Title

Studies on the disruption of heart, red blood cell, and vasculature development by pro-IGF-I E-peptide in fish

Date of Completion

January 2004

Keywords

Biology, Molecular|Agriculture, Fisheries and Aquaculture

Degree

Ph.D.

Abstract

E-peptides are produced from pre-pro-IGF-I by proteolytic cleavage after translation. Previous studies conducted in our laboratory showed that rainbow trout (rt) Ea4- and human (h) Eb-peptides possessed unique anti-tumor activities in established human and fish cancer cells. In this dissertation, I investigated the potential biological effects of E-peptide on fish embryonic development. Trout or human pro IGF-I E-peptide was introduced into newly fertilized fish, medaka (Oryzias latipes) or zebrafish ( Danio rerio) embryos. Embryos carrying the secreted form of E-peptide transgene showed abnormal heart, red blood cell and vasculature development as observed under a microscope. Three heart defective phenocopies were classified as group I, II, and III in medaka and group I and II in zebrafish. Group I defective embryos showed heart development arrested at cardiacmyocyte stage with trace amounts of red blood cells and incomplete vasculature developed on the yolk sac. Group II defective embryos showed heart development arrested at the heart tube stage, and few red blood cells and little vasculature development observed on the yolk sac. Group III defective embryos showed heart development arrested at the heart looping stage with a reduced amount of red blood cells and vasculature development. Red blood cells in defective embryos were stained by diaminofluorene (DAF) which revealed a drastic reduction of red blood cells in defective embryos, suggesting that, in addition to heart defects, embryonic red blood cell development may also be disrupted by the E-peptide. The heart defects in zebrafish embryos expressing rtEa4-peptide were further characterized by confocal microscopy, and 3D-images of heart from normal heart and defective heart were constructed. The temporal embryonic expression patterns of genes involved in heart development (GATA 5 and NKX2.5), hematopoiesis (GATA 1 and GATA 2), and vasculogenesis (VEGF) were determined during embryonic development in zebrafish, by reverse transcription-polymerase chain reaction (RT PCR). To quantify the expression levels of those genes in E-peptide electroporated embryos and normal embryos, quantitative real-time RT-PCR analysis was employed. Results of these studies revealed that overexpression of E-peptides as a transgene may disrupt embryonic heart development, hematopoiesis and vasculogenesis during fish embryonic development by lowering the mRNA levels of NKX2.5, GATA 5, GATA 1, GATA 2 and VEGF genes. ^