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

• Main Authors: Shelly Cruz, 柯雪莉
• Other Authors: Pung-Pung Hwang
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/37340248577712299036

碩士 === 國立臺灣大學 === 漁業科學研究所 === 93 === 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.