Cardiac related tbx1, tbx5 and tbx20 gene expression are down-regulated during heart development in raldh2 knock-down zebrafish embryos

  • Dr Jia Hou, Children`s Hospital of Fudan University, China
  • Yonghao Gui, Cardiovascular Center, Children's Hospital of Fudan University, Shanghai, China
  • Dr Yuexiang Wang, Shanghai Medical School & Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, China
  • Miss Houyan Song, Shanghai Medical School & Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, China

    • Objectives:
    To set up a model of retinal dehydrogenase type 2 (raldh2) gene knock-down zebrafish embryos, and to study the effect of insufficient retinoid acid (RA) signaling on the early embryonic cardiac development, especially on the regulation of cardiac related tbx1, tbx5 and tbx20 gene expression.

    Methods:
    We designed morpholino antisense oligonucleotides (MO) targeting zebrafish raldh2 gene to knock-down its expression and constructed raldh2-EGFP plasmid to verify the effectiveness and specificity of raldh2-MO. The embryonic cardiac phenotype and function were analyzed and compared between wild-type and raldh2-MO group. And we described the expression pattern of tbx1, tbx5 and tbx20 gene in zebrafish embryos, and the regulation of their expression when raldh2 gene was knocked down to elucidate the underlying mechanism of RA signaling in the cardiac development..

    Results:
    Raldh2-MO microinjection could effectively knock-down raldh2 gene expression, the mortality and abnormal embryo rate rose with the increase of raldh2-MO dosage. raldh2-MO embryos exhibit abnormal cardiac phenotype, including swollen pericardial cavity, tubular heart, incomplete D-loop, abnormal atria and ventricle development, blood regurgitation, slow blood flow and weak heart beat. The results of whole-mount in situ hybridization of tbx1, tbx5 and tbx20 probes revealed that these genes had distinct expression patterns, but all of them expressed in the embryonic heart. To some extent, the expression of tbx5, tbx1 and tbx20 was reduced in raldh2 knock-down embryos.

    Conclusions:
    The RA deficiency zebrafish embryo model can be effectively established by raldh2-MO microinjection. RA signaling plays a critical role in several key stages of cardiac development and may interact with cardiac related transcriptional factors to regulate cardiogenesis and development. The expression of tbx1, tbx5 and tbx20, which play important roles during embryogenesis, can be regulated by RA signaling during cardiac development, and the underlying mechanism still needs further investigation.