Fig. 3.

Blood IOL phenotype is caused by heme-deficient RBCs via Slc40a1. (A) Workflow of blood iron measurement in control, alas2^−/− and alad^−/− at 36 hpf by ICP-MS or iron colorimetric assay kit. (B,C) Quantification of the blood iron content by ICP-MS (B) and iron colorimetric assay kit (C). n=5 experimental replicates. (D) Schematic of bulk RNA-seq of flow cytometry-sorted gata1⁺ RBCs in alas2 and alad morphants at 36 hpf on Illumina NovaSeq 6000 platform. (E,F) GO analysis showing the enrichment of upregulated (E) and downregulated (F) overlapping terms in heme-deficient RBCs of alas2 and alad morphants. (G) Heatmap analysis of selected overlapping DEGs in iron transportation-related GO terms. The iron exporter Slc40a1 is highlighted with red dashed rectangle. (H) The expression of slc40a1 is detected by WISH in control, alas2^−/− and alad^−/− at 36 hpf. The RBC-enriched yolk regions are denoted by red arrowheads. (I) Statistical analysis of the relative slc40a1 expression in H by ImageJ. n (embryos)=10. (J) Relative mRNA level of slc40a1 in the flow cytometric-sorted RBCs (gata1⁺) of control, alas2^−/− and alad^−/− at 36 hpf examined by qRT-PCR. n=3 experimental replicates. (K) Expression of HSPC marker gene runx1 in control, alas2^−/− and alad^−/− with or without VIT2763 treatment at 36 hpf examined by WISH. The AGM regions for marker gene-positive cell counting are denoted by red arrowheads. (L) Quantification of the runx1-positive HSPCs in K. n=3 experimental replicates. Number of samples are indicated. Data are mean±s.d. ***P<0.001 (one-way ANOVA, Tukey's multiple comparisons). n.s., not significant. Scale bars: 250 μm (H); 100 μm (K).