CD9 surface expression identifies circulating nucleated primitive erythroid cells. (A) The tetraspanin CD9 is expressed on circulating EryP/H2B-GFP. (B) Immunocytochemical staining of PB from 13.5 embryos using anti-CD9. (C) Expression of Cd9 in EryP throughout development. GFP⁺ cells were sorted from E8.5-12.5 embryos and real-time RT-PCR analysis of Cd9 mRNA expression performed. (D) CD9 is redistributed onto expelled EryP nuclei. Side scatter (SSC) and GFP can be used to identify 3 populations: SSC(high) GFP⁺ EryP (green), SSC(low) GFP⁺ free nuclei (red) and SSC(low) GFP⁻ enucleated cells (blue) (left panel). CD9 expression on each population is shown (right panel). CD9 is present on nucleated EryP and at lower levels on free nuclei; it is not expressed on enucleated EryD. (E) CD9 expression is altered by loss of Eklf. While all E13.5 WT and heterozygous EryP express CD9, Eklf-null GFP⁺ cells show more heterogeneous CD9 expression, with a CD9-negative population. (F) Nucleated EryP can be isolated on the basis of CD9 expression. CD9⁺ and CD9⁻ cells were sorted from E14.5 blood. Both wet preparations (middle panels) and Giemsa stains (bottom panels) clearly document that the CD9⁺ cells are EryP. (G) Developmental progression of CD9 expression on circulating WT erythroid cells. CD9 is expressed on EryP but not on definitive erythroid cells. Images were captured in AxioVision using a Zeiss AxioCam camera mounted on a Zeiss Axioplan 2 microscope and outfitted with a 63×/Plan neofluar/1.25 NA oil objective (B) or a Zeiss Axiovert 25 inverted microscope outfitted with a 32×/LD-A-Plan/0.3 NA objective (F). Scale bars, 10 μm.