Figure 1
From: Endothelial cell-derived GABA signaling modulates neuronal migration and postnatal behavior
Endothelial Gabrb3 regulates telencephalic development. (A) GABA expression (red) in E11 Tie2-GFP dorsal telencephalon with specific labeling in Tie2-GFP+ endothelial cells (co-label in yellow). White arrows illustrate high magnifications (20×) of endothelial cells showing individual and merged images of GFP and GABA. (B) A high-magnification image of GABA labeling of endothelial cells in a periventricular vessel from E12 neocortex obtained by DAB immunohistochemistry (60×). (C) Individual isolectin 4, GABA, DAPI and merged image of a periventricular endothelial cell (pv ec, 60×). (D) Co-labeled image of isolectin 4, GABRB3 and DAPI labeling of pv ecs (40×). (E) In vivo expression of GABRB3 in periventricular endothelial cells of Tie2-GFP telencephalon at E13. White arrow illustrates the region of high-magnification images (20×), which show GFP-positive endothelial cells lining a vessel, co-labeled with GABRB3. (F) Individual Isolectin 4, GABRB3, DAPI and merged image of a Gabrb3fl/fl pv ec (60×). (G) No GABRB3 expression in pv ecs was detected in Gabrb3ECKO embryos (60×). (H-J) Fewer isolectin B4+ vessels in E13 Gabrb3ECKO telencephalon (yellow asterisk, I) compared to Gabrb3fl/fl telencephalon (white asterisk, H). (J) Morphometric analysis of isolectin B4 labeling revealed significant reduction in vessel densities in E13 Gabrb3ECKO telencephalon; Data represent mean ± SD (n = 8, *P < 0.05, Student's t-test). (K) While the tube-like plexus of periventricular vessels, labeled by isolectin B4, in the ganglionic eminence and dorsal telencephalon was continuous and well formed in Gabrb3fl/fl telencephalon, (white arrows), it was discontinuous and irregular (yellow arrows) in Gabrb3ECKO telencephalon. (L, M) GAD65/67 immunoreactivity showed decreased stream of GABA neurons in E15 Gabrb3ECKO telencephalon (yellow asterisk, M) when compared to Gabrb3fl/fl telencephalon (white asterisk, L). (N) High-magnification image (40×) revealing fewer GAD65/67 cells in Gabrb3ECKO dorsal telencephalon versus Gabrb3fl/fl telencephalon. (O-T) H&E stainings revealed no marked changes in cortical lamination in E18 Gabrb3ECKO dorso-lateral telencephalon (P) in comparison with Gabrb3fl/fl telencephalon (O). However, morphological abnormalities were observed in medial Gabrb3ECKO telencephalon (red asterisk, P). Striatal compartments were enlarged in Gabrb3ECKO telencephalon (yellow asterisk, P). The corpus callosum (blue arrow), hippocampus oriens layer (orange arrow), triangular septal nucleus (black arrow) and ventral hippocampal commissure (brown arrow) were normally formed in Gabrb3fl/fl telencephalon (Q) but perturbed in Gabrb3ECKO telencephalon (R). The two limbs of the anterior commissure (ac) crossed at the midline in both Gabrb3fl/fl and Gabrb3ECKO embryos (Q, R). Ventricular defects (blue asterisk, S) and reduced hippocampus (red arrow, S) were observed in E18 Gabrb3ECKO telencephalon in comparison to Gabrb3fl/fl telencephalon (blue arrow, S). (T) High-magnification images of hippocampus from S. (U, V) Fewer isolectin B4+ vessels in E18 Gabrb3ECKO pallium (yellow asterisks, V) compared with Gabrb3fl/fl pallium (white asterisks, U). (W) Significant reduction in cortical vessel densities in E18 Gabrb3ECKO embryos; Data represent mean ± SD (n = 8, *P < 0.05, Student's t-test). (X) Gabrb3ECKO mice at P0 were smaller in size than Gabrb3fl/fl mice. (Y) Weight chart of Gabrb3ECKO mice compared to Gabrb3fl/fl mice from P1 to P30; Data represent mean ± SD (n = 12, *P < 0.05, Student's t-test). Scale bars: A, 60 μm (applies to N); B, 30 μm (applies to D); C, 15 μm; (applies to F, G), E, 100 μm; (applies to H, I, K-M, O-S, U, V); T, 40 μm, high-magnification insets in A and E, 30 μm.
