Characterization of the Hb-eGFP-positive cell population. Chick embryos were processed for cVEGFR2 whole mount in situ hybridization at HH5 (A) or co-electroporated at HH3-4 with the Hb-eGFP reporter construct (PCR2) and the ubiquitous reporter pCAGGS-RFP (B-J) and imaged as whole mounts at HH5 (B), HH7 (C), HH9 (D) and HH11 (E), criosectioned through the yolk sac region at HH11 (F, H-J), or processed for microarray analysis at HH5-6 (G). (B-D) Overlay of bright field, Hb-eGFP green fluorescence and RFP red fluorescence images. (E) RFP fluorescence (left), Hb-eGFP fluorescence (middle) and overlay (right). (F, H-J) Top left: bright field and DAPI (F), bright field alone (H), or bright field and RFP (in blue; I and J); top right: Hb-eGFP green fluorescence; bottom left: RFP red fluorescence (F) or immunolabeling of cVEGFR2 (H), red blood cells (RBC; I) and smooth muscle actin (SMA; J); bottom right: overlay of bright field and fluorescence images. At HH5, cVEGFR2 expression is detected in the posterior extraembryonic population of hemangioblasts (A). At this early stage, Hb-eGFP fluorescence is specifically observed in the same cell population (B). At later stages, eGFP-fluorescent cells aggregate in the extraembryonic region (C), give rise to blood islands (D), and integrate the vascular plexus of the yolk sac (E). Note that the control RFP reporter is ubiquitously expressed, whereas Hb-eGFP fluorescence is restricted to the blood islands (E and F). At early stages, the gene expression profile of Hb-eGFP-positive cells revealed that known hemangioblast markers are enriched, whereas genes expressed in other tissues are downregulated in this cell population (G; see also Additional file 2). At later stages, Hb-eGFP fluorescence is detected in cVEGFR2-positive endothelial cells (H; arrow) and blood cells or erythroblasts (I; arrows), but not in smooth muscle cells (J; arrowheads).