細胞膜鈣離子幫浦與上皮鈣離子通道在斑馬魚內耳發育所扮演的角色

Role of Plasma Membrane Calcium ATPase (PMCA) and Epithelial Calcium Channel (ECaC) in the Inner Ear Development of Zebrafish (Danio rerio)

柯雪莉(Shelly Cruz)

指導教授 ： 黃鵬鵬

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.01341

摘要

研究魚類內耳的發育提供了瞭解動物相關疾病的絕佳材料。魚類的內耳同時具備生存所需的聽覺與平衡感的功能。內耳中的耳石，其成份超過99%是碳酸鈣。其次，產生聽覺與平衡感的基本構造，感覺毛細胞，也需要鈣離子以產生神經衝動。然而鈣離子如何進入內淋巴液仍有很大的爭議。關於鈣離子進入內淋巴液的解釋，目前有兩種理論：一者是藉由與鈣相關的離子運輸蛋白主動運輸，另一則是藉由被動擴散經細胞間隙通透。
鈣離子幫浦(Plasma Membrane Ca2+ ATPases, PMCA)的主要功能是維持細胞內低鈣濃度的環境。本實驗於斑馬魚的內耳組織中發現6種鈣離子幫浦(zPMCA)，與表皮性鈣離子通道(zebrafish Epithelial Ca Channel, zECaC)，並且對其功能進行瞭解。RT-PCR的結果顯示，zPMCA1a是所有的鈣離子幫浦中表現量最多的一型。mRNA in situ hybridization的結果顯示，zPMCA1a的mRNA不僅存在於成體內耳的離子細胞與感覺毛細胞之外，於仔魚期的內耳與側線感覺毛細胞中亦可發現。免疫組織化學染色法的結果也證實zPMCA蛋白質確實表現在內耳的感覺毛細胞與離子細胞。
藉由注射antisense-morpholino oligos以抑制zPMCA1a與zECaC蛋白質的表現，結果造成感覺毛細胞與三半規管發育異常。若以zPMCA1a mismatched antisense-morpholino oligos注射胚胎，則完全不影響內耳發育。無論是抑制單一zPMCA或是zECaC的蛋白質表現，或是同時抑制兩種蛋白質的表現，皆會造成耳石成長遲緩，但是仔魚對鈣離子的吸收與體內鈣離子的含量卻不受影響。此結果顯示仔魚的總鈣量與鈣吸收速率並非是造成耳石成長遲緩的原因。缺乏zPMCA1a或是zECaC蛋白質也會造成仔魚無法維持平衡，對外界刺激沒有反應，無法維持正常游泳姿勢，這些缺陷的發生率與注射的antisense-morpholino oligos的量呈現高度正相關。這些結果顯示zPMCA1a與zECaC不僅負責斑馬魚內耳的鈣離子運輸，對內耳的發育也扮演很重要的功能。
此外利用DASPEI (2-(4-dimethyl-aminostyryl)-N-ethylpyridinium iodide)浸泡斑馬魚仔魚的方式，以瞭解感覺毛細胞的機械性傳導功能是否還是正常運作。缺乏zPMCA1a或是zECaC蛋白質的仔魚，其感覺毛細胞無法正常吸收，甚至完全無法吸收DASPEI，此結果表示zPMCA1a與zECaC是感覺毛細胞發育不可或缺的蛋白質。
耳石鈣化所需的元素由內淋巴液提供。缺乏zPMCA1a或是zECaC，甚至兩者皆喪失的仔魚，耳石成長緩慢，可能是因為缺少了zPMCA1a與zECaC所提供的鈣來源，但是耳石既然還能夠形成，這表示經由其他管道讓鈣離子進入內淋巴液的機制可能還是存在，例如經由細胞間隙，讓鈣離子通透進入內淋巴液中。
本研究發現zPMCA1a與zECaC在斑馬魚內耳發育過程中的必要性。尤其是對感覺毛發育，三半規管的形成，耳石的定位與鈣化速度等影響最為重要。此結果對研究斑馬魚內耳發育與鈣離子運輸提供了更進一步嶄新的知識與研究方向。

關鍵字

斑馬魚 ； 內耳 ； 鈣離子幫浦 ； 上皮鈣離子通道

並列摘要

In fish inner ear study, it creates a very conducive animal related disease model. Teleosts’ inner ear functions as auditory and vestibular system for survival purposes. The otolith contained in the otic vesicle of inner ear composed of more than 99% calcium carbonate. The energy source that is Ca2+-driven hair-bundle motility of the hair cells helps in enhancing hearing sensitivity. However, the mechanism of transepithelial transport of calcium into endolymph remains controversial. Two proposed calcium transport pathway exist nowadays: cells proximal to the otolith actively extrude cytosolic free calcium ions by ATP-dependent calcium pump and passive transport by paracellular pathway.
The primary role of Ca2+ ATPases is to maintain the very low cytosolic free calcium ions critical for intracellular signaling and cellular processes. PMCA primarily contributes to the supply of calcium in the system and essential for the inner ear’s morphological and physiological aspect. In this present study the 6 zebrafish plasma membrane calcium ATPase (zPMCA) isoforms, as well as zebrafish epithelial calcium channel (zECaC) were determined to present in the inner tissue of adult zebrafish followed by functional studies. zPMCA1a isoform showed most abundant expression compared to other zPMCA isoforms through RT-PCR (Reverse transcript – PCR). The gene expression of zPMCA1a among adult zebrafish inner ear chamber was traced in ionocytes and hair cells as well as in larvae otic placode and lateral-line neuromast via mRNA in situ hybridization. Moreover, immunohistochemical staining using PMCA 5F10 monoclone localized zPMCA in the hair cells and ionocytes of the inner ear.
Blocking the protein translation of zPMCA1a isoform and zECaC by antisense-morpholino oligodeoxynucleotide showed malformation and suppressed production of hair cells as well as absent or lessened outgrowth of semicircular canal (zPMCA1a mismatched morpholino oligos was used for control group, showed normal development). Either zPMCA1a, zECaC or combined knock-down morphant’s otolith accretion was significantly reduced. Whole body calcium content did not show significant difference between zPMCA1a morphants and wild type until 7 day post fertilization (dpf). However, the otolith size between wild-type and zPMCA1a morphants was significantly different starting from 2 dpf, which means whole body Ca2+ content is not the reason to cause the difference of otolith size. Frequency of defective morphants in maintaining balance, response to the disturbance and inability to swim occurred in dose dependent manner depending on the quantities of injected antisense morpholino oligodeoxynucleotide. These results indicate relative importance of zPMCA1a and zECaC on normal development of the auditory and vestibular system as well as in the calcium transport system.
The function of hair cells in mechanotransduction was tested by using a vital mitochondrial dye named DASPEI (2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide). Since development of hair cells was affected among zPMCA1a morphants showed very few hair cells able to absorb the dye compared to wild-type. Indeed, zPMCA1a role is critical for cells development in-order to function properly.
Otolith accretion depends on acellular medium endolymph which composed all the components for otolith mineralization. Smaller otolith formed among zECaC, zPMCA1a and combined knock-down morphants may be resulted from the impairment of calcium uptake into the endolymph via active transport in the cells expressing zECaC and zPMCA1a. Apparently, otolith formation still resumes among morphants, therefore the possibility of paracellular pathway should not be excluded to be involved in calcium transport into endolymph.
The present study indicates that the expression and function of zPMCA1a and zECaC are essential for the development of inner ear hair cells, semicircular-canal non-sensory cells outgrowth, as well as otolith localization and accretion, providing a better understanding in the inner ear calcium transport system and development of zebrafish.

並列關鍵字

zebrafish ； inner ear ； PMCA ； ECaC

參考文獻

Adams L. A. 1940 Some characteristics of American ostariophysi. Journal of Morphology 66:497-527

Anken R. H., E. Edelmann, and H. Rahman 2000 Fish inner ear otoliths stop calcium incorporation after vestibular nerve transection. Auditory and Vestibular Systems 11(13):2981-3

Anken R. H., M. Ibsch, J. Breuer, and H. Rahmann 2001 Effect of hypergravity on the Ca/Sr composition of developing otoliths of larval cichlid fish (Oreochromis mossambicus). Comparative Biochemistry and Physiology 128A: 369-77

Bang P. I., W. F. Sewell, and J. J. Malicki 2001 Morphology and cell type heterogeneities of the inner ear epithelia in adult and juvenile zebrafish (Danio rerio). The Journal of Comparative Neurology 438:173-190

Borelli G., N. Mayer-Gostan, H. De Pontual, G. Boeuf, and P. Payan 2001 Biochemical relationships between endolymph and otolith matrix in the trout (Oncorhynchus mykiss) and turbot (Psetta maxima). Calcified Tissue International 69:356-64