VEGF-B-Neuropilin-1 signaling is spatiotemporally indispensable for vascular and neuronal development in zebrafish

Lasse D Jensen,Masaki Nakamura,Lars Bräutigam,Xuri Li,Yizhi Liu,Nilesh J Samani,Yihai Cao

doi:10.1073/pnas.1510245112

Abstract

Physiological functions of vascular endothelial growth factor (VEGF)-B remain an enigma, and deletion of the Vegfb gene in mice lacks an overt phenotype. Here we show that knockdown of Vegfba, but not Vegfbb, in zebrafish embryos by specific morpholinos produced a lethal phenotype owing to vascular and neuronal defects in the brain. Vegfba morpholinos also markedly prevented development of hyaloid vasculatures in the retina, but had little effects on peripheral vascular development. Consistent with phenotypic defects, Vegfba, but not Vegfaa, mRNA was primarily expressed in the brain of developing zebrafish embryos. Interestingly, in situ detection of Neuropilin1 (Nrp1) mRNA showed an overlapping expression pattern with Vegfba, and knockdown of Nrp1 produced a nearly identically lethal phenotype as Vegfba knockdown. Furthermore, zebrafish VEGF-Ba protein directly bound to NRP1. Importantly, gain-of-function by exogenous delivery of mRNAs coding for NRP1-binding ligands VEGF-B or VEGF-A to the zebrafish embryos rescued the lethal phenotype by normalizing vascular development. Similarly, exposure of zebrafish embryos to hypoxia also rescued the Vegfba morpholino-induced vascular defects in the brain by increasing VEGF-A expression. Independent evidence of VEGF-A gain-of-function was provided by using a functionally defective Vhl-mutant zebrafish strain, which again rescued the Vegfba morpholino-induced vascular defects. These findings show that VEGF-B is spatiotemporally required for vascular development in zebrafish embryos and that NRP1, but not VEGFR1, mediates the essential signaling.

Highlights

Physiological functions of vascular endothelial growth factor (VEGF)-B remain an enigma, and deletion of the Vegfb gene in mice lacks an overt phenotype
In addition to tyrosine kinase receptors (TKRs), various heparinbinding isoforms of each member in the VEGF family have been reported to bind to neuropilins (NRPs), which is crucial for angiogenesis, lymphangiogenesis, axon guidance, cell survival, migration, and invasion [11,12,13,14]
There are five structurally and functionally related members in the VEGF family, which includes VEGF-A, -B, -C, and -D and placental growth factor (PlGF) [4]. These factors bind primarily to three membrane tyrosine kinase receptors (TKRs), i.e., VEGFR1, VEGFR2, and VEGFR3, to display their biological functions [4]. According to their receptor-binding patterns and biological functions, members of the VEGF family are divided into three subgroups: (i) VEGFA as the VEGFR1- and VEGFR2binding ligand [5]; (ii) VEGF-B and PlGF that exclusively bind to VEGFR1 [4, 6,7,8]; and (iii) VEGF-C and VEGF-D as VEGFR3and VEGFR2-binding ligands [9]

Summary

Introduction

Physiological functions of vascular endothelial growth factor (VEGF)-B remain an enigma, and deletion of the Vegfb gene in mice lacks an overt phenotype. Gain-of-function by exogenous delivery of mRNAs coding for NRP1-binding ligands VEGF-B or VEGF-A to the zebrafish embryos rescued the lethal phenotype by normalizing vascular development. Independent evidence of VEGF-A gain-of-function was provided by using a functionally defective Vhl-mutant zebrafish strain, which again rescued the Vegfba morpholino-induced vascular defects These findings show that VEGF-B is spatiotemporally required for vascular development in zebrafish embryos and that NRP1, but not VEGFR1, mediates the essential signaling. For the first time to our knowledge, that downregulation of VEGF-B in zebrafish but not in mouse embryos produces a lethal phenotype owing to vascular defects These findings indicate that different species use distinct mechanisms by the same factor for vascular development. Differential targeting of the same VEGF-B in pathological and physiological angiogenesis may be potentially achieved by understanding spatiotemporal mechanisms of VEGF-B in relation to VEGF-A

Methods

Results

Conclusion