Heightened activation of eMks and platelets in Pdpn^−/− and Clec-2^−/− vasculature during midgestation. (A-B) Confocal images of diencephalon sections with an antibody to phospho-Syk (Y519/520) (A) or an antibody to phospho-PLCγ2 (Y1217; arrows) (B). 4′,6-Diamidino-2-phenylindole is the nuclear stain. (C-D) Pseudocolored TEM of eMks (C) or platelets (D) in the PNVP vessel lumen of the lower diencephalon. eMk demarcation membrane system (asterisks), microparticles (red arrows), and lamellipodia (pink arrows) are indicated (C). Platelet filopodia projections (orange arrow) and granule secretion (red arrow) are indicated (D). (E) Confocal images of diencephalon sprouts and COL-1 expression. Dashed lines outline each sprout. (F) Confocal images of eMks with α-granules labeled with TSP-1 in the lower diencephalon. Insets show eMks in the midbrain. Bar graph (bottom) represents the percentage of fetal Mks with high or low TSP-1 threshold levels. (G) Confocal images of eMks isolated from E11.5 peripheral blood, that were either plated on a slide through cytospin as a negative activation control or incubated on a slide coated with COL-1 or PDPN for 30 minutes. The α-granules were labeled with TSP-1. The bar graph (right) represents eMk α-granule intensity. Data represent mean ± standard error of the mean and 3 experimental repeats with at least 3 technical repeats each. Scale bars, 10 μm (A-B); 2 μm (C); 500 nm (D); 20 μm (E-F); and 5 μm (G). **P < .01.