First published online April 8, 2004
Journal of Experimental Biology 207, 1729-1739 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00930
Laser scanning cytometry and tissue microarray analysis of salinity effects on killifish chloride cells
Raquel N. Lima and
Dietmar Kültz*
University of California, Davis, One Shields Avenue, Davis, CA 95616,
USA
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Fig. 1. Salinity acclimation regimens used in this study. Killifish were subjected
to either acute transfer from freshwater (FW) to 1x seawater (SW, 1000
mosmol kg-1, green circles) or to gradual transfer from FW to
2.4x SW (2400 mosmol kg-1). During gradual transfers the
salinity was increased either at a rate of 300 mosmol kg-1 per day
(red triangles) or 250 mosmol kg-1 per day (black triangles) until
it reached 2400 mosmol kg-1. FW controls were transferred once on
the first day to FW (blue diamonds).
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Fig. 2. (A-C) Laser scanning cytometry (LSC) analysis of chloride cells (CC) from
killifish acclimated for 10 days to FW (blue), 1x SW (green) and
2.4x SW (red). CC were identified based on DASPMI fluorescence with the
LSC. CC area and maximal intensity of DASPMI fluorescence were recorded for
each CC during scanning by WinCyte software and plotted as scattergrams. (A)
FW; (B) 1x SW; (C) 2.4x SW. The boxes enclose all accepted data
and the circles enclose events that were used for generating image galleries
with the LSC relocation function. (D-F) Additional scattergrams showing the
relationship between CC area and integrated DASPMI fluorescence per CC were
also recorded. (D) FW; (E) 1x SW; (F) 2.4x SW. CCs have higher
DASPMI fluorescence than other cell types and are colored in all panels.
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Fig. 4. Laser scanning images (A-C) and corresponding LSC histogram (D) of chloride
cells (CC) from killifish exposed to FW, 1x SW or 2.4x SW for 1
week. Image galleries were acquired using the relocation function of the LSC.
Cells shown in image galleries represent CC from within the circled areas in
Fig. 2A-C. (A) CC from FW fish;
(B) CC from 1x SW fish; (C) CC from 2.4x SW fish. (D) Histogram of
CC area from fish acclimated to FW (blue), 1x SW (green) and 2.4x
SW (red). The arrow points to a population of small cells that may represent
accessory cells in the SW groups. Bars, 50 µm.
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Fig. 5. Laser scanning images (A-C) and corresponding LSC histogram (D) of chloride
cells (CC) from killifish exposed to FW, 1x SW or 2.4x SW for 2
weeks. Image galleries were acquired as described for
Fig. 4. (A) CC from FW fish;
(B) CC from 1x SW fish; (C) CC from 2.4x SW fish. (D) Histogram of
CC area from fish acclimated to FW (blue), 1x SW (green) and 2.4x
SW (red). The arrow points to a population of small cells that may represent
accessory cells. Note the shift of CC to a larger area in the SW treatment
groups compared to Fig. 4.
Bars, 50 µm.
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Fig. 6. Laser scanning images (A-C) and corresponding LSC histogram (D) of chloride
cells (CC) from killifish exposed to FW, 1x SW or 2.4x SW for 5
weeks. Image galleries were acquired as described for
Fig. 4. (A) CC from FW fish;
(B) CC from 1x SW fish; (C) CC from 2.4x SW fish. (D) Histogram of
CC area from fish acclimated to FW (blue), 1x SW (green) and 2.4x
SW (red). The arrow points to a population of small cells that may represent
accessory cells. Note the shift of CCs to a larger area in the 2.4x SW
treatment group compared to Fig.
5. Bars, 50 µm.
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Fig. 3. Chloride cell (CC) number and size determined by LSC analysis from gill
epithelial cell suspensions obtained after 10 days of acute and gradual
acclimation from FW to SW. (A) CC number does not increase significantly after
acclimation to 1x SW or 2.4x SW (P>0.05 in each case).
(B) CC volume increases significantly after acclimation to 1x and
2.4x SW (P<0.05; aFW vs. 1x SW,
bFW vs. 2.4x SW, c1x SW
vs. 2.4x SW). N=4 animals for each salinity and
time.
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Fig. 7. Mean chloride cell (CC) number and size determined by LSC analysis from
gill epithelial cell suspensions after acute or gradual acclimation from FW
(blue diamonds) to 1x SW (green circles) or 2.4x SW (red
triangles), respectively, versus acclimation period. (A) In the
1x SW group, CC volume is significantly larger at all the times analyzed
(P<0.05) but does not continue to increase after 2 weeks. In
contrast, during 5 weeks of 2.4x SW acclimation CC become significantly
larger only after 2 weeks, but continue to increase (P<0.05). (B)
CC number does not significantly increase until 2 weeks for 2.4x SW and
until 5 weeks for 1x SW acclimation (P<0.05; aFW
vs. 1x SW, bFW vs. 2.4x SW,
c1x SW vs. 2.4x SW). N=4 animals for
each salinity and time.
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Fig. 8. Quantification of Na+/K+-ATPase per chloride cell
(CC) and CC area dependence on salinity and acclimation period. (A) Tissue
microarray containing 1 mm cores of gill filament from fish acclimated to FW
or SW for different times. Bar, 1 mm. (B) Na+/K+-ATPase
antibody combined with PacificBlue-conjugated secondary antibody specifically
labels CC. (C) Automatic laser scanning cytometer (LSC) contouring of CC in
the same area as shown in A. (D) Quantification of
Na+/K+-ATPase content per CC based on LSC analysis (FW,
blue diamonds; 1x SW, green circles; 2.4x SW, red triangles).
Na+/K+-ATPase in CC is significantly but transiently
elevated only at 1 week for fish acclimated to 1x SW
(P<0.05). In contrast, Na+/K+-ATPase in CC
is significantly elevated at all times measured in fish acclimated to
2.4x SW (P<0.05) (E) CC area based on contouring of
Na+/K+-ATPase fluorescence as shown in B. Although
elevated at all times for both 1x and 2.4x SW groups the increase
is statistically significant only at 1 week (P<0.05;
aFW vs. 1x SW, bFW vs.
2.4x SW, c1x SW vs. 2.4x SW).
N=4 animals for each salinity and time.
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Fig. 9. Total Na+/K+-ATPase content in all gill CC of F.
heteroclitus acclimated to FW (blue diamonds), 1x SW (green
circles), or 2.4x SW (red triangles) expressed as relative fluorescence
units (RFU). (A) Time dependence of total Na+/K+-ATPase
content at different salinity acclimation regimens (P<0.05;
aFW vs. 1x SW, bFW vs.
2.4x SW, c1x SW vs. 2.4x SW). (B)
Salinity-dependence of total Na+/K+-ATPase content after
5 weeks of acclimation. The data fit an exponential regression with
r2=0.981. N=4 animals for each salinity and
time.
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© The Company of Biologists Ltd 2004
