Fig. 4. Gata factor and Smad binding motifs regulate Eklf expression in vivo. (A) Schematic of the Peklf-GFP reporter construct and its four derivatives, which were targeted to the Ainv18 ES cell line, that carry two GATA motif mutations (2xG/A) in the upstream enhancer (Peklf-2xG/A-GFP), or one GATA motif mutation in the proximal promoter (Peklf-G/A-GFP), two Smad binding motif deletions (2xSBM) in the upstream enhancer (Peklf-2xSBM-GFP), or an insertion of Eklf intron 1 (Peklf-intron-GFP). Positions of Eklf cis-regulatory elements and translation start codons (ATG) are indicated and color-coded as in Fig. 3. (B-E) qRT-PCR analysis of GFP transgene expression in the four derivative Ainv18 ES cell reporter clones between days 4 and 7 or 8 of EB differentiation. Expression levels were normalized to 18S rRNA or Gapdh. For Eklf and GFP, normalized levels are presented relative to a value of 1 on EB day 4, whereas for Gata2 (G2) and globin βH1 (bh1) the maximum expression level per gene is set to 1. Arithmetic mean ±s.d.; ^*, 0.01<P<0.05, n=3; ^**, 0.001<P<0.01, n=3. (Left) Endogenous Eklf (red) and GFP (green) expression from the Peklf-GFP clone, differentiated in parallel in each experiment, is shown for cross-comparison. (Right) Endogenous Eklf (red) and GFP (green) expression from each mutant clone (Peklf-2xG/A-GFP, Peklf-G/A-GFP, Peklf-2xSBM-GFP, Peklf-intron-GFP) is shown along with that of Gata2 (black) and globin βH1 (orange). Within each panel, high levels in Gata2 expression are indicative of a progenitor population in differentiating EBs prior to day 5.5, after which the onset of globin βH1 expression is indicative of erythroid commitment (expression patterns as in Fig. 2A).