Figure 2.

Figure 2. Wnt/β-catenin signaling deficiency does not cause any apparent defect in fenestrated capillary formation in the neurohypophysis (NH) and organum vasculosum of the lamina terminalis (OVLT).

(A) Schematic diagram of vasculature in the ventral brain at around 5–10 days post fertilization (dpf), illustrating the locations of the OVLT, the NH, and distinct blood vessels used for quantifications. HyA: hypophyseal artery, HyV: hypophyseal veins, Hy loop: hypophyseal loop, PLA: palatocerebral arteries, OA: optic artery, CrDI: cranial division of the internal carotid artery. (B) Dorsal view of 120 hours post fertilization (hpf) Tg(plvap:EGFP);Tg(glut1b:mCherry) ventral brain shows strong Tg(plvap:EGFP) expression in the Hy loop, HyA, and PLA compared to its fainter signals in the HyV. (C–D”) Dorsal views of the 10 dpf Tg(plvap:EGFP) head immunostained for Glut1. Glut1 immunoreactivity was undetectable in a rostral portion of the Tg(plvap:EGFP)⁺ HyA that lies in proximity to the OVLT (C–C”) and in the Tg(plvap:EGFP)⁺ Hy loop (D–D”). Faint signals were detected in a caudal portion of the HyA that resides close to the Hy loop (D–D”). (E–H) Dorsal views of 120 hpf wild-type (WT) (E), gpr124^-/- (F), reck^-/- (G), and wnt7aa^-/- (H) ventral brain vasculature visualized by Tg(kdrl:EGFP) expression. gpr124^-/-, reck^-/-, or wnt7aa^-/- larvae formed vasculature in the NH/OVLT regions similar to WT. (I–K) Quantification of ventral brain vessel formation at 120 hpf (the number of animals examined per genotype is listed in the panel). No significant difference was detected in gpr124^-/-, reck^-/-, or wnt7aa^-/- larvae compared to WT. Each data point shown in magenta represents individual animal’s vessel formation score. (L) Experimental workflow of tracer dye injections and subsequent imaging and tracer permeability analysis. Tg(kdrl:EGFP) larvae at 6 dpf were co-injected with 3 kDa and 10 kDa dextran dyes conjugated with different fluorophores. (M–P) Merged images of 3 kDa tetramethylrhodamine-conjugated dextran dye (magenta) and Tg(kdrl:EGFP)⁺ vasculature at 6 dpf. Unlike the midbrain parenchyma (M) where a functional blood-brain barrier (BBB) is established, a higher amount of tracer accumulation was detected in tissues around the diencephalic choroid plexus (dCP), NH, and OVLT brain regions (N–P). (Q) Quantification of normalized tracer intensity across the different brain regions at 6 dpf reveals a significant increase in tracer accumulation around the dCP, NH, and OVLT brain regions compared to the midbrain parenchyma. **** indicates p<0.0001 by one-way analysis of variance (ANOVA) followed by Tukey’s HSD test. Statistical significance was calculated for each dye tracer across different brain regions and represents differences in the graph. Scale bars: 50 µm in (B), in (H) for (E–H), in (M–N); 25 µm in (C”) for (C–C”), in (D”) for (D–D”), in (O–P).