Knockdown of cul4a in zebrafish significantly reduces the number of erythrocytes.ao-dianisidine staining showed depletion of erythrocytes in cul4a-morphant embryos, compared with controls. Zebrafish cul4a mRNA rescued primitive erythropoiesis in cul4a-morphant embryos. b Expression of the embryonic hemoglobin, hbbe3, was analyzed by WISH at 24 hpf in embryos injected with CoMO or cul4a-MO, or MO coinjected with zebrafish cul4a mRNA. Lateral views are shown with anterior to the left. c Relative mRNA level of hbbe3 was assayed by qRT-PCR in embryos injected with CoMO or cul4a-MO, or MO coinjected with cul4a mRNA at 24 hpf. d WISH was performed with hbbe3 probes in embryos at 24 hpf injected with CoMO or cul4a-MO1. e Relative mRNA level of hbbe3 was measured by qRT-PCR in embryos injected with CoMO or cul4a-MO1. fo-dianisidine staining showed decreased erythrocytes in cul4a−/− and double knockout, but not in cul4b−/− knockout embryos. Embryos shown are lateral views with anterior to the left. g The images of WISH with hbbe3 mRNA probes in control (cas9-tail injected), cul4a−/−, cul4b−/−, and double knockout mutants at 24 hpf. h Relative mRNA level of hbbe3 was assayed by qRT-PCR in control (cas9-tail injected), cul4a−/−, cul4b−/−, and double knockout mutants at 24 hpf. The number in the top right-hand corner indicates the phenotypic embryos/total embryos. qRT-PCR experiments were performed in triplicate. ***p < 0.001. All scale bars represent 250 μm
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