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

Abstract

BackgroundIn 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 Danio rerio and in situ hybridization used to assess the site-specific expression of the insulin 3-like gene in D. rerio testis.ResultsUp to six relaxin family genes were identified in each teleost species. Comparative syntenic mapping revealed that fish possess two paralogous copies of human RLN3, which we call rln3a and rln3b, an orthologue of human RLN2, rln, two paralogous copies of human INSL5, insl5a and insl5b, and an orthologue of human INSL3, insl3. Molecular evolutionary analyses indicated that: rln3a, rln3b and rln are under strong evolutionary constraint, that insl3 has been subject to moderate rates of sequence evolution with two amino acids in insl3/INSL3 showing evidence of positively selection, and that insl5b exhibits a higher rate of sequence evolution than its paralogue insl5a suggesting that it may have been neo-functionalized after the teleost whole genome duplication. Quantitative PCR analyses in D. rerio indicated that rln3a and rln3b are expressed in brain, insl3 is highly expressed in gonads, and that there was low expression of both insl5 genes in adult zebrafish. Finally, in situ hybridization of insl3 in D. rerio testes showed highly specific hybridization to interstitial Leydig cells.ConclusionsContrary to previous studies, we find convincing evidence that teleosts contain orthologues of four relaxin family peptides. Overall our analyses suggest that in teleosts: 1) rln3 exhibits a similar evolution and expression pattern to mammalian RLN3, 2) insl3 has been subject to positive selection like its mammalian counterpart and shows similar tissue-specific expression in Leydig cells, 3) insl5 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) rln is evolving under very different selective constraints from mammalian RLN. The results presented here should facilitate the development of hypothesis-driven experimental work on the specific roles of relaxin family genes in teleosts.

Evolution of the Relaxin/Insulin-like Gene Family in Placental Mammals: Implications for Its Early Evolution

“…The initial stages of this process would have occurred early in the evolution of vertebrates as orthology among tetrapod and teleost fish members of the family has been well established in previous studies (Good-Avila et al 2009;Park et al 2008a).…”

“…However, the most recent phylogenetic studies did not compare the competing evolutionary scenarios in a phylogenetic framework (Good-Avila et al 2009;Park et al 2008a;Wilkinson et al 2005). According to the divergence post-WGD model of evolution (Fig.…”

“…The RLN2 gene apparently originated between 29 and 18 mya (Goodman et al 1998;Steiper and Young 2009) in the last common ancestor of apes (Figs. 3, 4;Good-Avila et al 2009;Park et al 2008a;Wilkinson et al 2005), whereas the INSL4 gene arose from an RLN ancestor prior to the divergence of Euarchontoglires (Figs. 2, 3, 4).…”

“…There are two RLN and two INSL genes in the western clawed frog (Xenopus tropicalis), but only one RLN and one INSL gene in chicken (Gallus gallus) (Park et al 2008a, b). Ray-finned fish underwent an additional round of WGD, and as a result, possess duplicated RLN/INSL clusters and duplicated copies of INSL5 and RLN3 genes relative to tetrapods (Good-Avila et al 2009;Park et al 2008a). On the other hand, the RNL1 gene was lost in zebrafish (Danio rerio), but is found as a single-copy gene in other rayfinned fish species (Good-Avila et al 2009;Park et al 2008a), as is the case with the INSL3 gene, which has been found as a single-copy gene in the zebrafish, the spotted green pufferfish (Tetraodon nigroviridis), and the fugu (Takifugu rubripes) (Good-Avila et al 2009).…”

Transcriptome Changes Associated with Protective Immunity in T and B Cell-Deficient <i>Rag1<sup>-/-</sup> </i> Mutant Zebrafish

“…Relaxins are hormones that regulate the migration of leukocyte to sites of inflammation, and increases substrate adhesion [71]. Teleost RLN3a and RLN3b paralogues display similarities in evolution and expression to the mammalian counterparts [72].…”