EKLF and KLF2 have compensatory roles in embryonic beta-globin gene expression and primitive erythropoiesis

Abstract

The Krüppel-like C2/H2 zinc finger transcription factors (KLFs) control development and differentiation. Erythroid Krüppel-like factor (EKLF or KLF1) regulates adult beta-globin gene expression and is necessary for normal definitive erythropoiesis. KLF2 is required for normal embryonic Ey- and betah1-, but not adult betaglobin, gene expression in mice. Both EKLF and KLF2 play roles in primitive erythroid cell development. To investigate potential interactions between these genes, EKLF/KLF2 double-mutant embryos were analyzed. EKLF(-/-)KLF2(-/-) mice appear anemic at embryonic day 10.5 (E10.5) and die before E11.5, whereas single-knockout EKLF(-/-) or KLF2(-/-) embryos are grossly normal at E10.5 and die later than EKLF(-/-)KLF2(-/-) embryos. At E10.5, Ey- and betah1-globin mRNA is greatly reduced in EKLF(-/-)KLF2(-/-), compared with EKLF(-/-) or KLF2(-/-) embryos, consistent with the observed anemia. Light and electron microscopic analyses of E9.5 EKLF(-/-)KLF2(-/-) yolk sacs, and cytospins, indicate that erythroid and endothelial cells are morphologically more abnormal than in either single knockout. EKLF(-/-)KLF2(-/-) erythroid cells are markedly irregularly shaped, suggesting membrane abnormalities. EKLF and KLF2 may have coordinate roles in a common progenitor to erythroid and endothelial cells. The data indicate that EKLF and KLF2 have redundant functions in embryonic beta-like globin gene expression, primitive erythropoiesis, and endothelial development.

Figure 1

E10.5 wild-type and EKLF^−/−KLF2^−/− whole-mount embryos. Panels A and B are wild-type (WT), and panels C and D are EKLF^−/−KLF2^−/− E10.5 embryos. In panels A and C, embryos are surrounded by yolk sacs. Panels B and D are the same embryos, respectively, with yolk sac teased away, but vitelline and umbilical vessels still intact. Photographs were taken at 16× magnification. See “Analysis of EKLF^−/−KLF2^−/− embryos” for more image information.

Figure 2

Mouse embryonic globin gene expression in yolk sacs from EKLF^−/−KLF2^−/− compared with wild-type and single knockouts. (A) Ey-globin mRNA, (B) βh1-globin mRNA, (C) ζ-globin mRNA. Glycophorin A (GPA) mRNA was used as an internal standard for quantitative RT-PCR. The globin-to-GPA mRNA ratio for wild-type (□) was taken as 100%, and for the other genotypes is expressed compared with 100%. To maintain similar genetic backgrounds between test and control samples, all mutants are compared with wild-type littermates. The other genotypes are EKLF ^+/−KLF2 ^+/− (▨, n = 8), KLF2^−/− (▩, n = 7), EKLF^−/− (, n = 4), and EKLF^−/−KLF2^−/− (■, n = 6). n represents the number of embryonic yolk sacs of each genotype used to determine the mean globin-to-GPA mRNA ratio. Error bars represent the standard deviation from the mean. The asterisks in panels A and B indicate a significant reduction in Ey- and βh1-globin mRNA (P < .025) in double- compared with single-knockout embryos. The asterisk in panel C indicates a significant reduction in ζ-globin mRNA in EKLF^−/− compared with wild-type. (D) Mouse glycophorin A (GPA) gene expression in E10.5 EKLF^−/−KLF2^−/− yolk sacs. Cyclophilin A mRNA was used as an internal standard for quantitative RT-PCR. Wild-type (□, n = 5) and EKLF^−/−KLF2^−/− (■, n = 5) embryonic yolk sac RNAs were tested to determine the mean GPA-to-cyclophilin A mRNA ratio. Error bar is standard deviation, and wild-type was taken as 100%.

Figure 3

Morphology of WT, KLF2^−/−, EKLF^−/−, and EKLF^−/−KLF2^−/− E9.5 circulating blood cells. The total number of embryo samples examined for each genotype is indicated in the Figure 4 legend. (A-D) Representative cytospins of Giemsa-stained primitive erythroid cells. Cytospins were prepared from blood samples collected from E9.5 yolk sacs and embryos. (A) Wild-type. (B) KLF2^−/−. (C) EKLF^−/−. (D) EKLF^−/−KLF2^−/−. Photographs were taken at 1000 × magnification. See “Analysis of EKLF^−/−KLF2^−/− embryos” for more image information. (E) Semiquantitative assessment of abnormal cytoplasmic and nuclear morphology in WT, KLF2^−/−, EKLF^−/−, and EKLF^−/−KLF2^−/− cytospins. Ten primitive blood cells from each sample were scored from 1 to 3, based on severity of cytoplasmic pleiomorphism and nuclear atypia (white and black bars, respectively). A score of 1 indicates the most normal, and of 3 the most abnormal morphology. Error bars indicate standard deviation from the mean. For WT, EKLF^−/− and EKLF^−/−KLF2^−/−, n = 40 cells, and for KLF2^−/− samples, n = 20. * indicates a significant difference from WT at P < .001, and ** is significantly different from WT, KLF2^−/−, and EKLF^−/− at P < .001 using Student t test of comparison of means.

Figure 4

Representative double-labeled FITC-CD71 and PE-TER119 histoplots from E9.5 blood. Flow cytometry was used to determine CD71 and TER119 staining intensity of blood cells from E9.5 yolk sac and embryos. Mean percentages plus or minus standard deviations are indicated in each gate R1-R5 for each genotype. Regions R1 through R5 are defined respectively as CD71^medTER119^low, CD71^highTER119^low, CD71^highTER119^high, CD71^medTER119^high, and CD71^lowTER119^high. R1 contains predominantly progenitor cells, R2 contains proerythroblasts and early basophilic erythroblasts, R3 has early and late basophilic erythroblasts, R4 has chromatophilic and orthochromatophilic erythroblasts, and R5 has late orthochromatophilic erythroblasts and reticulocytes. (A) Wild type, mean percentages calculated from n = 32 embryos. (B) KLF2^−/−, n = 8. (C) EKLF^−/−, n = 10. (D) EKLF^−/−KLF2^−/−, n = 8.

Figure 5

E9.5 EKLF^−/−KLF2^−/− yolk sac blood islands have abnormal morphology. Panel A is a representative section from a wild type, panels B and C are from 2 different EKLF/KLF2 double knockout, and panel D is from a KLF2^−/− embryonic yolk sac. The abbreviations used are columnar epithelium (Ep), erythroid cells (Ery), endothelial cells (En), mesothelial cells (Me), endothelial/mesothelial layer (En + Me L), and ingrowth of epithelial cells (IGEp). Photographs were taken at 400 × magnification. See “Analysis of EKLF^−/−KLF2^−/− embryos” for more image information.

Figure 6

Electron micrographs of E9.5 yolk sac cells. (A) Representative erythroid cell from EKLF/KLF2 double knockout. (B) Wild-type and (C) EKLF/KLF2 double-knockout sections indicating the cell types in the yolk sac. (D) Abnormal ingrowth of microvilli in epithelial cells in EKLF/KLF2 double knockout. The abbreviations used are erythroid cells (Ery), cytoplasmic projections (CP), mesothelial cells (Me), intercellular space (IC) between the mesothelial (Me L) and endothelial layers (En L), basal lamina (B Lm), microvilli (MV), and ingrowth of epithelial cells (IGEp). Scale is shown in each photograph. See “Analysis of EKLF^−/−KLF2^−/− embryos” for more image information.