Establishment of new approaches of ion-homeostasis study with zebrafish mutant

1)从环境中吸收Na^+是包括斑马鱼在内的淡水鱼生存不可或缺的策略，因为它们很容易从血浆中失去Na^+到稀释的环境中。然而，参与Na^+摄取的分子的位置仍然知之甚少。在这项研究中，我们利用Na^+依赖的荧光试剂钠绿，提供了直接证据，证明Na^+吸收发生在斑马鱼幼虫卵黄囊表面的富线粒体细胞（MRCs）亚群中。结合免疫组织化学，我们发现Na^+吸收MRCs异常丰富的液泡型H^+- atp酶（H^+- atp酶），但适度丰富的Na^+/K^+- atp酶。我们还研究了fox3a的功能，fox3a是一种在富含H^+- atpase的MRCs中特异性表达的转录因子。通过利用反义morpholino寡核苷酸从斑马鱼胚胎中去除foxi3a，我们证明了foxi3a在MRC分化中是必不可少的，并且MRC是Na^+吸收的主要位点。此外，foxi3a的表达开始于原肠期的推定外胚层；因此，我们认为fox3a是控制MR细胞分化的关键基因。2)我们测试了斑马鱼幼虫期内分泌系统对环境盐度挑战的响应能力。我们发现斑马鱼幼体有一个内分泌系统，包括心房钠肽（anp）、肾素、催乳素、生长激素和甲状旁腺激素1 （pth1）在内的几种内分泌基因在转录水平上响应环境盐度的变化，并且这些响应是基因特异性的。anp和肾素在淡淡水培养基中均上调，而在浓淡水培养基中下调。另一方面，催乳素和生长激素的表达在稀释培养基中得到强烈增强，但在高盐度条件下几乎没有变化。有趣的是，正如在哺乳动物中观察到的那样，pth1的表达取决于Ca^<2+>浓度。因此，结合模式生物的优势，包括遗传方法的可及性，我们提出斑马鱼幼虫对离子和渗透稳态内分泌系统调节机制的综合研究是有用的。少

1)Uptake of Na^+ from the environment is an indispensable strategy for the survival of freshwater fish including zebrafish, as they easily lose Na^+ from the plasma to a diluted environment. Nevertheless, the location of, and molecules involved in Na^+ uptake remain poorly understood. In this study, we utilized Sodium Green, a Na^+-dependent fluorescent reagent, to provide direct evidence that Na^+ absorption takes place in a subset of the mitochondrion-rich cells (MRCs) on the yolk sac surface of zebrafish larvae. 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. We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H^+-ATPase-rich MRCs. By depletion of foxi3a from zebrafish embryos using antisense morpholino oligonucleotides, we demonstrated that foxi3a is indispensable for MRC differentiation and MRCs are … More primary site for Na^+ absorption. Additionally, foxi3a expression is initiated at gastrula stage in the presumptive ectoderm ; thus we propose that foxi3a is a key gene in the control of MR cell differentiation.2)We tested the capability of the endocrine system in zebrafish to respond to environmental salinity challenges during larval stages. We reveal that the zebrafish larvae has a system in which several endocrine genes, including atrial natriuretic peptide (anp), renin, prolactin, growth hormone and parathyroid hormone 1 (pth1), respond at the transcription level to changes in environmental salinity and that the responses are gene specific. Both anp and renin are upregulated in larvae raised in dilute freshwater medium but are downregulated in concentrated medium. On the other hand, expression of prolactin and growth hormone is strongly enhanced in the dilute medium, but shows little or no change under higher salinity conditions. Interestingly, pth1 expression depends on Ca^<2+> concentration, as observed in mammals. Thus, taken together with the advantages of a model organism, including accessibility to genetic approaches, we propose that zebrafish larvae are useful for a comprehensive study of the regulatory mechanisms of the endocrine system in ionic and osmotic homeostasis. Less