All ETDs from UAB

Advisory Committee Chair

Thomas M Ryan

Advisory Committee Members

Christopher A Klug

Anupam Agarwal

Ching-Yi Chen

Tim M Townes

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The mammalian erythrocyte is a highly specialized blood cell that differentiates via an orderly series of committed progenitors in the bone marrow in a process termed erythropoiesis. During erythroid development, hemoglobin synthesis increases from early erythroid progenitors to mature enucleated red blood cells (RBCs). Although hemoglobin is the most extensively studied protein in history, the role, if any, that hemoglobin plays in erythroid development remains obscure. In this study, I ask the question what happens during erythropoiesis in the absence of hemoglobin. I demonstrate that my original hypothesis that excess free heme would accumulate in the absence of globin chain production resulting in the early death of erythroid progenitors is incorrect. To test this hypothesis, mouse embryos and embryonic stem (ES) cells that have all their adult &alpha and &beta globin genes deleted (Hb Null) are generated and the Hb Null ES cells are used to produce Hb Null chimeric mice. I demonstrate that Hb Null embryos are extremely anemic, have a defect in their primitive erythropoiesis, and die in midgestation. Surprisingly, in Hb Null chimeric mice all stages of nucleated definitive erythroid development is normal, and Hb Null erythroid cells can even enucleate and form reticulocytes. Furthermore, the level of total heme in Hb Null erythoblasts is dramatically reduced. In chimeric mice the number of Hb Null reticulocytes is reduced more than 90%. Expression of the heme binding protein, human myoglobin, in Hb Null erythroid cells does not prevent reticulocyte loss. Bone marrow transplantation of donor Hb Null derived hematopoietic cells into lethally irradiated wild type mice results in reconstituted mice with pure Hb Null derived erythropoietic tissues and up to 1% Hb Null reticulocytes in the peripheral blood. These data suggest that hemoglobin reduction in erythroid cells unlikely causes free heme level elevation. This novel Hb Null model provides a unique experimental system to test future hypotheses on the role played by hemoglobin in erythroid cell enucleation, cytoskeleton maturation, and heme and iron regulation.

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