Sorting, imaging, and genotyping of single 3-dpf zebrafish WT, weh/+, and weh/weh embryos demonstrate feasibility of sorting erythroid cells from single embryos; iron supplementation ameliorates the effects of FPN1 deficiency. (A) Sorting of GFP⁺ erythroid cells from single zebrafish embryos onto slides and followed by benzidine and Hoechst, or Giemsa staining. Both Hoechst and Giemsa staining revealed that weh/weh erythroid cells were heme deficient and smaller than WT cells. This phenotype was largely reversed by iron supplementation. (B) weh/weh mutant embryos did not have significantly decreased numbers of erythroid cells at 72 hours post fertilization (hpf). (C) Analysis of forward scatter (FSC) confirmed that weh/weh erythroid cells are significantly smaller than WT and weh/+. This abnormality was reversed by iron supplementation. (D) ImageJ analysis of benzidine stained cells indicated a significant decrease in heme staining in weh/weh erythroid cells. Iron supplementation (15 μM) was required for restoration of hemoglobinization levels to WT levels (supplemental Figure 6). (E) ImageJ analysis of Giemsa-stained cells revealed that weh/weh erythroid cells had smaller nuclei than WT, which was corrected by iron supplementation. (F) The ratio of cytoplasmic/nuclear area was decreased in weh/weh erythroid cells and these phenotypes could not be reversed by iron supplementation, and cell morphology remained qualitatively different from WT cells (see supplemental Figure 7). Scale bar, 5 μm. ∗∗∗ Indicates >95% significance by multiple comparisons with Tukey-Kramer correction.