Figure 4

Abolishing endothelial GABA release and its effect on telencephalic angiogenesis (A) A low-magnification co-labeled image of Isolectin B4, VGAT and DAPI labeling of periventricular endothelial cells (pv ecs). (B) High-magnification image of Isolectin B4, VGAT and DAPI labeling of a pv ec (60×). (C) Different morphologies of Isolectin B4 (IB4) and Tie2-GFP-labeled ecs expressing VGAT (60×). (D) Low- and high-magnification images showing specifically in vivo expression of VGAT in endothelial cells of E13 Tie 2-GFP telencephalon. White arrows point to cells that were magnified. (E) No VGAT expression was detected in Vgat^ECKO pv ecs (60×). (F, G) Low- and high-magnification images showing that expression of GABA (F) and GAD65/67 (G) was not affected in Vgat^ECKO pv ecs. (H) Successful elimination of GABA secretion from embryonic Vgat^ECKO pv ecs measured by ELISA; Data represent mean ± SD (n = 6, ^*P < 0.05, Student's t-test). (I-K) Isolectin B4 labeling revealed a significant reduction in vessels in E13 Vgat^ECKO telencephalon (yellow asterisk, J) when compared to Vgat^fl/fl telencephalon (I). (K) Quantification of vessel densities; Data represent mean ± SD (n = 6, ^*P < 0.05, Student's t-test). (L) The migratory behavior of Qdot-labeled Vgat^ECKO pv ecs was decreased (yellow asterisk) compared to Vgat^fl/fl pv ecs. Representative images from the transwell migration assay are shown. (M) Quantification of the number of migrated cells per field from each group (n = 8, ^*P < 0.05, mean ± SD. Student's t-test). (N) Vgat^fl/fl pv ecs showed robust tube formation in an angiogenesis assay on matrigel (white arrows) reflecting their high angiogenic potential. (O) Vgat^ECKO pv ecs failed to form robust tubes (yellow arrows), signifying impaired angiogenesis. (P-R) Quantification of number of junctions and tubules analyzed by Wimasis and quantification of the angiogenesis score²⁸; Data represent mean ± SD (n = 10, ^*P < 0.05, Student's t-test). (S, T) Claudin 5 expression was decreased in E16 Vgat^ECKO dorsal telencephalon (T) when compared to Vgat^fl/fl(S) telencephalon, illustrating loss of tight junctions (n = 10). (U, V) Images of IgG staining from E17 Vgat^fl/fl and Vgat^ECKO dorsal telencephalon. While IgG was localized to Vgat^fl/fl vessels (white arrows, U), IgG leakage was observed from Vgat^ECKO vessels in dorsal and medial telencephalon (yellow arrows, V). (W) High-magnification images of IgG leakage (yellow arrows) from Vgat^ECKO vessels in the dorsal telencephalon. (X, Y) E18 Vgat^ECKO and littermate controls were given a trans-cardiac perfusion of biotinylated dextran. Vgat^ECKO tissue sections stained with streptavidin-Alexa 594 showed increased fluorescence (X) which was quantified and permeability relative to control was graphed (Y; n = 10, ^*P < 0.05, mean ± SD, Student's t-test). Scale bars: A, 50 μm (applies to S, T, W, X), B, 15 μm (applies to C, E, G), D, 100 μm (high-magnification inset 30 μm); F, 75 μm, I, 100 μm (applies to I, J, L, N, O, U, V).