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L. Wang et al.
this hypothesis, we divided the 8.5 kb zebrafish pu.1 pro- moter into four fragments, which were inserted separately into a luciferase reporter vector.13 The luciferase expres- sion in all of these four constructs was inhibited when co- transfected with irf2bp2b in HEK293T cells. The most prominent repression was found within the fragment nearest to the transcription start site (-1.7 kb) (Figure 4B). Next, a series of in vivo experiments was performed. The 8.5 kb pu.1 promoter was cloned into a mCherry reporter vector (pu.1:mCherry, Tol2 backbone), which was co- injected with Tol2 transposase mRNA into wildtype
zebrafish embryos with or without irf2bp2b mRNA. Overexpression of irf2bp2b led to significantly reduced expression of mCherry (Figure 4C-D). Moreover, pu.1 MO was injected into irf2bp2b-/- embryos, and effective rescue of aberrant myelopoiesis was obtained (Figure 4E-J, O). These observations suggest that the level of pu.1 expres- sion might be elevated in NMP in irf2bp2b mutants. To fur- ther demonstrate that Irf2bp2b regulates zebrafish NMP cell fate choice through repression of pu.1, we took advan- tage of a zebrafish pu.1G242D mutant line, in which the level of pu.1 transcripts is normal but its protein stability is dra-
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Figure 3. Biased myelopoiesis in irf2bp2b-deficient adult zebrafish. (A, B) Hematoxylin & eosin staining for morphological analysis of the kidney marrow collected from 3-month old adult wildtype (1 male and 1 female) and irf2bp2b-/- (2 males and 1 female) zebrafish. One representative image of each group is shown. Green and red arrows indicate typical neutrophils and macrophages, respectively. (C) Statistical results for A, B. Error bars represent the mean ± standard deviation (SD) of at least 15 images. ***P<0.001 (Student t test). (D) FACS analysis of whole kidney marrows from Tg(mpx:eGFP) and irf2bp2b-/-//Tg(mpx:eGFP) lines in 3-month old adults. The myeloid cells in the R5 gate were analyzed with fluorescence. (E) Statistical results for D. Error bars represent the mean ± SD of three replicates. ****P<0.0001 (Student t test). (F-G’) Whole-mount in situ hybridization (WISH) using mpx and mfap4 to monitor neutrophil and macrophage development in embryos injected with wildtype or irf2bp2b mRNA. (I-K) WISH analyses of the pan-myeloid marker l-plastin in embryos injected with wildtype, irf2bp2b-/-, and irf2bp2b mRNA embryos. (H, L) Statistical results for F-G’, I-K. Error bars represent the mean ± SD of at least 15-30 embryos. ns: not statistically significant; **P<0.01; ***P<0.001 (Student t test).
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haematologica | 2020; 105(2)