In situ identification of Eng⁺ cells in WT E7.5 embryos. (A) Confocal images of whole E7.5 embryos associated with close-up images shown in panels B through D. Immunostaining shows expression for Eng (red) and Flk-1 (green) and their merge (yellow-orange; 10× objective). DAPI is shown in blue. Eng is strongly expressed in the allantois (al) and the chorion (c). Some allantoic cells also express Flk-1. In addition to the extraembryonic region, Eng⁺Flk-1⁺ cells are also detected in the PS and anterior mesoderm (AM). (B) High magnification of E7.5 extraembryonic region shown in panel A (left) confirms coexpression (arrowhead) of Eng (red) and Flk-1 (green) in the developing BI next to the visceral endoderm (VE). Rare Eng⁻Flk-1⁺ cells are also observed in the BI region (arrow; 63× objective). (C-D) Serial cryosections of E7.5 embryos (panels C and D depict the square area shown in panel A right) stained with Abs to Eng and Flk-1 (C) or GATA1 (D). Extraembryonic mesoderm expresses Eng and Flk-1 (C), whereas primitive erythrocytes, characterized by the expression of GATA1 in the serial section (arrow in panel D) are Eng^lowFlk-1⁻ cells (arrow in panel C).