Fig. 5

Fig. 5

a Expression of runx1 in HE at 32hpf in wild-type (wt), gata2b MO-injected (7.5 ng) wt embryos, gata2a^Δi4/Δi4 mutants and gata2b MO-injected (7.5 ng) gata2a^Δi4/Δi4 mutants. b Quantification of the runx1 in situ hybridization (ISH) signal in wt, gata2b morphants, gata2a^Δi4/Δi4 mutants and gata2a^Δi4/Δi4 mutants injected with gata2b MO. runx1 expression is decreased in gata2a^Δi4/Δi4 mutants (µ_wt = 41.9, µ_mut = 28.9; F = 44.641, d.f. = 3, 62.3; p < 0.001). Gata2b MO knockdown significantly decreases runx1 in the DA of gata2a^Δi4/Δi4mutants (µ_mut = 28.9, µ_mut+MO = 17.2), but not wt embryos at 32hpf (µ_wt = 41.9, µ_MO = 40.1; p = 0.89, p = 0.89, Games-Howell post-hoc test, Welch’s ANOVA). n = 27, wt; n = 27, gata2a^Δi4/Δi4; n = 33, wt + gata2b MO; n = 32, gata2a^Δi4/Δi4 + gata2b MO. c Scoring cmyb expression at 4dpf in wt, gata2a^Δi4/Δi4 mutants and gata2a^Δi4/Δi4 mutant embryos injected with gata2b MO as wt (blue) or reduced (red). Gata2b MO knockdown (7.5 ng) inhibits the haematopoietic recovery of gata2a^Δi4/Δi4 mutants. (Χ² = 18.784, d.f. = 2, p < 0.001). d Quantification of the runx1 ISH signal, from 28hpf wt embryos (blue), gata2a^Δi4/Δi4 mutants (red) and their siblings injected with a gata2a-i4-450bp:gata2b construct (shaded blue and red). Ectopic expression of gata2b increases runx1 expression in the HE of wt embryos (µ_wt = 38.8, µ_wt+gata2b = 53.4; p < 0.01) and rescues runx1 expression in the DA of gata2a^Δi4/Δi4 mutants to wt levels (µ_mut = 17.9, µ_mut+gata2b = 33.2; p < 0.001; µ_wt = 38.8, µ_mut+gata2b = 33.2; p = 0.31, Tukey HSD post-hoc test). n = 25, wt; n = 33, gata2a^Δi4/Δi4;n = 18, wt + gata2a-i4-450bp:gata2b construct; n = 17, gata2a^Δi4/Δi4 + gata2a-i4-450bp:gata2b construct. e Quantification of the runx1 ISH signal at 36hpf in embryos treated with a suboptimal dose (25 μM) of the Notch inhibitor DAPM. 25 μM DAPM showed no effect on runx1 expression in wt compared to DMSO-treated embryos (µ_DMSO = 40.5, µ_DAPM = 38; p = 0.735, Tukey HSD post-hoc test). DMSO-treated gata2a^Δi4/Δi4 mutants show a decrease in runx1 expression (µ_DMSO = 40.5, µ_mut+DMSO = 31.5; F = 25.774, d.f. = 3, 91; ANOVA). DAPM treatment significantly reduced runx1 expression in the DA gata2a^Δi4/Δi4 mutants (µ_mut+DMSO = 31.5, µ_mut+DAPM = 19.4). n = 27, wt +DMSO; n = 20, gata2a^Δi4/Δi4 + DMSO; n = 30, wt + DAPM; n = 20, gata2a^Δi4/Δi4 + DAPM. f Representative images of the average runx1 expression at 36hpf in wt and gata2a^Δi4/Δi4 mutants treated with 25 μM DAPM. Error bars: mean ± SD. **p < 0.01; ***p < 0.001. See also Supplementary Fig. 5.