Irf8 is downstream of Pu.1. (A-B) WISH indicates irf8 RNA expression in the yolk sac of 18 hpf control embryo (n = 35/35) and pu.1 morphant (n = 28/29). (C-D) WISH shows pu.1 RNA expression in the yolk sac of 18 hpf control embryo (n = 31/31) and irf8 MO^sp morphant (n = 27/27). (E) RT-PCR analysis confirms a similar level of pu.1 expression in 18 hpf control embryos (left lane) and irf8 MO^sp morphants (right lane). (F-G) Anti-Myc staining reveals a significant induction of Irf8^myc protein expression in the heat shock-treated (HS) pu.1 morphant (n = 26/31) compared with the untreated (No-HS) pu.1 morphant (n = 33/33). (H-J) WISH indicates csf1r expression in 26 hpf untreated control embryo (n = 41/41), untreated (No-HS) pu.1 morphant (n = 45/46), and heat shock-treated (HS) pu.1 morphant (n = 51/51). (H-J) Insets are higher magnification (×20) views of the boxed region (blue). (K-M) WISH reveals apoeb RNA expression in the brain of 3 dpf untreated control embryo (n = 43/43), untreated (No-HS) pu.1 morphant (n = 37/37), and heat shock-treated (HS) pu.1 morphant (n = 41/41). (A-D,H,K) Blue arrows indicate csf1r⁺ macrophages. (H-J) Black arrowheads represent neural crest-derived pigment cells positive for csf1r. (N-P) DsRed shows macrophages loaded with E coli (red, white arrows) in the CHT of 40 hpf untreated control embryo (n = 27/30), untreated (No-HS) pu.1 morphant (n = 25/29), and heat shock-treated (HS) pu.1 morphant (n = 30/32).