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Deletion of the i4 enhancer in <italic>gata2a</italic><sup>Δi4/Δi4</sup> mutants leads to reduced levels of <italic>gata2a</italic> mRNA in the endothelium.a, b A significant majority of gata2aΔi4/Δi4 mutants have reduced levels of gata2a mRNA in the dorsal aorta (arrows) at 28hpf, compared to wild-type siblings, as detected with in situ hybridization. (Χ2 = 10.720, d.f. = 1, p < 0.01). The expression in the neural tube appears unaffected. c, d In situ hybridization for the endothelial marker kdrl at 28hpf reveals no difference between gata2aΔi4/Δi4 mutants and wild-type siblings. The dorsal aorta (arrows) appears unaffected. e, f Live images of the trunks of 48hpf Tg(kdrl:GFP) and Tg(kdrl:GFP); gata2aΔi4/Δi4 embryos show normal vascular morphology in the mutants. The endothelium of the dorsal aorta (arrows) appears normal in the gata2aΔi4/Δi4 embryos. gKdrl:GFPhigh and kdrl:GFP− cells were sorted from non-mutant (WT, blue) and gata2aΔi4/Δi4 (red) embryos carrying the Tg(kdrl:GFP) transgene. h, i qRT-PCR on RNA isolated from the sorted kdrl:GFPhigh or kdrl:GFP- cells (panel g) shows decreased levels of gata2a mRNA in the endothelium of gata2aΔi4/Δi4 mutants at 23hpf (t = 20.026,d.f. = 5, p < 0.001) compared to wild-type. At 30hpf this difference is not statistically significant (t = 2.146, d.f. = 4, p = 0.098). There is no difference in gata2a mRNA levels in non-endothelial cells between wild-type and gata2aΔi4/Δi4 mutants (23hpf: t = 0.69, d.f. = 5, p > 0.5; 30hpf: t = 0.618, d.f. = 4, p > 0.5). N = 4 for gata2aΔi4/Δi4 at 23hpf, N = 3 for other samples. Note different scales of expression levels. ***p < 0.001. See also Supplementary Fig. 2.
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