Figure 3.
Figure 3. BBB phenotype of LSR embryos. (A) Quantification of Sulfo-NHS biotin leakage through the BBB of wild-type embryos at different ages. At E12.5 and E13.5, biotin leaks through the BBB. From E14.5 and onwards, leakage of Sulfo-NHS biotin is greatly restricted. (B) Quantification of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr +/−, and Lsr −/− embryos at E12.5, E13.5, and E14.5. At E12.5 and E13.5, Sulfo-NHS biotin leakage is comparable between the genotypes. However, at E14.5 Sulfo-NHS biotin leakage is greatly diminished in Lsr+/+ and Lsr+/−, but not Lsr−/−, mice. Data represent mean ± SEM (error bars). ***, P < 0.0001. (C) Visualization of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr+/−, and Lsr−/− E14.5 embryos. PFA fixed tissue sections of E14.5 Sulfo-NHS biotin-injected embryos were stained with fluorophore (Alexa Fluor 488)-conjugated streptavidin. Lsr+/+ (left) and Lsr+/− (middle) embryos present minimal leakage of Sulfo-NHS biotin from the blood vessels into the SC, whereas the dye is observed throughout the neural tissue of Lsr−/− (right) embryos. Bar, 20 µm. (D) Fresh-frozen tissue sections of E13.5 mouse SC from Lsr+/+ (top), Lsr+/− (middle), and Lsr−/− (bottom) embryos were labeled with an anti–claudin 5 antibody (red) and CD31 to stain blood vessels (green). There was no discernable difference in the claudin 5 expression level or localization between genotypes. Bar, 20 µm. (E) Quantification of TJ protein (LSR, Zo1, Occludin, Cldn5) expression in E14.5 CNS Lsr+/+, Lsr+/−, and Lsr−/− embryos. This graph shows no significant differences for levels of Zo1, occludin, and claudin 5 between genotypes. Data represent mean ± SD (error bars). n is between 3 and 6. (F) Quantification of albumin leakage through the BBB of Lsr+/+, Lsr+/−, and Lsr−/− embryos from E12.5 to E14.5. There was no significant difference in leakage of albumin into the brain between genotypes at any age. Data represent mean ± SD (error bars). n is between 3 and 8. (G) Electron micrograph of SC blood vessels from E14.5 Lsr+/+ and Lsr−/− embryos. Arrowheads indicate kissing points of TJs. There were no discernable differences in the ultrastructure of TJs between genotypes. Bar, 50 nm.

BBB phenotype of LSR embryos. (A) Quantification of Sulfo-NHS biotin leakage through the BBB of wild-type embryos at different ages. At E12.5 and E13.5, biotin leaks through the BBB. From E14.5 and onwards, leakage of Sulfo-NHS biotin is greatly restricted. (B) Quantification of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr +/−, and Lsr −/− embryos at E12.5, E13.5, and E14.5. At E12.5 and E13.5, Sulfo-NHS biotin leakage is comparable between the genotypes. However, at E14.5 Sulfo-NHS biotin leakage is greatly diminished in Lsr+/+ and Lsr+/−, but not Lsr−/−, mice. Data represent mean ± SEM (error bars). ***, P < 0.0001. (C) Visualization of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr+/−, and Lsr−/− E14.5 embryos. PFA fixed tissue sections of E14.5 Sulfo-NHS biotin-injected embryos were stained with fluorophore (Alexa Fluor 488)-conjugated streptavidin. Lsr+/+ (left) and Lsr+/− (middle) embryos present minimal leakage of Sulfo-NHS biotin from the blood vessels into the SC, whereas the dye is observed throughout the neural tissue of Lsr−/− (right) embryos. Bar, 20 µm. (D) Fresh-frozen tissue sections of E13.5 mouse SC from Lsr+/+ (top), Lsr+/− (middle), and Lsr−/− (bottom) embryos were labeled with an anti–claudin 5 antibody (red) and CD31 to stain blood vessels (green). There was no discernable difference in the claudin 5 expression level or localization between genotypes. Bar, 20 µm. (E) Quantification of TJ protein (LSR, Zo1, Occludin, Cldn5) expression in E14.5 CNS Lsr+/+, Lsr+/−, and Lsr−/− embryos. This graph shows no significant differences for levels of Zo1, occludin, and claudin 5 between genotypes. Data represent mean ± SD (error bars). n is between 3 and 6. (F) Quantification of albumin leakage through the BBB of Lsr+/+, Lsr+/−, and Lsr−/− embryos from E12.5 to E14.5. There was no significant difference in leakage of albumin into the brain between genotypes at any age. Data represent mean ± SD (error bars). n is between 3 and 8. (G) Electron micrograph of SC blood vessels from E14.5 Lsr+/+ and Lsr−/− embryos. Arrowheads indicate kissing points of TJs. There were no discernable differences in the ultrastructure of TJs between genotypes. Bar, 50 nm.

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