Eklf-deficient primitive erythroblasts show dramatic alterations in cell morphology but can complete their maturation. (A) Wet preparations of freshly harvested E14.5 PB cells reveal large Eklf-null mutant EryP that fail to achieve the discoidal shape observed for WT cells. (B) Detail of Giemsa-stained cytospin preparations of PB from E13.5 and E14.5 mutant embryos, showing some macrocytic cells that lack nuclei (arrows). (C) Left panel, H2B-GFP expression in an E8.0 transgenic embryo. Right panel, wet preparation of PB from an E14.5 ϵ-globin::H2B-GFP embryo. An overlay of bright field and green fluorescence is shown. Expression of the fluorescent reporter is restricted to the EryP nucleus. (D) Expression of Eklf mRNA in developing EryP cells. GFP⁺ cells were sorted from E8.5-12.5 embryos and real-time RT-PCR analysis of Eklf was performed; expression was normalized to that of ubiquitin b. (E) Eklf expression in definitive versus primitive erythroid cells. GFP⁺ EryP from E11.5 embryos or CD71⁺Ter119⁺ definitive erythroid cells from adult mouse BM were sorted and the expression of Eklf mRNA assessed by real time RT-PCR; expression levels are relative to ubiquitin b. A representative experiment (3 independent samples) is shown. (F) Histograms of ϵ-globin::H2B-GFP fluorescence intensity in E13.5 PB cells harvested from Eklf WT, Eklf^+/−or Eklf^−/− embryos. Images were acquired in AxioVision v4.6 using a Zeiss AxioCam digital color camera mounted on a Zeiss Axiovert 25 inverted microscope outfitted with a 20× (LD-A-Plan/0.3 NA) or 32× (LD-A-Plan/0.4 NA) objective (A-C right panel) or on a Leica MZ12 stereomicroscope outfitted with a plan apo 1.0× objective (C, left panel). Scale bars: A, 20 μm; B, 10 μm; C, left panel, 100 μm, and right panel, 10 μm.