bloc1s2 deficiency promotes HSPC production in zebrafish.
(A-C) The results of (A) whole mount in situ hybridization (WISH), (B) qRT-PCR and (C) immunoblotting (Figure 5—source data 1) showed that the expression of bloc1s2 at the RNA and protein levels in bloc1s2 mutants was decreased but not abolished compared to that in control embryos at 24 hpf. Each bar represents the mean ± s.e.m of three independent experiments. **p<0.01, ***p<0.001 (Student’s t-test). (D) The Runx1 protein level was increased in bloc1s2 mutants (Figure 5—source data 2). The right panel is the quantitative analysis of the western blotting results. Each bar represents the mean ± s.e.m of three independent experiments. ***p<0.001. (E) The WISH results showed that the expression of runx1 at 24 hpf and 36 hpf, and cmyb expression at 36 hpf were increased in bloc1s2 mutants. Black arrowheads mark expression of runx1 and cmyb in the AGM region. (F) qRT-PCR results from the dissected trunk region showed that expression of cmyb at 36 hpf was significantly increased in bloc1s2 mutants. Each bar represents the mean ± s.e.m of three independent experiments. *p<0.05. (G) We generated the bloc1s2 mutants in a Tg (flk1:mcherry;cmyb:GFP) background. The upper panels show that the number of flk1+cmyb+ cells in outcrossed embryos was increased. White arrowheads mark flk1+cmyb+ cells in the AGM region at 36 hpf. The lower panel is the quantitative analysis of the western blotting results. Each bar represents the mean ± s.e.m of seven embryos. **p<0.01. (H) rag1 expression was increased in the thymus region, but gata1 and pu1 expression was decreased in the caudal hematopoietic tissue (CHT) region in bloc1s2 mutants at 4.5 dpf. Black dashed circles mark rag1 expression in the thymus region. Black arrowheads mark expression of gata1 and pu1 in the CHT region.