Inhibition of platelet aggregation causes a “nonseparation” phenotype. (A) E13.5 podoplanin^+/+ embryo whose mother was injected with rabbit anti-podoplanin IgG that blocks podoplanin-platelet interaction (left panel). Subtle vascular malformations (arrowheads) similar to those in podoplanin^−/− mice are locally observed. Immunofluorescence staining (middle panel) demonstrates dermal Lyve-1⁺ lymphatic vessels (green) containing erythrocytes (red; white arrow). Cell nuclei are stained with DAPI (blue). Right panel shows macroscopically normal vasculature in podoplanin^+/+ embryo whose mother was injected with irrelevant rabbit IgG. (B) E14.5 podoplanin^+/+ embryo whose mother was treated with acetyl salicylic acid (ASA; 25 mg/kg/d) to inhibit platelet aggregation. Vascular lesions reminiscent of those found in podoplanin^−/− mice are locally observed (left panel; inset shows higher magnification). Immunofluorescence staining (right panel) shows that the dermal vessels are Lyve-1⁺ lymphatics (green) and contain erythrocytes (Ter119 in red). Cell nuclei are stained with DAPI (blue). (C) Kindlin-3^−/− E14.0 embryo that shows blood-containing vascular malformations in the skin (left panel). Double immunofluorescence of dermal vessel (right panel); endothelium is positive for Prox1 (red nuclei) and podoplanin (green), and the vessel is filled with erythrocytes (yellow). Arrowheads mark extravasated erythrocytes (fluorescent images were taken on an Olympus AX70 with a 20× objective lens; all other images with the Olympus SZ40). Scale bars in panels A and B equal 50 μm; in panel C, 20 μm.