Chloride uptake mechanism and the functional regulation in zebrafish
Date Issued
2016
Date
2016
Author(s)
Wang, Yi-Fang
Abstract
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.
Subjects
zebrafish
chloride
homeostasis
hormone
Type
thesis
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