The Experimental Research For Contribution Of Insulin-like Growth Factor Binding Protein-2 During Zebrafish Embryonic Cardiovascular Development

The Insulin-like Growth Factor Binding Proteins(IGFBPs) constitute a secretary protein family of evolutionarily highly-conserved proteins which serve an endocrine function by regulating half-life time of circulating IGFs and modulating cellular responses to IGF signaling. IGFBPs genes abundantly expressed in many tissues and organs during embryonic developmental stage, which may suggested to play a critical role in the process of embryogenesis. It has been validated that IGFBPs are required for the induction of special cells strain into mature cardiomyocytes or vascular endothelial cells in vitro and IGFBP-4 is also a positive regulator to embryonic cardiogenesis of Xenopus in vivo. Further investigation to more precise function of IGFBPs in the process of embryonic cardiovascular system development can surely get a better understanding to the mystery of human born defect, particularly to the pathogenesis and molecule mechanisms of Congenital heart disease, thus lead us to some brand new ideas and more effective ways to prevent and manage this life-threatening disease.In recent years, zebrafish has emerged as an ideal animal model for studying vertebrate development and it is especially suitable for early stage development of cardiovascular system. First, gene expression map of zebrafish has already been identified and the development process of its cardiovascular system shared great similarity with mammals and human. Second, embryos of this species fertilize and develop externally, this optical clarity permits easy and real-time visualization of heart development; More importantly, zebrafish embryos are almost independent on well-developed and fully-functional cardiovascular system at early developmental stage. Without blood circulation even heart beat, zebrafish embryos can still take enough oxygen by passive diffusion to survive and continue to develop in a relatively normal way in the first week time. These unique advantages make zebrafish an outstanding model animal for the investigation to embryonic cardiovascular development and cardiogenesis. 复旦大学博士学位论文英文摘要PartⅠThe Evolutional Conservatism of Insulin-like Growth Factor Binding Protein-2 & The Spatiotemporal Expression of IGFBP-2 Gene during Early Stage of Zebrafish Embryonic DevelopmentTo prove the structure and function of IGFBP-2 gene and protein are extremely conserved during biological evolutionary process by bioinformatics method. Whole mount in situ hybridization with antisense RNA probe revealed the spatiotemporal expression pattern of IGFBP-2 gene in early stage of zebrafish embryonic development. IGFBP-2 gene expressed in turn at eyes, central nervous system (mainly at midbrain) and primitive liver. After 72hpf, IGFBP-2 gene predominantly expressed at zebrafish embryos'livers. There was no IGFBP-2 gene expression at local developing cardiovascular system.PartⅡEstablish IGFBP-2 Gene Morpholino Knock-down Zebrafish Model & The Heart Abnormal Phenotype Induced by IGFBP-2 Gene Down-regulationTo investigate the impact of IGFBP-2 gene down-regulation on the development of zebrafish embryonic heart and vasculature, well designed and synthesized morpholino modified antisense oligonucleotide which specific inhibits the initiating site of IGFBP-2 gene translation was microinjected into zebrafish embryos at one to four cells stage to block the IGFBP-2 gene expression. Four different concentration gradients:0.05,0.10,0.25 and 1.0mmol/L were set as IGFBP-2 MO injection groups with 0.25mmol/L Standard Control Morpholino (Con-MO) injection group and Wild type(Wt) group as controls.Contribution to the incidence of heart abnormal phenotypes and mortality rate induced by 4 different IGFBP-2 concentration injection group was recorded and compared with 2 control groups. The hearts of zebrafish embryos in IGFBP-2 MO group had defects in cardiac morphology and contractility in an IGFBP-2 Morpholino dose-dependent manner.0.25mmol/L concentration of IGFBP-2 MO microinjection resulted in heart malformation in nearly 60% of all injected zebrafish embryos, the abnormal phenotypes included slow heart beat, pericardial edema, weak systolic ventricle contraction, heart tube looping disorder; some of them represented atria dilation, blood regurgitation and ciculation obstruction.To validated the knocking-down effectiveness of IGFBP-2 MO, IGFBP-2 EGFP recombinant plasmid was microinjected solely or coinjected with IGFBP-2 MO and Con-MO, respectively.Wt zebrafish embryos that received single injection of IGFBP-2 EGFP recombinant plasmid or coinjection with Con-MO presented strong enhanced green fluorescence at 12hpf, meanwhile embryos coinjected with IGFBP-2 MO revealed that the EGFP expression was greatly attenuated. This strongly validated the gene specific knock-down effectiveness of IGFBP-2 MO.PartⅢIGFBP-2 gene MO knock-down influenced the Atrium specific gene expression, Peripheral angiogenesis & Ventricle specific green fluorescence of Vmhc-EGFP transgenic zebrafishSince IGFBP-2 gene knocking-down has great impact on the development of zebrafish embryonic cardiovascular system, we suppose that the cardiogenesis related gene and vascular development should also be interfered. In agreement with this notion, further investigation was carried on.Amhc was down-regulated at 48hpf in IGFBP-2 MO group. IGFBP-2 gene down-regulation to Vmhc-EGFP transgenic zebrafish also resulted in attenuated expression of ventricle specific enhanced green fluorescence at 48hpf. Intersegmental blood vessels of the IGFBP-2 MO group by microangiography at 60hpf demonstrated an sparsate and chaos image, suggesting IGFBP-2 also participate in normal peripheral vascular development of zebrafish embryo.