PDPN reduces COL-1–induced ANGPT-1 secretion from fetal Mks. (A) RNAscope confocal images of diencephalon Angpt1 expression. Shown is colocalization with CD41⁺ cells (left; pink arrows) in the lower diencephalon. High-magnification images (right) show CD41⁺ cells colocalized with Angpt1 in the diencephalon neural tissue and the PNVP. (B) Overview of eMk isolation, culture, and static adhesion assay on recombinant PDPN/human IgG Fc chimera protein (PDPN-Fc), COL-1, or both. Plate coated with IgG Fc used as negative control (Fc). (C-E) Confocal images of ANGPT-1 levels of WT (C) or Unc13d^−/− (E) eMks after incubation for 30 minutes on PDPN, COL-1, or both, or with Clec-2^−/− eMk after incubation for 30 minutes on isotype IgG Fc, COL-1, or COL-1+PDPN (D), with comparison with WT. Bar graphs (right) represents the percentage of Mks with high, medium, or low ANGPT-1 levels. Data represent mean ± standard error of the mean; n, number of experimental repeats, with 3 technical repeats each. Scale bars, 50 μm (A, left); 20 μm (A, right); and 10 μm (C-E). **P < .01.