Multiple Defects of Both Primitive and Definitive Erythrocytes in EKLF-Deficient Mice

• Published in: Blood 2007-11, Vol.110 (11), p.1234-1234
• Main Authors: Pilon, Andre M., Beaupre, Jacqueline, Bieker, James J., Gallagher, Patrick G., Bodine, David M.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V110.11.1234.1234

Erythroid Krüppel-Like Factor (EKLF) is the founding member of the mammalian Krüppel subfamily of transcription factors with 3 C2H2-type zinc fingers. In mice, loss of EKLF is lethal at day 14 of gestation (dE14), due to severe anemia. To study the physiological properties of the mixed population of circulating primitive and definitive erythrocytes in dE13.5 mice, we developed an osmotic fragility assay that compares the relative amount of embryonic (primitive) and adult (definitive) hemoglobin in the supernatant and pellet of cells exposed to increasing concentrations of NaCl. We found that both primitive and definitive EKLF-deficient cells were resistant to osmotic stress, consistent with the thalassemia-like phenotype of the cells. Flow cytometric analysis of WT dE13.5 fetal liver (FL) cells with TER119 and CD71 identified 5 previously described populations: R1+R2, composed of pro- and basophilic normoblasts, and R3+R4+R5, composed of more mature polychromatic and orthochromatic normoblasts and reticulocytes, respectively. Analysis of dE13.5 EKLF-deficient FL cells showed that R3+R4+R5 were absent, and morphological examination demonstrated that R1 and R2 were homogeneous populations of pro- and basophilic normoblasts. Colony-forming assays revealed an increase in the absolute number of BFU-E and CFU-E in EKLF-deficient R1 and R2 FL cells. Although the EKLF-deficient colonies required 24–72 more hours of culture than WT colonies to mature, they were composed of definitive erythroid cells as they expressed only adult β-globin. EKLF-deficient FL also contained 2-fold more CFU-Meg colonies (p