Krox20 is down-regulated following triazole in vitro embryonic exposure: a polycompetitor-based assay

This study was conducted in order to analyse gene-expression alterations in rat embryos following exposure to triazoles, using an easy-handling approach. Triazole derivatives have been shown to alter the morphology of cranio-facial structures and to induce abnormalities in hindbrain patterning and neural crest cell migration. Specification of hindbrain segments is regulated by retinoic acid and the hox code. Krox20 was chosen as molecular marker for its specific distribution in the anterior neural tube. In fact, this zinc-finger protein is expressed in rhombomere 3 and 5. Mis-regulation of Krox20 levels have shown to induce severe alterations in the correct patterning of the rhomboencephalon and the derived structures. In order to analyse Krox20 mRNA levels in rat embryos exposed in vitro to the triazole derivative triadimefon, a semi-quantitative approach utilising the competitive RT-PCR was chosen. A lambda phage-based plasmid construct that could compete with target and internal standard gene at the same time during enzymatic reaction was generated. Results were confirmed by real-time RT-PCR analysis on the same samples. Our data show a down-regulation of Krox20 transcript levels after exposure to the triazole derivative, implying a key role of this molecule in the pathogenic pathway induced by triazole exposure.