Podoplanin and CLEC-2 drive cerebrovascular patterning and integrity during development

Abstract

Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplanin(fl/fl) mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin- and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet α-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage.

Figure 1

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.

Figure 2

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.

Figure 3

Loss of podoplanin in the neural tube causes cerebral hemorrhages. (A) Pdpn^fl/flNes-Cre embryos develop hemorrhages in the brain by E12.5 (n = 4), whereas Pdpn^fl/fl littermates appear normal (n = 3). Scale bars, 1 mm. (B) H&E coronal sections from E12.5 embryo heads of Pdpn^fl/fl (n = 3) and Pdpn^fl/flNes-Cre (n = 4) embryos. White boxes in the middle panel magnify areas in the bottom panel. Yellow arrows indicate reticulated red blood cells in the ventricles, and yellow asterisks (*) marks an area of hemorrhage in the neuro-epithelium. Scale bars, 50 μm.

Figure 4

Altered pericyte recruitment to cerebral blood vessels in CLEC-2- and podoplanim-deficient mice. (A) Immunostaining of frozen sections of embryonic heads at E11.5 show pericytes (NG2, blue) adhering to the surface of blood vessels (PECAM-1, green) within the Pdpn (Pdpn, red) expressing neuro-epithelium in wild-type (^+/+, includes Pdpn^fl/fl littermates; n = 8), Clec-2^−/− (n = 3), Pdpn^fl/flPGK-Cre (n = 3), and Pdpn^fl/flNes-Cre (n = 3) embryos. White arrowheads in Clec-2^−/− embryos indicate where blood vessels are distended from the surrounding neuro-epithelium. Scale bars, 10 μm. (B) A reduction in the total number of pericytes in association with blood vessels was observed in Clec-2^−/−, Pdpn^fl/flPGK-Cre, and Pdpn^fl/flNes-Cre embryos. For each n = 1, a minimum of 4 different sections with 8 images per section were analyzed. Statistical significance was measured by a 1-way analysis of variance with a Tukey posttest, where *P < .05 and **P < .01. Error bars show means ± standard error of the mean.

Figure 5

Ultrastructural analysis of Clec-2^−/−, Pdpn^fl/flPGK-Cre, and Pdpn^fl/flNes-Cre microvessels at E11.5 by electron microscopy. In wild-type embryos (^+/+, includes Pdpn^fl/fl littermates; n = 4), endothelial cells enclose a vascular lumen containing a nucleated fetal erythrocyte and are connected by tight junctions (yellow arrowheads). The endothelium is closely associated with surrounding pericyte foot processes and neuro-epithelial cells. In Clec-2^−/− (n = 3), Pdpn^fl/flPGK-Cre (n = 4), and Pdpn^fl/flNes-Cre (n = 4) microvessels, vascular lumens appear expanded, and their surrounding endothelium is enriched in vacuoles. Inter-endothelial junctions remain tightly closed and secured by endothelial cell flaps (yellow arrowheads). Large gaps between the endothelium and overlying pericytes and neuro-epithelial cells are noticeable. NE, neuro-epithelial cell; BV, blood vessel; RBC, nucleated red blood cell; P, pericyte; E, endothelial cell. Original magnification: (left) ×7500; (right) ×15 000. Scale bar, 5 μm.

Figure 6

Evidence for a role for both platelet aggregation and secretion in maintaining cerebral vascular integrity. (A) Mice deficient in the major platelet integrin subinit αIIb present with cerebral hemorrhaging at E12.5 (black arrows; 6 of 6 αIIb^−/− embryos). Hemorrhages were also observed in 6 of 8 embryos at E12.5 from mice in which both platelet α-granule and dense-granule secretion is abolished (black arrows; Nbeal-2^−/−Unc13d^−/−). Scale bars, 1 mm. (B) H&E coronal sections from E12.5 embryo heads show the accumulation of erythrocytes in the ventricles of αIIb^−/− (n = 6) and Nbeal-2^−/−Unc13d^−/− (n = 6 of 8) embryos (red arrows), whereas only a minor hemorrhage was observed in 1 of 17 wild-type littermate controls (^+/+). White boxes show magnified areas in the lower panel. Scale bars, 50 μm.