The Effect Of Folic Acid Deficiency On The Development Of Zebrafish Cardiovascular System

Folic acid , a member of the vitamin B family plays an essential role in embryonic development. Analysis of a large case-control study among women who took drugs which are folic acid antagonisms that inhibit dihydrofolate reductase (DHFR) during early pregnancy increases the risk of heart defects. The disturbance in folate metabolism leads to multiple malformations including cardiovasculr abnormality has been shown in different animal models. These animal models showed delayed heart development, fewer embryonic myocardium and reduced thickness of cardiac ventricular compact walls, and conotruncal heart defects. However, the underlying mechanisms involved in the malformation induced by folic acid deficiency remain unclear.1．Establishing the folic acid deficiency model in zebrafishTo investigate the underlying mechanism by which folic acid deficiency affects the development of cardiovascular system, we establish a folic acid deficiency zebrafish model by exposing zebrafish embryos to methotrexate (the folic acid antagonist) or knock-down dhfr by using morpholino antisense oligonucleotides (MO) . Phenotypes of the cardiovascular system in this folic acid deficiency model are stable, such as the various malformation in heart, the cardiac function damage, the out flow tract abnormal and the vascular formation disturbed. Taking advantage of this folic acid deficiency model, we carry out the following investigations.2．The effect of folic acid deficiency on expressions of cardiovascular development related genes(1) Folic acid deficiency leads to the decreased expressions of some cardiovascular development related genesWe find that folic acid deficiency has no effect on expressions of vmhc, amhc ,mef2a, and ephB4, but leads to the decreased expressions of hand2, mef2c,bmp2b, nkx2．5, has2 ,flk-1 and ephrinB2．This results indicates that folic acid deficiency may induces cardiovascular malformation by decreasing these related genes' expressions.(2) The interactions among hand2, mef2c, bmp2b, nkx2．5 and has2There are complicated interactions among hand2,mef2c,bmp2b,has2 and nkx2．5．We find that mef2c and bmp2b can regulate the hand2 expression, and hand2 can affect expressions of both nkx2．5 and has2．Folic acid deficiency may disturb the above network to induce cardiac malformation.(3) The interaction between dhfr and hand2 during zebrafish cardiac developmentIn this study, we find that dhfr regulates hand2 expression during the heart development. dhfr knock-down induces the cardiac malformation by down regulating hand2 expression. hand2 over-expression can rescue the cardiac malformation phenotype of dhfr knock-down embryos, and this results can provide a clue to the study of genetic therapy on the congenital malformation induced by folic acid deficiency.3．The effect of folic acid deficiency on Hedgehog and Nodal passwayWe detect expressions of foxa2 and ptc1 (down-stream factors of Hedgehog passway), ntl and gsc (down-stream factors of Nodal passway) to investigate the possible reason by which folic acid deficiency induces cardiac malformation and dystopia. Our results show that folic acid deficiency disturbs Hedgehog passway but has no evident effect on Nodal passway. Our results also show that folic acid deficiency disrupts the development of mesoderm.4．Folic acid deficiency and apoptosisFolic acid deficiency can not only affect the gene expressing level, it also can affect the stability of DNA chains. In this part, we detect the apoptosis in embryos by using TUNEL methods. The results show that folic acid deficiency can induces the DNA chains broken then leads to embryonic over apoptosis.