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，因为它们很容易将Na^+从血浆中流失到稀释的环境中。然而，对于Na^+摄取的位置和分子的了解仍然很少。在这项研究中，我们利用钠绿色（一种Na^+依赖性荧光试剂）提供了直接证据，证明Na^+吸收发生在斑马鱼幼鱼卵黄囊表面的富含钙离子的细胞（MRC）亚群中。结合免疫组化结果，我们发现，Na^+吸收的MRCs异常富含空泡型H^+-ATP酶（H^+-ATP酶），但Na^+/K^+-ATP酶中等丰富。我们还研究了foxi 3a的功能，foxi 3a是一种在富含H^+-ATP酶的MRC中特异性表达的转录因子。通过使用反义吗啉代寡核苷酸从斑马鱼胚胎中去除foxi 3a，我们证明了foxi 3a对于MRC分化是必不可少的，并且MRC是 ...更多信息 Na^+吸收的主要部位。此外，foxi 3a的表达起始于外胚层原肠胚阶段，因此我们认为foxi 3a是控制MR细胞分化的关键基因。2）我们测试了斑马鱼内分泌系统在幼体阶段对环境盐度挑战的反应能力。我们揭示了斑马鱼幼鱼具有一个系统，其中包括心房钠尿肽（anp）、肾素、催乳素、生长激素和甲状旁腺激素1（pth 1）在内的几个内分泌基因在转录水平上对环境盐度的变化做出反应，并且这种反应是基因特异性的。在稀释的淡水培养基中，幼鱼的心钠素和肾素均上调，而在浓缩的培养基中则下调。另一方面，催乳素和生长激素的表达在稀培养基中强烈增强，但在较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，pth 1的表达依赖于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是有用的离子和渗透稳态内分泌系统的调节机制的全面研究。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