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Fig. 3 Spatial and temporal expression of claudin-5 in the developing BRB. Tg(flk1:EGFP) embryos and larvae from 2 dpf to 4 dpf were stained with mouse anti-claudin-5. Confocal images of whole mount were analyzed for claudin-5 (red) (A-I; Alexa Fluor 568) expression in hyaloid blood vessels (green) (A′-D′ and G′-I′; EGFP). All the panels are dorsal views except G&G′. The claudin-5 signal in the hyaloid vessels at 2 dpf (A&A′) is minimal. At 2.5 dpf (B&B′), and 3 dpf (C&C′) the staining in the hyaloid vasculature is increased. Claudin-5 is also expressed in the hyaloid artery (asterisks) and outer limiting membrane of the retina (shaded arrow) and inner plexiform layer (shaded arrowhead). (D&D′) At 3.5 dpf, the wider cone-shape staining is lost and the claudin-5 signal overlaps completely with the HA (asterisks). The vessel connecting the HV and the inner optic circle (IOC) also has a strong claudin-5 signal (blue arrows in D&D′, G&G′). (E&F) In the retina, the claudin-5 signal does not clearly outline the polygonal RPE (arrow) until 3 dpf. The insert is an enlarged view of the dashed square. At 4 dpf (G-I), the HV (arrows in G&G′, H&H′) and the HA, but not the choroidal vasculature (such as the IOC, indicated by arrowhead in G′), express claudin-5. The claudin-5 is also expressed around the foramen (opening) through which the HA penetrates the retina (arrow in I). The panel J is a schematic illustrating expression of claudin-5 in optic vasculature of zebrafish. Scale bars: 50 μm.