Figure 7.
Figure 7. The paralleled and vertical Pu.1−Spi-b cascades control macrophage development via a common downstream factor, Irf8. (A-B) WISH of irf8 expression in 20-hpf siblings (n = 35/35) and hypomorphic pu.1G242D mutants (n = 11/11). (C) Quantitative RT-PCR for irf8 expression in purified macrophages isolated from pu.1∆839;Tg(mpeg1:LRLG) and pu.1Δ839;spi-b∆232;Tg(mpeg1:LRLG) at 20 dpf. Expression level of target genes was normalized with elf1a expression. Error bars represent standard error (SE). (D-I) Fluorescence imaging of peripheral macrophages in the trunk and CHT regions of Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants at indicated developmental stages. (H′-I′) Magnified images of the boxed regions as indicated in panels H and I. (J) Quantification of tissue macrophages in the trunk and CHT regions (as shown in panels D-I) Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants at indicated developmental stages. ***P < .001, Student t test; mpeg1+ sib (3 dpf, mean/SE/n)= 14.3/15/0.7, mpeg1+irf8 mut (3 dpf, mean/SE/n) = 0.5/15/0.2, mpeg1+sib (8 dpf, mean/SE/n) = 33.3/10/1.3, mpeg1+irf8 mut (8 dpf, mean/SE/n) = 9.1/10/0.7, mpeg1+sib(15 dpf, mean/SE/n) = 90.2/10/1.4, mpeg1+irf8 mut (15 dpf, mean/SE/n) = 47.3/12/3.1). Error bars represent SE. (K-N) WISH of pu.1 and spi-b expression in 20-hpf siblings (pu.1, n = 40/40; spi-b, n = 38/38) and irf8∆171 mutants (pu.1, n = 15/15; spi-b, n = 12/12). (O) Quantitative RT-PCR for pu.1 and spi-b expression in purified macrophages isolated from wild-type Tg(mpeg1:LRLG) and irf8∆171;Tg(mpeg1:LRLG) at 20 dpf. Expression level of target genes was normalized with elf1a expression. Error bars represent SE. (P-Q) Costaining of Lcp1 antibody and mpeg1-DsRed on whole-mount brain sections of 15-dpf Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants. Dashed circles indicate the brain region. (R-S) Costaining of Lcp1 antibody and mpeg1-DsRed on brain sections of Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants at 30 dpf. White arrows in panel S indicate mpeg1-DsRed+/Lcp1+ microglia in irf8∆171 mutants. (T-U) Costaining of Lcp1 antibody and mpeg1-DsRed on the brain sections of adult (3-month-old) Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants. Blue and white arrows in panel U indicate branched microglia and rounded microglia-like cells in irf8∆171 mutants, respectively. Images are selected as representatives. (V) Quantification of Lcp1 and mpeg1-DsRed double-positive microglia in the midbrain of 3-month-old wild-type siblings and irf8∆171 mutants. **P < .01, Student t test; sib (mean/SE/n) = 201.7/15.8/4, irf8∆171 (mean/SE/n) = 21.4/2.2/3). Error bars represent SE.

The paralleled and vertical Pu.1−Spi-b cascades control macrophage development via a common downstream factor, Irf8. (A-B) WISH of irf8 expression in 20-hpf siblings (n = 35/35) and hypomorphic pu.1G242D mutants (n = 11/11). (C) Quantitative RT-PCR for irf8 expression in purified macrophages isolated from pu.1∆839;Tg(mpeg1:LRLG) and pu.1Δ839;spi-b∆232;Tg(mpeg1:LRLG) at 20 dpf. Expression level of target genes was normalized with elf1a expression. Error bars represent standard error (SE). (D-I) Fluorescence imaging of peripheral macrophages in the trunk and CHT regions of Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants at indicated developmental stages. (H′-I′) Magnified images of the boxed regions as indicated in panels H and I. (J) Quantification of tissue macrophages in the trunk and CHT regions (as shown in panels D-I) Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants at indicated developmental stages. ***P < .001, Student t test; mpeg1+sib (3 dpf, mean/SE/n)= 14.3/15/0.7, mpeg1+irf8 mut (3 dpf, mean/SE/n) = 0.5/15/0.2, mpeg1+sib (8 dpf, mean/SE/n) = 33.3/10/1.3, mpeg1+irf8 mut (8 dpf, mean/SE/n) = 9.1/10/0.7, mpeg1+sib(15 dpf, mean/SE/n) = 90.2/10/1.4, mpeg1+irf8 mut (15 dpf, mean/SE/n) = 47.3/12/3.1). Error bars represent SE. (K-N) WISH of pu.1 and spi-b expression in 20-hpf siblings (pu.1, n = 40/40; spi-b, n = 38/38) and irf8∆171 mutants (pu.1, n = 15/15; spi-b, n = 12/12). (O) Quantitative RT-PCR for pu.1 and spi-b expression in purified macrophages isolated from wild-type Tg(mpeg1:LRLG) and irf8∆171;Tg(mpeg1:LRLG) at 20 dpf. Expression level of target genes was normalized with elf1a expression. Error bars represent SE. (P-Q) Costaining of Lcp1 antibody and mpeg1-DsRed on whole-mount brain sections of 15-dpf Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants. Dashed circles indicate the brain region. (R-S) Costaining of Lcp1 antibody and mpeg1-DsRed on brain sections of Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants at 30 dpf. White arrows in panel S indicate mpeg1-DsRed+/Lcp1+ microglia in irf8∆171 mutants. (T-U) Costaining of Lcp1 antibody and mpeg1-DsRed on the brain sections of adult (3-month-old) Tg(mpeg1:LRLG) siblings and Tg(mpeg1:LRLG);irf8∆171 mutants. Blue and white arrows in panel U indicate branched microglia and rounded microglia-like cells in irf8∆171 mutants, respectively. Images are selected as representatives. (V) Quantification of Lcp1 and mpeg1-DsRed double-positive microglia in the midbrain of 3-month-old wild-type siblings and irf8∆171 mutants. **P < .01, Student t test; sib (mean/SE/n) = 201.7/15.8/4, irf8∆171 (mean/SE/n) = 21.4/2.2/3). Error bars represent SE.

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