Schematic depiction of the mechanisms involved in separation of lymph sacs from the cardinal veins, resulting in the “separation phenotype” and “nonseparation phenotype.” The developmental default “separation” phenotype in podoplanin^+/+ mice (right column) starts with formation of lymph sacs from the cardinal vein around E12. Endothelial cells of the lymph sacs acquire the lymphatic phenotype and express podoplanin (depicted by red “Y”-shaped objects) on their surface that binds and activates circulating platelets (depicted in green). Platelet thrombi develop around the orifice of the lymph sacs and lead to separation of the lymphatic sac from the cardinal vein either directly by an occluding platelet aggregate or indirectly by release of vasoconstrictive substances or growth factors released from activated platelets recruiting mural cells. At the stage of E14 and later, the separated lymph sacs are fully sequestered from the cardinal veins and sprout centrifugally to form a separate lymphatic circulatory system. The “nonseparation” phenotype in podoplanin^−/− mice (left column) is characterized by persisting patent connections between the venous and the lymphatic systems. This is due to the absence of podoplanin on the lymphatic sac's endothelial cells, and thus the lack of local platelet activation and aggregation at the lymph sac's orifices. This results in blood perfusion of the outgrowing lymphatic vessels.