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Dynamics of Pavement Cell–Chloride Cell Interactions During Abrupt Salinity Change in FUNDULUS HETEROCLITUS

K. Daborn, R. R. F. Cozzi and W. S. Marshall^*

Biology Department, Saint Francis Xavier University, PO Box 5000, Antigonish, Nova Scotia, Canada B2G 2W5

View larger version (163K): [in a new window]   Fig. 1. Representative electron micrograph of the apical surface of a control opercular epithelium of a fully acclimated seawater killifish. Apical crypts (arrows) occur at high density. Scale bar, 10µm.

View larger version (169K): [in a new window]   Fig. 2. Representative electron micrograph of the apical surface of the opercular epithelium of a fully acclimated seawater killifish subjected to a hypotonic shock on the basolateral surface for approximately 1h. This opercular epithelium shows a considerably lower density of apical crypts (arrows) than the paired control (Fig.1). Scale bar, 10µm.

View larger version (183K): [in a new window]   Fig. 3. Representative electron micrograph of the apical surface of a control opercular epithelium from a freshwater-acclimated specimen showing no apical crypts between the pavement cells. Scale bar, 10µm.

View larger version (162K): [in a new window]   Fig. 4. Representative electron micrograph of a freshwater-acclimated killifish opercular epithelium treated with basolateral hypertonic shock. This epithelium shows a significantly higher density of apical crypts (arrows) than the freshwater control (Fig.3). Scale bar, 10µm.

View larger version (156K): [in a new window]   Fig. 5. Representative electron micrograph of a seawater-acclimated opercular epithelium pretreated with cytochalasin D, then treated with a hypotonic shock to the basolateral side. Note the failure of the apical crypts to close over and the high crypt density similar to that of seawater controls (Fig.1). This micrograph also shows characteristic angular gaps between the cells (arrows). Scale bar, 10µm.

View larger version (126K): [in a new window]   Fig. 6. Confocal microscope images of wet mounts of seawater-adapted killifish opercular epithelium. All are paraformaldehyde/glutaraldehyde-fixed, permeabilized and stained with Oregon Green phalloidin with or without Mitotracker Red pretreatment. (A) Fluorescence image of the opercular membrane at 1.5µm below the plane of the pavement cells. Note the actin rings (arrows) of apical crypts. Scale bar, 5.0µm. (B) The same frame as for A but 6.0µm deeper into the tissue. Arrows are in the same locations and indicate apical crypts over mitochondria-rich chloride cells stained with Mitotracker Red. The asterisk indicates an adjacent cell. (C) Compare this image of opercular membrane fixed in paraformaldehyde with 0.1% glutaraldehyde at the plane of the pavement cells with D, which shows the same frame but at a focal plane 2.0µm below the plane of the pavement cells. Scale bar, 5.0µm. Note the thick actin ring just below the opening of the apical crypt (arrow). (E) Opercular epithelium stained with Oregon Green phalloidin. The image was collected at the plane of the microridges of pavement cells. This tissue was pretreated with cytochalasin D and has typical angular gaps and holes between pavement cells (arrows), but the actin cords are for the most part intact. Scale bar, 5.0µm.

View larger version (14K): [in a new window]   Fig. 7. Regression of the total epithelial conductance of the opercular epithelium on the density of apical crypts as detected by scanning electron microscopy for freshwater-acclimated animals (N=11). Epithelia have been exposed to basolateral hypertonicity. Extrapolation to the y-intercept gives an estimate of the conductance of an epithelium lacking apical crypt openings.