Partial recovery of macrophages in <i>irf8</i> mutants begins by ~7 dpf in the head region.

<p>Diagrams of zebrafish showing region of quantification (pink box), which was taken using the same magnification and field size of view at all stages. The field of view in older stages covers a relatively smaller region of the head because the fish are larger (comparing B with A). Red dotted box shows the region of the fluorescent images. (A) Expression of macrophage reporter <i>mpeg1</i>:<i>EGFP</i> at 6 and 7 dpf. Arrows show macrophages. At 6 dpf, most <i>irf8</i> mutants have no macrophages in the CHT (n = 7/12), while most have a few macrophages in the head (n = 10/13); image shows the head region of a mutant at 6 dpf with no macrophages. Panels show the tail CHT region of the <i>irf8</i> mutants at 6 and 7 dpf with no macrophages; a few autofluorescent pigment cells are present after PTU treatment. (B) Distribution of macrophages expressing <i>mpeg1</i>:<i>EGFP</i> at 14 dpf and 31 dpf. Arrows point to macrophages. (C) Quantification of the number of macrophages per field in the head region over time (pink box in left diagram). (D) Quantification of the number of macrophages per field in the tail region over time (pink box in right diagram). At 6 dpf, n = 20 for siblings and n = 13 for mutants; at 14 dpf, n = 8 for siblings and n = 7 for mutants; at 31 dpf, n = 7 for siblings and n = 5 for mutants. Statistical significance was determined by two-tailed Student’s t-test. Error bars represent S.E.M. ***, p < 0.001; **, p < 0.01; *, p < 0.05; CHT, caudal hematopoietic tissue; E, eye. All scale bars are 50 um. <i>irf8</i>^<i>-/-</i> in this figure represents trans-heterozygous <i>irf8</i>^{<i>st95/st96</i>} mutants.</p

Specific expression of <i>irf8</i> in macrophage or neutrophil lineage is sufficient to restore macrophage fates in <i>irf8</i> mutants.

<p>(A) Neutral red staining for microglia at 4 dpf in <i>irf8</i> siblings and mutants in control uninjected conditions or after tol2-mediated expression of <i>irf8</i> driven by tissue specific regulatory sequences. Expression of <i>mpeg1</i>:<i>irf8</i> or <i>lyz</i>:<i>irf8</i> was sufficient to restore some microglia in <i>irf8</i> mutants (arrows), but not expression in the neurons (<i>huc</i>) or skin (<i>krt4</i>). (B) Analysis of total macrophage population by <i>mfap4</i> RNA expression at 2.5 dpf after expression of <i>irf8</i> in different tissues. Specific expression of <i>mpeg1</i>:<i>irf8</i> or <i>lyz</i>:<i>irf8</i> was also sufficient to restore macrophages on yolk sac and embryo proper (arrows) in <i>irf8</i> mutants. (C) Plot showing frequency of microglia rescue. (D) Plot showing frequency of macrophage recovery. Purple denotes >25 macrophages and green shows partial recovery of less than 25 but more than 1. Numbers below bar graphs represent <i>n</i>, total number of embryos analyzed. sib, <i>irf8</i>^<i>+/+</i> and <i>irf8</i>^<i>+/-</i>; mut, <i>irf8</i>^<i>-/-</i>.</p

<i>irf8</i> mutant embryos have no macrophages but produce excessive neutrophils.

<p>(A) <i>mfap4</i> RNA expression at 2 dpf shows a complete loss of macrophages in <i>irf8</i> mutant but abundant macrophages in heterozygous sibling (arrows). (B) <i>mpx</i> RNA expression at 2 dpf shows an overproduction of neutrophils in <i>irf8</i> mutants (arrows, bottom panels) compared with the sibling (arrow, top panels). Right column in A and B shows a higher magnification image of the trunk region from the same embryos depicted in the left column. (C) Neutrophils can first be detected on the yolk sac by 1 dpf. <i>irf8</i> mutants have many yolk sac neutrophils (arrows) compared with sibling. (D) At 2 dpf, <i>irf8</i> mutants continue to have more neutrophils on the yolk sac (arrows). Overall, <i>irf8</i> mutants appear to have more neutrophils throughout the body. All scale bars are 100 um.</p

Summary model for role of <i>irf8</i> in macrophage ontogeny in zebrafish.

<p><i>irf8</i> is required for development of all macrophages in the early developmental period until 5–6 dpf (solid orange bar). Coinciding with the onset of the adult phase hematopoiesis in the kidney starting at ~7 dpf, hematopoiesis relies on <i>irf8</i> and other factors for macrophage specification and differentiation (dotted orange bar). RBI, rostral blood island; PBI, posterior blood island; CHT, caudal hematopoietic tissue; AGM, aorta-gonad-mesonephros analog; hpf, hours postfertilization; dpf, days postfertilization.</p

<i>irf8</i> mutants have immature myeloid cells and excess myeloid cell death during development in the CHT.

<p>Diagram of a zebrafish larva showing the regions of analysis in the CHT: red box, region in A and B; blue box, region in C and D; and dotted lines in blue box indicate area of TUNEL quantification in E. (A-B) Lateral view. Right panels, higher magnification of the dotted box shown on the left. Analysis at 2.5 dpf (A) and 5 dpf (B) shows that siblings have many macrophages strongly expressing <i>mpeg1</i>:<i>EGFP</i>; these cells have elaborate processes and complex morphologies, and some have migrated into other tissues (white arrows). By contrast, <i>irf8</i> mutants have cells weakly expressing <i>mpeg1</i>:<i>EGFP</i> that appear immature and different from macrophages in siblings. A few strongly expressing cells are first detected in mutants at 5 dpf (B, blue arrows), indicating recovery of a few macrophages. (C-D) Early myeloid reporter <i>pu</i>.<i>1</i>:<i>gal4-UAS-GFP</i> at 5 dpf shows abnormally small cellular specks restricted to the CHT in all mutants (D, arrows, n = 9/9 at 3 and 5 dpf) but not in the siblings (C, n = 5/5 at 3 and 5 dpf). Bottom, TUNEL labeling of apoptotic cells at 5 dpf. The small <i>pu</i>.<i>1</i> reporter expressing cellular specks in the CHT are similar in size and appearance to small-sized dying cells labeled by TUNEL, which are shown in another 5 dpf stage-matched <i>irf8</i> mutant larva (compare arrows in top panel with arrowheads in bottom panel from different larvae). TUNEL labeling (middle) and area traces of the TUNEL+ nuclei (bottom) are shown for each genotype. (E) Quantification of TUNEL assay as represented in C-D shows a significant increase in total dying cells in the CHT of <i>irf8</i> mutants (n = 3) compared with siblings (n = 6; p = 0.0039). This is largely accounted for by a significant increase in very small-sized TUNEL+ cells measuring less than 26 pixels in area (p = 0.0017). No significant difference was found in larger TUNEL+ cells (≥ 26 pixels in area, p = 0.16). Error bars represent S.E.M. Statistical significance was determined by two-tailed Student’s t-test. **, p < 0.01; *, p < 0.05; n.s., not significant; CHT, caudal hematopoietic tissue. All scale bars are 50 um and are the same for each set of panels.</p

TALEN-induced <i>irf8</i> mutations <i>st95</i> and <i>st96</i> eliminate embryonic microglia but allow survival to adulthood.

<p>(A) TALE nucleases target region near <i>irf8</i> translational start site, creating frameshift mutations <i>st95</i> and <i>st96</i>, which introduced premature stop codons as shown (bottom). Top panels show representative neutral red staining for microglia in <i>irf8</i>^{<i>st95/st95</i>} mutant that lacks all microglia, compared with a heterozygous sibling that has a wildtype microglial population (arrow). (B) Analysis of <i>apoe</i> RNA expression by in situ hybridization shows presence of microglia in <i>irf8</i> sibling but no microglia in <i>irf8</i> mutants at 3 and 6 dpf. (C) Images of heterozygous and homozygous mutant larvae at 5 dpf, showing that the mutants have normal overall morphology. Images of the whole adult zebrafish were compiled from two tiled images of the same fish. At 3 months of age, the <i>irf8</i> mutant zebrafish grew to a similar size as its sibling. All images represent the <i>st95</i> allele. Scale bars are shown below each set of panels or for each individual panel.</p