The experimental research for contribution of insulin-like growth factor binding protein-2 during zebrafish embryonic cardiovascular development / 复旦学报（医学版）

ABSTRACT

Objective To establish a zebrafish IGFBP-2 gene knock-down model by morphilino modified antisense oligonucleotide injection, so as to investigate the abnormal phenotypes of heart and vessels in early stage of zebrafish development and the expression of zebrafish cardiogenesis related genes. Methods The spatiotemporal expression of IGFBP-2 gene in early stage of zebrafish development was testified by whole mount in situ hybridization with antisense RNA IGFBP-2 probe. The IGFBP-2 morpholino (IGFBP-2 MO) that especially inhibited the gene promoter and standard control morpholino (Con-MO) were designed and synthesized by Gene-tools Corporation. Four different concentration gradients (0.05, 0.10, 0.25 and 1.0 mmol/L) were set as IGFBP-MO injection groups with 0.25 mmol/L Con-MO injection group and wild type group as controls. Contribution to the incidence of heart abnormal phenotypes and mortality rate induced by 4 different IGFBP-2 concentrations injection group was recorded and compared with 2 control groups. Heart abnormal phenotypes at different developmental stages in 0.25 mmol/L IGFBP-2 injection group were observed in detail. To validate the effectiveness of IGFBP-2 MO, the expression of enhanced green fluorescence presented by wild type zebrafish embryos at 12hpf which received single injection of IGFBP-2 EGFP recombinant plasmid and those co-injected with Con-MO or IGFBP-2 MO were detected. To investigate the regulation relationship between IGFBP-2 gene and other cardiogenesis related genes, expression of atrium specific marker gene Amhc was detected in IGFBP-2 MO and wild type group by in situ hybridization. Ventricle specific green fluorescence of Vmhc-EGFP transgenic zebrafish embryos whose IGFBP-2 gene was knocked-down were compared with those untreated. Zebrafish peripheral vascular development in the IGFBP-2 MO group was also checked out by micro-angiography. Results Whole mount in situ hybridization revealed that IGFBP-2 gene expressed in turn at eyes, midbrain and then focused on liver in early stage of zebrafish development. The micro-injection of 0.25 mmol/L IGFBP-2 MO resulted in heart malformation in nearly 60% of all injected zebrafish embryos. Heart malformation phenotypes included slow heart beat, pericardial edema, weak ventricle systole contraction and heart tube looping disorder. Some of them represented atria dilation, blood regurgitation and ciculation obstruction. Wild type zebrafish embryos that received single injection of IGFBP-2 EGFP plasmid DNA or co-injected with Con-MO presented strong enhanced green fluorescence at 12hpf, meanwhile, the fluorescence was barely seen in the embryos co-injected with IGFBP-2 MO. This strongly validated the gene specific knock-down effect of IGFBP-2 MO. Amhc was down-regulated at 48hpf in IGFBP-2 MO group. Vmhc-EGFP transgenic zebrafish down-regulated by IGFBP-2 gene also resulted in attenuated expression of ventriclar-specific green fluorescence protein at 48hpf. Intersegmental blood vessels of IGFBP-2 MO group by micro-angiography at 60hpf demonstrated an sparsate and chaos image, which suggested that IGFBP-2 gene expression was involved in the regulation of normal vascular development. Conclusions Micro-injection of IGFBP-2 MO is an efficient way to knock-down IGFBP-2 gene in zebrafish embryos. IGFBP-2 gene expression down-regulation leads to heart and vessels maldevelopment and have an impact on the expression of cardiogenesis related genes of zebrafish embryos as well. In short, IGFBP-2 plays a critical role in the normal cardiovascular development of zebrafish embryos.