洞察自然：從分子至演化層級探討離子壓力、視覺與聽覺訊號作用機制之整合性跨領域研究-洞察自然：從分子至演化層級探討離子壓力、視覺與聽覺訊號作用機制之整合性跨領域研究

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Title:	洞察自然：從分子至演化層級探討離子壓力、視覺與聽覺訊號作用機制之整合性跨領域研究-洞察自然：從分子至演化層級探討離子壓力、視覺與聽覺訊號作用機制之整合性跨領域研究
Other Titles:	Making Sense in the Natural World: an Inter-Discipline Approach on Mechanisms in Ionic Stresses and Visual and Sound Signaling
Authors:	林惠真,林良恭,劉薏雯,卓逸民,范聖興
Contributors:	東海大學生命科學系行政院國家科學委員會
Date:	2012
Issue Date:	2014-03-07T07:02:37Z (UTC)
Abstract:	子計劃一：呼吸空氣魚類與兩棲類離子調節細胞之形態與功能探討呼吸空氣魚類與兩棲類是兩類群可以穿梭在水域與陸域兩個生態系主要介質的脊椎動物。由於這兩類動物多分布於淺水層或短暫的水體，對於水域與空氣環境的變化非常敏感。在行為與生理上，都需要在很短的時間內調整，以因應包括水中離子濃度、含氧量、陸域溫度等變化。過去本團隊分別在呼吸空氣的攀鱸亞目魚類與兩棲類的生理生態學有多年的基礎，本研究計劃以攀鱸亞目魚類與食蟹蛙蝌蚪為研究離子的壓力的模型動物，將探討水中離子濃度變化對牠們的各對鰓結構與功能的變異。子計畫二：腎素-血管緊縮素如何在成長中的斑馬魚調控全身電解質平衡及粒線體動態變化腎素-血管收縮素系統(Renin-Angiotensin System, RAS)是我們體內負責調控血壓及電解質平衡的系統，為心血管及腎臟疾病用藥之重要標靶。對於水生的魚類而言，RAS 是否存在？它所扮演的生理功能為何？與哺乳類系統的RAS 又有何不同？這些是在生理及演化上都未解而有趣的問題。本計畫將利用斑馬魚模型研究RAS 在硬骨魚的發育過程及生理調節上之角色。我們將以遺傳學及藥物學的方法來抑制成長中斑馬魚的腎素，再將腎素被抑制的斑馬魚與正常斑馬魚在不同生長階段同時以一系列鹽度處理後，分析斑馬幼魚對環境鹽度之適應是否受腎素之調控，並分析腎素如何藉由調節類固醇的分泌、電解質平衡相關蛋白質之表現、以及粒線體的功能來調控幼魚之鹽水平衡。本計畫之進行將配合兩種轉基因斑馬魚株的建立，以期在活體觀測類固醇荷爾蒙作用及粒線體型態之動態變化，並建立斑馬魚為RAS 用藥及粒線體疾病之臨床前試驗模型。子計劃三：節肢動物彩色視覺的行為、生態與演化學整合性研究視覺是動物溝通系統中運用最廣泛的一種，因此視覺溝通系統在動物的生存與適應上扮演重要的角色。在色彩感知生態學研究中受矚目的焦點之ㄧ是探討為什麼動物演化、並使用眾多不同色彩感知系統來觀看這個世界。使用不同的光接受器是否為適應的結果? 而利用這些光接受器是否真的為這些生物帶來適存度上的優勢, 進而造成感知系統演化上的重大突破? 這種種問題須要一個整合性的生態與演化研究來解決。過去節肢動物彩色視覺的研究均建立在授粉者蜜蜂、植食性蝴蝶、或是雜食性果蠅的視覺系統上。本計畫結合行為生態、視覺電生理、分子演化、和生物資訊分析來探討豆娘和跳蛛的彩色視覺系統。我們將利用電顯技術重建豆娘和跳蛛複眼感光色素細胞的空間排列組成，以胞內光譜測量和微光譜顯微測量其感光細胞的吸收光譜，並使用色差分析和操控行為實驗來檢測豆娘和跳蛛身體的色彩(翅膀、翅痔、頭胸腹部、前額等) 是否作為物種辨識、雄性競爭、或是擇偶時的視覺信號。最後我們將重建豆娘和跳蛛視覺蛋白基因親緣樹來分析其視覺系統的演化歷史，和檢測選汰壓力如何作用在色蟌的視覺蛋白基因上，並以生物資訊學方法來瞭解視覺色素的最大吸收光譜值λmax 的表型。本計畫特別以具有鮮豔體色的視覺捕食者? 豆娘和跳蛛的角度來探討昆蟲的視覺演化生態，結果將提供研究生物視覺系統ㄧ個嶄新的面向。子計畫四：視覺傳導作用時視紫質（rhodopsin）之表現與運送的分子機制視覺的形成係透過光線照射感光細胞的視紫紅質後，將11-cis-3-hydrox retinal 轉變成all-trans-3-hydroxretinal，促使opsin 結構改變成為有活性的視紫紅質（metarhodopsin）。Metarhodopsin 透過與G protein 的作用並活化Phospholipase C，當 Phospholipase C 被活化後會促使TRP 和TRLP 鈣離子通道打開，而造成感光細胞產生去極化（depolarization）並引發神經衝動而將神經訊號傳入腦中產生視覺。當光訊息傳遞結束後，活化的視紫質會經由arrestin 蛋白進行endocytosis 過程而使metarhodopsin 去活化。過去研究顯示，感光細胞中光傳遞訊號基因或arrestin 基因突變時則導致感光細胞的退化。因此，瞭解感光細胞中視紫紅質的活化與分解，將有助於瞭解視覺成像與功能。Rhodopsin 基因缺失或調控失常會造成人類視神經退化。本實驗室過去著重在視覺器官的發育調控，在果蠅探討orthodenticle （otd）幼蟲時期調控單眼的發育的分子機制。目前的研究發現，otd 突變會造成果蠅成蟲視神經退化。從微晶片研究發現，otd 會調控果蠅rhodopsin 及arrestin 2 的表達。otd 及它的人類同源基因otx1, otx2 及crx 眼睛發育伴演重要的角色。otx2 及crx 突變會造成人類視神經退化疾病，例如利伯氏先天性黑蒙(Leber's congenital amaurosis，LCA) 及桿錐細胞退化症(Cone-rod dystrophy)。 otd/crx/otx2 突變會造成視神經退化，但造成神經退化的分子機制仍不清楚。我們推測 otd/otx2/crx 可能透過調控rhodopsin 視覺分子表達，影響生物體內視神經完整的結構與正常生理的維持。研究發現otd 基因會在蛹時期調控Rh3 及Rh5 基因的起始轉錄 (specification)，但otd 是否參與成蟲時期rhodopsin 轉錄的維持（maintenance）仍不清楚。 Tubby 及Tubby-like proteins 屬於一個小的蛋白家族，分別存在於哺乳動物、線蟲、果蠅(Ronshaugen et al., 2002)、鳥類及植物中，研究顯示Tubby 基因可能是一種轉錄調節因子並參與細胞內運輸的調節，以免疫螢光染色法發現果蠅Dtulp 表現在發育的神經細胞中，而且本實驗室的初步研究結果亦顯示果蠅Dtulp 會表現在感光細胞中，因此，本計畫將研究（1）dtulp 在視覺傳遞過程中所扮演的角色（2）dtulp 是否參與rhodopsin 的endocytosis 的過程（3）dtulp 透過何種機制調控rhodopsin 的endocytosis。(4) otd 是否參與維持rhodopsin 基因在果蠅成蟲中的轉錄調控? (5) otd 突變株是否因rhodopsin 的轉錄失調，而導致視神經細胞的退化與視神經功能失調。(6) otd 突變株造成的視神經退化的機制。子計畫五：蝙蝠聽覺之回聲定位研究：CF-FM 及FM 蝙蝠之耳蝸毛細胞顯微結構與行為反應之差異蝙蝠具有的回聲定位功能，其聽覺系統的結構出現了明顯的特化現象，高頻音波波動使耳蝸基底膜(basilar membrane)上方科蒂氏器(organ of Corti)的纖毛細胞(hair cells)彎曲而將聲波的訊息轉換成神經訊號。蝙蝠有關耳蝸纖毛細胞的超顯微研究有限，且FM 與CF-FM的蝙蝠之差異並不清楚，內纖毛則狀況不明。最近研究更顯示纖毛細胞上Prestin 基因編碼的蛋白，其對聲音頻率的敏感性和選擇性有關。回聲定位蝙蝠分為“調頻蝙蝠” （Frequency modulated bats，簡稱FM蝙蝠）和“?頻蝙蝠”（Constant frequency- frequency modulated bats，簡稱CF-FM蝙蝠F）兩類，兩者音型所代表的訊息表現作用有所差異。為此，本研究將針對同樣棲息於洞穴的FM 與CF-FM蝙蝠的耳蝸與其基底膜的毛細胞超微結構進行比較觀察。子計劃一：呼吸空氣魚類與兩棲類離子調節細胞之形態與功能探討 Air-breathing fishes and amphibians are two groups of vertebrates that can live between aquatic and terrestrial ecosystems. Because both groups of animals mostly live in shallow or temporary water bodies, they are extremely sensitive to changes in water and land. The animals have to adjust both behaviorally and physiologically within a short period of time in response to changes, such as ion concentrations and dissolved oxygen in water and temperature on land. From our several years of experience in the physiogical ecology in both anabantoid fishes and amphibians, we intend to use anabantoid fishes and crab-eating frog as animal models for ionic stresses. This study will focus on the effects of ion concentrations on the structure and functions of their gills. 子計畫二：腎素-血管緊縮素如何在成長中的斑馬魚調控全身電解質平衡及粒線體動態變化 The renin-angiotensin system plays a critical role in the control of blood pressure and electrolyte homeostasis in mammals, and is an important target for developing pharmaceuticals in cardiovascular and renal medicine. It remains largely unclear whether a functionally equivalent RAS exists in the aquatic animals, which is an interesting issue in terms of both comparative physiology and evolution. In this project, we aim to explore the role of RAS in the development and physiology of teleosts, by using the zebrafish model system. RAS will be inhibited in the developing zebrafish through genetic as well as pharmaceutical approaches. The RAS-inhibited zebrafish will be subject to a systematic series of cellular and histological analyses to determine whether and how RAS plays a role in the electrolyte homeostasis and mitochondrial dynamics in the developing zebrafish. Two kinds of stable transgenic zebrafish lines will be established, for the real-time and in-vivo monitoring of steroid action and mitochondrial morphology. Prospectively, we will be able to establish zebrafish as a preclinical pharmaceutical testing platform for RAS- as well as mitochondrion-related diseases. 子計劃三：節肢動物彩色視覺的行為、生態與演化學整合性研究 Vision is one of the most widespread communication systems among animals and plays an important role for organism’s survival and adaptation to the natural world. One majorfocus of sensory ecology has been the question why animals evolve and see the world through many different color receptors. Whether using different color receptors represent results of adaptation to different photic environment? Whether animals adopting these color receptors result in fitness increase, and further provide an evolutionary breakthrough in their sensory systems? Using an ecological and evolutionary approach is necessary to understand why different animals see the world in different colors. Previous vision research of arthropods was built mainly on studies of pollinators (honeybees) and plant-feeders (butterflies, flies). The proposed research combines vision physiology, behavioral ecology, molecular evolution, and bioinformatic analyses to investigate the color vision in two groups of visual predators ? metalwing damselflies (Calopterygidae) and jumping spiders (Salticidae). We will first use transmission electron microscopy (TEM) to build 3D distribution of photoreceptor cells within the ommatidia of the damselfly and spider’s compound eyes. Intracellular recordings and microspectrophotometry (MSP) will be used to characterize the spectral sensitivity of the damselfly and spider’s photoreceptors. We will then study the color pattern and color contrast of body parts in damselflies and spiders using color analyses and manipulative field experiments. Finally, the evolutionary history, molecular evolution, and phenotypes of opsin genes in damselflies and spiders will be investigated using phylogenies, statistical methods, and bioinformatic tools. 子計畫四：視覺傳導作用時視紫質（rhodopsin）之表現與運送的分子機制 Rhodopsin, the major component involving in phototransduction, consists of the protein moiety opsin and a reversibly covalently bound cofactor, retinal. Isomerization of 11-cis-retinal into all-trans-retinal by light induces a conformational change in opsin that activates the associated G protein and phospholipase C. The activation of phospholipase C then induces opening of TRP and TRLP channel and makes depolarization of photoreceptor cell. After photoactivation, the activated rhodopsin, metarhodopsin, is deactivated by arrestin through endocytotic pathway. It has been shown that mutation of arrestin induces retinal degeneration suggesting that synthesis and recycling of rhodopsin is critical in phototransduction pathway. rhodopsin mutants or mis-regulated Rhodopsin caused retinal degeneration in human. In our previous studies, we focus on the early eye development.We found orthodenticle (otd) can specify oculus fate in eye disc during the third instar larval stage. Recently, it has been shown that photoreceptors are degenerated in the otduvimutant, a hypomorph allele. Expression of rhodopsin 3, 4, 5 and arrestin2 are reduced in the otduvi mutant in the microarray analysis. otd and its vertebrate orthologs, otx1, otx2, and crx play important roles in the retinal development, especially crx. Mutation in crx or ctx2 may results in Leber's congenital amaurosis or Cone-rod dystrophy in human. The molecular mechanism of retinal degeneration induced by crx and ctx2 is still unclear. It has been showed otd specifies rh3, 4 and 5 transcription in R7 and R8 during pupal development. It is still unclear whether otd plays a role in the maintenance of otd expression in the adult stage. Tubby and Tubby-like proteins belong to a small family protein that exists in mammals, bird, Drosophila, nematode and plants. The functions of Tubby and Tubby-like proteins are known as transcription factor and may also involve in vesicle transport. Immunocytochemistry indicates that Drosophila Dtulp expresses in developing neuron and photoreceptor cells. In this proposal, we will address the following issues: (1) The role of dtulp in phototransduction (2)Whether dtulpparticipate in deactivation of rhodopsin through endocytotic pathway (3) How does dtulp regulate rhodopsin endocytosis (4) Does otd maintain the transcription of rh3, 4, and 5 directly in the adult stage? (5) Is the retinal degeneration induced by otd mutants due to loss of rhodopsin expression in the adult stage? (6) To verify the possible molecular mechanism of retinal degeneration induced by otd mutants. 子計畫五：蝙蝠聽覺之回聲定位研究：CF-FM 及FM 蝙蝠之耳蝸毛細胞顯微結構與行為反應之差異 The remarkable high-frequency sensitivity and selectivity of the mammalian auditory system has been attributed to the evolution of mechanical amplification, in which sound waves are amplified by outer hair cells in the cochlea. The bat cochlear hair cells were observed by the scanning and transmission electron microscope. This process is driven by the recently discovered protein prestin, encoded by the gene Prestin. Insectivorous bats typically emit two types of ultrasonic signals. They are frequency modulated (FM) signals and signals containing a constant frequency (CF) component plus a short FM component. Bats using these different signals for hunting can be called FM and CF-FM bats. The purpose of this study is to investigate the specialized features of the bat’s hair cellson cave bats with FM and CF-FM types.
Relation:	計畫編號：NSC99-2632-B029-001-MY3 研究期間：2012-08~ 2013-07
Appears in Collections:	[生命科學系所] 國科會研究報告

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