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Figure 1 (A) Diagram of the tracer leakage assay. Fluorescently conjugated tracers (turquoise) were injected intracardially into transgenic fish that express mCherry in the vasculature (magenta; Tg(kdrl:mCherry)) and allowed to circulate for 1 hr before imaging. (B) Dorsal view maximum intensity projection of the larval brain vasculature at 3 dpf. Left image is pseudo-colored to demarcate the midbrain (violet) and the hindbrain (gold) vasculature. Right image shows the NHS tracer (turquoise) in the entire larval brain, with a large number of tracer-filled parenchymal cells in the midbrain. (C) Representative dorsal view maximum intensity projections of larval zebrafish midbrains at different developmental stages reveal increased permeability at 3 and 4 dpf compared to 5, 6 and 10 dpf. The increased early 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. (D) Quantification of normalized parenchymal tracer intensity in the midbrain between 3 and 10 dpf reveals a significant decrease in tracer leakage at five dpf. There was no difference observed between different tracers at any time point. There was no significant change from 3 to 4 dpf or from 5 to 10 dpf, suggesting that the midbrain barrier seals around 5 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, ns is not significant by two-way ANOVA.