zSpi-1l, and the P/S/T residues (31.8% in mSPI-B and 37.5% in zSpi-1l) are highlighted by the yellow background. (D-E) Fluorescence imaging of VDA-labeled mpeg1-GFP⁺ and unlabeled mpeg1-DsRed⁺ macrophages on the trunk of 8-dpf siblings (D) and spi-b^∆232 mutants (E). (F) Quantification of VDA-labeled macrophages (GFP⁺) in the total macrophage population (mpeg1⁺). n.s., not significant, Student t test; GFP⁺/mpeg1⁺_sib (mean/standard error [SE]/n) = 0.40/0.02/6, GFP⁺/mpeg1⁺_spi-b∆232 (mean/SE/n) = 0.44/0.03/4). Error bars represent SE. (G-H) Sudan Black B (SB) staining of 4 dpf siblings and spi-b^∆232 mutants.