Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis

<p>Abstract</p> <p>Background</p> <p>In recent years, the relaxin family of signaling molecules has been shown to play diverse roles in mammalian physiology, but little is known about its diversity or physiology in teleosts, an infraclass of the bony fishes comprising ~ 50% of all extant vertebrates. In this paper, 32 relaxin family sequences were obtained by searching genomic and cDNA databases from eight teleost species; phylogenetic, molecular evolutionary, and syntenic data analyses were conducted to understand the relationship and differential patterns of evolution of relaxin family genes in teleosts compared with mammals. Additionally, real-time quantitative PCR was used to confirm and assess the tissues of expression of five relaxin family genes in <it>Danio rerio </it>and <it>in situ </it>hybridization used to assess the site-specific expression of the insulin 3-like gene in <it>D. rerio </it>testis.</p> <p>Results</p> <p>Up to six relaxin family genes were identified in each teleost species. Comparative syntenic mapping revealed that fish possess two paralogous copies of human <it>RLN3</it>, which we call <it>rln3a </it>and <it>rln3b</it>, an orthologue of human <it>RLN2</it>, <it>rln</it>, two paralogous copies of human <it>INSL5</it>, <it>insl5a and insl5b</it>, and an orthologue of human <it>INSL3</it>, <it>insl3</it>. Molecular evolutionary analyses indicated that: <it>rln3a, rln3b </it>and <it>rln </it>are under strong evolutionary constraint, that <it>insl3 </it>has been subject to moderate rates of sequence evolution with two amino acids in <it>insl3/INSL3 </it>showing evidence of positively selection, and that <it>insl5b </it>exhibits a higher rate of sequence evolution than its paralogue <it>insl5a </it>suggesting that it may have been neo-functionalized after the teleost whole genome duplication. Quantitative PCR analyses in <it>D. rerio </it>indicated that <it>rln3a </it>and r<it>ln3b </it>are expressed in brain, <it>insl3 </it>is highly expressed in gonads, and that there was low expression of both <it>insl5 </it>genes in adult zebrafish. Finally, <it>in situ </it>hybridization of <it>insl3 </it>in <it>D. rerio </it>testes showed highly specific hybridization to interstitial Leydig cells.</p> <p>Conclusions</p> <p>Contrary to previous studies, we find convincing evidence that teleosts contain orthologues of four relaxin family peptides. Overall our analyses suggest that in teleosts: 1) <it>rln3 </it>exhibits a similar evolution and expression pattern to mammalian <it>RLN3</it>, 2) <it>insl3 </it>has been subject to positive selection like its mammalian counterpart and shows similar tissue-specific expression in Leydig cells, 3) <it>insl5 </it>genes are highly represented and have a relatively high rate of sequence evolution in teleost genomes, but they exhibited only low levels of expression in adult zebrafish, 4) <it>rln </it>is evolving under very different selective constraints from mammalian <it>RLN</it>. The results presented here should facilitate the development of hypothesis-driven experimental work on the specific roles of relaxin family genes in teleosts.</p