The Mechanisms Of Ethanol-mediated Early Developmental Toxicity In Zebrafish

It has been demonstrated that maternal drinking during pregnancy had serious adverse effects on the health of the newborns. Fetal alcohol syndrome (FAS) is the most important developmental abnormality caused by maternal alcohol abuse during pregnancy. Clinically, it is characterized by head and facial abnormalities, cardiovascular malformation, and permanent nervous system damage. A lot of experimental models have been developed to study the effect of ethanol on embryonic development, but different models have different advantages and shortcomings. As an ideal vertebrate model system, zebrafish have developmental processes which share high evolutionary conservation with humans. The transparency of the embryos and their rapid development make zebrafish widely used in the study of developmental genetics and environmental toxicology. Since the pioneering work of Laale, it is known that ethanol can induce cyclopia (fusion of the two eyes) in zebrafish. Besides, there are many other complex phenotypes manifested in ethanol-treated zebrafish embryo, including pericardial edema, circulatory defect, axial malformation, bilateral liver, and abnormal development of forebrain. By now the underlying mechanisms involved in ethanol-mediated developmental toxicity remain unclear.1. Ethanol Disrupts Cardiovascular Development in Zebrafish EmbryosCongenital cardiopathy and cardiovascular defects appear in up to 50% of FAS children. It is characterized by atrial septal defect, ventricular septal defect, lung artery agenesis, and aortic stenosis. However, the FAS-associated congenital cardiopathy should be confirmed by cardiac catheterization clinically, which makes the final diagnosis difficult. Thus, relatively few attentions have been paid to the FAS-associated congenital cardiopathy for a long period of time. In our study, we demonstrated that ethanol of gradient concentrations can interfere with the establishment of circulatory system in embryonic zebrafish. Theeffective concentration to cause 50% malformations (EC50) was 182.5 mmol/L. The ethanol pulse exposure experiment displayed that dome stage during embryogenesis is the sensitive time window to ethanol. It is found that 400 mmol/L ethanol pulse exposure can induce circulatory defects in 43% treated embryos. We ruled out the possibility that ethanol can interfere with the process of hematopoiesis in zebrafish. By employing in situ hybridization with endothelial biomarker (Flk-1), we revealed that ethanol disrupts the establishment of trunk axial vasculature, but has no effect on cranial vessels. Combined with the results of semi-thin histological sections, the in situ hybridization experiments with arterial and venous biomarkers (ephrinB2, ephB4) suggested that ethanol mainly interrupts the development of dorsal aorta while has little effect on axial vein. Further study indicated the negative influence of ethanol on the development of hypochord in zebrafish. The consequent lack of vasculogenic factors including Radar and Ang-1 partly explains the defects in formation and integrity of dorsal aorta. These results provide important clues to the study of adverse effects of ethanol on the cardiovascular development in human fetus.2. Proteomic analysis of zebrafish embryos at early developmental stageProteomes of zebrafish embryos at early stages, including zygote stage, dome stage, shield stage and 5-somite stage, were separated by two-dimensional electrophoresis(2-DE). The optical information of silver-stained gels was captured by the Imagescanner instrument, and submitted to Image Master 2D software. 226 protein spots were detected at zygote stage, 307 spots at dome stage, 327 spots at shield stage, and 574 spots at 5-somite stage. It is shown that there is only a little increment of protein spots in the 2D maps from zygote stage to shield stage. To 5-somite stage, the protein spots showed a rapid increment, which suggests that the protein abundance in embryos is closely related to morphogenesis and organogenesis. 48% of early embryonic proteins still exist in the proteome of embryos at 5-somite stage, which indicates the parent proteins contribute greatly to the embryonic development of zebrafish. Furthermore, three high-abundance protein spots found in all four different developmental stages were selected for mass spectrumanalysis. The results showed that all of the three spots are identified to be different proteolytically cleaved fragments of vitellogenin. We made an improvement on the technology related to the proteomic analysis of embryonic zebrafish, and established the 2-DE maps of zebrafish embryos at early stages. It has provided an important platform for the further research of functional proteomics of zebrafish.Separate the total proteins of normal control and ethanol-treated embryos at 12hpf by using 2-DE, and then use Image Master 2D software to analyze the difference between the 2D images of these two groups. We found three protein spots were downregulated and seven spots upregulated in ethanol-treated groups. The three downregulated proteins were identified by mass spectrometry to be proenkephalin, collagen 2al, and TAR DNA binding protein. It provides an important clue to elucidate the mechanisms of ethanol-induced teratology.3. Ethanol interferes with Nodal signaling pathway We demonstrated that the ethanol-induced axial malformation is due to its influence on the development of axial mesendoderm, while the cyclopia is due to the migrational defect of prechordal plate caused by ethanol. We also validated the result obtained from 2-DE analysis by in-situ hybridization, and found that ethanol impairs the expression of col2a1 mRNA in axial mesendoderm. It is found that ethanol can also disrupt the left-right laterality, and this phenomenon is considered to be triggered by Nodal signaling influence. To further test the hypothesis, we injected Nodal mRNA into zygote to induce the ectopic expression of downstream genes—gsc and ntl, and examined whether the ectopic expression of gsc and ntl induced by Nodal signaling could be impaired by ethanol. From the results, we demonstrated that Nodal signaling pathway was involved in the ethanol-induced teratogenic effect on zebrafish embryos. Previous research reported that ethanol was able to decrease the shh expression in the developing embryos, but there was no experiment which could prove that ethanol acts directly on the shh signaling transduction. We injected shh mRNA into zygote to induce the ectopic expression of downstream genes—foxA2 and nkx2.2a, and examined whether the ectopic expression of foxA2 and nkx2.2a induced by shh signaling could be impaired by ethanol. From the results, we demonstrated that ethanol barely interfered with the shh signaling pathway directly.The long-range effect of Nodal signaling is crucial for maintaining the Nodal concentration gradient in the blastoderm. Sqt participates in the long-range secretion, while cyc is in charge of short-range secretion. We found that ethanol has different interferential effect on the two different Nodal signaling pathways. Sqt or cyc mRNA was injected into a single cell of 128-cell-stage zebrafish embryos, and the injected embryos were subsequently submitted to ethanol treatment. From the results, we demonstrated that ethanol mainly impacts on the activity range of sqt, but has little effect on that of cyc. Since ethanol disrupts the long-range effect of Nodal signaling, the Nodal concentration gradient fails to be established well in the blastoderm, which leads to malformation of mesendoderm and its auxiliary organs.