中国科学院昆明动物研究所机构知识库

绝大多数蝙蝠完全依赖回声定位获取外界信息。因此，听觉反馈在回声定位蝙蝠的日常生活中显得尤为重要。耳蜗作为听觉系统的终端感受器，不仅涉及被动的听任务而且是主动定位过程中不可缺少的一部分。此外，为适应复杂的捕食生境，蝙蝠的回声定位声波同时具备高频和高强度的特性。因此，在长期的适应过程中，回声定位蝙蝠耳蜗的相对大小、圈数及其听觉上皮（基底膜长度和外毛细胞结构等）都经历了适应性进化。然而，缺乏回声定位功能适应方面的研究。因此，本论文将以蝙蝠为研究对象，从蛋白、细胞和器官水平揭示回声定位的分子基础。表型趋同的分子机制一直是研究的热点。关键蛋白的功能趋同是表型趋同的分子基础。我们首先以调频蝙蝠为研究对象，通过进化分析和功能实验相结合的研究方法揭示听力关键基因prestin在调频蝙蝠中的适应进化。我们的研究表明尽管在物种树中调频蝙蝠（FM）呈并系分布。但在调频蝙蝠中，高频听力相关的马达蛋白prestin的功能是相似的。点突变实验证明：在调频蝙蝠中，prestin蛋白308号氨基酸位点从天冬酰胺到丝氨酸（N308S）的转变对其功能的相似起着重要作用。此外，我们进化分析结果强烈支持，308号位点的突变是由于祖先多态性位点的不完全谱系分选（incomplete lineage sorting）造成的。该位点仅在假吸血蝠科（Megadermatidae），菊头蝠科（Rhinolophidae）和Yangochiroptera亚目调频蝙蝠最近的共同祖先枝（MRCA）上发生过一次替代，而随后在调频蝙蝠中该祖先位点的多态性被分别固定下来。基于回声定位蝙蝠独特的生理特性（完全暴露在高强度的高频噪声中）及已有的研究，我们假定回声定位蝙蝠耳蜗毛细胞具有特殊抗损伤机制。通过氨基糖苷类抗生素体内注射实验，我们发现相对非回声蝙蝠，回声定位蝙蝠具有更高的耐受性。回声和非回声蝙蝠内耳差异表达基因的分析表明：在回声定位蝙蝠中过表达的基因主要富集在与听力相关的功能类别，证明这些高表达的基因在回声定位中起着重要的作用。此外，差异表达基因的互作网络分析表明，ISL1和GATA3是两个与听力相关的hub基因。因此，我们在体外进行实验验证，结果证明，ISL1转录因子可以提高小鼠耳蜗听觉上皮外植体抗新霉素的能力（外毛细胞的存活率叫对照组显著增加）。本研究首次揭示回声定位蝙蝠耳蜗毛细胞抗损伤的机制。耳蜗的相对大小取决于处理声音信号的功能需求。回声定位蝙蝠中，恒频蝙蝠声波的音频和强度普遍偏高。研究表明恒频蝙蝠的耳蜗显著大于调频蝙蝠及非回声定位的哺乳动物。为揭示恒频蝙蝠内耳发育机制，我们以中华菊头蝠为研究对象，昆明小鼠为对照，结合胚胎发育比较与转录组分析展开研究。结果表明：中华菊头蝠和昆明小鼠经历了相同的胚胎发育期使内耳发育为成体型（即和成体具有相同的基底膜圈数）。但中华菊头蝠的内耳发育发生了适应性的进化：首先，中华菊头蝠基底膜长度发生显著延伸的胚胎期远远多于昆明小鼠。其次，中华菊头蝠耳蜗基底膜圈数增长最快的时期与昆明小鼠不一致。再次，中华菊头蝠耳蜗感觉上皮的发育（即Sox2蛋白的表达）晚于昆明小鼠，但感觉上皮附属结构（前庭阶和鼔阶）的发育早于昆明小鼠。综上所述，本论文结合进化分析、转录组分析和功能实验验证等方法，旨在揭示回声定位蝙蝠高频听力功能趋同及内耳保护和发育进化的分子机制。以期对哺乳动物高频听力的适应性进化提供补充。

Most of bats rely entirely on echolocation for getting information from surroundings. Therefore, auditory feedback plays a key role in the daily life of echolocation bats. As a terminal sensor of the auditory system, cochlea involves both passive listening and active localization tasks. In addition, in order to adapting to the complex predation habitats, bats have evolved high frequency and high intensity echolocation calls. Therefore, during the long-term adaptation, the relative size, number of circles and auditory epithelium (basement membrane length and outer hair cell structure, etc.) of cochlea from echolocation bats have undergone adaptive evolution. However, studies on the functional adaptation of echolocation are lacking. Therefore, this paper will take bats as the research object to reveal the molecular basis of echolocation from protein, cell and organ levels.The molecular mechanism of phenotypic convergence is a research hotspot. Functional convergence of key proteins is the molecular basis of phenotypic convergence. We combined the evolutionary analysis and functional experiments to reveal the adaptive evolution of hearing gene prestin in FM bats. Here, we report that although the FM bats is paraphyletic in the species tree, prestin exhibits similar functional changes between FM bats. Site-directed mutagenesis experiments show that the 308th amino acid (N308S) substitution in FM bats is responsible for the similar functional changes of prestin. Additionally, evolution analysis strongly supports that the mutation on 308th amino acid is a case of hemiplasy, caused by incomplete lineage sorting of an ancestral polymorphism.Echolocating bats are naturally exposed to intensively high frequency calls from their own and the neighbors, which could cause cochlear hair cell damage in nonecholocating mammals. Here we hypothesize that echolocating bats have evolved a special protective mechanism against the noise damage. We found that echolocating bats have higher tolerance compared to nonecholocating bats through a in vivo injection experiment with aminoglycoside antibiotics. The transcriptome analysis of echolocating and nonecholocating bats cochleas indicate that the up-regulated genes in echolocating bats are significantly enriched in some functional categories associated with hearing, suggesting their important roles on bat echolocation. Analyzing the functional network for the genes differently expressed between echolocating and nonecholocating bats reveals two hearing-related hub genes (GATA3 and ISL1) and overexpression of ISL1 significantly improves the survival of cochlear hair cells when being treated with aminoglycosides. Taken together, our findings are the first demonstration of protective mechanism of cochlear hair cells in echolocating bats.The relative size of the cochlea depends on the functional requirements for processing sound signals. In echolocation bats, the echolocation calls of CF bats are more intense. Previous studies have shown that the cochleas of CF bats are significantly larger than that of FM bats and non-echolocation mammals. In order to reveal the development mechanism of inner ear in CF bats, we conducted a study by comparing embryonic development and transcriptome analysis with Rhinolophus sinicus and Kunming mouse. The results showed that Rhinolophus sinicus and Kunming mice experienced the same embryonic stages to develop as an adult-like type inner ear (that is, with the same turns as the adult). However, the development of the inner ear from the Rhinolophus sinicus has undergone adaptive evolution. First, compared to Kunming mouse, the embryonic stages with basement membrane extension in Rhinolophus sinicus are more. Second, the stage with the fastest increased in the cochlear turns is not consistent with that of Kunming mouse. Third, the development of cochlear sensory epithelium (i.e., Sox2 protein expression) is later in Rhinolophus sinicus than that in Kunming mouse, but the accessory structure of sensory epithelium (scala vestibuli and scala tympani) is delayed in Kunming mouse.In conclusion, we combined evolutionary analysis, transcriptome analysis and functional experimental to reveal the molecular mechanism of function convergence in high-frequency hearing gene prestin and of inner ear protection and development evolution in echolocating bats. This will be a supplement to adaptive evolution of mammalian high frequency hearing.