Figure 7: Inhibition of endo-lysosomal trafficking in vascular endothelial cells in vivo.

(a) Blood-CNS barrier defects are revealed in the cortex and hippocampus of Cre-activated Rosa26 LSL-VPS4 EQ mice transcardially perfused with Sulfo-NHS-biotin by staining with Streptavidin coupled to Alexa 555. (b) Barrier defects in the molecular layer of the cerebellum. (c) Retinal sections show failure of intraretinal vascularization and strongly increased PLVAP expression in VPS4 EQ expressing mice. These defects recapitulate hallmark phenotypes of impaired Norrin/FZD4 signalling in the retinal vasculature. NFL, nerve fiber layer; IPL, inner plexiform layer; OPL, outer plexiform layer. (d) Confocal projections of the retinal vasculature in P10 whole mount retinas stained with IsolectinB4-Alexa488. Characteristic Norrin LOF phenotypes are observed. Progression towards the rim of the retina (indicated by white dashed line) is delayed (distance from the vascular front to the rim of the retina indicated by white bar) and the microscopic appearance of the vascular front is altered (arrowheads). (e,f) Optical sections of the boxed areas in d show separate representations of the vascular plexuses located at different depth levels of the retina. Normal vasculature is present in the nerve fiber layer (NFL) and outer plexiform layer (OPL) in control mice, whereas intraretinal capillaries in the OPL of VPS4 EQ expressing mice are absent. (g) Colour depth projections show the vascular beds of the boxed areas in d as single projection, in which distinct depth levels are colour coded. These projections reveal the characteristic glomeruloid vascular malformations (yellow structures, arrowheads) in the nerve fiber layer vasculature, similar to the defects described in Fzd4, Ndp, Lrp5 and Tspan12 mutant mice.