Fig. S2

BBB functionality shown with FD4 (4 kDa) as a fluorescent tracer [A-B] Casper embryo, injected with fluorescent FD4 at 2 dpf via the caudal vein. Distribution of the tracer was monitored with confocal microscopy and shows leakage to the ventricles within the first 30 minutes post injection. [Panel A] Overview of the head of the embryo 90 minutes post injection with accumulation of the tracer in the ventricular system indicating that the BBB is not formed yet at this time point, scale bar = 150 µm, [Panel B] Enlargement of the midbrain ventricle and the hindbrain ventricle with dye accumulation, scale bar = 60 µm. [C-D] Casper embryo, injected with FD4 at 3 dpf via the caudal vein. [Panel C] Overview of the head of the embryo 90 minutes post injection. A clear pattern of the blood vessels is visible with minimal leakage of fluorescent dye to the ventricular system, Scale bar = 150 µm. [Panel D] Enlargement of Panel C, same region as Panel B, Scale bar = 60 µm. [E-F] Casper embryo, injected with FD4 at 4 dpf via the caudal vein. [Panel E] Overview of the head of the embryo 90 minutes post injection. A clear pattern of the blood vessels is visible without leakage of fluorescent dye to the ventricular system, indicating that the BBB is functional at 4 dpf in limiting traversal of molecules of 4 kDa and larger, scale bar = 150 µm. [Panel F] Enlargement of Panel E, same region as Panel B and D, scale bar = 60 µm.