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L. Wang et al.
cell-related markers runx1 and c-myb was relatively unchanged in irf2bp2b-deficient embryos (Online Supplementary Figure S3M-R, X). The erythroid marker hbαe1 (Online Supplementary Figure S3S-T), and the lym- phoid marker rag1 (Online Supplementary Figure S3U-V) were also unaffected.
As for myelopoiesis, a significant decrease in multiple neutrophil markers, including c/ebp1 (a marker of neutrophil progenitors)29 and mpx/lyz (a marker of mature neu- trophils),30 and a simultaneous increase of monocyte and macrophage markers such as csf1r (a monocyte/macrophage marker)30 and mfap4/mpeg1.1 (an early embryonic macrophage marker)31,32 were observed from 36 hours post fertilization (hpf) to 5 days post fertilization (dpf) in irf2bp2b-deficient mutants compared to controls (Figure 2A- I). The decreased neutrophil population was further con- firmed by Sudan black staining33 at 3 dpf in the ventral wall of the dorsal aorta (Figure 2J-J’, M), as well as in irf2bp2b-/- //Tg(mpx:eGFP) embryos at 5 dpf in caudal hematopoietic tissue (Figure 2K-K’, M). Similarly, an expanded macrophage population was found in irf2bp2b-/-//Tg(mpeg1.1:eGFP) embryos at 5 dpf (Figure 2L-L’, M). Flow cytometry analysis was performed to quantify the numbers of neutrophils and macrophages, and the results showed a 34.9% reduction of eGFP-positive cells in irf2bp2b-/-//Tg(mpx:eGFP) embryos and a 21.4% increase in irf2bp2b-/-//Tg(mpeg1.1:eGFP) embryos (Figure 2N-P). The irf2bp2b-/- zebrafish were not only viable but also fertile, which made the myelopoiesis study possible in adults. Morphological staining of the 3- month old adult zebrafish kidney marrow further con- firmed the expanded macrophages and reduced neutrophils (Figure 3A-C). Meanwhile, FACS analyses were also done with whole kidney marrow from Tg(mpx:eGFP) and irf2bp2b-/-//Tg(mpx:eGFP) lines in 3-month old adults. The myeloid cell populations were analyzed, and many fewer neutrophils were found in irf2bp2b-/-//Tg(mpx:eGFP) zebrafish than in controls (29.7% mpx+ vs. 84.0% mpx+) (Figure 3D, E).
An opposite phenotype emerged when irf2bp2b mRNA was injected into one-cell stage wildtype embryos (Figure 3F-H). It is worth noting that the overall numbers of cells positive for the pan-myeloid marker l-plastin30 (which is a marker of both neutrophils and macrophages), were com- parable among irf2bp2b-deficient mutants, irf2bp2b-over- expressing embryos and wildtype embryos (Figure 3I-L). In addition, embryos injected with a specific irf2bp2b mor- pholino (MO) exactly phenocopied the aberrant myelopoiesis that occurs in irf2bp2b knockout embryos (Online Supplementary Figure S4A-D, I).
All of the abnormalities in irf2bp2b-deficient and mor- phant embryos could be effectively rescued with the wildtype zebrafish irf2bp2b mRNA, confirming the speci- ficity of the phenotype (Online Supplementary Figure S4E- F, I). It should be noted that zebrafish irf2bp2a mRNA did not rescue the defects of myelopoiesis, indicating that the two paralogs might have distinct roles (data not shown). Accordingly, loss of irf2bp2a resulted in a quite different phenotype in zebrafish myelopoiesis, which could not be rescued by irf2bp2b mRNA, either (experiments ongoing). Moreover, human IRF2BP2b mRNA, but not IRF2BP2a mRNA, could rescue the biased myelopoiesis in zebrafish irf2bp2b-deficient mutants, suggesting that human IRF2BP2b is the functional ortholog of zebrafish irf2bp2b in this process (Online Supplementary Figure S4G-H, I and data not shown).
Irf2bp2b regulates neutrophil-macrophage progenitor fate by repressing pu.1 expression
The imbalanced proportion of neutrophil and macrophage populations in irf2bp2b-defective mutants can result from either abnormalities in apoptosis or prolifera- tion rate. To distinguish between these possibilities, termi- nal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and antiphosphohistone H3 (pH3) antibody staining assays were performed to assess the apoptosis and proliferation status of neutrophils and macrophages, respectively. Neither TUNEL nor pH3 assays revealed dis- cernable differences in the percentages of double-positive stained cells (TUNEL+GFP+, pH3+GFP+) in irf2bp2b-/- //Tg(mpx:eGFP) and irf2bp2b-/-//Tg(mpeg1:eGFP) embryos compared to the percentage in controls, indicating that there is no change in the status of either apoptosis or pro- liferation of each lineage in irf2bp2b-deficient embryos (Online Supplementary Figure S5). Moreover, the fact that l- plastin-positive cell numbers remained unchanged in both irf2bp2b-overexpressing and -deficient embryos suggest that irf2bp2b might participate in regulating neutrophil ver- sus macrophage commitment.
The relative levels of the master regulators PU.1 and C/EBPα are critical in macrophage versus neutrophil cell fate specification.6 To ensure balanced commitment of NMP, the endogenous levels of PU.1 and C/EBPα must be appropriately tuned to a proper range. Overexpression of PU.1 can bias myeloid output to macrophages, whereas overexpression of C/EBPα has an opposite effect. Thus either pu.1 upregulation or c/ebpα downregulation within NMP could be the cause of biased myelopoiesis toward macrophages in irf2bp2b mutants. We tried to examine the expression levels of pu.1 and c/ebpα by WISH analysis. No obvious difference was observed between the wildtype and irf2bp2b-/- embryos. However, considering that Pu.1 is expressed in multiple hematopoietic cell lineages, such as hematopoietic stem cells, common lymphoid progenitors, and common myeloid progenitors,34 and C/ebpα is also widely expressed in hematopoietic stem cells and myeloid cells,35 changes in their levels of expression within NMP might be difficult to show. Due to the lack of a lineage cell detection cocktail for the zebrafish hematopoietic system, we were unable to isolate the NMP subpopulation by flow cytometry to compare the endogenous expression levels of pu.1and c/ebpα. RT-qPCR was performed to detect the expression of c/ebpα and pu.1 in wildtype and irf2bp2b-deficient whole embryos, and no obvious changes were observed (Online Supplementary Figure S6), suggesting that changes occurring in NMP might be masked. To resolve this problem, we used a murine myeloid progenitor cell line 32Dcl3 retrovirally transduced with human IRF2BP2b. RT-qPCR analyses revealed that the transcript level of Pu.1 was downregulated, whereas that of C/ebpα was unaffected (Figure 4A). Meanwhile, expression of multiple monocyte differentiation-related genes such as Mcsfr, Mmp1, Tlr2, and Irf8 was reduced, whereas expression of neutrophil differentiation-related genes, including Gcsfr, Ltf, Prtn3, and Elane, was induced (Figure 4A). These observations imply that an alteration of pu.1 expression, rather than that of c/ebpα, might account for the shift in the balance of neutrophil and macrophage populations in irf2bp2b-deficient zebrafish embryos. Since IRF2BP2 is a negative transcription regulator, we won- dered whether pu.1 is a direct target of Irf2bp2b, which could be upregulated in irf2bp2b-deficient NMP. To test
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haematologica | 2020; 105(2)