Figure 2. NPC endfeet appose the vasculature in the developing tectum. A, Schematics of the X. laevis experimental system. Brain regions are indicated in the sketch of the dorsal view of the tadpole head (top). The heart and intracardial dextran injection are shown in a sketch of the ventral view (lower left) and labeled vasculature in a drawing of the dorsal tectum (lower right). B, A representative image (right) of dextran-labeled blood vessels (magenta) and NPCs (green) labeled by electroporation of pCAG::GFP. 42.7% of labeled NPCs (n = 511) have endfeet (arrows) that are apposed to the vasculature. Scale bar: 50 μm. C, C’, D, D’, Examples of pCAG::GFP-labeled NPCs (green) whose endfeet (arrows) are apposed to the vasculature (magenta). C’, D’, 3D rotations of regions with NPC endfeet highlighted by arrows in C, D, respectively, showing vascular apposition. Scale bars: 50 and 20 μm. E, Projection of a confocal stack through tectum with pCAG::GFP-labeled NPCs (green) and dextran-labeled vasculature (magenta). Automated vessel and endfoot tracing (white) on a representative cell, with the shortest distance between the endfoot centroid and vessel edge (yellow), computed in 3D. Scale bar: 20 μm. F, Histogram of the distances, computed in 3D, between endfoot centroids and the closest vessel edge (n = 200 NPCs). G, Representative in vivo time lapse images of a pSox2bd::mGFP-labeled NPC endfoot, collected at two time points: 0 min (green) and 4 min (magenta). The overlay in the right panel highlights rapid extensions (arrows) and retractions (asterisk) of the NPC endfoot’s filopodia. Scale bar: 10 μm. H, DiI-labeled NPCs (green) in late stage 46 Flk1:GFP (magenta) transgenic X. laevis tadpoles. Scale bar: 100 μm. H’, Magnification of the boxed region in H showing the association between blood vessels (magenta) and NPC endfeet (green) in single orthogonal sections of cross-sectional planes in XY, YZ, and XZ. Scale bar: 10 μm.