Podoplanin is expressed throughout the neural tube in development and its loss results in hemorrhaging. (A) Immunostaining shows podoplanin in the neuro-epithelium on frozen sections of wild-type embryonic mouse heads at E11.5 (n > 4) and E14.5 (n = 3); white boxes in the left panel highlight magnified areas in the right panel. NE, neuroepithelium; V, ventricle; CP, choroid plexus. Scale bars, 100 μm. (B) Pdpn^fl/fl mice were generated on a C57BL/6 background at Taconic Artemis by insertion of loxP sites flanking exon 3 of the podoplanin gene. Pdpn^fl/fl mice were crossed to mice expressing PGK-cre recombinase resulting in constitutive deletion of exon 3 creating a nonfunctional podoplanin gene. Gray arrows mark primer binding sites. (C) Pdpn^fl/flPGK-Cre embryos (E10.5, n = 3; E11.5, n > 10; and E12.5, n > 10) develop hemorrhages in the brain between E10.5 and E11.5 (arrows), whereas Pdpn^fl/fl littermates appear normal (E10.5, n = 5; E11.5, n = 7; and E12.5, n > 10). Scale bars, 1 mm. (D) H&E coronal sections from E12.5 embryo heads of Pdpn^fl/fl (n = 3) or Pdpn^fl/flPGK-Cre (n = 4) embryos. White block or dashed boxes in the left panel show magnified areas in the middle and right panels, respectively. Yellow arrows mark blood vessels closely associated with surrounding neuro-epithelial cells in wild-type embryos compared with dissociations in Pdpn^fl/flPGK-Cre mice (middle). Eosin-stained red erythrocytes from large hemorrhages are seen to invade matrix tissue into neighboring vascular beds in Pdpn^fl/flPGK-Cre embryos (right). Scale bars, 50 μm.