title: Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis.
abstract: BACKGROUND
  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 approximately 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.
  
  
  RESULTS
  Up 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.
  
  
  CONCLUSIONS
  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) 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.
authors.affiliation: Department of Biology, University of Winnipeg, Winnipeg, Manitoba, R3E 2H9, Canada. s.good-avila@uwinnipeg.ca
authors.forename: Sara V
authors.initials: SV
authors.lastname: Good-Avila
authors.forename: Sergey
authors.initials: S
authors.lastname: Yegorov
authors.forename: Scott
authors.initials: S
authors.lastname: Harron
authors.forename: Jan
authors.initials: J
authors.lastname: Bogerd
authors.forename: Peter
authors.initials: P
authors.lastname: Glen
authors.forename: James
authors.initials: J
authors.lastname: Ozon
authors.forename: Brian C
authors.initials: BC
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005399:Fishes / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D017403:In Situ Hybridization; D007328:Insulin / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics; D012065:Relaxin / Q000235:genetics*; D016415:Sequence Alignment; D026801:Synteny; D013737:Testis / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; D029961:Zebrafish Proteins; C534578:rln3a protein, zebrafish; D012065:Relaxin