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Irf2bp2b regulates zebrafish NMP cell fate choice
out and wildtype embryos. The overexpression of c/ebpα mRNA induced biased myelopoiesis toward neutrophils in control embryos (Figure 8E, F, I, J, M), but had no effect on myelopoiesis in irf2bp2b-/- embryos (Figure 8G, H, K- M).
Meanwhile, to elucidate whether gfi1 could also be a secondary determinant of C/ebpα, gfi1 mRNA was inject- ed into wildtype embryos. Although gfi1 overexpression did give rise to a remarkable expansion of the neutrophil population, the macrophage population was unaffected (data not shown). Moreover, this overexpression did not have any rescue effect in irf2bp2b-deficient embryos (Online Supplementary Figure S8E, F, I).
In summary, these data indicate that Irf2bp2b plays a pivotal role in mediating the antagonistic function of C/ebpα on pu.1 transcription regulation, which fine tunes the level of pu.1 expression in NMP and determines the
choice of NMP cell fate in order to maintain a normal neu- trophil and macrophage population ratio (Figure 8N).
Discussion
Although multiple regulators involved in hematopoietic lineage restriction have been characterized, the molecular details of NMP differentiation are still under debate. The relationship between the master regulators PU.1 and C/EBPα in myelopoiesis is complicated, being not only synergistic, but also antagonistic.41 On the one hand, C/EBPα can stimulate PU.1 expression by directly binding to its promoter.42,43 On the other hand, C/EBPα can interact directly with PU.1 and block its function, or inhibit PU.1 indirectly through activation of the transcription repressor GFI1,44,45 which in turn inhibits PU.1 activity through a
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Figure 8. Irf2bp2b mediates the antagonistic effect of C/ebpα on pu.1. (A) Schematic diagram of the zebrafish irf2bp2b promoter (-2.2 kb) (top panel). C/ebpα acti- vation on the irf2bp2b promoter was measured by a luciferase activity assay (bottom panel). Error bars represent the mean ± standard deviation (SD) of at least three replicates. ****P<0.0001 (Student t test). (B, C) Representative fluorescent images of transient mCherry expression at 20 hours post-fertilization (hpf) of wild- type (WT) and c/ebpα-overexpressing embryos injected with an irf2bp2b:mCherry construct (bottom panel). Corresponding bright field images (top panel). (D) Schematic diagram of the zebrafish irf2bp2b promoter (-2.2 kb), in which two putative C/ebpα binding sites are predicted (CS1, CS2) (top panel). Luciferase activity assays of C/ebpα activation on the irf2bp2b CS1 and CS2 mutant promoters (bottom panel). Data shown are the mean ± SD of at least three independent experi- ments. Error bars represent the mean ± SD of at least three replicates. ***P<0.001 (Student t test). (E-L) C/ebpα mRNA overexpression in WT and irf2bp2b-/- mutant embryos. Mpx and mfap4 probes were used in whole-mount in situ hybridization analysis to investigate any rescue effect. (M) Statistical results for E-L. Error bars represent the mean ± standard error of mean of 15-30 embryos. ns: not statistically significant; *P<0.1; **P<0.01; ***P<0.001 (analysis of variance followed by the least significant difference post-hoc test for multiple comparisons). (N) Schematic depiction of the regulation of neutrophil and macrophage fate in WT (left panel) and irf2bp2b-/- (right panel) zebrafish.
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