Mfrn1 is primarily required for erythroid cell maturation; neither mfrn1 (frs) nor fpn1 (weh) mutants have significant increases in the thrombocytic lineage that indicate lineage shifts in the MEP population. (A-B) scRNA-seq analysis indicates that the primary phenotype of mfrn1 deficiency is in formation of the mature erythroid lineage, which is gata1a⁻/globin⁺. There is also a significant defect in the development of gata1a⁺ erythroid cells in frs/frs mutants. (C) frs/frs mutants had normal proportions of gata1a⁺ cells at 24 hpf, indicating normal formation of erythroid precursors. (D) Vehicle-treated frs/frs mutants had a significant decrease in the proportion of gata1a⁺ cells at 72 hpf that was rescued to WT levels with 15 μM Fe-hinokitiol. (E-F) frs/frs mutant embryos have a significantly higher proportion of gata1a⁺:lcr^-−cells and lower proportion of gata1a⁺:lcr⁺ cells at 72 hpf that is rescued to WT levels with 15 μM Fe-hinokitiol. (G) At 96 hpf, there was no significant difference in the proportion of CD41⁺ cells between vehicle-treated WT, frs/+, or frs/frs embryos. (H) frs and frs/frs mutants did not experience alterations in the development of cd41^low cells, indicating that development of the adult hematopoietic stem cell population was unaltered. (I) frs and frs/frs mutants did not experience alterations in the development of CD41^high cells, indicating that development of the thrombocyte population was unaltered. (J) At 96 hpf, FPN1 mutation status or iron treatment did not alter percentages of total CD41⁺ cells. (K) fpn1 mutants did not experience alterations in the development of CD41^low cells, indicating that development of the adult hematopoietic stem cell population was unaltered. (L) fpn1 mutants did not experience alterations in the development of CD41^high cells, indicating that development of the thrombocyte population was unaltered. ∗∗P < .01; ∗∗∗P < .001. mut, mutant; UMAP, uniform manifold approximation and projection.