TGFβ1/BMP signaling is active in Eng-expressing cells at E7.5. (A) Clustering of genes associated with the TGFβ superfamily among the E⁻F⁻, E⁻F⁺, E⁺F⁺, and E⁺F⁻ fractions showing high expression of these TGFβ superfamily members in Eng-expressing cells. (B-D) Confirmatory qPCR analyses for selected TGFβ superfamily members, including ligands (B), type I receptors (C), and type II receptors (D). Transcripts are normalized to Gapdh. Error bars indicate the SEM from 3 independent biologic datasets. (E) E7.5 cryosectioned embryos were stained for pSMAD1/5 and analyzed by confocal microscopy. pSMAD1 was found to be highly expressed in the PS, and extraembryonic mesoderm (underlying visceral endoderm). Top panel shows low magnification of the whole embryo shown in the bottom panels (10× objective). We observed a gradient of staining for pSMAD1/5 (top panel), with the highest expression in the same areas where Eng and/or Flk-1 are present, as demonstrated in Figure 1. Middle and bottom panels show confocal images of E7.5 serial sections demonstrating abundant expression of phosphorylated SMAD1/5 (pSMAD1/5, arrows in middle panels) in extraembryonic E⁺F⁺ cells (arrows in bottom panels). Chorionic E⁺F⁻ cells show dim expression of pSMAD1/5. Note that visceral endoderm cells also show strong staining for pSMAD1/5 (63× objective). A indicates amnion; c, chorion; and VE, visceral endoderm.