Molecular characterization of chloride cells and their mechanism of differentiation

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We mainly focused on the chloride cells that are very unique in the structure and function and play key roles in adapting to low or high salt conditions and acidic or alkaline conditions. Our analyses contributed to pushing the forefront of not only osmoregulation of aquatic animals but also fluid homeostasis of the mammalian system. Furthermore mitochondrion-rich nature of chloride cells led us to explore the mitochondrial morphology and to discover a novel regulator of mitochondrial fusion and fission factors. (1) We have clarified the molecular mechanism underlying the acid adaptation of Osorezan dace living in a pH 3.5 lake by identifying molecules associated with gross morphological and functional changes of chloride cells. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism. (2) We succeeded in visualizing mitochondrion-rich cells (MRCs) … More responsible for uptake of Na^+ in freshwater using a fluorescent dye, Sodium Green. Combined with immunohistochemistry, we revealed that the Na^+-absorbing MRCs were exceptionally rich in vacuolar-type H^+-ATPase (H^+-ATPase) but moderately rich in Na^+/K^+-ATPase. (3) Through molecular, functional, and immunohistochemical characterization of ammonia transporters in fish for the first time, we demonstrated a tight link of ammonia excretion with MRCs and suggested a functional coupling between ion homeostasis and nitrogen metabolism. (4) We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H^+-ATPase-rich MR cells. When foxi3a was depleted from zebrafish embryos by antisense morpholino oligonucleotide injection, differentiation of the MRCs was completely blocked and Na+ influx was severely reduced, indicating that MRCs are the primary sites for Na^+ absorption. Considering these results with onset of the expression at gastrula stage, we proposed that foxi3a is a key gene for the control of MRC differentiation. The delta-notch signaling system has also been demonstrated to be involved in the differentiation and distribution of MRCs. (5) Mitochondria frequently fuse and divide to form a dynamic network in various eukaryotic cells. We showed that MARCH5 is a mitochondrial outer membrane protein and plays a crucial role in control of mitochondrial morphology by regulating Mfn2 (a fusion factor) and Drp1 (a fission factor) activities. Less