Erythropoiesis occurs first in the yolk sac as a transit “primitive” form, then is gradually replaced by the “definitive” form in the fetal liver (FL) during fetal development and in the bone marrow (BM) postnatally. While it is well known that differences exist between primitive and definitive erythropoiesis, the similarities and differences between FL and BM definitive erythropoiesis have not been studied. Here we performed comprehensive comparisons of erythroid progenitors and precursors at all maturational stages sorted from E16.5 FL and adult BM. We found that FL cells at all maturational stages were larger than their BM counterparts. We further found that FL BFU-E cells divided at a faster rate and underwent more cell divisions than BM BFU-E. Transcriptome comparison revealed that genes with increased expression in FL BFU-Es were enriched in cell division. Interestingly, the expression levels of glucocorticoid receptor Nr3c1, Myc and Myc downstream target Ccna2 were significantly higher in FL BFU-Es, indicating the role of the Nr3c1-Myc-Ccna2 axis in the enhanced proliferation/cell division of FL BFU-E cells. At the CFU-E stage, the expression of genes associated with hemoglobin biosynthesis were much higher in FL CFU-Es, indicating more hemoglobin production. During terminal erythropoiesis, overall temporal patterns in gene expression were conserved between the FL and BM. While biological processes related to translation, the tricarboxylic acid cycle and hypoxia response were upregulated in FL erythroblasts, those related to antiviral signal pathway were upregulated in BM erythroblasts. Our findings uncovered previously unrecognized differences between FL and BM definitive erythropoiesis and provide novel insights into erythropoiesis.

N ⁶ -methyladenosine (m ⁶ A) is a prevalent modification of mammalian mRNA. Increasing evidence has documented diverse roles of m ⁶ A in normal cell physiology and diseases. However, its functional role in erythropoiesis remains poorly understood. In this study, we found that deletion of Mettl3 using the EpoR-Cre mouse led to microcytic/hypochromic anemia due to defective erythropoiesis along with impaired hemoglobin biosynthesis. Mechanically, Mettl3 deficiency disrupted nucleotide biosynthesis, which induced DNA damage, leading to apoptosis of colony-forming unit–erythroid cells and cell-cycle arrest of erythroblasts. Integrated m ⁶ A-seq and RNA-seq analysis along with biochemical studies identified Mthfd1, a key enzyme involved in nucleotide biosynthesis, as a Mettl3 direct target gene. Furthermore, deletion of Mettl3 led to decreased expression of Mthfd1 , accompanied by a shortage of nucleotides deoxythymidine monophosphate and inosine monophosphate, in erythroid cells. Additionally, inhibition of METTL3 in human erythroid cells led to similar phenotypic and molecular changes, indicating a conserved role of METTL3 in human and murine erythropoiesis. Our findings have identified an METTL3-m ⁶ A- MTHFD1 axis that plays a critical role in erythropoiesis by maintaining genome stability of erythroid cells via regulation of nucleotide biosynthesis. These findings provide important insights into the regulatory mechanisms of erythropoiesis and may have implications for underlying the mechanisms of anemias.