Functional Diversity of Chloride Cells in Iono-and Osmo-Regulation in Fish

1.为阐明皮质醇参与鳃氯细胞功能分化的机制，采用原位杂交和免疫细胞化学染色方法，研究了淡水和海水环境下鲑鱼幼鱼鳃氯细胞皮质醇受体的基因表达和定位。用原位杂交和抗皮质醇受体血清特异性免疫组化方法检测了分布于鳃丝和鳃板的两种氯细胞中皮质醇受体基因的表达，表明皮质醇可能是调节氯细胞功能的重要因素之一。皮质醇受体的基因表达的丝状氯化物细胞，这是高度活化的SW适应鱼苗，是显着更大的鱼苗适应SW比FW适应鱼苗。皮质醇受体也存在于板层间区的未分化细胞中，这也表明皮质醇参与了氯细胞的功能分化.氯细胞形态的变化进行了研究，在卵黄囊膜莫桑比克罗非鱼胚胎和幼虫从FW转移到海水SW。标记氯细胞与DASPEI，一个线粒体特异性的荧光探针，我们观察在体内的顺序变化，在单个氯细胞的共聚焦激光扫描显微镜。受精后3d从FW移植到SW的胚胎中，96h后75%的氯细胞存活，每个细胞的大小显着增加。与此相反，细胞大小没有改变胚胎和幼虫保持在FW。在FW和SW中观察到相同的氯细胞周转率。免疫荧光观察显示，单个氯细胞扩大，并经常被新分化的辅助细胞缩进SW适应过程中形成多细胞复合物。这些结果表明，FW型单氯细胞转化为SW型多细胞复合体在SW适应。

1. To clarify the involvement of cortisol in functional differentiation of branchial chloride cells, cellular gene expression and localization of cortisol receptor were examined in chum salmon fry in fresh water (FW) and those adapted to seawater (SW) by jn situ hybridization and immunocytochemical staining. The receptor gene expression examined by in situ hybridization and specific immunostaining with anti-cortisol receptor serum were found in two types of chloride cells distributed in the gill filaments and lamellae, indicating that cortisol may be one of the important factors regulating chloride cell functions. Gene expression of cortisol receptor in filament chloride cells, which were highly activated in SW-adapted fry, was significantly greater in the fry adapted to SW than in FW-adapted fry. Cortisol receptors were also present in undifferentiated cells in the interlamellar regions, also suggesting the involvement of cortisol in the functional differentiation of chloride cells.2. Changes in chloride cell morphology were examined in the yolk-sac membrane of Mozambique tilapia embryos and larvae transferred from FW to seawater SW. Labeling chloride cells with DASPEI, a fluorescent probe specific for mitochondria, we observed jn vivo sequential changes in individual chloride cells by confocal laser scanning microscopy. In embryos transferred from FW to SW 3 days after fertilization, 75% of chloride cells survived 96 h later, and each cell showed a remarkable increase in size. In contrast, the cell size did not change in embryos and larvae kept in FW. The same rate of chloride cell turnover was observed in both FW and SW.Immunofluorescence observations revealed that single chloride cells enlarged and were frequently indented by newly-differentiated accessory cells to form multicellular complexes during SW adaptation. These results indicate that FW-type single chloride cells are transformed into SW-type multicellular complexes during SW adaptation.