Despite the critical role of endothelial Wnt/β-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/β-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/β-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/β-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function.DOI: http://dx.doi.org/10.7554/eLife.06489.001

Wnt signaling is key to many developmental, physiological, and disease processes in which cells seem able to discriminate between multiple Wnt ligands. This selective Wnt recognition or "decoding" capacity has remained enigmatic because Wnt/Frizzled interactions are largely incompatible with monospecific recognition. Gpr124 and Reck enable brain endothelial cells to selectively respond to Wnt7. We show that Reck binds with low micromolar affinity to the intrinsically disordered linker region of Wnt7. Availability of Reck-bound Wnt7 for Frizzled signaling relies on the interaction between Gpr124 and Dishevelled. Through polymerization, Dishevelled recruits Gpr124 and the associated Reck-bound Wnt7 into dynamic Wnt/Frizzled/Lrp5/6 signalosomes, resulting in increased local concentrations of Wnt7 available for Frizzled signaling. This work provides mechanistic insights into the Wnt decoding capacities of vertebrate cells and unravels structural determinants of the functional diversification of Wnt family members.

The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful blood-borne factors. Although BBB dysfunction is a hallmark of several neurological disorders, therapies to restore BBB function are lacking. An attractive strategy is to repurpose developmental BBB regulators, such as Wnt7a, into BBB-protective agents. However, safe therapeutic use of Wnt ligands is complicated by their pleiotropic Frizzled signaling activities. Taking advantage of the Wnt7a/b-specific Gpr124/Reck co-receptor complex, we genetically engineered Wnt7a ligands into BBB-specific Wnt activators. In a “hit-and-run” adeno-associated virus–assisted CNS gene delivery setting, these new Gpr124/Reck-specific agonists protected BBB function, thereby mitigating glioblastoma expansion and ischemic stroke infarction. This work reveals that the signaling specificity of Wnt ligands is adjustable and defines a modality to treat CNS disorders by normalizing the BBB.

SUMMARYContinuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/b-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina. and a transgenic male. The latter was selected beforehand as having a single transgene insertion site (as inferred by mendelian ratios in its progeny) in order to ensure homogeneous levels of GFP expression in the offspring. Construction of the LEF1-VP16 and LEF1-EnR transgenesis vectors has been described previously (Denayer et al., 2008) and transgenic X. tropicalis lines (LEF1-VP16Tg and LEF1-EnRTg) were generated as described (Sekkali et al., 2008). These constructs are fused with the dexamethasone-responsive hormone-binding domain of the human glucocorticoid receptor (GR). Expression constructs and morpholinospCS2- TCF3-VP16GR and pCS2-dnTCF3-GR (de Croze et al., 2011), pCS2-Ihh-CD2 [previously called Bhh (Locker et al., 2006)], pCS2-Smo-M2 , pCS2-cyclinA2 and pCS2-cdk2 (Decembrini et al., 2006) and pCS2-GFP (a gift from David Turner, University of Michigan, Ann Arbor, USA) were described previously. pCS2-Shh-CD2 and pCS2-Dhh-CD2 (previously called Chh) were generated by subcloning the N-terminal coding regions (devoid of the C-terminal cleavage product) of Shh and Dhh cDNAs (Ekker et al., 1995) Microinjection and in vivo DNA lipofectionCapped mRNAs encoding TCF3-VP16GR and GFP were transcribed from pCS2 plasmids after NotI digestion using the mMessage mMachine SP6 Kit (Ambion). Then, 400 pg of each mRNA was injected into two blastomeres of four-cell stage embryos. Gli3, Sfrp-1 or standard control morpholino oligonucleotides (Gene Tools) were injected into one blastomere at the one-cell stage (30 ng). Their efficacy was tested by analysing in vivo GFP fluorescence following co-injection of a chimeric GFP construct fused downstream of the morpholino-complementary sequence (supplementary material Fig. S1).Lipofection experiments were performed by cotransfecting the indicated pCS2 constructs together with pCS2-GFP at stage 18 into the presumptive region of the retina, as previously...