Figure 3.
Sorting, imaging, and genotyping of single 3-dpf zebrafish WT, frs/+, and frs/frs embryos demonstrate feasibility of sorting erythroid cells from single embryos; iron supplementation significantly ameliorates the effects of mfrn1 deficiency. (A) Sorting of GFP+ erythroid cells from single zebrafish embryos onto slides and followed by benzidine and Hoechst, or Giemsa staining. Genomic DNA was extracted from unsorted cells/debris and subjected to PCR with primers flanking the mutation region; the isolated PCR product was sequenced. Heterozygotes had overlapping nucleotide peaks at the mutation site. GFP+ WT cells were benzidine stained, indicating that sorted cells were from the erythroid lineage; iron treatment showed some evidence of toxicity (wrinkled morphology, decreased benzidine staining of individual cells). Both Hoechst and Giemsa staining revealed that frs/frs erythroid cells had very enlarged nuclei. This phenotype was largely reversed by iron supplementation. (B) Both frs/+ and frs/frs mutant embryos had a significantly decreased number of erythroid cells. Erythroid cell proportion in frs/frs were significantly increased by iron treatment. (C) FSC analysis indicates that frs/frs erythroid cells are significantly larger than WT and frs/+. This abnormality was largely reversed by iron supplementation. (D) ImageJ analysis of benzidine stained cells indicated a significant decrease in heme staining in both frs/+ and frs/frs erythroid cells (see supplemental Figure 3). Iron supplementation (15 μM) was required for restoration of hemoglobinization levels to WT. (E) ImageJ analysis of Giemsa-stained cells revealed that frs/+ and frs/frs erythroid cells had enlarged nuclei. These phenotypes could be reversed by iron supplementation. (F) The ratio of cytoplasmic/nuclear area was decreased in frs/+ and frs/frs erythroid cells and these phenotypes could be reversed by iron supplementation though cell shapes qualitatively remained more variable than WT cells (see supplemental Figure 4). ∗∗∗ Indicates >95% significance. FSC-A, forward scatter.

Sorting, imaging, and genotyping of single 3-dpf zebrafish WT, frs/+, and frs/frs embryos demonstrate feasibility of sorting erythroid cells from single embryos; iron supplementation significantly ameliorates the effects of mfrn1 deficiency. (A) Sorting of GFP+ erythroid cells from single zebrafish embryos onto slides and followed by benzidine and Hoechst, or Giemsa staining. Genomic DNA was extracted from unsorted cells/debris and subjected to PCR with primers flanking the mutation region; the isolated PCR product was sequenced. Heterozygotes had overlapping nucleotide peaks at the mutation site. GFP+ WT cells were benzidine stained, indicating that sorted cells were from the erythroid lineage; iron treatment showed some evidence of toxicity (wrinkled morphology, decreased benzidine staining of individual cells). Both Hoechst and Giemsa staining revealed that frs/frs erythroid cells had very enlarged nuclei. This phenotype was largely reversed by iron supplementation. (B) Both frs/+ and frs/frs mutant embryos had a significantly decreased number of erythroid cells. Erythroid cell proportion in frs/frs were significantly increased by iron treatment. (C) FSC analysis indicates that frs/frs erythroid cells are significantly larger than WT and frs/+. This abnormality was largely reversed by iron supplementation. (D) ImageJ analysis of benzidine stained cells indicated a significant decrease in heme staining in both frs/+ and frs/frs erythroid cells (see supplemental Figure 3). Iron supplementation (15 μM) was required for restoration of hemoglobinization levels to WT. (E) ImageJ analysis of Giemsa-stained cells revealed that frs/+ and frs/frs erythroid cells had enlarged nuclei. These phenotypes could be reversed by iron supplementation. (F) The ratio of cytoplasmic/nuclear area was decreased in frs/+ and frs/frs erythroid cells and these phenotypes could be reversed by iron supplementation though cell shapes qualitatively remained more variable than WT cells (see supplemental Figure 4). ∗∗∗ Indicates >95% significance. FSC-A, forward scatter.

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