Hematopoietic progenitors are restricted to the Eng⁺ fraction. (A) Flow cytometric analyses of WT E7.5 embryos. Eng expression is shown on the x-axis. Percentage represents the fraction of cells that express Eng. Gates represent Eng⁻ (red) and Eng⁺ (green) fractions that were sorted for further in vitro characterization (B-D). The y-axis represents PerCP, which provides a measure of autofluorescence. (B) Eng⁻- and Eng⁺-sorted cell fractions (A) were assessed for their hematopoietic activity (CFUs). Hematopoietic colonies were detected exclusively in the Eng⁺ fraction. Eng⁻ cells failed to produce any colonies. Error bars indicate the SEM from 3 independent experiments. (C) Emergence of robust hematopoietic outgrowths after the coculture of Eng⁺ (right panel) cells sorted from E7.5 embryos on OP9 cells for 5 days. Hematopoietic growth was virtually absent in Eng⁻ cocultures (left panel). Photographs are representative of at least 3 independent experiments. (D) FACS characterization of Eng⁺- and Eng⁻-derived OP9 cocultures after a 5-day in vitro expansion. Representative profiles are shown for c-Kit and CD41 (left panels), CD45 and Ter119 (middle panels), and Gr-1 and Mac-1 (right panels). Fluorescence intensity for c-Kit, CD45, or Gr-1 is indicated on the y-axis and CD41, Ter119, or Mac-1 on the x-axis. Plots are representative of at least 3 independent experiments.