Erythropoiesis in the Absence of Adult Hemoglobin

Abstract

During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucleated erythrocytes. Although Hb is one of the most extensively studied proteins, the role of Hb in erythroid lineage commitment, differentiation, and maturation remains unclear. In this study, we generate mouse embryos and embryonic stem (ES) cells with all of the adult α and β globin genes deleted (Hb Null). While Hb Null embryos die in midgestation, adult globin genes are not required for primitive or definitive erythroid lineage commitment. In vitro differentiation of Hb Null ES cells generates viable definitive proerythroblasts that undergo apoptosis upon terminal differentiation. Surprisingly, all stages of Hb Null-derived definitive erythroblasts develop normally in vivo in chimeric mice, and Hb Null erythroid cells undergo enucleation to form reticulocytes. Free heme toxicity is not observed in Hb Null-derived erythroblasts. Transplantation of Hb Null-derived bone marrow cells provides short-term radioprotection of lethally irradiated recipients, whose progressive anemia results in an erythroid hyperplasia composed entirely of Hb Null-derived erythroblasts. This novel experimental model system enables the role played by Hb in erythroid cell enucleation, cytoskeleton maturation, and heme and iron regulation to be studied.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01734-12.

ACKNOWLEDGMENTS

We thank Y. Huo for critical discussions, M. Alberti and D. Zhou for help with the EGFP-EKLF construct, C. Sun, K. Pawlik, D. Zhou, and L. Wu for recombineering help, J. Ren for microinjection training, J. Yu and L. Chow for mCherry-C1 plasmid, L. Gartland, E. Keyser, and M. Spell at the UAB Flow Cytometry Core, M. Chimento at the UAB High Resolution Imaging Facility, T. Schoeb at the UAB Comparative Pathology Laboratory, and T. Townes, L. Chow, and K. Popov for equipment use.

S.L. designed experiments, generated constructs and targeted ES cells, and generated all mice by microinjection, breeding, and transplantation, produced and analyzed data, and wrote the manuscript. S.C.M. assisted in mouse sample preparation and real-time PCR analyses. T.M.R. generated Hb Null ES cells, conceived, instructed, and supervised the project, and wrote the paper.

We declare no competing financial interests.

This work was supported by National Institutes of Health grants R01 HL072351 (T.M.R.), R01 HL073440 (T.M.R.), and T32 GM008111 (S.C.M.). Support was also provided by Cooley's Anemia Foundation, UNICO Foundation Inc., J. Ruisi and the BMG Thalassemia Action Group, and the Carmichael Scholarship (S.C.M.).