Haploinsufficiency of Eklf results in the absence of Ter119 in the primitive but not the definitive erythroid lineage. (A) FACS analysis of CD71 expression on WT, Eklf^+/−, and Eklf^−/− embryos. CD71 is expressed on the Eklf mutant EryP, though at modestly reduced levels (approximately 4-fold) on null mutant cells. (B) Eklf dose-dependent expression of Ter119 on E14.5 EryP. WT GFP⁺ cells (EryP) expressed Ter119 at higher levels than WT GFP⁻ cells (EryD). In contrast, deletion of one allele of Eklf results in loss of expression of Ter119 on GFP⁺ EryP but not on the GFP⁻ EryD. Ter119 is absent from any of the null mutant cells. (C) Representative FACS histogram of Ter119 fluorescence intensity on GFP⁺ EryP from Eklf WT or Eklf^+/− PB, gated from samples in panel B. Expression of Ter119 on the surface of Eklf heterozygous cells was greatly reduced, compared with WT EryP. (D) Expression of Glycophorin A (Gypa) in E13.5 Eklf^+/+, Eklf^+/−, and Eklf^−/− EryP/H2B-GFP as measured using real-time RT-PCR. mRNA expression was normalized using 18S RNA as a control and was then normalized again, setting WT Eklf levels at 1.0. (E) Ter119 staining of PB cells from E13.5 ϵ-globin:H2B-GFP; Eklf^+/− embryos. Cells were cytospun onto glass slides, fixed, and permeabilized before immunostaining. No Ter119 reactivity was detected in EryP (green fluorescent nuclei) compared with the smaller Ter119⁺ enucleated definitive erythrocytes. Images were acquired using a Zeiss AxioCam camera mounted on a Zeiss Axioplan 2 microscope outfitted with a 63×/Plan neofluar/1.25 NA oil objective. Scale bar, 20 μm.