Purpose: 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.
Methods: 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.
Results: 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.
Conclusion: 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.