Department of Biology, University of Winnipeg, Winnipeg, Manitoba, R3E 2H9, Canada.
BMC Evol Biol. 2009 Dec 16;9:293. doi: 10.1186/1471-2148-9-293.
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.
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.
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.
近年来，松弛素家族信号分子已被证明在哺乳动物生理学中发挥着多样化的作用，但人们对其在硬骨鱼类中的多样性或生理学知之甚少，硬骨鱼类是包含大约 50%现存脊椎动物的骨鱼纲的一个亚纲。在本文中，通过从 8 种硬骨鱼类的基因组和 cDNA 数据库中搜索，获得了 32 个松弛素家族序列;进行了系统发育、分子进化和基因图谱分析，以了解松弛素家族基因在硬骨鱼类与哺乳动物中的关系和进化的差异模式。此外，实时定量 PCR 用于证实和评估 5 种松弛素家族基因在斑马鱼中的组织表达，并通过原位杂交评估胰岛素 3 样基因在斑马鱼睾丸中的特定部位表达。
在每种硬骨鱼类中都鉴定出多达 6 种松弛素家族基因。比较基因图谱显示，鱼类具有两种与人 RLN3 同源的基因，我们称之为 rln3a 和 rln3b，与人类 RLN2 同源的 rln，与人类 INSL5 同源的两个基因，insl5a 和 insl5b，以及与人 INSL3 同源的 insl3。分子进化分析表明：rln3a、rln3b 和 rln 受到强烈的进化约束，insl3 经历了适度的序列进化速率，insl3/INSL3 中的两个氨基酸显示出正选择的证据，而 insl5b 的序列进化速度高于其同源基因 insl5a，表明它可能在硬骨鱼全基因组复制后具有新的功能。在斑马鱼中的定量 PCR 分析表明，rln3a 和 rln3b 在脑中表达，insl3 在性腺中高度表达，而两种 insl5 基因在成年斑马鱼中的表达水平较低。最后，在斑马鱼睾丸中的 insl3 原位杂交显示与间质 Leydig 细胞高度特异性杂交。
与先前的研究相反，我们有令人信服的证据表明，硬骨鱼含有四种松弛素家族肽的同源物。总的来说，我们的分析表明，在硬骨鱼中：1) rln3 的进化和表达模式与哺乳动物的 RLN3 相似，2) insl3 经历了与哺乳动物对应物相似的正选择，并在 Leydig 细胞中表现出相似的组织特异性表达，3) insl5 基因在硬骨鱼基因组中高度表达，序列进化速度相对较高，但在成年斑马鱼中表达水平较低，4) rln 在进化上受到与哺乳动物 RLN 非常不同的选择约束。这里提出的结果应该有助于对松弛素家族基因在硬骨鱼中的特定作用进行假设驱动的实验研究。
