mcm10 is expressed in the hemogenic endothelium (A) WISH for mcm10 at different stages of zebrafish embryonic development (24–36 hpf). (B) Double WISH for mcm10/runx1 at 26 hpf. (C) Experimental outline of qPCR analysis after dissection of heads, trunks, and tails from 26 hpf flk1:GFP transgenic animals (around 50 embryos), comparing with whole embryos. Data represent biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Scale bars: 200 μm (A), 100 μm (B).

The loss of mcm10 affects HSCs emergence

(A) WISH for runx1 expression at 28 hpf in mcm10^−/− embryos and their siblings and after mcm10 knockdown by morpholino injection.

(B) WISH for cmyb expression along the aorta at 36 hpf in control and mcm10-morphants.

(C) Statistical analysis was completed using Fisher’s exact test, ^∗∗∗∗p < 0.0001 (n = number of total embryos from three independent experiments).

(D) WISH for cmyb expression in the CHT at 48 hpf in control and mcm10 morphants.

(E) Statistical analysis was completed using unpaired two-tailed t test, ^∗∗∗p < 0.001 (n = number of total embryos from three independent experiments). Scale bars: 100 μm (A, B, and D).

mcm10 deficiency induces apoptosis in developing HSCs

(A) Fluorescence imaging of the dorsal aorta in flk1:mCherry;cmyb:GFP embryos injected with control or mcm10-MOs.

(B) Quantification of HSCs. Statistical analysis: unpaired two-tailed t test, ^∗∗∗p < 0.001 (n = number of total embryos from three independent experiments).

(C) Anti-GFP and TUNEL stainings of cmyb:GFP embryos at 32 hpf, after injection of either control or mcm10 morpholinos (mcm10-MO).

(D) Quantification of the number of GFP+ and TUNEL+ cells in control and mcm10-morphants. Center values denote the mean, error values denote SEM, and statistical analysis was completed using an unpaired two-tailed t test. ^∗∗∗p < 0.001 (n = number of total embryos from three independent experiments). Scale bars: 50 μm (A), 100 μm (C).

mcm10 overexpression increases the expression HSC marker

(A) runx1 expression in 28-hpf embryos, either non-injected or injected with mcm10 full-length mRNA.

(B) Statistical analysis was completed using Fisher’s exact test. ^∗∗∗p < 0.001 (n = number of total embryos from three independent experiments).

(C) WISH against cmyb at 36 hpf in embryos, either non-injected or injected with mcm10 full-length mRNA.

(D) Statistical analysis was completed using Fisher’s exact test. ^∗∗p < 0.01 (n = number of total embryos from three independent experiments).

(E) cmyb expression in 48-hpf embryos, either non-injected or injected with mcm10 full-length mRNA.

(F) Quantification of the number of cmyb-positive cells in the CHT per embryo. Statistical analysis was completed using an unpaired two-tailed t test. ^∗∗p < 0.01 (n = number of total embryos from three independent experiments).

(G) WISH against cmyb in 5-dpf embryos, either non-injected or injected with mcm10 full-length mRNA.

(H) Statistical analysis was completed using Fishers exact test. ^∗∗∗p < 0.001.

(I) WISH against rag1 at 5 dpf in embryos, either non-injected or injected with mcm10 full-length mRNA.

(J) The area of the thymus was measured for each embryo. Statistical analysis was completed using an unpaired two-tailed t test. ^∗∗p < 0.01 (n = number of total embryos from three independent experiments). Scale bars: 100 μm (A, C, E, G, and I).

Mcm10 overexpression increases the proliferation of emerging HSCs at the level of the hemogenic endothelium

(A) Fluorescence imaging of dorsal aorta in 32-hpf kdrl:mcherry/cmyb:GFP double-transgenic embryos, either non-injected or injected with mcm10 full-length mRNA.

(B) The number of double-positive cells was reported for each condition. Statistical analysis was completed using an unpaired two-tailed t test. ^∗∗∗p < 0.0001. Center values denote the mean, and error values denote SEM (n = number of total embryos from three independent experiments).

(C) An immunofluorescence against GFP and phospho-histone 3 (pH3) was performed on 32-hpf flk1:GFP transgenic embryos, either injected with control or mcm10 morpholinos, and with mcm10 full-length mRNA. The aorta region was imaged and (D) the number of double-positive cells (representing the proliferating endothelial cells) was scored for all embryos in each condition. Center values denote the mean, error values denote SEM, and statistical analysis was completed using ANOVA multiple t test. ^∗p < 0.01, ^∗∗p < 0.001 (n = number of total embryos from three independent experiments). Scale bar: 50 μm (A–C).

mcm10-deficient embryos accumulate DNA damages in nascent HSCs

(A) Western blot to quantify γH2A.X in control or mcm10 morphants.

(B) Statistical analysis of the ratio γH2A.X/actin was completed using an unpaired two-tailed t test. ^∗p < 0.01 (three independent experiments, with >30 embryos pooled per condition, per experiment).

(C) Anti-GFP and γH2A.X stainings performed on 32-hpf cmyb:GFP embryos, either injected with control or mcm10 morpholinos.

(D) Quantification of the number of double-positive cells in the aorta floor of control and mcm10 morphants. Center values denote the mean, error values denote SEM, and statistical analysis was completed using an unpaired two-tailed t test. ^∗∗∗p < 0.001 (n = number of total embryos from three independent experiments). Scale bar: 50 μm (C).

p53 knockdown rescues the loss of HSCs in mcm10-deficient embryos

(A) runx1 expression at 28 hpf in mcm10^−/− mutant embryos and their siblings, either injected with control or p53 morpholinos.

(B) runx1 expression at 28 hpf in embryos co-injected with mcm10 and p53 morpholinos.

(C) Statistical analysis was completed using Fisher’s exact test. ^∗p < 0.01; ^∗∗p < 0.001; ^∗∗∗p < 0.0001 (n = number of total embryos from three independent experiments). Scale bars: 200 μm (A), 100 μm (B).