Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development.
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. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed 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 or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-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 was obviously 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-down embryos.


CONCLUSIONS
DHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.
Sun
Shu-na
SN
Children's Hospital, Fudan University, Shanghai 200032, China.
Gui
Yong-hao
YH
Wang
Yue-xiang
YX
Qian
Lin-xi
LX
Jiang
Qiu
Q
Liu
Dong
D
Song
Hou-yan
HY
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000502:physiology; D005260:Female; D006321:Heart / Q000196:embryology*; D006330:Heart Defects, Congenital / Q000209:etiology*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000502:physiology*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics* / Q000502:physiology
D051792:Basic Helix-Loop-Helix Proteins; D013762:Tetrahydrofolate Dehydrogenase; D029961:Zebrafish Proteins; C410915:hand2 protein, zebrafish