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

aGata1 was analyzed by WISH in embryos at 6 somite injected with CoMO, cul4a-MO, cul4b-MO, cul4a/cul4b-MO, or cul4a-MO coinjected with zebrafish cul4a mRNA. b Relative gata1 expression was analyzed by qRT-PCR in embryos injected with CoMO, cul4a-MO, cul4b-MO, cul4a/cul4b-MO, or MO coinjected with cul4a mRNA, respectively. ccul4a-MO was injected into Tg (gata1: EGFP) transgenic embryos and decreased EGFP-positive cells were observed. Coinjection of cul4a mRNA rescued the reduction in gata1⁺ cells caused by cul4a MOs. d–f Effects of gata1 mRNA in rescuing hematopoietic defects in cul4a-morphant embryos as demonstrated by staining of o-dianisidine (d), WISH (e) and qRT-PCR (f) of hbbe3 probes. 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

aPu.1 was analyzed by WISH at 24 hpf in embryos injected with CoMO or cul4a-MO. b Relative expression of pu.1 mRNA was analyzed by qRT-PCR in embryos injected with CoMO or cul4a-MO. c, d Expression of pu.1 in control and cul4a^−/−, cul4b^−/−, and double knockout mutants was assayed by WISH (c) and by qRT-PCR (d). e, fMpo was analyzed by WISH (e) and qRT-PCR (f) at 24 hpf in embryos injected with CoMO or cul4a-MO. g–jrunx1 and c-myb expression were analyzed by WISH (g, i) or qRT-PCR (h, j) in embryos injected with CoMO or cul4a-MO. k, l Expression of runx1 in control and cul4a^−/−, cul4b^−/−, and double knockout embryos. The number in the top right-hand corner indicates the phenotypic embryos/total embryos. qRT-PCR experiments were performed in triplicate. All scale bars represent 250 μm

a, b WISH was performed with scl (a) or lmo2 (b) probes in embryos at 6 somite and 24 hpf, respectively. Embryos at 6-somite stage were in poster order view with anterior to the top. The ICM regions of embryos were lateral views with anterior to the left. c, d qRT-PCR analyzed the expression of scl and lmo2 in embryos injected with CoMO or cul4a-MO, or MO coinjected with zebrafish cul4a mRNA at 6 somite and 24 hpf. e, f The images of WISH with scl or lmo2 mRNA probes in control (cas9-tail injected), cul4a^−/−, cul4b^−/−, and double knockout mutants at 24 hpf. g, h Relative expression of scl and lmo2 mRNA were analyzed by qRT-PCR in control and cul4a^−/−, cul4b^−/−, and double knockout mutants. 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

a The relative expression of the two isoforms of scl, scl-α and scl-β, were measured by qRT-PCR in embryos injected with CoMO or cul4a-MO. bo-dianisidine staining showed depletion of erythroid cells in cul4a-morphant embryos compared with controls. Zebrafish scl-α, but not scl-β, mRNA rescued the reduction in primitive erythropoiesis of cul4a-morphant embryos. c, d WISH was performed with gata1 or hbbe3 probes in 24 hpf embryos injected with CoMO or cul4a-MO, or MO coinjected with zebrafish scl-α or scl-β mRNA. e, f Relative expression of gata1 or hbbe3 in embryos injected with CoMO or cul4a-MO, or MO coinjected with zebrafish scl-α or scl-β mRNA at 24 hpf. g WISH was performed with lmo2 probes in embryos at 6 somite and 24 hpf, respectively. Reduced expression of lmo2 in cul4a-morphant embryos were rescued by coinjection of zebrafish scl-α, but not scl-β mRNA. h Relative expression of lmo2 was measured by qRT-PCR in embryos injected with CoMO or cul4a-MO, or MO coinjected with zebrafish scl-α or scl-β mRNA at 6 somite and 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

a-f Expression of cul4a was analyzed via WISH at 1 cell (a), 6 hpf (b), 12 hpf (c and d), 24 hpf (e) and 48 hpf (f) stages with the cul4a antisense probe. Red arrowheads indicate PLPM and ICM regions. Lateral views are shown with anterior to the left (c, e, f), and dorsal views are shown with anterior to the top (d). g Diagram of the target site in the zebrafish cul4a and cul4b genome. h Representative sequences from control and cul4a or cul4b mutants. All scale bars represent 250 μm.