Hemorrhages develop within tortuous vascular networks in CLEC-2- and podoplanin-deficient mice. Whole mount immunostaining of PECAM-1⁺ blood vessels in whole embryonic heads of Clec-2^−/− and Pdpn^fl/flPGK-Cre embryos at (A) E10.5 and (B) E12.5 (Clec-2^−/−; E10.5, n = 3; E12.5, n = 3; Pdpn^fl/flPGK-Cre; E10.5, n = 4; E12.5, n = 3) and littermate controls (E10.5, n = 4; E12.5, n = 5). PECAM-1⁺ vessels in the whole embryonic head are shown as a sagittal section (z-dimension = 100 μm) taken from a 3-dimensional reconstructed image of optical sections (A, left). Red boxes in the left panel mark areas of higher magnification depicted in the middle panel where a 3-dimensional image shows abnormally patterned, tortuous capillaries branching off a large cerebral vessel in Clec-2^−/− and Pdpn^fl/flPGK-Cre mice (z-dimension = 650 μm). Red boxes in the second panel depict a further magnified area displayed as a single sagittal section in the third panel (z-dimension = 250 μm). (B) At E12.5, sagittal sections taken from 3-dimensional movies (supplemental Videos 1-3) show hemorrhages located throughout the fore-, mid-, and hindbrain in CLEC-2-deficient and Pdpn^fl/flPGK-Cre mice only (left, red arrows). A 3-dimensional reconstruction of optical sections shows a clear well-patterned PECAM-1-stained vascular network in wild-type mice compared with the disorganized vascular network in Clec-2^−/− and and Pdpn^fl/flPGK-Cre mice (middle, red arrows mark hemorrhages; z-dimension = 1500 μm). Red boxes in the middle panel mark areas of higher magnification depicted in the third panel of cerebral capillaries branching off a major vessel leading to hemorrhage (outlined by a red dashed line) in Clec-2^−/− and Pdpn^fl/flPGK-Cre mice (z-dimension = 300 μm). Images were processed using Imaris software. Scale bars, 100 μm.