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First published online December 26, 2008
Journal of Experimental Biology 212, 287-296 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.022822

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Occludin and hydromineral balance in Xenopus laevis

Helen Chasiotis^* and Scott P. Kelly

Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3

View larger version (12K): [in this window] [in a new window]   Fig. 1. Distribution of occludin mRNA in Xenopus tissues by RT-PCR analysis. Xenopus β-actin (bottom panel) was used as a loading control. A negative control for each gene was also run. Amplicon size for occludin and β-actin was 351 and 361 bp, respectively.

View larger version (79K): [in this window] [in a new window]   Fig. 2. Occludin and Na+,K+-ATPase immunolocalization in cross-sections of Xenopus (A) stomach, (B) anterior intestine, (C) posterior intestine and (D) rectum. Occludin (red) immunolocalized to apicolateral membranes of surface epithelial cells lining the stomach mucosa and enterocytes of the small intestine and rectum. Enhanced occludin immunostaining was also observed in the stomach between cells at the base of the gastric pits (arrow). Na+,K+-ATPase (green) was undetectable in the stomach but immunolocalized basolaterally in the small intestine and rectum. Control sections, probed with secondary antibody only, are shown in E and F for the stomach and rectum, respectively. L, lumen. Scale bars, 20 µm.

View larger version (130K): [in this window] [in a new window]   Fig. 3. (A) Longitudinal section of the Xenopus kidney stained with haematoxylin and eosin. A medial band of glomeruli (*) separates the ventromedial zone from the dorsolateral zone of the Xenopus kidney. Proximal tubules and collecting ducts are contained within the dorsolateral zone, while distal tubules are located within the ventromedial zone. (B) Na+,K+-ATPase immunostaining (green) within a longitudinal section of the Xenopus kidney. Within the dorsolateral zone, only collecting ducts show Na+,K+-ATPase immunoreactivity. Within the ventromedial zone, distal tubules (both early and late) show immunofluorescence labelling for Na+,K+-ATPase. (C–F) Dual-immunofluorescence labelling of occludin and Na+,K+-ATPase within cross-sections of the (C) proximal tubule, (D) early distal tubule, (E) late distal tubule and (F) collecting duct of the Xenopus nephron. Occludin (red) and Na+,K+-ATPase (green) were differentially expressed in the nephron. Occludin was undetectable in proximal tubules, but immunolocalized to apical membranes of renal epithelial cells of distal tubules (early and late) and the collecting duct. Na+,K+-ATPase weakly immunolocalized basally in proximal tubules and exhibited robust basolateral localization in the distal and collecting segments. A control section, probed with secondary antibody only, is shown in G. L, lumen. Scale bars for A and B, 300 µm; scale bars for C–G, 20 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 4. The effects of brackish water (BW) acclimation on Xenopus serum (A) osmolality, (B) urea, (C) Na+ and (D) Cl– as well as (E) muscle moisture content. Data are expressed as mean values ± s.e.m., N=8-16 per group. *Significant difference (P0.05) from freshwater (FW) group. Significant difference (P0.05) from all other BW-acclimated groups.

View larger version (17K): [in this window] [in a new window]   Fig. 5. (A) Occludin mRNA distribution in the Xenopus gastrointestinal (GI) tract by quantitative real-time PCR (qRT-PCR) analysis. Occludin mRNA expression increased along the longitudinal axis of the GI tract (i.e. stomach < anterior and posterior intestine < rectum). (B–D) The effects of BW acclimation on occludin mRNA expression in Xenopus (B) anterior intestine, (C) posterior intestine and (D) rectum. Occludin mRNA expression was normalized to β-actin mRNA expression (a.u., arbitrary units). Data are expressed as means ± s.e.m., N=6–7 per group. *Significant difference (P0.05) from FW group. Significant difference (P0.05) from 2 BW-acclimated group.

View larger version (8K): [in this window] [in a new window]   Fig. 6. The effects of BW acclimation on occludin mRNA expression in the Xenopus (A) kidney and (B) urinary bladder. Occludin mRNA expression was normalized to β-actin mRNA expression. Data are expressed as means ± s.e.m., N=4–6 per group. *Significant difference (P0.05) from FW group. Significant difference (P0.05) from 2 BW-acclimated group.

View larger version (7K): [in this window] [in a new window]   Fig. 7. BW acclimation and occludin mRNA expression in Xenopus (A) dorsal skin and (B) ventral skin. Occludin mRNA expression was normalized to β-actin mRNA expression. Data are expressed as means ± s.e.m., N=6–7 per group.

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