Background Folic acid is very important for embryonic development and dihydrofolate reductase is one of the keyenzymes in the process of folic acid performing its biological function.Therefore,the dysfunction of dihydrofolatereductase can inhibit the function of folic acid and finally cause the developmental malformations.In this study,weobserved the abnormal cardiac phenotypes in dihydrofolate reductase(DHFR)gene knock-down zebrafish embryos,investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2(HAND2)and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations.Methods Morpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR orHAND2.Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpressHAND2.The cardiac morphologies,the heart rates and the ventricular shortening fraction were observed and recordedunder the microscope at 48 hours post fertilization.Whole-mount in situ hybridization and real-time PCR were performedto detect HAND2 expression.Results DHFR or HAND2 knock-down caused the cardiac malformation in zebrafish.The expression of HAND2 wasobviously reduced in DHFR knock-down embryos(P<0.05).Microinjecting HAND2 mRNA into fertilized eggs can induceHAND2 overexpression.HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-downembryos.Conclusions DHFR plays a crucial role in cardiac development.The down-regulation of HAND2 caused by DHFRknock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.Chin Med J 2007;120(13):1166-1171 Background Folic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Before this, the dysfunction of dihydrofolatereductase can inhibit the function of folic acid and finally cause the developmental malformations.In this study, weobserved the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos, investigated the effect of DHFR on the expression of heart and neural crest organs expressed transcript 2 (HAND2) and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations. Methods Morpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or AND2.Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpressHAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recor dedunder the microscope at 48 hours post fertilization. Well-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression. Results DHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 wasobviously reduced in DHFR knock- down embryos (P <0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-downembryos. Conclusions DHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFRknock -down is the possible mechanism of DHFR knock-down inducing the cardiac malformation. Chin Med J 2007; 120 (13): 1166-1171