A number of non-visual responses to light in vertebrates, such as circadian rhythm control and pupillary light reflex, are mediated by melanopsins, G-protein coupled membrane receptors, conjugated to a retinal chromophore. In non-mammalian vertebrates, melanopsin expression is variable within the retina and extra-ocular tissues. Two paralog melanopsin genes were classified in vertebrates, Opn4x and Opn4m. Snakes are highly diversified vertebrates with a wide range of daily activity patterns, which raises questions about differences in structure, function and expression pattern of their melanopsin genes. In this study, we analyzed the melanopsin genes expressed in the retinas of 18 snake species from three families (Viperidae, Elapidae, and Colubridae), and also investigated extra-retinal tissue expression. Phylogenetic analysis revealed that the amplified gene belongs to the Opn4x group, and no expression of the Opn4m was found. The same paralog is expressed in the iris, but no extra-ocular expression was detected. Molecular evolutionary analysis indicated that melanopsins are evolving primarily under strong purifying selection, although lower evolutionary constraint was detected in snake lineages (ω = 0.2), compared to non-snake Opn4x and Opn4m (ω = 0.1). Statistical analysis of selective constraint suggests that snake phylogenetic relationships have driven stronger effects on melanopsin evolution, than the species activity pattern. In situ hybridization revealed the presence of melanopsin within cells in the outer and inner nuclear layers, in the ganglion cell layer, and intense labeling in the optic nerve. The loss of the Opn4m gene and extra-ocular photosensitive tissues in snakes may be associated with a prolonged nocturnal/mesopic bottleneck in the early history of snake evolution. The presence of melanopsin-containing cells in all retinal nuclear layers indicates a globally photosensitive retina, and the expression in classic photoreceptor cells suggest a regionalized co-expression of melanopsin and visual opsins.

中文翻译：

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昼夜蛇的黑色素蛋白基因（Opn4x）的鉴定。

脊椎动物对光的许多非视觉响应，例如昼夜节律控制和瞳孔光反射，是由与视网膜发色团结合的黑素（G蛋白偶联膜受体）介导的。在非哺乳动物脊椎动物中，黑素蛋白的表达在视网膜和眼外组织内是可变的。在脊椎动物中将两个旁系黑素蛋白基因分类为Opn4x和Opn4m。蛇是高度多样化的脊椎动物，具有多种日常活动模式，这引发了有关其黑视蛋白基因的结构，功能和表达模式差异的疑问。在这项研究中，我们分析了来自三个科（Vi蛇科，天牛科和Colubridae）的18种蛇的视网膜中表达的黑素蛋白基因，并调查了视网膜外组织的表达。系统发育分析表明，扩增的基因属于Opn4x基团，未发现Opn4m的表达。虹膜中表达相同的旁系同源物，但未检测到眼外表达。分子进化分析表明，黑素视蛋白主要在强纯化作用下进化，尽管与非蛇类Opn4x和Opn4m（ω= 0.1）相比，在蛇谱系（ω= 0.2）中检测到了更低的进化限制。对选择性约束的统计分析表明，与物种活动模式相比，蛇的系统发育关系对黑视蛋白的进化产生了更强的影响。原位杂交揭示了黑素蛋白在外核层和内核层，神经节细胞层中的细胞内存在，并且在视神经中有强烈的标记。蛇进化过程中，Opn4m基因和眼外光敏组织的丢失可能与夜间/近视瓶颈延长有关。在所有视网膜核层中都存在含有黑视蛋白的细胞，表明该区域具有整体光敏性视网膜，而经典感光细胞中的表达表明，黑视蛋白和视觉视蛋白存在区域性共表达。