(A) Representative dorsal view maximum intensity projections of larval zebrafish hindbrains at different developmental stages reveals tracer permeability into the hindbrain parenchyma at 3 dpf that decreases at 4 dpf and remains low until 10 dpf. This low permeability was observed with two injected tracers of different sizes, a 1 kDa NHS (turquoise) and a 10 kDa Dextran (white), as well with an 80 kDa transgenic serum protein DBP-EGFP (green). Parenchymal tracer intensity outside of the vasculature (magenta) was measured and normalized to the blood vessel tracer intensity in each fish. Scale bars represent 50 µm. (B) Quantification of normalized parenchymal tracer intensity in the hindbrain between 3 and 10 dpf reveals a decrease in tracer uptake beginning at 4 dpf. There was no significant change from 4 to 10 dpf, suggesting that the hindbrain barrier seals around 4 dpf. N = 14–21 fish, each represented as a single dot on the plot. The mean and the standard error are drawn in black for each tracer and stage. ****p<0.0001, **p=0.0034, *p=0.0115, ns is not significant by two-way ANOVA.
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