Abstract
Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation. BBB formation and modulation are not only important for development, but also relevant for vascular and neurodegenerative diseases. However, there is little understanding of how Wnt signaling contributes to brain angiogenesis and BBB formation. Here we show, using high resolution in vivo imaging and temporal and spatial manipulation of Wnt signaling, different requirements for Wnt signaling during brain angiogenesis and BBB formation. In the absence of Wnt signaling, premature Sphingosine-1-phosphate receptor (S1pr) signaling reduces VE-cadherin and Esama at cell-cell junctions. We suggest that Wnt signaling suppresses S1pr signaling during angiogenesis to enable the dynamic junction formation during anastomosis, whereas later S1pr signaling regulates BBB maturation and VE-cadherin stabilization. Our data provides a link between brain angiogenesis and BBB formation and identifies Wnt signaling as coordinator of the timing and as regulator of anastomosis.
Highlights
Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation
We analyzed during which stages of brain capillary angiogenesis active Wnt signaling can be observed using confocal live imaging and two different transgenic Wnt signaling reporter lines, which express short-lived fluorophores controlled by Wntresponsive promoters: Tg(axin2BAC:Venus-Pest)mu[288] and Tg (14TCF:loxP-STOP-loxP-destabilized GFP (dGFP))mu20233
With the onset of lumen formation about 2 h after cell–cellcontact formation, the dGFP signal decreased to a baseline level, which was maintained in the perfused Central arteries (CtAs), pointing to a postdetermination role of Wnt signaling
Summary
Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation. Single-cell analysis of brain vascular development through live imaging approaches in zebrafish further revealed that the control of pre-tip cell function by the Gpr124-Reck complex operates within the parental vessels (PHBCs) at pre-invasive stages This early function of Wnt signaling has so far largely precluded the analysis of Wnt signaling functions at later stages of CNS vascular development, including the invasion process itself and subsequent patterning events. Experiments using mice and tissue culture revealed that VE-cadherin is subject to extensive post-transcriptional regulation, including intracellular complex formation and trafficking, membrane localization as well as association in cell–cell junctions This dynamic regulation of VE-cadherin in adherens junctions is crucial for vascular patterning, and for the function of mature blood vessels, e.g., for maintenance of EC integrity or during leukocyte extravasation[25,26], and can be regulated by Sphingosine-1-phosphate (S1p) signaling[27,28]. The contribution of neither S1pr nor VE-cadherin signaling has been addressed during brain capillary angiogenesis, leaving a missing link between brain EC angiogenesis and BBB formation
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