A repeatedly evolved mutation in Cryptochrome-1 of subterranean animals alters behavioral and molecular circadian rhythms

The repeated evolution of similar phenotypes in independent lineages often occurs in response to similar environmental pressures, through similar or different molecular pathways. Recently, a repeatedly occurring mutation R263Q in a conserved domain of the protein Cryptochrome-1 (CRY1) was reported in multiple species inhabiting subterranean environments. Cryptochromes regulate circadian rhythms, and glucose and lipid metabolism. Subterranean species show changes to their circadian rhythm and metabolic pathways, making it likely that this mutation in CRY1 contributes to adaptive phenotypic changes. To identify the functional consequences of the CRY1 R263Q mutation, we generated a mouse model homozygous for this mutation. Indirect calorimetry experiments revealed delayed locomotor activity, energy expenditure and feeding patterns of mutant mice in the dark phase, but no further metabolic phenotypes unlike a full loss of function of CRY1. Gene expression analyses showed altered expression of several canonical circadian genes in the livers of the mutant mice, fortifying the notion that CRY1 R263Q impacts metabolism. Our data provide the first characterization of a novel mutation that has repeatedly evolved in subterranean environments, supporting the idea that shared environmental constraints can drive the evolution of similar phenotypes through similar genetic changes. We collected brain and liver tissues at two different zeitgeber times (ZT, ZT12 and ZT22) from wild-type and CRY1 R263Q homozygote mice from both sexes, and then extracted and sequenced the mRNA. Samples from each ZT were collected, sequenced and analyzed separately. Comparisons were made within the same ZT, tissue type and sex, and between genotypes.独立演化谱系中相似表型的反复演化现象，往往是物种响应相似环境压力，并通过相似或不同分子通路实现的演化结果。近日，研究人员在多个栖息于地下环境的物种中，报道了一种反复出现的、位于隐花色素1（Cryptochrome-1, CRY1）保守结构域内的突变R263Q。隐花色素可调控昼夜节律与糖脂代谢过程。地下栖息物种的昼夜节律及代谢通路均会发生改变，据此推测CRY1的该突变可能参与了适应性表型演化。为探究CRY1 R263Q突变的功能效应，本研究构建了该突变的纯合小鼠模型。间接量热实验结果显示，突变小鼠在黑暗周期中的运动活性、能量消耗与进食模式均出现延迟，但与CRY1完全功能缺失的表型不同，本研究未观测到其他代谢表型。基因表达分析显示，突变小鼠肝脏内数种经典昼夜节律基因的表达水平发生改变，进一步支持CRY1 R263Q可影响代谢过程的结论。本研究的数据首次对地下环境中反复演化出的该新型突变进行了功能表征，支持“共同的环境约束可通过相似的遗传改变驱动相似表型演化”这一观点。研究人员从雌雄两性的野生型与CRY1 R263Q纯合小鼠中，于两种不同的授时时间（zeitgeber time, ZT，即ZT12与ZT22）采集脑组织与肝组织，随后提取mRNA并进行测序。不同授时时间的样本分别完成采集、测序与分析。研究在相同授时时间、相同组织类型与相同性别组内，以及不同基因型之间开展了对比分析。