Doi Takashi, Puri Prem, Bannigan John, Thompson Jennifer
National Children's Research Centre, Our Lady's Children's Hospital, Dublin 12, Ireland.
Pediatr Surg Int. 2011 Feb;27(2):205-9. doi: 10.1007/s00383-010-2779-y.
Administration of heavy metal cadmium (Cd) after 60-h incubation induces omphalocele spectrum in the chick embryo. Although previous studies have shown that the earliest detectable histological changes in the chick Cd model occurs commencing at 4-h post-treatment (4H). However, the molecular mechanism by which Cd acts in the critical period of early embryogenesis still remains unclear. Zic3, a Gli superfamily transcription factor, is expressed in somites and plays an important role in vertebrate development, including somitogenesis and thus ventral body wall formation. Gli3 is also expressed in somites and interacts with Zic3 physically and functionally. It has been reported that Gli3 homozygous double mutants display omphalocele. Zic3 mutant mice have also been known to result in omphalocele phenotype. We designed this study to test the hypothesis that Gli3 and Zic3 gene expression is downregulated during the critical period of very early embryogenesis in the Cd-induced omphalocele in the chick model.
After 60-h incubation, chick embryos were exposed to either saline or 50 μM cadmium and divided into two groups: control and Cd (n = 24 for each group). Real-time reverse transcription polymerase chain reaction was performed to evaluate the relative mRNA expression levels of Gli3 and Zic3 in the Cd-induced omphalocele chick model. Differences between the two groups at each time point were analyzed statistically and the significance was accepted at p < 0.05. Immunohistochemistry was also performed to evaluate the expression/distribution of those proteins in chick embryo.
The relative mRNA expression level of Gli3 and Zic3 was significantly decreased in the Cd group at 4H when compared with controls (p < 0.05). However, there were no significant differences at the other time points. At 4H, immunoreactivity of GLI3 and ZIC3 was also markedly decreased in Cd-treated embryos, whereas strong expression of them was seen in the somite in controls.
We provide evidence, for the first time, that Gli3 and Zic3 gene expression is downregulated during the narrow window of very early embryogenesis in Cd chick model. Disruption of Gli3-Zic3 interaction in the critical period for ventral body wall formation may contribute to omphalocele phenotype in Cd chick model.
在孵化60小时后给予重金属镉（Cd）可诱导鸡胚出现脐膨出谱系。尽管先前的研究表明，在鸡Cd模型中最早可检测到的组织学变化始于处理后4小时（4H）。然而，Cd在早期胚胎发育关键期发挥作用的分子机制仍不清楚。Zic3是一种Gli超家族转录因子，在体节中表达，在脊椎动物发育中发挥重要作用，包括体节发生以及腹侧体壁的形成。Gli3也在体节中表达，并在物理和功能上与Zic3相互作用。据报道，Gli3纯合双突变体表现出脐膨出。已知Zic3突变小鼠也会导致脐膨出表型。我们设计本研究以检验以下假设：在鸡模型中，Cd诱导的脐膨出在非常早期胚胎发育的关键期，Gli3和Zic3基因表达下调。
孵化60小时后，将鸡胚暴露于生理盐水或50μM镉中，并分为两组：对照组和Cd组（每组n = 24）。进行实时逆转录聚合酶链反应以评估Cd诱导的脐膨出鸡模型中Gli3和Zic3的相对mRNA表达水平。对两组在每个时间点的差异进行统计学分析，p < 0.05时差异具有统计学意义。还进行了免疫组织化学以评估这些蛋白质在鸡胚中的表达/分布。
与对照组相比，Cd组在4H时Gli3和Zic3的相对mRNA表达水平显著降低（p < 0.05）。然而，在其他时间点没有显著差异。在4H时，Cd处理的胚胎中GLI3和ZIC3的免疫反应性也明显降低，而在对照组的体节中可见它们的强表达。
我们首次提供证据表明，在鸡Cd模型中，Gli3和Zic3基因表达在非常早期胚胎发育的狭窄窗口期下调。在腹侧体壁形成的关键期，Gli3 - Zic3相互作用的破坏可能导致鸡Cd模型中的脐膨出表型。
