The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.

Intercellular junctions mediate adhesion and communication between adjoining cells. Although formed by different molecules, tight junctions (TJs) and adherens junctions (AJs) are functionally and structurally linked, but the signalling pathways behind this interaction are unknown. Here we describe a cell-specific mechanism of crosstalk between these two types of structure. We show that endothelial VE-cadherin at AJs upregulates the gene encoding the TJ adhesive protein claudin-5. This effect requires the release of the inhibitory activity of forkhead box factor FoxO1 and the Tcf-4-beta-catenin transcriptional repressor complex. Vascular endothelial (VE)-cadherin acts by inducing the phosphorylation of FoxO1 through Akt activation and by limiting the translocation of beta-catenin to the nucleus. These results offer a molecular basis for the link between AJs and TJs and explain why VE-cadherin inhibition may cause a marked increase in permeability.

SummaryPlanar cell polarity (PCP) signaling controls tissue morphogenesis by coordinating collective cell behaviors. We show a critical role for the core PCP proteins Celsr1 and Vangl2 in the complex morphogenetic process of intraluminal valve formation in lymphatic vessels. We found that valve-forming endothelial cells undergo elongation, reorientation, and collective migration into the vessel lumen as they initiate valve leaflet formation. During this process, Celsr1 and Vangl2 are recruited from endothelial filopodia to discrete membrane domains at cell-cell contacts. Celsr1- or Vangl2-deficient mice show valve aplasia due to failure of endothelial cells to undergo rearrangements and adopt perpendicular orientation at valve initiation sites. Mechanistically, we show that Celsr1 regulates dynamic cell movements by inhibiting stabilization of VE-cadherin and maturation of adherens junctions. These findings reveal a role for PCP signaling in regulating adherens junctions and directed cell rearrangements during vascular development.

Tissue and vessel wall stiffening alters endothelial cell properties and contributes to vascular dysfunction. However, whether extracellular matrix (ECM) stiffness impacts vascular development is not known. Here we show that matrix stiffness controls lymphatic vascular morphogenesis. Atomic force microscopy measurements in mouse embryos reveal that venous lymphatic endothelial cell (LEC) progenitors experience a decrease in substrate stiffness upon migration out of the cardinal vein, which induces a GATA2-dependent transcriptional program required to form the first lymphatic vessels. Transcriptome analysis shows that LECs grown on a soft matrix exhibit increased GATA2 expression and a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including VEGFR3. Analyses of mouse models demonstrate a cell-autonomous function of GATA2 in regulating LEC responsiveness to VEGF-C and in controlling LEC migration and sprouting in vivo. Our study thus uncovers a mechanism by which ECM stiffness dictates the migratory behavior of LECs during early lymphatic development.