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

1. 为了阐明皮质醇在鳃氯细胞功能分化中的作用，采用原位杂交和免疫细胞化学染色的方法检测了淡水鲑鱼（FW）和海水鲑鱼（SW）鱼苗中皮质醇受体的细胞基因表达和定位。原位杂交和抗皮质醇受体血清特异性免疫染色检测了分布在鳃丝和鳃片中的两种氯细胞的受体基因表达，提示皮质醇可能是调节氯细胞功能的重要因素之一。在SW适应的鱼苗中被高度激活的氯丝细胞中皮质醇受体的基因表达在SW适应的鱼苗中显著高于fw适应的鱼苗。皮质醇受体也存在于层间区未分化的细胞中，也提示皮质醇参与了氯细胞的功能分化。研究了莫桑比克罗非鱼胚和幼鱼卵黄囊膜中氯离子细胞形态的变化。我们用线粒体特异性荧光探针DASPEI标记氯细胞，通过共聚焦激光扫描显微镜观察单个氯细胞的体内序列变化。受精后3 d从FW转移到SW的胚胎，96 h后75%的氯细胞存活，且每个细胞的大小都显著增加。相反，在FW中保存的胚胎和幼虫细胞大小没有变化。在FW和SW中观察到相同的氯化物细胞更新速率。免疫荧光观察显示，在SW适应过程中，单个氯细胞扩大，并经常被新分化的辅助细胞压痕形成多细胞复合物。这些结果表明，在SW适应过程中，fw型单氯细胞转化为SW型多细胞复合物。

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.