Molecular characterization of chloride cells and their mechanism of differentiation

我们主要关注在结构和功能上非常独特的氯离子细胞，它们在适应低盐或高盐条件以及酸性或碱性条件方面发挥着关键作用。我们的分析不仅推动了水生动物渗透调节的前沿，也推动了哺乳动物系统的液体动态平衡。此外，氯细胞富含线粒体的特性使我们探索了线粒体的形态，并发现了一种新的线粒体融合和分裂因子的调节因子。(1)我们通过鉴定与氯细胞大体形态和功能变化相关的分子，阐明了生活在pH 3.5湖泊中的水华鱼酸适应的分子机制。在不同组织中也观察到了谷氨酸脱氢酶的表达增加，这表明谷氨酰胺分解代谢产生的氨和碳酸氢盐起着重要作用。(2)成功实现了富线粒体细胞(Mrcs)…的可视化更多的是使用荧光染料钠绿来吸收淡水中的钠离子。结合免疫组织化学，我们发现吸收Na~+的MCs特别富含液泡型H^-ATPase(H^-ATPase)，但中等富含Na~+/K~+-ATPase。(3)首次通过对鱼类氨转运蛋白的分子、功能和免疫组织化学特征的研究，证实了鱼类体内氨转运蛋白与细胞排氨的紧密联系，并提出了离子稳态与氮代谢之间的功能耦合关系。(4)我们还研究了富含H^-ATPase的MR细胞中特异表达的转录因子foxi3a的功能。当注射反义吗啉寡核苷酸去除斑马鱼胚胎中的foxi3a时，mRCs的分化被完全阻断，Na内流严重减少，表明mRCs是吸收Na+的主要部位。考虑到这些结果是在原肠胚期开始表达的，我们认为foxi3a是控制MRC分化的关键基因。Delta-Notch信号系统也被证明参与了MRC的分化和分布。(5)在各种真核细胞中，线粒体频繁融合分裂，形成动态网络。我们发现March5是一种线粒体外膜蛋白，通过调节Mfn2(融合因子)和Drp1(分裂因子)的活性，在线粒体形态调控中发挥关键作用。较少

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