Objective To establish a model of retinal dehydrogenase type 2 (raldk2) gene knock-down zebrafish embryos, and to explore the effect of insufficient retinoid acid ( RA) signaling on the early embryonic cardiac development, especially on the regulation of cardiac development-related nppa and tbx20 gene expression. Methods A morpholino anti-sense oligonucleotides (MO) targeting zebrafish raldh2 gene was designed to knock down its expression and constructed a raldh2-EGFP plasmid to verify the effectiveness and specificity of raldh2-MO. The embryonic heart phenotype and cardiac function were analyzed and compared between wild-type and raldh2-MO groups. Then, the expression pattern of nppa and (6x20 genes in the zebrafish embryos, and the regulation of their expression when raldh2 gene was knocked down, were determined to elucidate the underlying mechanism of RA signaling in the cardiac development. Results raldh2-MO microin-jection effectively knocked down the raldh2 gene expression, and the mortality and abnormal embryo rate rose with the increase of raldh2-MO dosage. Raldh2-MO embryos exhibited abnormal cardiac phenotype, including tubular heart, incom-plete D-loop, abnormal atria and ventricle development, blood regurgitation, and cardiac dysfunction. The results of whole-mount in situ hybridization of nppa and tbx20 probes revealed that nppa expression was obviously reduced in the atria, while tbx20 expression was down-regulated in the heart, especially in cardiac outflow tract and atria in the fish of raldh2-MO group. Conclusions raldh2 gene plays a critical role in several key stages of cardiac development and may interact with cardiac related transcription factors to regulate cardiogenesis and development. The expression of nppa and (6x20, which play important roles during embryogenesis, can be regulated by RA signaling during cardiac development, however, the underlying mechanism still needs further investigation.%目的 通过显微注射吗啡啉修饰的反义寡核苷酸(MO)阻抑视黄醛脱氢酶2(raldh2)基因表达,探讨raldh2基因阻抑对斑马鱼胚胎心脏发育的影响及可能的分子机制.方法 根据斑马鱼raldh2基因起始密码区域序列设计合成吗啡啉修饰的反义寡核苷酸,采用显微注射方法阻抑斑马鱼胚胎raldh2基因表达.构建raldh2-EGFP重组质粒进一步验证MO的特异性和有效性.分析raldh2基因阻抑后对胚胎发育,尤其心脏表型和功能的影响.通过胚胎整体原位杂交,分析心脏相关nppa和tbx20基因表达模式以及raldh2阻抑后对其表达的影响.结果显微注射raldh2-MO能有效地特异地阻抑斑马鱼胚胎raldh2基因表达,raldh2-MO对胚胎发育影响呈剂量依赖性.raldh2基因阻抑可导致胚胎心脏发育畸形,干扰正常的房室分化和向右环化,导致房室瓣血液反流.与野生型胚胎比较,raldh2基因阻抑组胚胎心率和心室收缩分数降低(P＜0.05),心功能受损.整体原位杂交结果显示raldh2基因阻抑后nppa基因表达改变,心室部位nppa表达清晰,而心房部位表达减弱.tbx20基因在心脏、运动神经元、顶盖及视网膜表达,raldh2基因阻抑后,tbx20表达下调,在心脏表达减弱,以心房和流出道部位更显著.结论raldh2基因在心脏早期发育的多个环节发挥重要作用,影响房室分化、心管环化和心肌收缩等.在心脏发育过程中nppa和tbx20基因表达受到raldh2基因调控,可能参与RA信号缺乏导致心脏畸形的潜在分子机制.
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