视黄酸缺乏对斑马鱼心脏房室分化的影响

Effect of Retinoic Acid Deficiency on the Cardiac Atrial-Ventricular Differentiation in Zebrafish Embryos

目的通过化学遗传学方法建立视黄酸缺乏的斑马鱼模型,探讨视黄酸缺乏对斑马鱼胚胎心脏前后轴发育即房室分化的影响。方法在斑马鱼胚胎孵育的5 hpf,用不同浓度梯度的视黄醛脱氢酶2抑制剂DEAB(1×10-6、5×10-6、10×10-6、25×10-6mol/L)处理斑马鱼胚胎,实时观察斑马鱼胚胎发育的全过程。通过给予斑马鱼胚胎外源性视黄酸,观察其对DEAB的拮抗作用。应用胚胎整体原位杂交观察视黄酸缺乏对心脏特异基因vmhc和amhc表达的影响。结果斑马鱼胚胎的生存率随着DEAB处理浓度的增加而降低,当DEAB浓度≥5×10-6mol/L时,斑马鱼的畸胎率达100%。5×10-6mol/L DEAB的致畸作用能够被1×10-9mol/L外源性视黄酸所拮抗。整体原位杂交结果显示视黄酸缺乏会导致斑马鱼胚胎心脏房室分化异常,表现为vmhc表达细胞的范围增大,amhc表达细胞的范围缩小。结论通过外源性DEAB处理能有效地建立视黄酸缺乏的斑马鱼模型,DEAB影响胚胎发育存在剂量依赖性。视黄酸在斑马鱼心脏前后轴发育过程中起重要调控作用,心脏发育早期视黄酸缺乏会抑制心房的发育而支持心室的发育,出现房室分化异常。

Objective To establish a zebra-fish model with retinoic acid（RA） deficiency by a chemogenetic approach, and to study the effect of RA deficiency on the cardiac atrial-ventricular differentiation and development along the anterior-posterior axis in zebra_fish embryos. Methods Zebrafish embryos were treated with 4-diethylaminobenzaldehyde （ DEAB）, an inhibitor of retinal dehydrogenase type 2, at various concentrations including 1×10^-6 , 5×10^-6 , 10×10^-6 , 25×10^-6 mol/L from 5 hours post fertilization. The effects of DEAB on the embryonic development were assessed under the Olympus dissection microscope. Exogenous retinoic acid was then added to detect the antagonistic effect against DEAB. The expression of specific cardiac genes, vmhc and amhc, were monitored by whole-mount in situ hybridization to demonstrate the cardiac atrial and ventricular dififfer entiation. Results The survival of zebrafish embryos declined with the increase of DEAB concentration. The percentage of abnormal embryos reached one hundred percent when the concentration of DEAB was higher than 5×10^-6 mol/L. 1×10^-9 mol/L of exogenous RA could eliminate the teratogenic effect of 5×10^-6 mol/L DEAB. The result of whole-mount in situ hybridization showed that the vmhc expression cell domain was expanded while amhc expression cell domain was reduced in DEAB-treated group. Conclusions A zebra-fish model of RA deficiency has been effectively established by exogenous DEAB treatment. The effect of RA deficiency on the zebrafish embryoic development is dose-dependent. The occurrence of RA deficiency at the early stage of cardiac development may act as a contributor to ventricular development, and an inhibitor to atrial development. RA plays a critical role in the cardiac differentiation along, the anterior-posterior axis.