GFP expression from the ϵ-globin::H2B-EGFP transgene marks the primitive erythroid lineage in the blood islands of the YS and can be used to identify and isolate EryP progenitors. (A) GFP expression in ϵ-globin::H2B-EGFP primitive streak to early somite–stage transgenic embryos. To visualize the emergence and expansion of GFP(+) EryP cells, time-lapse videos of cultured ϵ-globin::H2B-EGFP embryos were acquired. GFP(+) cells appear in a narrow band of 3-5 cell diameters in the proximal YS during mid-to-late gastrulation (MS/LS stage). Scale bar, 500 μm. MS, midstreak; LS, late streak; EB, early bud; LHF, late headfold; ss, somite stage; ESom, early somite. (B) Selected snapshots from time-lapse video (supplemental Video 1) of an ϵ-globin::H2B-EGFP embryo cultured in vitro under physiologic conditions from the LS to the ESom stage. Early-bud-stage embryos were imaged over a period of 14 hours, from the time before transgene induction (∼ EB, t = 0) through the ESom stages (t = 860 minutes; supplemental Video 1). Confocal images were acquired as sequential optical x-y sections taken at 4-μm z intervals. Images were taken at 20-minute intervals (total imaging time: 14.5 hours). (C) Flow cytometric histogram profiles of dispersed ϵ-globin::H2B-EGFP transgenic embryos reveals a clearly identifiable GFP(+) population. (D) Cells from whole E7.5 or E8.5 embryos were FACS sorted to GFP(+) and GFP(−) populations. Left panel, Giemsa-stained cytospun cells from FACS sort. Scale bar, 20 μm. EryP-progenitor numbers were measured using a clonogenic assay. Virtually all progenitor activity was recovered in the GFP(+) population. Characteristic EryP colonies (right panels) showed red pigmentation (hemoglobin) and GFP fluorescence. Scale bar, 50 μm. (E) Real-time RT-PCR expression of endogenous embryonic ϵy- and βh1-globin genes in GFP(+) and GFP(−) FACS-sorted cells from E7.5 ϵ-globin::H2B-EGFP transgenic embryos. Expression was normalized to ubiquitin b (Ubb). (F) EryP numbers at the YS stages of development.
