斑馬魚之氯離子吸收機制與其功能方面之調控

Chloride uptake mechanism and the functional regulation in zebrafish

王怡方(Yi-Fang Wang)

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/博士(2016年)

https://doi.org/10.6342/NTU201602000

摘要

脊椎動物為了確保生理機能的正常運作必須維持體內氯離子的恆定，在由水生生物演化為陸生的過程中發展出一套複雜且精細的調控氯離子恆定的機制，以適應環境中氯離子濃度的變化。魚類和陸生脊椎動物具有相似的調控氯離子機制，然而，非哺乳類的脊椎動物其調控氯離子的詳細機制則尚不明確。之前的研究發現鈉氯共同運輸蛋白2b (NCC2b)負責斑馬魚在鰓上以及表皮的氯離子吸收，然而，詳細的氯離子吸收的運輸途徑則仍屬未知。
內分泌系統的功能一直被視為在生物面對環境變化時負責維持正常的生理機能，然而，我們對於魚類內分泌系統對於氯離子吸收的機制上所知甚少。因此，本研究旨在以斑馬魚為模式物種調查和表現NCC2b 的離子細胞相關的氯離子吸收途徑，並進一步研究和氯離子吸收相關的賀爾蒙及其調控的機制與角色為何。
第一章主要研究 CLC 氯離子通道在氯離子吸收扮演的角色。藉由原位雜和反應及免疫螢光染色實驗發現CLC-2c 表現在NCC2b 離子細胞上，當clc-2c 基因表現缺失時，會造成斑馬魚胚胎體內氯離子含量下降，顯示CLC-2c 和氯離子吸收相關。並且，當clc-2c 基因表現缺失時會刺激ncc2b mRNA 的表現，反之亦然。由分子與細胞生理的實驗結果證明CLC-2c 和NCC2b 共同負責斑馬魚鰓上及表皮的氯離子吸收功能。綜合演化上的分析，魚類特有的NCC2b 和CLC-2c可能是共同參與魚類鰓上的氯離子吸收機制而共同演化保留至今。
第二章主要研究 CGRP 和其受器CRLR1 對於氯離子吸收機制的調控。適應在高氯的環境中會刺激cgrp 和crlr1 mRNA 的表現，當cgrp 基因缺失的時候會刺激ncc2b RNA 的表現同時造成表現NCC2b 的離子細胞的密度增高，反之，當CGRP過量表現時會抑制ncc2b mRNA的合成同時造成斑馬魚胚胎氯離子吸收的減少。本實驗首次證實CGRP 會經由抑制NCC2b 的合成和降低表現NCC2b 離子細胞的分化來調控降低血液中的氯離子含量，了解CGRP 在調控氯離子吸收方面扮演的角色有助於我們更加了解脊椎動物相關的生理功能。
第三章主要研究 AVT 對於氯離子吸收調控的機制。當avt 基因功能缺失時會造成NCC2b mRNA 和蛋白質的表現下降，同時造成表現NCC2b 的離子細胞的密度降低並且造成胚胎體內氯離子含量的下降，實驗結果顯示AVT 藉由調控離子運輸蛋白的轉抑或是轉錄機制進而達到調控氯離子吸收的功能。此外，avt基因功能缺失亦會造成cgrp 及crlr1 mRNA 表現量下降，顯示了AVT 和CGRP之間有交互作用以便共同調控氯離子吸收的路徑，維持生物體內的氯離子恆定。

關鍵字

斑馬魚 ； 氯離子 ； 體液恆定 ； 賀爾蒙

並列摘要

Vertebrates need to maintain body fluid Cl- homeostasis to ensure normal operation of physiological process; the transition from aquatic to terrestrial environments necessitated the development of sophisticated mechanisms to ensure Cl- homeostasis in the face of fluctuating Cl-levels. The homeostatic mechanism of Cl- in aquatic fish appears to be similar to that of terrestrial vertebrates; however, the mechanism in non-mammalian vertebrates is poorly understood. Previous studies in zebrafish identified Na+-Cl- cotransporter (NCC) 2b-expressing cells in the gills and skin as the major ionocytes responsible for Cl- uptake. However, the mechanism by which basolateral ions exit from NCC2b-expressing cells is still unclear. Endocrine system is considered as the primary system to response to the environmental changes and maintain the normal physiological functions in vertebrates. However, less is known about the endocrine control of Cl- uptake mechanism in fish. The aims of this study are to use zebrafish as a model to investigate the Cl- uptake pathway of NCC2b-expressing ionocytes and identify hormones involved in the Cl- uptake mechanisms and to elucidate the mechanism in the regulation of Cl- uptake function.
In the first chapter, the role of CLC Cl- channels in the Cl- uptake mechanism were examined. Doubled in situ hybridization/immunocytochemistry indicated colocalization of apical NCC2b with basolateral CLC-2c. Loss-of-function of clc-2c resulted in a significant decrease in whole body Cl- content in zebrafish embryos, which suggests a role of CLC-2c in Cl- uptake. Translational knockdown of clc-2c stimulated ncc2b mRNA expression and vice versa, revealing cooperation between these two transporters in the context of zebrafish Cl- homeostasis. Several lines of molecular and cellular physiological evidences demonstrated the cofunctional role of apical NCC2b and basolateral CLC-2c in the gill/skin Cl- uptake pathway. Taking the phylogenetic evidence into consideration, fish-specific NCC2b and CLC-2c may have coevolved to perform extra-renal Cl- uptake during the evolution of vertebrates in an aquatic environment.
In the second chapter, the roles of calcitonin gene-related peptide (CGRP) and its receptor, calcitonin receptor-like receptor (CRLR1), in the regulation of Cl- uptake mechanism were examined. Acclimation to high-Cl− artificial water stimulated the mRNA expression of cgrp and crlr1 when compared with low-Cl−. CGRP knockdown induced upregulation of the ncc2b, while overexpression of CGRP resulted in the downregulation of ncc2b mRNA synthesis and a simultaneous decrease in Cl− uptake in embryos. Consistent with these findings, knockdown of either cgrp or crlr1 was found to increase the density of NCC2b-expressing cells in embryos. This is the first demonstration that CGRP acts as a hypochloremic hormone through suppressing NCC2b expression and the differentiation of NCC2b-expressing cells. Elucidation of this novel function of CGRP in fish body fluid Cl− homeostasis promises to enhance our understanding of the related physiology in vertebrates.
In the third chapter, the role of arginine vasotocin (AVT) on Cl- uptake regulation was examined. The loss-of-function of avt significantly downregulated the mRNA and protein expressions of NCC2b. Moreover, NCC2b expressing cells were significantly decreased in avt morphants. The whole body Cl- content was also declined in avt morphants. These results suggest that AVT exerts its actions on Cl uptake pathway in zebrafish embryos through regulating the transcriptional and/or translational levels of the ion transporters. Notably, the mRNA expression of cgrp and crlr1 were downregulated in avt morphants, suggesting that the crosstalk between AVT and CGRP mediated the regulation signaling of Cl-uptake mechanisms in zebrafish.

並列關鍵字

zebrafish ； chloride ； homeostasis ； hormone

參考文獻

Background references

Breves, J.P., Serizier, S.B., Goffin, V., McCormick, S.D., Karlstrom, R.O., 2013. Prolactin regulates transcription of the ion uptake Na+/Cl- cotransporter (ncc) gene in zebrafish gill. Molecular and Cellular Endocrinology 369, 98-106.

Chou, M.Y., Hung, J.C., Wu, L.C., Hwang, S.P.L., Hwang, P.P., 2011. Isotocin controls ion regulation through regulating ionocyte progenitor differentiation and proliferation. Cellular and Molecular Life Sciences 68, 2797-2809.

Costanzo, L.S., 1985. Localization of diuretic action in microperfused rat distal tubules: Ca and Na transport. Am J Physiol 248, F527-535.

Gamba, G., Saltzberg, S.N., Lombardi, M., Miyanoshita, A., Lytton, J., Hediger, M.A., Brenner, B.M., Hebert, S.C., 1993. Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter. Proc Natl Acad Sci U S A 90, 2749-2753.