Table_3_New Insights Into the Evolution of Corticotropin-Releasing Hormone Family With a Special Focus on Teleosts.xlsx

Corticotropin-releasing hormone (CRH) was discovered for its role as a brain neurohormone controlling the corticotropic axis in vertebrates. An additional crh gene, crh2, paralog of crh (crh1), and likely resulting from the second round (2R) of vertebrate whole genome duplication (WGD), was identified in a holocephalan chondrichthyan, in basal mammals, various sauropsids and a non-teleost actinopterygian holostean. It was suggested that crh2 has been recurrently lost in some vertebrate groups including teleosts. We further investigated the fate of crh1 and crh2 in vertebrates with a special focus on teleosts. Phylogenetic and synteny analyses showed the presence of duplicated crh1 paralogs, crh1a and crh1b, in most teleosts, resulting from the teleost-specific WGD (3R). Crh1b is conserved in all teleosts studied, while crh1a has been lost independently in some species. Additional crh1 paralogs are present in carps and salmonids, resulting from specific WGD in these lineages. We identified crh2 gene in additional vertebrate groups such as chondrichthyan elasmobranchs, sarcopterygians including dipnoans and amphibians, and basal actinoperygians, Polypteridae and Chondrostei. We also revealed the presence of crh2 in teleosts, including elopomorphs, osteoglossomorphs, clupeiforms, and ostariophysians, while it would have been lost in Euteleostei along with some other groups. To get some insights on the functional evolution of the crh paralogs, we compared their primary and 3D structure, and by qPCR their tissue distribution, in two representative species, the European eel, which possesses three crh paralogs (crh1a, crh1b, crh2), and the Atlantic salmon, which possesses four crh paralogs of the crh1-type. All peptides conserved the structural characteristics of human CRH. Eel crh1b and both salmon crh1b genes were mainly expressed in the brain, supporting the major role of crh1b paralogs in controlling the corticotropic axis in teleosts. In contrast, crh1a paralogs were mainly expressed in peripheral tissues such as muscle and heart, in eel and salmon, reflecting a striking subfunctionalization between crh1a and b paralogs. Eel crh2 was weakly expressed in the brain and peripheral tissues. These results revisit the repertoire of crh in teleosts and highlight functional divergences that may have contributed to the differential conservation of various crh paralogs in teleosts.

促肾上腺皮质激素释放激素（Corticotropin-releasing hormone, CRH）最初因作为调控脊椎动物促肾上腺皮质激素轴的脑神经激素而被发现。后续研究在全头亚纲软骨鱼、基干哺乳类、多种蜥形纲动物以及一种非硬骨鱼辐鳍全骨类物种中，鉴定出了crh基因的另一旁系同源基因crh2——该基因是crh（后命名为crh1）经脊椎动物第二轮全基因组复制（2R WGD）事件产生的旁系同源物。有研究提出，crh2在包括硬骨鱼在内的部分脊椎动物类群中发生了反复丢失。本研究进一步探究了脊椎动物中crh1与crh2的演化命运，重点聚焦于硬骨鱼。
系统发育与共线性分析显示，多数硬骨鱼中存在由硬骨鱼特异性全基因组复制（3R WGD）产生的crh1旁系同源基因crh1a与crh1b。其中crh1b在所有已研究的硬骨鱼中均得到保留，而crh1a则在部分物种中独立发生了丢失。鲤科与鲑科鱼类中还存在额外的crh1旁系同源基因，这源于这些演化支特异性的全基因组复制事件。
本研究在更多脊椎动物类群中鉴定到了crh2基因，包括软骨鱼纲板鳃亚纲、肉鳍鱼类（含肺鱼与两栖类）以及基干辐鳍鱼纲类群多鳍鱼科与软骨硬鳞鱼亚纲。本研究同时证实，硬骨鱼中的海鲢类、骨舌鱼类、鲱形目与骨鳔总目均保留有crh2基因，而该基因在新真骨下纲（Euteleostei）及其他部分类群中发生了丢失。
为探究crh旁系同源基因的功能演化规律，本研究选取两种代表物种开展分析：拥有crh1a、crh1b与crh2共3种crh旁系同源基因的欧洲鳗鲡，以及拥有4种crh1型旁系同源基因的大西洋鲑。研究中我们比较了这些基因的一级结构与三维结构，并通过实时定量聚合酶链式反应（qPCR）分析了它们的组织表达分布。所有肽段均保留了人源CRH的结构特征。欧洲鳗鲡的crh1b与大西洋鲑的两种crh1b基因主要在脑组织中表达，这支持了crh1b旁系同源基因在硬骨鱼促肾上腺皮质激素轴调控中发挥核心作用的结论。与之相反，欧洲鳗鲡与大西洋鲑中的crh1a旁系同源基因主要在肌肉、心脏等外周组织中表达，这反映出crh1a与crh1b旁系同源基因之间存在显著的亚功能化现象。欧洲鳗鲡的crh2则在脑组织与外周组织中均呈低水平表达。
本研究结果重新梳理了硬骨鱼中crh基因的组成谱，并揭示了可能促成硬骨鱼中不同crh旁系同源基因差异化保留的功能分化机制。