Erythroid kruppel-like factor exhibits an early and sequentially localized pattern of expression during mammalian erythroid ontogeny

Erythroid Kruppel-like factor (EKLF) is an erythroid cell-specific transcription factor that mediates activation via binding to a 9 base pair sequence that encompasses the CACCC element, one of a trio of evolutionarily conserved sequence moths that are functionally important for transcription of red cell-specific genes. Molecular analyses have delineated the specificity of its interaction and activation through the CAC site at the adult P-globin promoter. However, its expression and distribution during murine ontogeny have not been established. To address these issues, we have focused on biological aspects of EKLF expression by examining the onset and localization of its mRNA during murine development by using reverse transcription/polymerase chain reaction (RT/PCR) analysis of differentiating embryonic stem cells and in situ analyses of normal developing embryos. In addition, we have monitored the presence of EKLF protein by blot analysis of whole-cell extracts derived from circulating cells and embryonic tissue. Our studies show that EKLF mRNA is first expressed at the neural plate stage (day 7.5) within primitive erythroid cells at the very beginning of blood island formation in the yolk sac. EKLF is then expressed by day 9 in the hepatic primordia and remains high in the liver, which becomes the sole source of EKLF mRNA in the 14.5 day fetus. Concomitantly with EKLF mRNA, EKLF protein is also expressed in primitive erythroid cells and in the fetal liver. Finally, EKLF expression in the adult spleen is strictly localized to the red pulp. These studies demonstrate that EKLF is a specific, early marker of erythroid differentiation consistent with its requirement for later globin (and possibly other red cell gene-specific) expression. In addition, EKLF exhibits alternate, sequentially active sites of expression within regions known to harbor hematopoietic precursors during murine ontogeny. Thus, EKLF expression exhibits biological properties that, in addition to previous molecular and more recent genetic studies, augment the evidence in favor of its important role in erythroid cell-specific expression. (C) 1996 Wiley-Liss, Inc.