【摘要】 目的观察不同浓度外源性视黄酸对斑马鱼早期胚胎和心血管系统发育的影响,为进一步研究视黄酸影响斑马鱼心脏前后轴(A-P轴)发育的分子机制提供形态学依据。方法选择斑马鱼胚胎孵育的3,6,9·5,12h四个时间点,用不同浓度视黄酸(1×10-6,1×10-7,4×10-8,1×10-8mol/L)处理斑马鱼胚胎,在解剖显微镜下实时观察斑马鱼胚胎心脏发育的全过程和视黄酸对斑马鱼心脏发育的影响。并采用胚胎整体原位杂交技术观察flk-1mRNA在斑马鱼胚胎的表达。结果1×10-6mol/L视黄酸可导致斑马鱼胚胎表现出多系统的严重畸形,胚胎很快死亡。在胚胎孵育的9·5、12h给与10-7~10-8mol/L浓度的视黄酸,胚胎只表现出心血管系统的畸形,其他系统无明显异常。胚胎整体原位杂交显示视黄酸对flk-1mRNA在斑马鱼胚胎血管的表达没有影响。结论视黄酸影响斑马鱼胚胎心脏发育有剂量依赖性和严格的时间窗,视黄酸影响心脏前后轴发育的关键时间是原肠胚晚期。视黄酸处理组胚胎的循环缺陷主要为心脏发育异常所致。10-7~10-8mol/L浓度视黄酸在9·5、12h处理斑马鱼胚胎可以作为研究心脏发育调控机制的动物模型。更多还原

【Abstract】 Objective In order to further provide morphological evidence for the molecular mechanism of cardiac development pattern along anterior-posterior axis, the effect of exogenous retinoic acid (RA) on the cardiovascular development of zebrafish embryos was investigated. Methods Zebrafish embryos were treated with RA at various concentrations including 1×10~ -8 , 4×10~ -8 , 1×10~ -7 , 1×10~ -6 mol/L et al. The effects of RA on the cardiac development at different stages from 3hpf to 12hpf was assessed under the Olympus dissection microscope. The expression of flk-1 mRNA in the developing vessels was monitored by whole-mount in situ hybridization. Results Zebrafish embryos were treated with 1×10~ -6 mol/L RA at various stages from 3hpf to 12hpf. These embryos showed shorter length, abnormal somite, no eye and no otic vesicle, in addition, cardiac rhythm was slower than that in untreated embryos. These embryos died very soon. The embryos treated at a dose of 10~ -8 mol/L～10~ -7 mol/L RA, also exhibited similar anomalities to those in the 1×10~ -6 mol/L RA treated group, but less serious and the mortality was reduced. By adding 10~ -7 ～10~ -8 mol/L RA at as early as 9.5 hpf and 12 hpf, only heart tube development was affected, without interfering with the rest of embryo. Whole-mount in situ hybridization with flk-1 showed no difference between the control and treated groups. Conclusion The effect of RA on zebrafish embryo is both dose-dependent and stage-dependent. The time window of RA is critical. Zebrafish heart tube is established during gastrulation, and the time period in which RA can affect the normal anterior-posterior axis polarity is also the end of gastrulation. Hemodynamic changes in the embryo are caused by the heart inefficiency rather than the vascular abnormality. Zebrafish embryo treated with 10~ -7 ～10~ -8 mol/L RA at the end of gastrulation or the early segmentation can be used as an experimental animal model to explore the mechanisms of cardiac A-P pattern. It may also provide further details regarding the gene expression pattern in the vertebrate heart development.更多还原