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First published online May 24, 2005
Journal of Experimental Biology 208, 2023-2036 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01611

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Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na⁺/K⁺-ATPase, Na⁺/K⁺/2Cl^- cotransporter and CFTR anion channel

Junya Hiroi^1,*, Stephen D. McCormick^2,3, Ritsuko Ohtani-Kaneko¹ and Toyoji Kaneko⁴

¹ Department of Anatomy, St Marianna University School of Medicine, Miyamae-ku, Kawasaki 216-8511, Japan
² USGS, Conte Anadromous Fish Research Center, Turners Falls, MA 01376, USA
³ Department of Biology, University of Massachusetts, Amherst, MA 01003, USA
⁴ Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

View larger version (33K): [in a new window]   Fig. 1. A typical type-I cell in the yolk-sac membrane of a tilapia embryo at 0 h of the freshwater-to-seawater transfer experiment. (A,C,E,G) X-Z optical sections, cut transversely at the horizontal lines indicated in H, I and J. (B,D,F,H,I,J) X-Y optical sections, cut transversely at the line indicated in G. The immunofluorescence for Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) is shown as separate channels (A-F), and the three channels are merged in G and H. The merged X-Y images are further merged with differential interference contrast images (DIC+flu, I,J). n, nucleus. Scale bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 2. A typical type-II cell (A-J), and those with a hairline-like apical opening (K-O) and with a wide apical opening (P-S) at 72 h of the seawater-to-freshwater transfer experiment. (A,C,E,G,K,P) X-Z optical sections. (B,D,F,H,I,J,L,M,N,O,Q,R,S) X-Y optical sections. n, nucleus; *, apical opening of type-II cell. Scale bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 3. A typical type-III cell at 0 h of the freshwater-to-seawater transfer experiment (A-J) and with noticeable apical NKCC staining at 72 h of the seawater-to-freshwater transfer experiment (K-U). (A,C,E,G,K,M,O,Q) X-Z optical sections. (B,D,F,H,I,J,L,N,P,R,S,T,U) X-Y optical sections. n, nucleus; ac, accessory cell; *, apical opening of type-III cell. Scale bar, 10 µm.

View larger version (93K): [in a new window]   Fig. 4. A typical type-IV cell (A-K) and two type-IV cells of similar size sharing an common apical opening (L-O) at 72 h of the freshwater-to-seawater transfer experiment. (A,C,E,G,L) X-Z optical sections. (B,D,F,H,I,J,K,M,N,O) X-Y optical sections. n, nucleus; ac, accessory cell; *, apical opening of type-IV cell. Scale bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 5. X-Y projection images of immunofluorescence staining for Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR), at 0 h (A-D), 24 h (E-H) and 72 h (I-L) of the freshwater-to-seawater transfer experiment, and at 0 h (M-P), 24 h (Q-T) and 72 h (U-X) of the seawater-to-freshwater transfer experiment. Arrowheads and arrows indicate apical NKCC staining and apical CFTR staining, respectively. I, type-I cell; II, type-II cell; III, type-III cell; IV, type-IV cell. Scale bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 6. Changes in the density of the four mitochondrion-rich cell types following transfer from freshwater to seawater (A) and vice versa (B). The data are expressed as the mean ±S.E.M. of four individuals. Columns with different letters are significantly different (P<0.0125, the Bonferroni/Dunn test). red, type-I cell; cyan, type-II cell; magenta, type-III cell; green, type-IV cell.

View larger version (32K): [in a new window]   Fig. 7. Size/frequency distributions of the mitochondrion-rich cell types existing at 0 h and 72 h of the freshwater-to-seawater transfer experiment (A) and seawater-to-freshwater transfer experiment (B). Data of four individuals were pooled for each histogram. red, type-I cell; cyan, type-II cell; magenta, type-III cell; green, type-IV cell.

View larger version (23K): [in a new window]   Fig. 8. Schematic diagrams of the four mitochondrion-rich cell types, showing intracellular localization of Na+/K+-ATPase (red), Na+/K+/2Cl- cotransporter (NKCC, cyan) and cystic fibrosis transmembrane conductance regulator (CFTR, green). The movements of ions are focused on Na+ and Cl-, and a K+ channel recycling K+ is omitted in order to simplify the diagrams. A Cl- channel (gray) is added on the basolateral membrane of the type-II cell, although its presence was not examined in this study. The presumed interrelationship among the four cell types are indicated by arrows A-E.