Molecular mechanism of Zic3 controlling embryonic cerebrovascular development via non-classical Wnt and VEGF signaling axes in zebrafish

Abstract

Objective To explore the effects of zinc finger transcription factor 3 (zic3) gene on cerebrovascular development in zebrafish at early stage and its mechanisms. Methods CRISPR/Cas9 technique was used to obtain the zebrafish with zic3 mutation. The proportion of cerebral hemorrhage in embryos of each genotype was counted at 72 h post fertilization (hpf), with wild-type AB zebrafish serving as control. The vasculature-labelled transgenic AB zebrafish (Tg flk1 ∶gfp) was crossed with zic3 mutants to obtain zic3+/-(Tg flk1 ∶gfp), which was subsequently inbred for littermate control, and the phenotypes of angiogenesis in embryos (n=30) were observed by fluoroscopy at 72 hpf. Finally, RT-PCR was applied to detect the expression of related genes. Results Zic3 homozygous mutants were successfully achieved and validated by DNA sequencing. The mutants manifested an obvious hemorrhagic phenotype and significantly up-regulated expression of plasmalemma vesicle associated protein (Plvap) gene and zic2 gene as compared with the control (P < 0.05). However, this phenotype was not observed in the heterozygous mutants, in which the angiogenesis of hindbrain was significantly inhibited, and the number of vessels was less than that of the wild type by 60% (P < 0.05), indicating an evident developmental defect. Meanwhile, the expression levels of non-classical Wnt signaling pathway genes (wnt5a, wnt7ba and wnt9b) and vascular endothelial growth factor (VEGF) signaling pathway genes (kdrl, vegfab and ephrlnB2) were all significantly increased in the heterozygous mutants (P < 0.05). Conclusion In zebrafish, function of zic3 can be compensated by zic2. Zic3 reduces cerebrovascular permeability in embryos by suppressing the expression of Plvap, and regulates cerebral angiogenesis by inhibiting non-classical Wnt and VEGF signaling pathways.

Keywords

• zebrafish
• zinc finger transcription factor 3
• non-classical wnt signaling
• vascular endothelial growth factor
• cerebrovascular development