Molecular and cellular mechanisms of functional modification of ionocytes during seawater acclimation in Japanese Medaka (Oryzias latipes)

• Main Authors: Hao-Hsuan Hsu, 徐浩軒
• Other Authors: Pung-Pung Hwang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/61585340097251492220

碩士 === 國立臺灣大學 === 漁業科學研究所 === 98 === Ionocytes, mitochondrion-rich cells (MRCs), are specialized epidermal cells which maintain ionic homeostasis of body fluid by actively taking up or excreting ions. The purpose of this study is to examine the regulatory mechanism of cell turnover and transporters expression of ionocytes in Japanese Medaka (Oryzias latipes) during seawater (SW) acclimation. Four types of ionocyte were identified in medaka embryo, the freshwater (FW) type were NHE cell, NCC cell and HA cell and only one SW type ionocyte. The NHE cells expressing apical Na+/H+ exchanger-3 (NHE3), cystic fibrosis conductance regulator (CFTR), and basolateral Na+/K+-ATPase (NKA) and constituted the majority of FW ionocytes. We also found that most of ionocytes were basolateral expressing NKCC1a and co-localized with NKA suggesting that they are belonged to NHE cell. The cell number of NCC cell expressing apical Na+/Cl- cotransporter (NCC) was in minority. Expressions of above-mentioned transporters were regulated in response to SW stimulation. NKCC1a signal was up-regulated and no apical NCC signal was found in SW-acclimated embryo. The gene expression of NHE3 (slc9a3) was down-regulated but CFTR (abcc7) was up-regulated in medaka gill during 24 hours SW transfer. In 2-days SW transfer medaka embryo, the density of NKA-immunoreactive ionocytes was not changed, but the cell size became larger. Ionocyte turnover was examined on medaka yolk sac membrane during short-term SW adaptation. P63 is an epidermal stem cell marker, immunocytochemistry of P63 showed no significant difference in the density of P63-cells after 1 hour and 7 hours SW transfer, but a little decrease showed after 1 day SW transfer. The result of TUNEL assay indicated that cell apoptosis was not significant at 12, 24 and 48 hours after SW transfer. By qRT-PCR, The expression of ionocyte differentiation marker foxi3 and gcm2 did not differ between FW and 24-hour SW transfer medaka gill. These results suggest that proliferation, differentiation and apoptosis of epidermal cells were not changed in short-term SW adaptation response. In mammalian, deleted in malignant brain tumor 1 (dmbt1) encodes Hensin which transform b-intercalated cell into a-intercalated cell in cortical collecting duct during systemic metabolic acidosis. In medaka, dmbt1 was also expressed in ionocytes. The protein expression of DMBT1 was down-regulated in 36 hours SW transfer embryo by immunocytochemistry. However, there was no difference in the gene expression of dmbt1 during 24 hours SW tranfer. Loss-of-function experiment showed higher mortality in dmbt1 morpholino-injected group than in control. Morpholino knock down accompany with SW transfer treatment showed obvious delay in the development of medaka morphant. We suggested dmbt1 might involve in transformation of ionocytes during SW acclimation. Taken together, we demonstrated a model of short-term SW adaptation in medaka which suggests that FW ionocytes directly transform to SW ionocytes and dmbt1 might involve in this process.