Knockdown and knockout of cbs or cth impair brain vascular development of larval zebrafish. (A) In situ hybridisation of whole zebrafish larvae at 3 dpf showing the ubiquitous expression of cbs and cth in the brain (lateral view). (B and C) Effects of morpholino oligonucleotide-mediated cbs or cth knockdown on brain vascular development. (B) Representative midbrain vessel structures reconstructed from confocal images of cbs or cth morphants at 3–5 dpf of the same larva. (C) Summary of data. The experiments were repeated three times, and six embryos were examined for each group at each time. (D and E) Effects of cbs or cth knockout (F0) on brain vascular development. (D) Representative midbrain vessel structures reconstructed from confocal images of the same F0 mutant larva at 3–5 dpf. (E) Summary of data. Six embryos were examined for each group. (F and G) Effects of cbs or cth homomutants on brain vascular development. (F) Representative midbrain vessel structures reconstructed from confocal images of the same cbs or cth homomutant larva at 3 and 5 dpf. (G) Summary of data. Six embryos were examined for each group. Scale bar, 300 µm (A), 25 µm (B, D, F). Error bars, SEM. *P<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student’s t-test).

Rescue effect of GYY4137 on the defects of brain vascular development in cbs and cth morphants. (A) Representative projected confocal images showing that GYY4137 treatment ameliorated the impaired brain vascular development in cbs and cth morphants. Confocal images were taken at 3–5 dpf of the same larva. (B) Summary of the rescue effect of GYY4137. Six embryos were examined for each group. Scale bar, 50 µm (A). Error bars, SEM. *P<0.05, **p<0.01, ***p<0.001 (one-way analysis of variance). MO, morpholino oligonucleotide.

Structure analysis of the midbrain vasculature in cbs and cth morphants. (A) Image of a 3-dpf larva showing the midbrain position delineated with dashed lines (upper left), and representative midbrain vasculature centerlines of 3-dpf larvae of Ctrl morpholino oligonucleotide (MO; upper right), cbs MO (lower left), and cth MO (lower right). C, caudal; L, lateral. (B–E) Summary of changes in the total vessel length (B), vessel segment number (C), weighted average segment Strahler order (D) and internal vessel loop number (E) of the midbrain vasculature in groups of Ctrl MO, cbs MO and cth MO. Six embryos were examined for each group. Error bars, SEM. ***P<0.001 (unpaired two-tailed Student’s t-test).

The nitric oxide synthase (NOS)/nitric oxide (NO) pathway is involved in cbs and cth knockdown-induced defects of brain vascular development. (A and B) Effects of cbs and cth knockdown on the NOS/NO signalling pathway. Summary of nos1, nos2a and nos2b RNA expression (A) and total NO production (B) in 3-days post fertilisation (dpf) embryos. (C and D) Rescue effect of nos2a mRNA on the brain vascular developmental defects in cbs and cth morphants. Summary of increased total NO production (C) and midbrain vessel density (D) in 3-dpf embryos coinjected with cbs or cth MO and nos2a mRNA in comparison with those injected with cbs or cth MO. Six embryos were examined for each group. (E and F) No effect of cbs and cth knockdown on the protein expression of p-ERK1/2, ERK1/2 and VEGF. Representative blots (E) and summary (F) of Western blotting data. Error bars, SEM. *P<0.05, **p<0.01 (unpaired two-tailed Student’s t-test).