hlx1 regulates hematopoietic stem cell formation and myeloid cell maturation in zebrafish. a–b Whole-mount in situ hybridization (WISH) for runx1 (a) and rag1 (b) in control or fli:hHLXOE zebrafish embryos at 36 or 96 hpf, respectively. Arrows indicate HSPCs. Numbers in the bottom right corner of panels indicate the number of zebrafish embryos with the indicated phenotype compared to the total number of zebrafish analyzed. Quantification of WISH was performed using FIJI software and statistical significance of three independent experiments in 12 zebrafish embryos was evaluated by Student’s t-test, *P < 0.05, **P < 0.01 (mean + s.d.). c WISH for the early myeloid marker pu.1 in control or fli:hHLXOE zebrafish embryos at 48 hpf. Numbers and WISH quantification was performed as described above (n = 3, in total 12 fish, Student’s t-test, *P < 0.05, mean + s.d.). d Zebrafish caudal hematopoietic tissue (CHT) smears stained with May–Grünwald–Giemsa stain in control or fli:hHLXOE embryos at 48 hpf. On the right, cell number counts of the indicated cell populations from 10 fish (n = 3, mean + s.d., Student’s t-test, *P < 0.05). e EDU assay at 48 hpf in control, hlx1 morphants (hlx1MO) or fli:hHLXOE zebrafish cells (n = 2). f Zebrafish CHT smears stained with May–Grünwald–Giemsa stain in control or Runx:hHLXOE embryos at 5 dpf. On the right, cell number counts of the indicated cell populations from 10 fishes, in two independent experiments (mean + s.d., Student’s t-test, **P < 0.01, ****P < 0.0001). g WISH for runx1 and rag1 in control or hlx1MO embryos at 36 and 96 hpf, respectively. h Representative images of Tg(Runx:mCherry) embryos at 72 hpf, injected or not with hlx1 morpholino and the indicated amounts of fli:hHLX construct. Numbers of mCherry-positive HSPCs from each embryo are represented in the graph (n = 10; mean + s.d., Student’s t-test, *P < 0.05, ***P < 0.001)
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