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. Author manuscript; available in PMC: 2018 May 3.
Published in final edited form as: Neuron. 2017 Apr 13;94(3):581–594.e5. doi: 10.1016/j.neuron.2017.03.043

Figure 3. The lipid transport function of Mfsd2a is required for the suppression of transcytosis and maintenance of BBB integrity in vivo. See also Figures S3 and S6; Table S4.

Figure 3

(A–D) Increased vesicular density in CNS endothelial cells of Mfsd2aD96A mutants (C–D) compared to their littermate controls (A–B). Electron microscopy analysis at P4 reveals increased number of vesicles in Mfsd2aD96A mutants along the luminal (arrows) and abluminal (Ab; black arrowheads) plasma membranes. Scale bar, 150 nm. (E–F) Quantification of vesicular density along the luminal (E) and abluminal (F) plasma membranes. n = 3 animals per condition, 20 capillary cross-sections per animal. (G–J) Increased transcytosis is evident in CNS endothelial cells of HRP-injected Mfsd2aD96A mutants at P90. HRP tracer (black) is confined with the vessel lumen (L) of littermate control animals (G–H). Tracer-filled vesicles are observed invaginating from the luminal plasma membrane (arrows), within the cytoplasm (asterisks), and along to abluminal plasma membrane (arrowheads) in Mfsd2aD96A mutants (I–J). n = 3 animals per condition. Scale bar, 100 nm. (K–L) Tight junctions are functional in CNS endothelial cells of HRP-injected Mfsd2aD96A mutants. HRP tracer fills the vessel lumen (L) and enters intercellular clefts but is sharply halted at tight junction “kissing points” (yellow arrows) in both Mfsd2aD96A mutants (L) and Mfsd2a+/+ littermate control animals (K) at P90. n = 3 animals per condition. Scale bar, 100 nm. (M–N) Mfsd2aD96A mutants display BBB leakage. 10kD TMR-dextran tracer (red) is completely confined within vessels (green, Isolectin B4 (IB4)) (arrows) in Mfsd2a+/+ littermate control animals (M) at P4. Tracer-filled parenchyma cells (arrowheads) surround vessels in the brain of Mfsd2aD96A mutants (N). Scale bar, 50 μm. (O) Permeability index of tracer leakage in brain, as quantified by area of tracer divided by area of vessels per image (value = 1 signifying no leakage). n = 4 animals per genotype. All data are mean ± s.e.m.; * P < 0.05, ** P < 0.01 (Student’s t-test).