Figure 3. Spock1 mutant leakage arises through increased endothelial vesicles.

(A-B) The neurovascular unit remains intact in spock1^hm41/hm41 mutants (B) with a continuous single layer of endothelial cells (pseudocolored magenta) enclosing the lumen (pseudocolored orange) and in close contact with pericytes (pseudocolored green). (C-H) The majority of tight junctions (white arrowheads) are functionally restrictive in the spock1^hm41 mutant endothelial cells (88%). Mutant endothelial cells displayed a significant increase in vesicular density, including both small flask shaped vesicles (yellow stars, G) and larger vesicles greater than 200 nm in diameter (outlined by a white dashed line in F, H). (I-K) While pericyte coverage is unaltered in spock1^hm41 mutants, the pericyte-endothelial cell interactions are altered in the mutants, with several instances of direct pericyte-endothelial cell contact (white arrows) and overall diminished average basement membrane thickness between the two vascular cells (K). Scale bars represent 1 μm (B) and 200 nm (J). N= 4 fish, each demarcated by a unique color, with 10 vessels analyzed per fish and shown as individual points. ** p=0.0029 (H), **** p<0.0001 by nested t test (G and K).