title: Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development.
abstract: 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.
authors.affiliation: Children's Hospital, Fudan University, Shanghai 200032, China.
authors.forename: Shu-na
authors.initials: SN
authors.lastname: Sun
authors.forename: Yong-hao
authors.initials: YH
authors.lastname: Gui
authors.forename: Yue-xiang
authors.initials: YX
authors.lastname: Wang
authors.forename: Lin-xi
authors.initials: LX
authors.lastname: Qian
authors.forename: Qiu
authors.initials: Q
authors.lastname: Jiang
authors.forename: Dong
authors.initials: D
authors.lastname: Liu
authors.forename: Hou-yan
authors.initials: HY
authors.lastname: Song
mesh_terms: 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
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D013762:Tetrahydrofolate Dehydrogenase; D029961:Zebrafish Proteins; C410915:hand2 protein, zebrafish