Molecular characterization of chloride cells and their mechanism of differentiation

我们主要关注氯离子细胞，它们在结构和功能上非常独特，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。我们的分析不仅有助于推动水生动物渗透调节的前沿，而且有助于推动哺乳动物系统液体稳态的前沿。此外，氯化物细胞富含线粒体的性质促使我们探索线粒体形态并发现线粒体融合和裂变因子的新型调节剂。 (1) 通过鉴定与氯细胞总体形态和功能变化相关的分子，我们阐明了生活在 pH 3.5 湖泊中的 Osorezan 鲮鱼酸适应的分子机制。在适应酸的鲮鱼的各种组织中也观察到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具有重要作用。 (2) 我们使用荧光染料钠绿成功地可视化了富含线粒体的细胞 (MRC)，这些细胞负责吸收淡水中的 Na^。结合免疫组织化学，我们发现吸收Na^的MRC异常富含液泡型H^-ATP酶（H^-ATP酶），但中等富含Na^/K^-ATP酶。 (3) 通过对鱼类氨转运蛋白的分子、功能和免疫组织化学表征，我们首次证明了氨排泄与 MRC 的紧密联系，并提出了离子稳态和氮代谢之间的功能耦合。 (4)我们还探讨了foxi3a的功能，foxi3a是一种在富含H^-ATP酶的MR细胞中特异性表达的转录因子。当通过反义吗啉代寡核苷酸注射从斑马鱼胚胎中耗尽foxi3a时，MRC的分化被完全阻断并且Na流入严重减少，表明MRC是Na + 吸收的主要位点。考虑到在原肠胚阶段开始表达的这些结果，我们提出foxi3a是控制MRC分化的关键基因。 delta-notch 信号系统也被证明参与 MRC 的分化和分布。 (5)线粒体在各种真核细胞中频繁融合和分裂，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（裂变因子）活性，在控制线粒体形态方面发挥着至关重要的作用。较少的

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