Laser scanning cytometry and tissue microarray analysis of salinity effects on killifish chloride cells
University of California, Davis, One Shields Avenue, Davis, CA 95616, USA
* Author for correspondence (e-mail: dkueltz{at}ucdavis.edu)
Accepted 10 February 2004
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Summary |
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Key words: chloride cell, salinity adaptation, killifish, Fundulus heteroclitus, gill epithelium, osmoregulation, tissue microarray, laser scanning cytometry, Na+/K+-ATPase
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Introduction |
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In this study we utilize laser scanning cytometry (LSC) and tissue microarrays (TMAs) for quantitative analysis of salinity effects on CC and Na+/K+-ATPase in gills of the euryhaline killifish Fundulus heteroclitus. We hypothesize that using this approach it is possible to compare salinity effects on chloride cell properties in situ and in dissociated gill cell suspensions and that this will enable us to better understand the kinetics of adaptation during acute and gradual salinity increase. A major advantage of the TMA-LSC approach is its high-throughput capability and extraordinary sensitivity in recording fluorescence-based properties of thousands of individual chloride cells in a very short time. In addition, because TMAs array all samples of one or more experiments on a single slide they can be stained and analyzed under identical conditions. Thus, immunohistochemistry and other fluorescence-based measurements using this technique are highly quantitative when comparing salinity effects in different samples.
Laser scanning cytometry (LSC) was developed as a hybrid-technology merging
the advantages of laser scanning fluorescence microscopy and flow cytometry
(Kamentsky, 2001). Tissue
microarrays (TMAs) are an ideal complementary technology to LSC because they
permit high-throughput analysis of many tissue samples simultaneously under
identical conditions, thus minimizing sample-to-sample variation. TMAs were
originally developed to accelerate the scoring of tumor tissues for clinical
pathology (Bubendorf et al.,
2001
). However, because of their versatility TMAs are increasingly
popular for many other applications in biological research and they promise to
become a powerful tool for comparative experimental biology. Using LSC and TMA
technology it is possible to perform large-scale analyses of proteins, e.g. by
immunophenotyping (Mocellin et al.,
2001
), of DNA, e.g. by nuclear DNA staining
(Buse et al., 1999
) and of RNA,
e.g. by fluorescence in situ polymerase chain reaction
(Pachmann et al., 2001
). Such
LSC/TMA-based analyses are much faster, less error-prone and more
cost-efficient than other approaches.
Using this novel approach we have investigated the kinetics of CC hypertrophy and proliferation as well as Na+/K+-ATPase abundance in CC of killifish gill epithelium after exposure of fish to acute and gradual salinity acclimation regimens.
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Materials and methods |
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Gill perfusion, cell dissociation and live cell staining
At each sampling point during the acclimation experiments fish were killed
by exposure to a lethal dose of MS-222 (0.4% aminobenzoic
acid-ethylether-methanesulfonate). The pericardium was opened and the gills
perfused via the bulbus arteriosus with ice-cold phosphate-buffered
saline (PBS: 146 mmol l-1 NaCl, 3 mmol l-1 KCl, 15 mmol
l-1 NaH2PO4, 15 mmol l-1
Na2HPO4, 10 mmol l-1 NaHCO3, pH
7.4; osmolality=330 mosmol kg-1). After blood had been removed and
gills had turned white (ca. 1-3 min) individual gill arches were dissected.
All four gill arches of the left side were rinsed in PBS and fixed in 4%
paraformaldehyde for immunohistochemistry (see below). All four gill arches of
the right side were rinsed with PBS and the gill epithelium was scraped off
the cartilage and cells dissociated as previously described
(Kültz and Somero, 1995).
Cell suspensions were stained with DASPMI (10 µmol l-1) for 30
min and washed twice in PBS. After resuspension in 200-300 µl PBS an 80
µl sample was placed on a microscope slide and covered with a 60x24
mm coverslip. Preliminary experiments established that 80 µl are optimal
for complete coverage of the entire area under the coverslip without any
excess liquid remaining in the uncovered area of the slide. Slides were then
evaluated using LSC (see below).
Tissue microarray construction and immunohistochemistry
Gill arches were fixed in 4% paraformaldehyde for at least 48 h and the
fixative was changed once before embedding the tissue in paraffin (Tissue Prep
2, Fisher Scientific, Pittsburgh, PA, USA). These paraffin blocks were then
used as donor blocks for tissue microarray (TMA) construction. An empty
paraffin block served as a recipient block for TMA. TMA construction was done
with a MTA-1 tissue microarrayer (Beecher Instruments, Sun Prairie, WI, USA)
equipped with a set of 1 mm diameter punches as described previously
(Kononen et al., 1998). Fixed
and paraffin-embedded specimens of gill tissue from all acclimation groups
were arrayed in the same recipient block and 4 µm sections of this block
were taken using a microtome (Bromma 2218 Historange, LKB, Uppsala, Sweden)
and floated onto poly-L-lysine coated slides. The slides containing
the arrayed samples were dried overnight. When completely dry they were
deparaffinized three times for 5 min each in xylene, twice for 5 min each in
100% ethanol, twice for 5 min each in 95% ethanol, and once for 5 min in 80%
ethanol. After deparaffinization the slides were incubated for 30 min in
blocking solution (PBS containing 1% bovine serum albumin) and for another 30
min in blocking solution containing 2% purified mouse IgG. Primary antibody
against avian Na+/K+-ATPase
-subunit developed by
Douglas M. Fambrough was obtained from the Developmental Studies Hybridoma
Bank instituted under the auspices of National Institute for Child Health and
Human Development (NICHD) and maintained by the University of Iowa, Department
of Biological Sciences, Iowa City, IA 52242, USA. The antibody was diluted in
blocking solution to a final concentration of 1% and the slides incubated in
this solution at room temperature for 60 min. Slides were rinsed three times
for 5 min each in PBS and incubated for 30 min with secondary goat anti-mouse
IgG antibody covalently bound to PacificBlue (P-10993, Molecular Probes,
Eugene, OR, USA) at a final concentration of 0.5% in blocking solution at room
temperature in the dark. Slides were rinsed again three times for 5 min each
in PBS, topped with a coverslip, and sealed with nail polish. They were stored
in the dark until analyzed by LSC.
Laser scanning cytometry
A Laser Scanning Cytometer (LSC, Compucyte, Cambridge, MA, USA) was used to
analyze suspensions of dimethylaminostyrylmethylpyridiniumiodine
(DASPMI)-stained gill cells and slides containing gill TMAs. For analysis of
chloride cells (CC) in gill cell suspensions we used a 20x objective
(UPlanFl 20x/0.50//0.17, Olympus, Melville, NY, USA) in
combination with the LSC argon laser (488 nm). For analysis of TMA slides
processed by immunohistochemistry we used a 40x objective (UPlanFL
40x/0.75/
/0.17, Olympus) in combination with the LSC UV laser
(400 nm). Contouring and event segmentation variables were adjusted for
optimal detection of CCs as identified by high DASPMI fluorescence in live
cell suspensions and by Na+/K+-ATPase/Pacific Blue
fluorescence in TMAs using WinCyte software (Compucyte). For each CC detected
during scanning we recorded the area, fluorescence integral and maximum pixel
fluorescence, among other variables using WinCyte software. Fluorescence
intensities are expressed as relative fluorescence units (RFU). Wincyte
software also automatically recorded the exact coordinates of each CC on the
slide and the number of CCs in the scan area. These features enabled us to
rapidly and reliably record the properties of thousands of CCs in isolated
cell suspensions and of hundreds of CCs in TMA sections. In addition, the
recorded coordinates for each CC made it possible to use the LSC relocation
function to visually inspect particular subpopulations of CCs after each scan.
For live CC analysis we used the same scan area of 8 mmx6 mm for each
sample. For TMA analysis of fixed gill tissue we scanned an area with a
diameter of 1 mm2 for each sample. All data were stored
automatically in electronic form in FCS file format.
Statistical analysis
CC numbers are expressed per fish and were calculated by multiplying the
cells counted by the LSC with the scan area factor, the cell suspension volume
factor and 2 (because only four of the eight gill arches were used for cell
dissociation). The scan area factor equals 1400 µm2 (effective
slide area)/64 µm2 (LSC scan area)=21.875. The cell suspension
volume factor equals the volume that was used to resuspend the cells after
DASPMI staining/80 µl (volume of cell suspension under the coverslip). Cell
sizes are expressed as cell volume (µm3) for dissociated CC
because they round up in suspension and volumes can be calculated based on the
area measured with the LSC using the sphere formula. For CC in situ,
cell sizes are expressed as the area measured in µm2. All data
are expressed as means ± standard error of the mean (S.E.M.)
and the number of replicates is four animals for each salinity and time.
Statistics software (KyPlot,
http://www.kyenslab.com)
was used to evaluate the significance of differences between treatment groups.
The F-test was used to assess statistical differences of standard
deviations. Depending on its result, either a paired t-test or the
Mann-Whitney test was used to test for statistical significance between means
of treatment groups. The significance threshold is P<0.05 for all
tests.
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Results |
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Effect of intermediate term salinity acclimation on chloride cell properties
We acclimated killifish gradually to 2.4x SW by increasing the
salinity by 250 mosmol kg-1 per day for a period of 10 days. Such
intermediate term salinity acclimation was also done for two additional
groups: acute transfer from FW to 1x SW and transfer from FW to FW,
which served as a control. At the end of the 10 day acclimation period CC
numbers and CC volumes from fish exposed to these different salinity
acclimation regimens were evaluated by LSC analysis. Interestingly, there is
no significant increase in CC number after acute or gradual SW acclimation
compared to FW controls, although a trend can be seen towards a higher number
of CC in the group acclimated to the highest salinity of 2.4x SW
(Fig. 3A). In contrast to CC
number, the size of CC increases significantly after 10 days acclimation to
SW, with the extent of the increase being proportional to the osmotic strength
of the SW (Fig. 3B). The size
of animals from the different groups was not significantly different and
therefore not a factor that could explain the lack of a significant and
salinity-dependent increase in CC numbers (data not shown). Therefore,
killifish respond to intermediate term hyperosmotic stress by CC hypertrophy
but not by an increase in CC number.
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Effect of long-term salinity acclimation on chloride cell properties
Based on the data presented in Fig.
3 we carried out another series of experiments during which
killifish were exposed to acute and gradual salinity acclimation for 5 weeks.
During these experiments fish were sampled at 1, 2 and 5 weeks to assess
intermediate- and long-term effects of acute and gradual salinity acclimation
on CC number and size. Histograms depicting CC size distributions illustrate
that salinity-induced CC hypertrophy was already apparent after 1 week
(Fig. 4D) but continued to
become more prominent after 2 weeks (Fig.
5D) and for the 2.4x SW group even more so after 5 weeks
(Fig. 6D). Notably, besides a
major peak for large CC in 1x and 2.4x SW acclimated fish a more
minor peak was consistently seen, falling within the same size range as the
peak for FW CC (arrows in Figs
5D and
6D). Cells forming this peak
were probably accessory cells. Image galleries depicting CC of mean size that
corresponded to the peaks in the histograms in Figs
4D,
5D and
6D were acquired using the
relocation feature of the LSC. Examples are shown in Figs
4A-C,
5A-C and
6A-C. Large CC size differences
were particularly apparent after 5 weeks of acclimation
(Fig. 6A-C). CC hypertrophy
occurred rapidly in fish acutely exposed to 1x SW and the size of CC did
not continue to increase significantly from 1 week to 5 weeks under these
conditions (Fig. 7A). In
contrast, CC hypertophy was absent after 1 week of gradual acclimation to
2.4x SW (the salinity at this time was 1200 mosmol kg-1; see
Fig. 1). Instead CC size in
fish gradually acclimated to 2.4x SW reached that of fish acutely
acclimated to 1x SW after 2 weeks and continued to increase dramatically
at 5 weeks (Fig. 7A).
Quantitative analysis of all samples revealed that CC numbers increased
moderately by about twofold after 1-2 weeks of acute and gradual acclimation
to 1x and 2.4x seawater (Fig.
7B). They continued to increase robustly and after 5 weeks CC
numbers were fourfold higher in fish acclimated to 1x SW and sevenfold
higher in fish acclimated to 2.4x SW compared to FW controls
(Fig. 7B). CC hypertrophy
displayed very different kinetics depending on the SW acclimation regimen.
Differences in CC numbers and CC hypertrophy cannot be explained by fish size
because total length of the animals did not differ significantly among
experimental groups (data not shown).
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Tissue microarray analysis of chloride cells during salinity acclimation
Pieces of intact (non-dissociated) gill filaments from fish exposed to FW,
1x SW or 2.4x SW for 1, 2 and 5 weeks were arrayed on a microscope
slide and stained with anti-Na+/K+-ATPase antibody and
fluorescent secondary antibody as described in Materials and methods. An
example of a Hematoxylin/Eosin-stained tissue microarray constructed from gill
tissue is shown in Fig. 8A. By
staining such arrays with a fluorescent antibody against
Na+/K+-ATPase CC were identified as distinct and
brightly fluorescent cells at the outer layer of gill filaments and at the
base of the secondary lamellae (Fig.
8B). We have used the LSC for automatically contouring all
fluorescent cells using the segmentation algorithm inherent in the LSC WinCyte
software (Fig. 8C). For each
sample on the slide the integral fluorescence per CC and the area of each CC
were automatically measured during the LSC scan. The data for
Na+/K+-ATPase abundance (= integral fluorescence) per CC
are plotted in Fig. 8D.
Similarly to the different kinetics of CC hypertrophy in fish exposed to
1x SW versus 2.4x SW (see
Fig. 7C) the kinetics of
CC-specific Na+/K+-ATPase abundance also differs greatly
in these two groups. While the Na+/K+-ATPase abundance
per CC increases rapidly and transiently in fish transferred acutely to
1x SW, it increases more slowly but permanently in fish acclimated
gradually to 2.4x SW (Fig.
8D). An advantage of LSC analysis is that the source of variation,
which is particularly high for Na+/K+-ATPase abundance
per CC after 5 weeks of gradual acclimation to 2.4x SW (±40.1%),
can be traced to the level of individual animals because within each animal
the cell-to-cell variation for Na+/K+-ATPase abundance
in chloride cells is much lower for 2.4x SW at 5 weeks (±9.9,
9.8, 12.1 and 10.0%). This suggests that there are large individual
differences in adaptability to very high salinities that approach the
tolerance threshold for this species of euryhaline fish.
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With regard to CC hypertrophy we observed the same trend in intact tissue specimens with the tissue microarray approach compared to evaluating dissociated suspensions of CC. The size of CC is smallest in FW fish and increases rapidly after acute transfer to 1x SW and more slowly but more steadily during gradual acclimation to 2.4x SW (Fig. 8E). However, differences between salinity groups are largely statistically insignificant and the overall effect is not as pronounced as observed in isolated cell suspensions. Because it was possible with the LSC/ TMA approach to accurately quantify Na+/K+-ATPase abundance per CC we calculated the total Na+/K+-ATPase abundance in all killifish CC by multiplying CC-specific Na+/K+-ATPase fluorescence with the number of CC for each sample (Fig. 9A). The resulting data show a comparable kinetics of increase in Na+/K+-ATPase abundance as for CC hypertrophy: a rapid increase that levels off within 1 week in fish exposed to acute 1x SW transfer and a slow increase that continues steadily for 5 weeks in fish exposed to gradual 2.4x SW transfer. After 5 weeks of acclimation Na+/K+-ATPase abundance is ca. 4.5-fold in 1x SW and 17-fold in 2.4x SW fish compared to FW controls. At that time the relationship between total gill Na+/K+-ATPase and environmental salinity is exponential suggesting that the amount of energy needed to maintain plasma ion homeostasis increases steeply when the salinity exceeds 1x SW (Fig. 9B).
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Discussion |
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Kinetics of changes in chloride cell properties
The results of our experiments on dissociated suspensions of CC visualized
with the vital stain DASPMI show that the kinetics and extent of CC
proliferation are only marginally affected by the salinity acclimation
regimen. In contrast, CC hypertrophy depends very strongly on the salinity
acclimation regimen. Acute transfer to 1x SW leads to rapid but
relatively modest hypertrophy of CC that is complete within 1 week while
gradual acclimation to 2.4x SW does not induce significant CC
hypertrophy during the first week but leads to a slow and constant increase in
CC size over 5 weeks. These differences in salinity-dependent kinetics of CC
hypertrophy may be physiologically important. Although it appears that most
natural environments are characterized by gradual changes in salinity we know
much more about CC responses to acute salinity transfer (e.g.
Kültz et al., 1992).
Interestingly, CC number in opercular epithelium in F. heteroclitus
does not change in response to acute transfer from FW to 1x SW
(Daborn et al., 2001
). These
authors suggest that only the area of apical CC exposure varies with salinity
in opercular epithelium. Thus, in F. heteroclitus salinity-dependent
CC proliferation may be regulated differently in gill and opercular epithelia.
Such differential regulation of CC proliferation in gill and opercular
epithelia contrasts to other euryhaline teleosts, for instance Oreochromis
mossambicus, where CC number increases in both types of epithelia
proportional to external salinity
(Kültz et al., 1992
). Our
data support the notion that CC hypertrophy is much more closely correlated
with salt secretory capacity than CC number
(Foskett et al., 1981
). Size
distribution profiles of CC obtained in this study are very similar to those
obtained previously in O. mossambicus
(Kültz et al., 1992
). In
FW-adapted fish there is only one population of small mitochondria-rich cells
(FW CC) while in SW there are two populations. A minor population is very
similar in size to the FW CC and the major population is significantly larger
and represents typical SW CC. We hypothesize that the population of small
mitochondria-rich cells in SW fish are accessory cells that form multicellular
complexes with CC because they display lower DASPMI and
Na+/K+-ATPase fluorescence
(Katoh et al., 2001
). An
elegant recent study on O. mossambicus larvae indicates that FW CC
are transformed into SW CC during salinity acclimation while accessory cells
represent a newly differentiated cell type unique to SW fish
(Hiroi et al., 1999
).
Interestingly, in some euryhaline teleosts such as Dicentrarchus
labrax the number of CC is lowest in 1x SW and increases upon
salinity transfer to FW and 2x SW
(Varsamos et al., 2002). In
this context, it is important to note that the energy requirements for
osmoregulation increase in very dilute environments, and are accompanied by
increases in CC number (Laurent and
Hebibi, 1989
; Perry and
Laurent, 1989
; Greco et al.,
1996
; Moron et al.,
2003
). For our experiments the FW was still relatively rich in
major ions (see Materials and methods), and the increased CC numbers in SW
relative to FW confirm previous observations under these conditions in other
euryhaline teleosts (Foskett et al.,
1981
; Langdon and Thorpe,
1984
; Karnaky,
1986
; Kültz et al.,
1992
).
Tissue microarrays enable quantitative tissue analysis by LSC
In addition to analyzing suspensions of single, dissociated CC we have used
LSC to analyze salinity effects on CC and Na+/K+-ATPase
in fixed gill tissue. To minimize variability associated with tissue
processing, staining and slide scanning and to increase the throughput of
samples we constructed tissue microarrays (TMAs) from the filament portion of
fixed and paraffin-embedded gills from killifish exposed to FW, 1x SW
and 2.4x SW for 1, 2, and 5 weeks. TMA technology was developed only a
few years ago and is currently almost exclusively used for clinical pathology
(Kononen et al., 1998;
Packeisen et al., 2003
). It is
an extremely useful tool for many areas of histology and immunohistochemical
analysis and holds great potential for applications in comparative
experimental biology. We decided to combine TMA technology with LSC to assess
salinity effects on gill tissue of killifish because the combination of the
two provides a very powerful means of quantitative tissue analysis
(Gandour-Edwards et al., 2002
).
This approach worked exceptionally well for quantification of CC-specific
Na+/K+-ATPase abundance in dependence of salinity
acclimation and it provides a useful tool for quantifying other proteins, DNA
and RNA in fish gills in the future.
Salinity-dependence of CC and Na+/K+-ATPase in situ
In fixed gill tissue CC can be identified based on their strong expression
of Na+/K+-ATPase (e.g.
Van der Heijden et al., 1999).
We used this property to automatically segment and quantify CC on tissue
microarrays of killifish gills using the LSC. On average 144±29 (mean
± S.E.M.) CC were quantified in each 1 mm diameter piece of
gill tissue on the array. This number was too low for accurate quantification
of CC because of their heterogeneous distribution. However, this number of CC
is sufficient to accurately determine the mean size and
Na+/K+-ATPase content of CC in dependence of the
different salinity acclimation regimens. Interestingly, CC display much less
hypertrophy compared to isolated cell suspensions. This is probably a result
of extensive packaging and infoldings of the basolateral membrane, which is
particularly pronounced in SW (Karnaky,
1986
). Such membrane infoldings are less pronounced after cell
dissociation because when isolated and without basement membrane support
epithelial cells round up. Therefore, CC size measurements are more accurate
when undertaken on suspensions of isolated cells. Despite these limitations
for analysis of CC properties the tissue microarray approach proved very
useful for quantification of salinity effects on
Na+/K+-ATPase content in individual CC and total
Na+/K+-ATPase content in killifish gills. In agreement
with our finding of different kinetics of CC hypertrophy after acute and
gradual transfer from FW to 1x and 2.4x SW we also observed very
similar kinetic differences for Na+/K+-ATPase content.
The rapid and transient increase in Na+/K+-ATPase
content per CC after acute salinity transfer is in agreement with biochemical
determinations of Na+/K+-ATPase activity in this species
(Mancera and McCormick, 2000
).
At the new steady state at 5 weeks after salinity transfer there is no
difference in Na+/K+-ATPase content per CC between FW
and 1x SW groups, consistent with an earlier report
(Katoh et al., 2001
). However,
taking into account the total number of CC, the
Na+/K+-ATPase content is ca. 4.5-fold higher in 1x
SW compared to FW fish. It should be emphasized that using the approach
described here the actual amount of Na+/K+-ATPase is
quantified rather than activity of this enzyme, which is reported more
frequently than its abundance. The exponential relationship between
Na+/K+-ATPase content and environmental salinity
suggests that the cost of osmoregulation in euryhaline killifish increases
steeply at salinities exceeding regular SW. This is an indirect notion,
however, which is based on the assumption that more
Na+/K+-ATPase in gills translates into higher
expenditure of energy for synthesizing this protein and faster ATP
breakdown.
In summary, in this paper we introduce laser scanning cytometry and tissue microarray as powerful tools for comparative experimental biology. Using this approach we have quantified CC properties and Na+/K+-ATPase content in killifish exposed to salinity stress and discovered that these parameters respond with different kinetics to acute and gradual increases in environmental salinity. Such differences in acclimation kinetics could be physiologically very important. They provide a good starting point for further dissection of the regulatory mechanisms underlying salinity adaptation in euryhaline teleosts.
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Acknowledgments |
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