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First published online October 10, 2003

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Short-term transformation and long-term replacement of branchial chloride cells in killifish transferred from seawater to freshwater, revealed by morphofunctional observations and a newly established `time-differential double fluorescent staining' technique

Fumi Katoh^* and Toyoji Kaneko

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan

View larger version (15K): [in a new window]   Fig. 1. Changes in plasma Na+ concentration (A) and branchial chloride cell size (B) following direct transfer from seawater to freshwater. Values are means ± S.E.M. (N=5). Asterisks indicate significant differences compared with the values of seawater-adapted fish (0 h).

View larger version (26K): [in a new window]   Fig. 2. Western blot analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) protein expressed in the gills of seawater-adapted killifish. The membrane was incubated with (A) or without (B) anti-CFTR. One specific protein band (arrowhead) was detected in lane A. The positions of molecular markers (kDa) are indicated on the left side of the figure.

View larger version (84K): [in a new window]   Fig. 3. Confocal laser scanning micrographs of whole-mount preparations of the gill filaments (A,C,E,G,I,K) and fluorescence microscope images of gill cryosections (B,D,F,H,J,L) in killifish at 0 h (A,B), 3 h (C,D), 12 h (E,F), 1 day (G,H), 7 days (I,J) and 30 days (K,L) after transfer from seawater to freshwater. Gill filaments were double stained with anti-cystic fibrosis transmembrane conductance regulator (green) and anti-Na+/K+-ATPase (red). Scale bar, 50 µm.

View larger version (145K): [in a new window]   Fig. 4. Scanning electron micrographs of gill filaments in killifish at 0 h (A,B), 3 h (C,D), 12 h (E,F), 1 day (G), 3 days (H,I), 7 days (J), 14 days (K) and 30 days (L,M) after transfer from seawater to freshwater. (B,D,F,I,M) Enlarged views of the apical region of chloride cells, showing an apical surface of an accessory cell next to the chloride cell (arrowhead in D), which is characteristic of the seawater-type chloride cell. Scale bars, 10 µm (A,C,E,G,H,J-L); 1 µm (B,D,F,I,M).

View larger version (142K): [in a new window]   Fig. 5. Transmission electron micrographs of branchial chloride cells (cc) in killifish at 0 h (A), 3 h (B), 12 h (C), 1 day (D), 3 days (E), 7 days (F), 14 days (G) and 30 days (H) after transfer from seawater to freshwater. Multicellular complexes were observed at 0 h and 3 h after transfer. Arrowheads indicate numerous mitochondria. ap, apical pit; pv, pavement cell; ac, accessory cell; *, microvilli. Scale bar, 1 µm.

View larger version (74K): [in a new window]   Fig. 6. Time-differential double fluorescent staining of branchial chloride cells in killifish transferred from seawater to freshwater (A,B), or maintained in freshwater (D,E) or in seawater (G,H). Gill chloride cells were double labeled in vivo with Rhodamine 123 (A,D,G) just before transfer (day 0) and MitoTracker (B,E,H) on day 3. Cells positive for both Rhodamine 123 and MitoTracker were pre-existing. Arrows indicate newly differentiated chloride cells, which are Rhodamine 123-negative (black arrows)/MitoTracker positive (white arrows). (C,F,I) Changes in chloride cell density on day 0 and day 3 in fish transferred from seawater to freshwater (C), and fish maintained in freshwater (F) or in seawater (I). Values are means ± S.E.M. Green and red portions of the columns indicate the density of pre-existing and newly differentiated chloride cells, respectively. Significant differences in frequency of newly differentiated chloride cells were detected between freshwater-transferred and two control groups (P<0.01). An asterisk indicates significant difference from the density of Rhodamine 123-positive, pre-existing cells on day 0 (P<0.01). Scale bar, 20 µm.

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