PRELIMINARY STUDIES ON THE MECHANISM OF BLOOD CIRCULATION DISTEMPEREDNESS IN GYNOGENETIC HAPLOID EMBRYOS OF GOLDFISH, CARASSIUS AURATUS

Haploidy refers to the presence of only one set of chromosomes in a cell. In vertebrates, haploid cells can be cultivated in vitro and proliferate continuously, but artificially induced fish and amphibian haploid embryos show lethal “haploid syndrome”, and its mechanisms remain incompletely understood. The developmental defect of the circulatory system is the common morphological features in haploid fish embryos. Gynogenetic haploid embryos were produced when eggs were fertilized by UV-irradiated sperm of common carp. Live analysis of embryonic development showed that the blood circulation of goldfish gynogenetic haploid embryos had different phenotypes at the 65% otic vesicle closure (OVC) stage. We sort embryos into three categories: A embryos have normal blood circulation; B embryos have poor blood circulation; and C embryos have no blood circulation. The proportion of B embryos was significantly higher than that of the other two categories. The results of o-dianisidine staining showed that all goldfish gynogenetic haploid embryos contained hemoglobin, but the staining area and intensity of hemoglobin were lower than those of diploid embryos in the same period. These results suggested that goldfish gynogenetic haploid embryos had different degrees of blood circulation distemperedness and erythropoiesis defects. In order to further study the molecular mechanism of developmental defects in circulatory system of goldfish gynogenetic haploid embryos, the expression patterns of scl (stem cell leukemia), gata-1 (gata binding protein-1) and flk-1 (fetal liver kinase-1) during goldfish embryogenesis were compared in gynogenetic haploid and inbred diploid embryos by antisense RNA whole-mount in situ hybridization. Like inbred diploid embryos, the expression of scl and gata-1, the key regulatory genes required for primitive hematopoiesis, could be detected in the posterior lateral-plate mesoderm (PLM) of gynogenetic haploid embryos during segmentation stages. However, at the 14-somite stage, scl and gata-1 expression in PLM shortened along the anterior-posterior axis of haploid embryos. From the 20-somite stage to the 25% OVC stage, scl and gata-1 expression in haploid embryos was reduced in the trunk domain and absent from the midline, which indicated that scl and gata-1 expressing PLM cells failed to migrate towards the midline and the formation of ICM was interfered. At 25% OVC stage, the expression pattern of vascular endothelial marker gene flk-1 was also different between gynogenetic haploid and inbred diploid embryos. In haploid embryos. expression of flk-1 was reduced in the trunk domain, the dorsal aorta and the posterior cardinal vein were structurally disordered and indistinguishable, the intersegmental vessels were arranged vertically with the primitive axis vessels and decreased in number, which could be lost on one side of the trunk domain or completely in the trunk domain. These results suggested that severe developmental defects of ICM and primitive trunk axial vessels caused by PLM abnormal migration to the midline, the decrease of erythrocyte differentiation and peripheral vascular dysplasia may be important reasons for poor blood circulation in goldfish gynogenetic haploid embryos. In addition, live analysis of embryonic development showed that haploid embryos had a range of gastrulation defects. Compared with diploid embryos, initiation and progression of epiboly were delayed in goldfish gynogenetic haploid embryos, which reached bud stage with a 30min-1h delay and had a comparatively shorter body axis. RNA in situ analysis showed that there were severe defects of convergence and extension movement in goldfish gynogenetic haploid embryos. Compared with diploid embryos, the expression region of prechordal plate marker gene gsc (Goosecoid) in haploid embryos was closer to the plant pole at 95%-epiboly stage, and the expression domains of N-cadherin (Neural cadherin) representing the neural plate and mesoderm on the dorsal of the haploid embryos was significantly wider at the 2-somite stage. These results showed that there were severe cell movement defects in gastrulation of goldfish gynogenetic haploid embryos. Combined with the aberrant migration of PLM cells to the midline during segmentation stages, we speculated that extensive abnormal cell movement and migration during embryogenesis may be an important reason for developmental malformations in goldfish gynogenesis haploid embryos.