a Schematic diagram showing the two major function domains of PDCD11: N-terminal Ribosome domain (1–1407) and C-Terminal Coil domain (1408–1871). b Gross morphology of 3 dpf WT and pdcd11 mutants. c qPCR confirmation of hyperactivated inflammatory pathway genes, including tnfa, il1b, and il6, in 22 hpf pdcd11 mutants, which could not be restored by combined p53 mutation. d–eIl6 and il1b expression in the brain of WT or pdcd11 mutants with p53 mutated or not examined by WISH at 52 hpf. f WISH analysis of l-plastin, mfap4, and csf1ra expression in 24 hpf WT and pdcd11 mutant embryos. Red arrows indicate csf1ra-positive cells in the brain. g Microglia development in 3 dpf WT and pdcd11 mutants by WISH assessment of apoeb and Neutral Red staining. hL-plastin and mfap4 expression pattern in 3 dpf WT and pdcd11 mutants. i Morphology of macrophages in the brain and caudal hematopoietic tissue (CHT) were examined using the Tg(mpeg1:GFP) transgenic line. White arrows indicate the vacuolated macrophages found in the pdcd11 mutant. j Increased tnfa mRNA expression in sorted macrophages Tg(mpeg1:GFP) from 60 hpf pdcd11 mutants as compared with WT controls. k qPCR examination of the expression of macrophage-related genes cxcr3.1 and cxcr3.2 in 22 hpf WT and pdcd11 mutants. l WISH analysis of cxcr3.1 and cxcr3.2 expression in 22 hpf WT and pdcd11 mutants. The number positioned in the lower right corner of d–h, l represent the number of zebrafish embryos shown positive phenotypes versus the total number of embryos examined. Scale bar: 100 μm. Black horizontal lines indicate mean ± standard error of mean (SEM). Means ± SEM are shown for three independent experiments. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 (Student’s t test).

qPCR (a), western blot (b) and WISH (c) examination of the expression levels of tgfb1a or Tgfβ1 in 22 hpf zebrafish. d Immunofluorescence assessment of Pdcd11 and Tgfβ1 expression in 22 hpf zebrafish embryos. White arrows indicate the co-expressed signals found on the YS. e Fold changes of Tgfβ1-regulated genes expression in macrophages sorted from the brains of 60 hpf pdcd11 mutants as compared with WT controls using Tg(mpeg1:GFP) transgenic line. f. Expression of csf1b and ctsba in 60 hpf WT and pdcd11 mutants by WISH. Red arrows indicate the positive cells. g Effect of zebrafish tgfb1a mRNA overexpression on csf1ra expression in WT and pdcd11 mutants. Effect of tgfb1a mRNA or combined mpeg1 driven smad7 overexpression on microglia markers apoeb (h) or Neutral Red staining (i) in WT and pdcd11 mutants. j The effect of LY364947 addition on apoeb expression in WT and pdcd11 mutants with tgfb1a mRNA overexpression. The number positioned in the lower right corner of f–j represent the number of zebrafish embryos shown positive phenotypes versus the total number of embryos examined. Scale bar: 100 μm. Means ± SEM are shown for three independent experiments. ^*P < 0.05 (Student’s t test).

a Schematic diagram showing different domains of PDCD11 consisting of Ribosome (Rib, 1-1407 aa), Coil (1408-1871 aa), and Terminal (Ter, 1742-1871 aa) domains. b Rescuing effects on apoeb positive microglia of pdcd11 mutants with the Rib or Coil domain mRNA overexpression. Red arrows indicate the apoeb positive cells. Scale bar: 100 μm. c qPCR assessment of inflammatory genes expression in pdcd11 mutants with Rib or Coil domain of PDCD11 overexpression. d Western blot examination of the 536-serine modified P65 expression in 22 hpf WT and pdcd11 mutants. e Western blot examination of nuclear P65⁵³⁶ contents with the Coil or Ter overexpression in HEK293T cells. f Immunofluorescence assay showing the expression pattern of cells co-transfected with N-terminal Flag fused Coil and P65-GFP. White arrowheads indicate the colocalized Coil and P65 in the cytoplasm or nucleolus. Scale bar: 20 μm. g Representative image of three independent experiments showing the effect of increasing Coil transfection on nuclear/cytoplasm P65 content. h Representative image of three independent experiments showing P65⁵³⁶ levels in WT and pdcd11 mutants with Coil domain (Coil), Terminal domain (Ter), or Ribosome domain (Rib) mRNA overexpression. i Appearance of mfap4 expressing macrophages in pdcd11 mutants treated with NF-κB inhibitor. Scale bar: 50 μm. j Rescuing effect of NF-κB inhibitors on apoeb positive microglia in pdcd11 mutants. Scale bar: 100 μm. k Representative image of three independent experiments showing the rescue effects of Tgfβ1 levels with Coil domain (Coil), Terminal domain (Ter), Ribosome domain (Rib) mRNA or NF-κB inhibitor addition in 22 hpf pdcd11 mutants. The number positioned in the upper right corner of b and lower right corner of i, j represent the number of zebrafish embryos shown positive phenotypes versus the total number of embryos examined. Means ± SEM are shown for three independent experiments. n.s not significant, ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 (Student’s t test).

a IF assay of Pdcd11 expression in Tg(NF-κB:mCherry) transgenic line. White arrowheads indicate the colocalized cells on the YS at 22 hpf. Scale bar: 100 μm. b In vitro transfection of plasmid containing NF-κB recognition motif driven GFP and Flag-Coil. White arrows indicate that cells with nuclear Coil expression displayed NF-κB activation. Scale bar: 20 μm. Representative image show cells co-transfected with Flag-Coil and c-Rel (c) or P105 (d). White arrows indicate that cells with nuclear expressed Coil and c-Rel (c) or P105 (d). Scale bar: 20 μm. e. Western blot showing the effect of increasing Coil transfection on nuclear/cytoplasm c-Rel content. f Influence of Coil and c-Rel transfection on luciferase activities driven by the NF-κB-binding site. Microglia numbers in WT and pdcd11 mutants examined by apoeb (g) or Neutral Red staining (h) with c-rel and p105 mRNA or combined mpeg1 promoter driven smad7 plasmid injection. Scale bar: 100 μm. qPCR (i) and western blot (j) assessment of tgfb1a or Tgfβ1 level in 22 hpf WT and pdcd11 mutants with c-Rel and p105 mRNA overexpression. k Microglia numbers in embryos with c-Rel inhibition (PTXF) or combined tgfb1a mRNA complementation. Scale bar: 100 μm. l Reporter assay showing the regulation of TGFB1 promoter by Coil and c-Rel transfection. TGFB1 WT promoter containing three c-Rel-binding sites (5′-ccGGGGcacccccc-3′, −761 bp to TSS; 5′-ggGGGGacgccccgt-3′, −776 bp to TSS; 5′-aaGGGAcccccctcg-3′, −1019 bp to TSS). The three binding motifs were respectively mutated and designated as M1 (5′-ccAAAAcacccccc-3′, −761 bp to TSS), M2 (5′-ggAAAAacgccccgt-3′, −776 bp to TSS), M3 (5′-aaAAACcccccctcg-3′, −1019 bp to TSS). The number positioned in the lower right corner of g, h, k represents the number of zebrafish embryos shown positive phenotypes versus the total number of embryos examined. Means ± SEM are shown for three independent experiments. n.s not significant, ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 (Student’s t test).

a Schematic diagram showing the domain structures of human P65, c-Rel, and PDCD11-Coil. b Immunoprecipitation (IP) assay of the interaction between P65-GFP and Flag-Coil. c IP assay of the interaction between c-Rel-GFP and Flag-Coil. d IP assay of the interaction between the Coil–coil fragment of PDCD11 with the RHD domain or TAD domain of P65. e IP assay of the interaction between the terminal (Ter) fragment of PDCD11 with the RHD domain or TAD domain of P65. f IP assay of the interaction between the Coil–coil fragment of PDCD11 with the RHD domain or TAD domain of c-Rel. g IP assay of the interaction between the terminal (Ter) fragment of PDCD11 with the RHD domain or TAD domain of c-Rel. h Western blot examination of the influential role of overexpression Coil, Coil–coil and Terminal of PDCD11 on P65ser536 expression in 22 hpf pdcd11 mutants. i Western bot show the effect of increasing Ter transfection on nuclear/cytoplasm P65 content in 293T cells. j Schematic diagram showing the working model for PDCD11-mediated regulation of c-Rel and P65. Under normal circumstances, on one hand, the Ter fragment of PDCD11 (yellow) binds and masks the phosphorylation site of P65 (pink), which is required for its nuclear translocation and transcriptional activation; on the other hand, the only existing interaction between the coil–coil of PDCD11 and the RHD domain of c-Rel contrarily promotes c-Rel (green) nuclear retention for activating TGFβ1 expression, which drives macrophage differentiation toward microglia with “M2” properties. However, cells deficient in PDCD11 show increased P65 nuclear translocation, which tends to lead to activation of inflammation-related genes, such as IL-1, IL-6, and TNFα. These cytokines then facilitate “M1” macrophage differentiation.

a WISH assessment of pdcd11 expression in zebrafish at 7-ss, 22 hpf, and 32;hpf. The red arrows indicate pdcd11 signals on the YS and pericardial cavity. b Immunofluorescence staining of Pdcd11 in Tg(pu.1:GFP) zebrafish. The red arrows indicate colocalized Pdcd11 and Pu.1 expressing cells on the YS at 10-ss. c Pdcd11 expression in 22 hpf and 32 hpf Tg(mpeg1:GFP) zebrafish assayed by immunofluorescence staining. d WISH examination of pdcd11 expression in pu.1 deficient embryos. The red dotted lines denote the pericardial cavity underneath the brain where pdcd11 expressed. e IF assessment of the co-expression between GFP driven by pdcd11 promoter and endogenous Pdcd11. White arrowheads indicate the colocalization signal on the 22 hpf YS. f The expression pattern of mfap4-positive macrophages in the 52 hpf brain with pdcd11 promoter driven Coil overexpression. g Rescue effects of apoeb positive microglia with Coil overexpressed under different promoters including Huc, mpeg1, mpo, and pdcd11. The number positioned in the lower left corner of Fig. 6f, g represent the number of zebrafish embryos shown positive phenotypes versus the total number of embryos examined. Scale bar: 100 μm.