Fig. 5. VEGF is essential for the survival of primitive erythrocytes. (A) Single-cell suspensions of dissected E9.0-E9.5 yolk sacs were stained with Annexin V-FITC (apoptosis) and TER119-PE (erythroid). Dead cells that stain with 7-ADD were excluded from the analysis. (B) KI67 immunostaining of E9.0-E9.5 Vegf^lo hypomorph yolk sacs. Homozygous Vegf^lo/lo yolk sacs contain few blood islands, harboring rare primitive erythroid cells (F,G) in contrast to heterozygote Vegf^lo/+ (D,E) and wild-type Vegf^+/+ (B,C) littermates. KI67-positive (brown precipitate), proliferating, primitive, erythroid cells (arrows) are found at a slightly reduced frequency in Vegf^lo/+ and Vegf^lo/lo yolk sacs. n represents the number of primitive erythrocytes scored: Vegf^+/+, n=137; Vegf^lo/+, n=237; Vegf^lo/lo, n=80. No staining was observed when the primary antibody was omitted (data not shown). Nuclei were counterstained with Methyl Green. Scale bar: 10 µm.