Mitochondrial Iron Transport Via MFRN1 Is Required for Erythroid Cell Cycle Progression Even Under Iron-Replete Conditions Sufficient for Hemoglobinization

Iron metabolism is a key driver of erythropoietic processes, including hemoglobinization, survival, proliferation, and morphological changes. How iron regulates development is poorly understood because mutations in iron metabolism genes are embryonic lethal in mammals. To determine how iron dysregulation disrupts erythropoiesis, we took advantage of the zebrafish, whose key developmental pathways are conserved with mammals. Zebrafish embryos develop externally, facilitating pharmacological, microscopic and in vivo labeling approaches. They acquire oxygen by passive diffusion, allowing survival of anemic mutants. However, small cell numbers preclude single embryo experiments, requiring investigators to sort and batch phenotypically similar animals for analysis. “Genotyping by phenotype” precludes understanding subtle heterozygote phenotypes that may be clinically relevant.

We developed methods to sort, image and genotype erythroid cells from single Tg(globin lcr:eGFP) reporter zebrafish. mfrn1 mutants have mitochondrial iron import defects, while fpn1 mutants have defective transport of yolk iron stores into the embryo, modeling whole body iron deficiency. Both mutants had decreased erythroid cell numbers and severe hemoglobinization and morphological defects. Notably, mfrn1 heterozygotes had a slight, but significant decrease in erythroid cell number, which was undetectable by visual phenotyping. To determine if these phenotypes were attributable to decreased iron, we treated the zebrafish with iron-hinokitiol, a lipophilic chelate which transports iron through lipid bilayers. Using rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester, a mitochondrial Fe²⁺ stain, we confirmed that iron-hinokitiol restored mitochondrial Fe²⁺ in the mutants to wild-type levels. Iron-hinokitiol restored hemoglobinization in both mutant lines. However, while it restored erythroid cell number in fpn1 mutants to wild-type levels, it did not fully restore erythroid cell count in mfrn1 mutants. Iron-hinokitiol did not fully rescue the morphological defects in both fpn1 and mfrn1 mutant erythrocytes.

To determine if decreases in erythroid cell number were caused by cell cycle defects, we stained zebrafish embryos with Dyecycle violet and EdU. Neither fpn1 nor mfrn1 mutant erythroid cells had increased cell death. However, mfrn1 mutant erythroid cells were arrested in G2/M, unlike fpn1 mutants, whose cell cycle status was similar to wild-type fish. These data indicated that the severe decrease in mfrn1 mutant erythroid cell number was caused by cell cycle arrest. Iron supplementation only slightly decreased the number of G2/M arrested mfrn1 erythroid cells.

As iron deficiency can promote megakaryocytic fate (at the expense of erythropoiesis) in hematopoietic progenitors, we quantitated hematopoietic lineages in mfrn1 mutants using scRNAseq, thrombocytes and erythroid progenitors using Tg(cd41:GFP) and Tg (gata1:dsRed) transgenic lines. We did not observe any change in the numbers of erythroid progenitors or thrombocytes indicating the phenotypes were not caused by lineage decision defects;. scRNAseq analysis suggested that differentiation defects in mfrn1 mutants were largely erythroid specific. mfrn1 mutant erythrocytes were arrested in the gata1a⁺ progenitor stage, in contrast to wild-type cells which had mostly lost gata1aexpression and expressed globin genes.

In summary, we have developed techniques that significantly improve the rigor and resolution of zebrafish experiments, providing fundamental insights into the regulation of erythropoiesis by iron transport. We demonstrate that mitochondrial iron transport through MFRN1 is required for cell proliferation during terminal erythropoiesis, even when mitochondrial iron is replete and sufficient for hemoglobinization. More broadly, our studies demonstrate that iron transporters are required developmental signaling, providing a potential mechanism for understanding the pathophysiology of iron refractory anemias.