Molecular mechanism of Zic3 controlling embryonic cerebrovascular development <italic style="font-style: italic">via</italic> non-classical Wnt and VEGF signaling axes in zebrafish

Shuaiting LI; Yunpeng WANG; Zhi YUAN; Yeqi WANG; Zhi CHENG; Huizhe HUANG

doi:10.16016/j.1000-5404.202104042

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

Neurosciences | 更新时间：2026-01-09

- Molecular mechanism of Zic3 controlling embryonic cerebrovascular development via non-classical Wnt and VEGF signaling axes in zebrafish
- Journal of Third Military Medical University Vol. 43, Issue 19, Pages: 1831-1838(2021)
- 作者机构：
  
  1.400010 重庆，重庆医科大学附属第二医院生殖医学中心，发育生物学研究室
  2.400030 重庆，重庆大学生物工程学院
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(31771619)
- DOI：10.16016/j.1000-5404.202104042
  CLC： R322.12;R329.1;R329.26
- Received：07 April 2021，
  
  Revised：2021-05-23，
  
  Published：15 October 2021
- 稿件说明：
移动端阅览
Shuaiting LI, Yunpeng WANG, Zhi YUAN, et al. Molecular mechanism of Zic3 controlling embryonic cerebrovascular development via non-classical Wnt and VEGF signaling axes in zebrafish[J]. Journal of Third Military Medical University, 2021, 43(19): 1831-1838.
DOI：

Shuaiting LI, Yunpeng WANG, Zhi YUAN, et al. Molecular mechanism of Zic3 controlling embryonic cerebrovascular development via non-classical Wnt and VEGF signaling axes in zebrafish[J]. Journal of Third Military Medical University, 2021, 43(19): 1831-1838. DOI： 10.16016/j.1000-5404.202104042.

摘要

目的

探究锌指转录因子3(

zic3

)基因对斑马鱼早期脑血管发育的影响及其机制。

方法

利用CRISPR/Cas9技术获得

zic3

基因突变体，以野生型斑马鱼(AB品系)为对照，统计各个基因型72 hpf(hour post fertilization)胚胎脑出血比例。利用血管标记转基因鱼AB(Tg flk1 ∶gfp)与

zic3

突变体杂交，将得到的

zic3

+/-

(Tg flk1 ∶gfp)进行自交得到同窝对照，使用激光共聚焦显

微镜观察72 hpf胚胎血管发育表型(

=30)，利用RT-PCR检测相关基因表达。

结果

成功获得了

zic3

纯合突变体并经基因测序验证。与对照组相比，

zic3

纯合突变体存在明显的出血表型，且质膜膜泡关联蛋白(plasmalemma vesicle associated protein，Plvap)基因与zic2基因的表达量明显升高(

0.05)，但杂合突变体未观察到此表型。在杂合突变体中，后脑血管发育出现明显抑制，血管数量相比野生型降低60%(

0.05)，存在明显发育缺陷。同时非经典Wnt信号通路基因(

wnt5a

、

wnt7ba

、

wnt9b

)以及血管内皮生长因子(vascular endothelial growth factor，VEGF)通路基因(

kdrl

、

vegfab

、

ephrlnB2

)表达量均明显上升(

0.05)。

结论

在斑马鱼中

zic3

功能可被zic2进行代偿，

zic3

通过抑制

plvap

的表达降低胚胎脑血管通透性，通过抑制非经典Wnt信号和VEGF信号通路调控胚胎脑血管发育。

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 (

=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 associate

d protein (Plvap) gene and

zic2

gene as compared with the control (

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% (

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 (

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.

关键词

Keywords

references

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⁰

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

DOI：10.16016/j.1000-5404.202104042

摘要

Abstract

关键词

Keywords

references