Fig. 7

A Frequency of CD41^lo HSPCs in Tg(−6.0itga2b:EGFP) embryos by flow cytometry at 72 hpf after treatment (Tx: 48–72 hpf) with NR (*P = 0.0448), Bafilomycin (*P = 0.0107), or both (P = 0.8705) vs control; NR vs both (*P = 0.0492) [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 12/condition, 8 clutches]. B Frequency of CD41^lo HSPCs in Tg(−6.0itga2b:EGFP) embryos by flow cytometry at 72 hpf after treatment (Tx: 48–72 hpf) with FCCP (**P = 0.0040), Bafilomycin (*P = 0.0273), or both (P = 0.2859) vs control; FCCP vs both (***P = 0.0008) [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 15/condition, 8 clutches]. C Frequency of CD41^lo HSPCs in Tg(−6.0itga2b:EGFP) embryos by flow cytometry at 72 hpf after treatment (Tx: 48–72 hpf) with NR (*P = 0.0154), bnip3lb-MO (*P = 0.0426), or both (P = 0.6133) vs control; NR vs both (P = 0.0581) [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 8/condition, 6 clutches]. D Representative WISH image of cmyb expression in the KM (black box) in control and NR-treated (Tx: 10 µM, 48–120 hpf) embryos at 120 hpf. Scale bar, 100 µM. E Phenotypic distribution of runx1/cmyb expression scored in embryos from (D) (**P = 0.0028) [Pearson’s chi-square (two-sided): n = 20/condition, 6 clutches]. F Frequency of CD41^lo HSPCs in Tg(−6.0itga2b:EGFP) embryos by flow cytometry at 120 hpf after NR treatment (Tx: 10 µM, 48–120 hpf; *P = 0.0341) [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 8/condition, 4 clutches]. G Frequency of Rag2+ lymphoid progenitor cells in Tg(rag2:GFP) embryos by flow cytometry at 120 hpf after NR treatment (Tx: 10 µM, 48–120 hpf; *P = 0.0165) [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 8/condition, 4 clutches]. H Schematic of Loh hiPSC hematopoietic differentiation protocol. CD34+ HSPCs utilized in serial-replating CFU assays. NR (1 mM) or FCCP (5 µM) treatment conducted at EHT stage from days 8 to 11. Created in BioRender. Walcheck, M. (2026) https://BioRender.com/kndj4o7. I Frequency of CD34+CD45+ iPSC-derived HSPCs on day 11 after treatment with NR (1 mM; *P = 0.0160) or FCCP (10 µM; P = 0.2881) during EHT [two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 3 technical replicates]. J CFU analysis NR or FCCP-treated iPSC-derived HSPCs showing stacked distribution of hematopoietic colony-types [E erythroid, G granulocyte, M macrophage, GM mixed granulocyte/macrophage, GEMM mixed granulocyte/erythroid/macrophage/megakaryocyte] scored after primary (left, day 14 post plating) and secondary (right) serial culture compared to control (Control vs NR *P = 0.0378; Control vs FCCP *P = 0.0117, two-tailed unpaired t test with Welch’s correction: mean ± SEM, n = 3 technical replicates). A–C, E–G, I, J Source data are provided as a Source Data file.