Precise Dose of Folic Acid Supplementation Is Essential for Embryonic Heart Development in Zebrafish

Simple Summary Folic acid is an essential vitamin for human beings. It has become a consensus to supplement folic acid during pregnancy. It is reported that 15~20% of people in the world supplement folic acid excessively. We found that excessive folic acid supplementation or insufficient folic acid intake could lead to abnormal heart development in zebrafish embryos. We elucidated the mechanism of folic acid on early cardiac development for the first time. These results provide a scientific basis for the important reasonable supplement dose of folic acid. At the same time, we constructed zebrafish mutants with abnormal folate metabolism, which provide a novel biological model for the study of folate acid metabolism. Folic acid, one of the 13 essential vitamins, plays an important role in cardiovascular development. Mutations in folic acid synthesis gene 5,10-methylenetetrahydrofolate reductase (MTHFR) is associated with the occurrence of congenital heart disease. However, the mechanisms underlying the regulation of cardiac development by mthfr gene are poorly understood. Here, we exposed zebrafish embryos to excessive folate or folate metabolism inhibitors. Moreover, we established a knock-out mutant of mthfr gene in zebrafish by using CRISPR/Cas9. The zebrafish embryos of insufficient or excessive folic acid and mthfr(-/-) mutant all gave rise to early pericardial edema and cardiac defect at 3 days post fertilization (dpf). Furthermore, the folic acid treated embryos showed abnormal movement at 5 dpf. The expression levels of cardiac marker genes hand2, gata4, and nppa changed in the abnormality of folate metabolism embryos and mthfr(-/-) mutant, and there is evidence that they are related to the change of methylation level caused by the change of folate metabolism. In conclusion, our study provides a novel model for the in-depth study of MTHFR gene and folate metabolism. Furthermore, our results reveal that folic acid has a dose-dependent effect on early cardiac development. Precise dosage of folic acid supplementation is crucial for the embryonic development of organisms.