Figure 4

Cu overload destroys embryonic HSPC cytoskeleton

(A) Schema for the experiments in (B) and (C).

(B) Enriched GO terms for down-regulated DEGs in Cu-stressed HSPCs at 33 hpf.

(C) Cytoskeleton protein immune-fluorescence for Cu-stressed HSPCs at 33 hpf. C1, C4, anti-β-tubulin staining; C2, C5, DAPI staining; C3, C6, merged; C7, the percentage of the Cu-stressed runx1GFP⁺ cells exhibiting destructive cytoskeleton; C8, calculation of the fluorescence intensity in each cell.

(D) Mitotic malformation of HSPCs in the cytoskeleton gene morphants (tuba1a-MO, stmn4-MO, tubb5-MO, and tmsb2-MO together) at the 33 hpf (AGM) and 72 hpf (CHT), respectively. D1, D5, D10, D14, DAPI staining; D2, D6, D11, D15, anti-α-tubulin staining; D3, D7, D12, D16, anti-GFP staining; D4, D8, D13, D17, merged. At least 10 mitotic HSPCs in more than 10 embryos were observed for each group. D9, D18, calculation of the length of spindles in metaphase runx1GFP⁺ cells at 33 hpf and 72 hpf, respectively.

(E) The protein level of Tuba1a (E1) and Stmn4 (E3) in tuba1a mutants and stmn4 mutants at 33 hpf, respectively. GAPDH was used as an internal control.

(F) Phenotypes of tuba1a (F2, F5) mutants with a 4-bp deletion and stmn4 (F3, F6) mutants with an 8-bp deletion at 33 hpf and 120 hpf, respectively.

(G) Expression of runx1 and cmyb in tuba1a mutants with or without Cu stresses at 72 hpf. G9, calculation of runx1 and cmyb expression in embryos from different groups.

(H) Expression of runx1 and cmyb in stmn4 mutants with or without Cu stresses at 72 hpf. H9, calculation of runx1 and cmyb expression in embryos from different groups. F1-F6, G1-G8, H1-H8, lateral view, anterior to the left, and dorsal to the up. Data are mean ± SD. t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. NS, not significant. Scale bars, 1.2 μm (C1-C6), 2 μm (D1-D8, D10-D17), 100 μm (D1-D3, F1-F6), and 200 μm (G1-G8, H1-H8).