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Developmental angiogenesis at the interface of the diencephalic choroid plexus (dCP) and pineal gland (PG), and vegf expression patterns during the vascularization of this interface. (A) Schematic representation of the dorsal view of the zebrafish larval head at 10 days post fertilization (dpf), indicating the location of the dCP and PG interface (red boxed area). (B–C”’) 3D (B), lateral (B’), and dorsal (C–C”’) views of the 10 dpf Et(cp:EGFP);Tg(kdrl:ras-mCherry) head immunostained for rhodopsin show the 3D spatial relationship between dCP epithelial cells (green), pineal photoreceptor cells (magenta), and blood vessels (white). (D) Dorsal view of 10 dpf Tg(plvap:EGFP);Tg(glut1b:mCherry) head shows strong Tg(plvap:EGFP) and undetectable Tg(glut1b:mCherry) expression in the prosencephalic artery (PrA), PG vessel (PGV), and anterior cerebral vein (ACeV). In contrast, the neighboring vessels mesencephalic vein (MsV) display strong Tg(glut1b:mCherry) expression. (E–E”) Dorsal views of the 75 hours post fertilization (hpf) Et(cp:EGFP) head immunostained for Claudin-5 show EGFP+ dCP epithelial cells (yellow arrows) outlined by the tight junction protein Claudin-5. (F–J) Dorsal views of 23 (F), 26 (G), 32 (H), 48 (I), and 75 (J) hpf Tg(kdrl:EGFP) rostral cranial vasculature show the developmental time courses of vascularization at the dCP/PG interface. (K) Schematic diagram of the vasculature at the dCP/PG interface shows the direction of blood flow at around 75–10 dpf. (L–L”) Dorsal views of a 26 hpf TgBAC(vegfab:EGFP);Tg(kdrl:ras-mCherry) head show TgBAC(vegfab:EGFP)+ cells at the midline where bilateral PrA connect. (M–N”) Dorsal (M–N”) and lateral (M’) views of a 75 hpf TgBAC(vegfab:EGFP);Tg(kdrl:ras-mCherry) head immunostained for Claudin-5. As compared to 26 hpf, an increased number of TgBACvegfab:EGFP + cells was observed at the PrA connection site and in its anterior brain regions (M, M’). Magnified images of the boxed area in (M) indicate TgBAC(vegfab:EGFP)+ and Claudin-5+ dCP epithelial cells (yellow arrows, N–N”). (O–O”) Dorsal views of a 26 hpf TgBAC(vegfc:EGFP);Tg(kdrl:ras-mCherry) head show TgBAC(vegfc:EGFP)+ vascular endothelial cells (vECs, white arrows) and separate cells at the PrA connection site. (P–Q”) Dorsal (P, Q–Q”) and lateral (P’) views of a 75 hpf TgBAC(vegfc:EGFP);Tg(kdrl:ras-mCherry) head immunostained for Claudin-5. TgBAC(vegfc:EGFP)+ cells were observed at the PrA connection site and in its posterior brain regions (P, P’). Magnified images of the boxed area in (P) indicate TgBAC(vegfc:EGFP)+ and Claudin-5+ dCP epithelial cells (yellow arrows, Q–Q”). (R–R”) Dorsal views of a 26 hpf TgBAC(vegfd:EGFP);Tg(kdrl:ras-mCherry) head show TgBAC(vegfd:EGFP)+ meningeal fibroblast-like cells that reside posterior to the dCP/PG interface. TgBAC(vegfd:EGFP)+ axonal projections were also visualized. (S–T”) Dorsal (S, T–T”) and lateral (S’) views of a 75 hpf TgBAC(vegfd:EGFP);Tg(kdrl:ras-mCherry) head immunostained for Claudin-5. TgBAC(vegfd:EGFP)+ cells were observed in meningeal fibroblast-like cells that reside posterior to the PGV/ACeV (S, S’). Magnified images of the boxed area in (S) show no obvious TgBAC(vegfd:EGFP) expression in dCP epithelial cells (T–T”). (U–U”) Dorsal views of a 26 hpf TgBAC(vegfaa:EGFP);Tg(kdrl:ras-mCherry) head show TgBAC(vegfaa:EGFP)+ vECs (white arrows). (V–W”) Dorsal (V, W–W”) and lateral (V’) views of a 75 hpf TgBAC(vegfaa:EGFP);Tg(kdrl:ras-mCherry) head immunostained for Claudin-5. Sparse TgBAC(vegfaa:EGFP)+ cells were observed at the lateral periphery of the dCP (W-W’). Magnified images of the boxed area in (V) show TgBAC(vegfaa:EGFP)+ and Claudin-5+ dCP epithelial cells at the periphery (W-W’). Scale bars: 30 µm in (B), (B’), (D), in (C”’) for (C–C”’), in (E”) for (E–E”), in (V’) for (M’), (P’), (S’); 50 µm in (F–J), in (U”) for (L–L”), (O–O”), (R–R”), (U–U”), in (V) for (M), (P), (S), in (W”) for (N–N”), (Q–Q”), (T–T”), (W–W”).
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