KLF2 is essential for primitive erythropoiesis and regulates the human and murine embryonic beta-like globin genes in vivo

Abstract

The Krüppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins that are important in controlling developmental programs. Erythroid Krüppel-like factor (EKLF or KLF1) positively regulates the beta-globin gene in definitive erythroid cells. KLF2 (LKLF) is closely related to EKLF and is expressed in erythroid cells. KLF2-/- mice die between embryonic day 12.5 (E12.5) and E14.5, because of severe intraembryonic hemorrhaging. They also display growth retardation and anemia. We investigated the expression of the beta-like globin genes in KLF2 knockout mice. Our results show that KLF2-/- mice have a significant reduction of murine embryonic Ey- and beta h1-globin but not zeta-globin gene expression in the E10.5 yolk sac, compared with wild-type mice. The expression of the adult beta(maj)- and beta(min)-globin genes is unaffected in the fetal livers of E12.5 embryos. In mice carrying the entire human globin locus, KLF2 also regulates the expression of the human embryonic epsilon-globin gene but not the adult beta-globin gene, suggesting that this developmental-stage-specific role is evolutionarily conserved. KLF2 also plays a role in the maturation and/or stability of erythroid cells in the yolk sac. KLF2-/- embryos have a significantly increased number of primitive erythroid cells undergoing apoptotic cell death.

Figure 1.

Effect of KLF2 on endogenous murine α- and β-like globin gene expression in E10.5 yolk sac. GPA was used as an internal control. The globin/GPA mRNA ratio for the wild type is taken as 100%, and for the other genotypes it is expressed compared with 100%. n represents the number of embryos of each genotype used to determine the mean globin/GPA mRNA ratio. Error bars represent the standard deviation from the mean. ^*Statistically significant difference of expression in KLF2^+/- or KLF2^-/- embryos from the wild type; P < .025. (A) Expression of the embryonic Ey-globin gene in KLF2^+/+, KLF2^+/-, and KLF2^-/- embryos; (B) expression of the embryonic βh1-globin gene in KLF2^+/+, KLF2^+/-, and KLF2^-/- embryos; and (C) expression of the embryonic ζ-globin gene in KLF2^+/+ and KLF2^-/- embryos.

Figure 2.

Effect of KLF2 on endogenous murine β-like globin gene expression at E12.5. The percentage of globin/GPA mRNA ratio was determined, and statistical analyses were performed as described in the legend of Figure 1. (A) Embryonic Ey-globin mRNA in the yolk sac, (B) embryonic βh1-globin mRNA in the yolk sac, and (C) adult β^maj-plus β^min-globin mRNA in the fetal liver.

Figure 3.

Effect of KLF2 on human β-like globin gene expression in E10.5 yolk sac in β-globin locus YAC transgenic mice. The percentage of globin/GPA mRNA ratio was determined, and statistical analyses were performed as described in the legend of Figure 1. (A) Embryonic ε-globin mRNA in KLF2^+/+globin⁺, KLF2^+/-globin⁺, and KLF2^-/-globin⁺ embryos; and (B) fetal γ-globin mRNA in KLF2^+/+globin⁺, KLF2^+/-globin⁺, and KLF2^-/-globin⁺ embryos.

Figure 4.

Effect of KLF2 on human β-like globin gene expression at E12.5, in KLF2⁺/+globin⁺, KLF2⁺/-globin⁺, and KLF2^-/-globin⁺ embryos. The percentage of globin/GPA mRNA ratio was determined, and statistical analyses were performed as described in the legend of Figure 1. (A) Embryonic ε-globin mRNA in the yolk sac, (B) fetal γ-globin mRNA in the yolk sac, and (C) adult β-globin mRNA in the fetal liver.

Figure 5.

Representative photographs of 1-μm thick plastic sections of E10.5 yolk sacs. (A) KLF2^+/+, (B) KLF2^+/-, and (C) KLF2^-/- embryos, stained with a solution of Toluidine Blue, Azure II, and Methylene Blue. EC indicates erythroid cells; BI, blood island; EL, epithelial layer; ML, mesothelial layer. Photographs were taken at 200 × magnification.

Figure 6.

Electron micrographs of ultrathin sections of E10.5 yolk sacs. (A) KLF2^+/+ and (B-C) KLF2^-/- embryos, stained with uranyl acetate and lead citrate. N indicates nucleus; C, cytoplasm. Photographs were taken at 21 000 × magnification.