Changes in globin gene mRNA accumulation in primitive erythroid cells. (A) E12.5 and (B) E15.5 of mouse gestation. Relative levels (mean ± SEM of at least 3 independent experiments) of the β-globin genes (ϵy, βH1, β1, and β2) and of the α-globin genes (ζ, α1/α2) quantified by qPCR from nucleated primitive erythroid cells are shown on the left. Total β- and total α-like globin transcript levels are each considered as 1. Globin transcript accumulation in circulating blood cells at E12.5 visualized by in situ hybridization is shown in the right panels of A. Isolation by FACS of primitive and definitive orthochromatic erythroblasts and reticulocytes from E15.5 peripheral blood is shown on the right panels in B. These populations can be distinguished by forward-side scatter and further isolated as Ter119-positive cells (not shown). Orthochromatic erythroblasts can be distinguished from reticulocytes after staining RNA with thiazole orange and DNA with Hoechst. Also shown are the patterns of β-globin gene expression in each purified population quantified by qPCR. Late-stage primitive erythroid cells at E15.5 express predominantly ϵy-globin transcripts, while late stage definitive erythroid cells express only β1- and β2-globin transcripts. Images were processed as described in “Materials and methods.”