Molecular genetic studies of several connexin genes in the zebrafish (Danio rerio)

This study explores the molecular genetic mechanisms that regulate the expression and function of five connexin (Cx) genes (Cx43, Cx44.1, Cx45.6, Cx48.5, and Cx30.3) in zebrafish (Dania rerio) embryonic development and adult tissues.;A new zebrafish Cx gene, Cx30.3, was identified within the Cx48.5 promoter. Sequence similarity and biophysical analyses suggest that zebrafish Cx30.3 is the ortholog of mammalian Cx26. Its transcriptional start site and full-length gene organization were identified using RACE technique. RT-PCR (reverse transcriptase polymerase chain reaction), real time PCR, and in situ hybridization techniques revealed that Cx30.3 is expressed in early embryos and in the skin, inner ear, lateral line, fin, heart, and retina of adults. This expression pattern may suggest the role of Cx30.3 in the development and function of hearing and balance in zebrafish. For future studies, zebrafish Cx30.3 could be employed to model human diseases of hearing and disorders in the skin.;The transcriptional start sites and 5'UTRs (untranslated regions) of these genes were identified using 5' RACE (rapid amplification of cDNA ends). Interestingly, most of these Cx genes have alternative 5' UTRs, differing only in their first exons, which are generated from different promoter usages and alternative pre-mRNA splicing. Comparative analyses of zebrafish and mouse orthologs suggest that these features of Cx gene expression are evolutionarily conserved. Preliminary in silico and in vivo comparative analyses of promoter activities of zebrafish Cx44.1 and mouse Cx50 suggest that transcription regulatory networks of homologous Cx genes might be conserved between species. In silico analyses confirmed very strong loop-stem secondary structures and frequent uORFs in their 5'UTRs. Sequence analyses also suggest that IRESs (internal ribosome enter sites) might be involved in their translational regulation. Interestingly, tandem alternative splicing was identified within 5' UTRs of Cx45.6, and this is the first report of this phenomenon in Cx genes. Overall the diversity of 5'UTRs reflects a complexity of post-transcriptional regulation for Cx genes, especially considering the fact that alternative transcripts actually encode the same protein product. Functional analyses using "transcript variant-specific morpholino knock-down" approaches in GFP (green fluorescent protein)-labelled zebrafish embryos are proposed to elucidate this question.