The erythroid phenotype of EKLF-null mice: defects in hemoglobin metabolism and membrane stability

Abstract

Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Kruppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in beta-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis.

FIG. 1.

Differentiation of primary erythroid progenitors. (A) Cell size distribution of wild-type and EKLF^−/− erythroblast cultures during differentiation. The size of wild-type cells decreases during differentiation and after 48 h enucleated cells appear (arrow). (B) Hemoglobin per cell volume was measured with intervals of 12 h in wild-type and EKLF^−/− cultures after induction of differentiation. (C) Morphological analysis of wild-type and EKLF^−/− erythroblasts during differentiation. Aliquots of the cultures were cytocentrifuged onto glass slides and stained with both cytological dyes and with neutral benzidine for hemoglobin (brownish stain). The absence of EKLF does not affect progenitors (T = 0). At T = 36 a decrease of cell size compared to cells at T = 0 is observed in the wild-type culture, but not in the EKLF^−/− culture. Only in the wild-type cultures, hemoglobin is detected, first at T = 54 (arrowheads), and these cells can complete the terminal differentiation to enucleated erythrocytes (black arrows). EKLF^−/− cells do not accumulate hemoglobin, and their nuclei become pycnotic (red arrows). Original magnification, ×100. T, time in hours; KO, knockout.

FIG. 2.

Expression of genes involved in heme synthesis in EKLF-deficient erythroid cells. (A) Expression of genes involved in heme synthesis was measured by real time PCR on cDNA of wild-type and EKLF KO cultures 36 h after induction of erythroid differentiation, and of fresh E12.5 fetal livers. Values were normalized to Hprt. The wild-type values (grey) were set to 100%, EKLF KO values are displayed in black. Error bars indicate the standard deviation. (B) Northern blot showing the expression of Alas2 in differentiating wild-type and EKLF KO cultures and in E12.5 fresh fetal livers of wild-type, heterozygous, and EKLF KO embryos. Gapdh was used as a loading control. T, time in hours; wt, wild-type; +/−, heterozygous for the EKLF knockout; KO, EKLF knockout. Abbreviations for the heme synthesis enzymes are given in the text.

FIG. 3.

Gene regulation by activating EKLF. EKLF null fetal liver cells carrying an EKLF-lbd transgene were cultured for 16 h. Gene expression was measured by real time quantitative PCR. The graph shows the difference in PCR cycles at which the PCR products reach the threshold level between T = 0 (fresh liver cells) and T = 16 (cultured cells). The medium of the cultured cells contained 4-OHT with (dark bars) or without (light bars) CHX. Expression of Hprt or Gapdh was used to normalize the data. Error bars indicate the standard deviation.

FIG. 4.

Ahsp expression in EKLF null erythroid cells. (A) Northern blot showing accumulation of Ahsp in wild-type cells during differentiation but not in EKLF^−/− cultures. Furthermore, Ahsp expression is EKLF-dependent in E12.5 fetal livers and in embryonic blood. Gapdh was used as a loading control. (B) Apparent inclusion bodies detected as blue spots (indicated by arrows) in EKLF^−/− embryonic blood cells in toluidine/methylene blue-stained semithin sections of fixed E12.5 yolk sacs. These blue spots were not detected in wild-type embryonic blood cells. Original magnification, ×100. Other details are as for Fig. 2.

FIG. 5.

Epb4.9 expression in EKLF null erythroid cells. (A) Western blot of whole cell protein extracts. Expression of Epb4.9 is detected in E12.5 fetal livers and in embryonic blood of wild-type embryos but not in EKLF^−/− embryos. Antibody against translation elongation factor eIF2α was used as a loading control. (B) Cytospins of primitive blood cells, stained with neutral benzidine and histological dyes, showing morphological abnormalities of EKLF KO cells. Membranes of wild-type blood cells appear smooth, and the cells are round shaped (arrowheads). The membranes of EKLF KO blood cells are wrinkled, and the cells are deformed (arrows). Other details are as for Fig. 2.

FIG. 6.

EKLF regulates genes necessary late in the erythroid differentiation program. The hematopoietic stem cell gives rise to all blood cell types, including the erythroid lineage. In the erythroid differentiation program, EKLF is dispensable for the expansion of erythroid progenitors. To complete the differentiation program, EKLF is required for the proper expression of genes involved in cell membrane stability (e.g., Epb4.9) and hemoglobin metabolism (e.g., β-globin, heme synthesis enzymes, and Ahsp), in order to enable appropriate terminal differentiation to erythrocytes.