Molecular cloning and gene expression of mummichog (Fundulus heteroclitus) Runx2 during embryogenesis

In our previous study, we clarified the toxicity of 2,2'-dipyridyldisulfide [(PS)2], one of photodegradation products of a metal pyrithione that is used as an alternative antifouling paint biocides to organotin compounds in Japan. In early life stage toxicity tests, we exposed the mummichog, (Fundulus heteroclitus) to (PS)2, and the hatched larvae subsequently displayed notochord undulations and skeletal deformities ( Mochida et al., 2012 ). Runx2, a transcription factor of the runt family, is a key regulator in skeletal development in mammals. It is possible that (PS)2 inhibits Runx2 gene expression, inducing the skeletal deformities in mummichog. In the present study, we cloned two Runx2 cDNAs (type I and type II) from mummichog embryos. The deduced amino acid sequences of type I and type II contain an open reading frame encoding 450 and 464 amino acid residues, respectively. The derived amino acid sequence of Fundulus Runx2 type I showed the highest identity (93.8%) with Takifugu Runx2 type I, and Fundulus Runx2 type II showed 94.6% homology with medaka Runx2. The expression level of Runx2 mRNA in the early stage series was measured using a real-time quantitative PCR assay. Expression levels tended to increase in both the blastula-gastrula and the retinal pigmentation stage. To examine the effect of toxic compounds on skeletal formation, mummichog embryos in the late blastula to retinal pigmentation stage were exposed to (PS)2. After exposure to (PS)2 for one week, the expression level of Runx2 mRNA was unchanged. These results suggest that there is no inhibition of Runx2 gene expression due to (PS)2 exposure.