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
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
* Author for correspondence (e-mail: j-hiroi{at}marianna-u.ac.jp)
Accepted 16 March 2005
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Summary |
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Key words: mitochondrion-rich cell, chloride cell, Na+/K+-ATPase, Na+/K+/2Cl- cotransporter, cystic fibrosis transmembrane conductance regulator, tilapia, Oreochromis mossambicus
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Introduction |
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Recently, by observing in vivo sequential changes in individual
MRCs in the yolk-sac membrane of Mozambique tilapia Oreochromis
mossambicus embryos and larvae, we have found that freshwater-type small
MRCs possess the ability to survive after direct transfer from freshwater to
seawater and to be transformed into seawater-type large MRCs
(Hiroi et al., 1999). This
implies that MRCs possess the plasticity to alter their ion-transporting
functions, from ion absorption to ion secretion. Although our previous study
focused on MRC morphology, it is still unclear whether these cells change the
direction of ion transport in response to salinity changes.
A currently accepted model for NaCl secretion by MRCs consists of the
cooperative action of three major ion transport proteins:
Na+/K+-ATPase,
Na+/K+/2Cl- cotransporter and Cl-
channel (Silva et al., 1977;
Marshall, 1995
). A
Na+/K+-ATPase, which is localized to the basolateral
membrane of MRCs, creates low intracellular Na+ and a highly
negative charge within the cell. The Na+ gradient is used to
transport Na+, K+ and 2Cl- into the cell
through a basolateral Na+/K+/2Cl-
cotransporter (NKCC). Cl- then leaves the cells down on an
electrical gradient through an apical Cl- channel, which is
homologous to human cystic fibrosis transmembrane conductance regulator
(CFTR). Na+ is transported back outside the cells via
Na+/K+-ATPase, and then secreted by a paracellular
pathway between chloride and accessory cells. K+ is considered to
be recycled by a basolateral K+ channel
(Suzuki et al., 1999
). This
NaCl secretory mechanism by teleost MRCs is comparable to that of mammalian
secretory epithelia, such as intestines, airways and salivary glands
(Haas and Forbush, 2000
).
Recently, basolateral localization of Na+/K+-ATPase
and NKCC and apical localization of CFTR in MRCs were immunocytochemically
demonstrated in seawater-acclimated Hawaiian goby Stenogobius
hawaiiensis, confirming the current model
(McCormick et al., 2003).
Therefore, colocalization of all three proteins to MRCs is expected to provide
functional evidence that the cells are involved in active ion secretion. In
the present study, we transferred Mozambique tilapia embryos directly from
freshwater to seawater or from seawater to freshwater, and examined changes in
the immunolocalization of Na+/K+-ATPase, NKCC and CFTR
within MRCs in the yolk-sac membrane using triple-color whole-mount
immunofluorescence staining.
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Materials and methods |
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Antibodies
To detect Na+/K+-ATPase, a rabbit polyclonal
antiserum was directed against a synthetic peptide corresponding to part of
the highly conserved region of the Na+/K+-ATPase
-subunit (NAK121; Katoh et al.,
2000
), which was based on the method described by Ura et al.
(1996
). The specific antibody
was purified by affinity chromatography, and conjugated to Alexa Fluor 546
using the Alexa Fluor Protein Labeling Kit (Molecular Probes, Eugene, OR,
USA).
The antibody to detect NKCC was mouse monoclonal antibody directed against
310 amino acids at the carboxyl terminus of human colonic NKCC1 (T4, developed
by Christian Lytle and Bliss Forbush III, and obtained from the Developmental
Studies Hybridoma Bank developed under the auspices of the National Institute
of Child Health & Human Development and maintained by The University of
Iowa, Department of Biological Sciences, Iowa City, IA, USA). The T4 antibody
has been shown to be specifically immunoreactive with NKCC from many
vertebrates including teleost fish (Lytle
et al., 1995; Wilson et al.,
2000a
; Pelis et al.,
2001
; Marshall et al.,
2002
; McCormick et al.,
2003
; Wu et al.,
2003
). The antibody for CFTR was mouse monoclonal antibody against
104 amino acids at the carboxyl terminus of human CFTR (R&D Systems,
Boston, MA, USA). To allow triple-color immunofluorescence staining, the mouse
monoclonal antibodies against NKCC and CFTR were directly labeled with Alexa
Fluor 647 and Alexa Fluor 488, respectively, using the Zenon Mouse IgG
Labeling Kits (Molecular Probes). In addition to triple-color direct
immunofluorescence staining, conventional single-color indirect
immunofluorescence staining with Alexa-conjugated secondary antibodies was
carried out for each of the primary antibodies, resulting in the same staining
patterns with the triple-color staining. Negative control experiments (without
primary antibody) showed no specific staining.
Triple-color whole-mount immunofluorescence staining
The embryos were anesthetized with 2-phenoxyethanol and fixed in 4%
paraformaldehyde in 0.1 mol l-1 phosphate buffer (pH 7.4) for 90
min at 4°C. The yolk-sac membrane was then incised, the yolk was carefully
removed, and the connective tissue and capillaries lining the yolk-sac
membrane were removed using sharp-pointed forceps under a dissecting
microscope. The membrane was further fixed overnight at 4°C, and preserved
in 70% ethanol.
After a rinse with 0.01 mol l-1 phosphate-buffered saline containing 0.2% Triton X-100 (PBST, pH 7.2) for 1 h, the fixed yolk-sac membrane was incubated simultaneously with Alexa-Fluor labeled anti-Na+/K+-ATPase, anti-NKCC and anti-CFTR for 12 h at 4°C. Anti-Na+/K+-ATPase was diluted 1:250. Anti-NKCC and anti-CFTR were used at concentrations of 0.8 µg ml-1 and 1.0 µg ml-1, respectively. The antibodies were diluted with PBST containing 0.02% keyhole limpet hemocyanin, 0.1% bovine serum albumin, 10% normal goat serum and 0.01% sodium azide. The membrane was then washed in PBST for 1 h, subjected to post-staining fixation with 4% paraformaldehyde for 15 min, washed briefly in PBST, and mounted on a slide with the SlowFade-Light antifade reagent (Molecular Probes).
Both confocal fluorescence and differential interference contrast (DIC) images were taken using a Carl Zeiss 510 META confocal laser scanning microscope with a C-apochromat 40x numerical aperture 1.2, water-immersion objective lens. The wavelengths of excitation and recorded emission for each Alexa dye are as follows: Alexa Fluor 488, 488 nm and 505-530 nm; Alexa Fluor 546, 543 nm and 560-615 nm; and Alexa Fluor 647, 633 nm and 649-756 nm. To avoid any crosstalk, a `multitrack' configuration was used, in which images were collected successively, rather than simultaneously, on three channels. The size of an optical section was 230 µm x 230 µm x 1 µm (X-Y-Z), and confocal images were taken at 0.5 µm intervals to generate Z-stacks. Ten images corresponding to 0.53 mm2 were obtained from each sample, while the total surface area of the yolk-sac membrane was approximately 3 mm2.
Statistics
The cell number and individual cell area of X-Y projection images were
measured for four individuals at each sampling time with Photoshop 7.0 (Adobe,
San Jose, CA, USA) and the public domain NIH image program (version 1.63,
http://rsb.info.nih.gov/nih-image/).
The temporal change in the number of each cell type was analyzed by a
one-way analysis of variance (ANOVA; among-groups degrees of freedom=4,
within-groups degrees of freedom=15) and the Bonferroni/Dunn post-hoc
test (Zar, 1999), using
Statview 5.0 (SAS Institute, Cary, NC, USA). No significant heterogeneity of
variances was detected (Bartlett's test, P>0.05). Since the ANOVA
and post-hoc test were repeated for four cell types, the
P-value was lowered from the widely used 0.05 to 0.0125 (=0.05/4) to
avoid excessive type-I errors.
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Results |
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Type-III cell
Type-III cells were Na+/K+-ATPase positive and CFTR
negative, like type I and type II. However, NKCC immunoreactivity was
detectable throughout the cell except for the nucleus, coinciding with the
staining pattern of Na+/K+-ATPase. In
Fig. 3G,H, the red color for
Na+/K+-ATPase and the blue for NKCC were merged into
magenta, indicating Na+/K+-ATPase and NKCC were
co-localized at a pixel level. The type-III cells possessed a distinct apical
opening (Fig. 3I), and were
mostly larger in size than the types I and II.
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Accessory cell
In contrast to type-I cells and type-II cells, type-III cells and type-IV
cells were frequently accompanied by one or more small cells that were
distinct from pavement cells. Based on its size, location and morphology, this
cell type was identified as an accessory cell. The nucleus of the accessory
cell was distinguishable by DIC observation, whereas the cytoplasm was
indistinct and immunonegative or slightly immunopositive for
Na+/K+-ATPase (Figs
3J,U and
4J,O). The accessory cells were
attached to the edge of type-III cells
(Fig. 3J,U), and were located
on the shoulder of type-IV cells resulting in a concave appearance of the
type-IV cells (Fig. 4G,J,K).
Two or more type-IV cells sharing a common apical opening were also
accompanied by accessory cells (Fig.
4O).
Transfer from freshwater to seawater
In embryos kept in freshwater (at 0 h), three types of MRCs without CFTR
immunoreactivity, type I, type II and type III, were observed
(Fig. 5A-D). Type-IV cells
started to appear at 12 h after transfer from freshwater to seawater, and were
frequently observed after 24 h. Apical CFTR immunostaining of type-IV cells
was not very bright at 12 h and 24 h (Fig.
5G), while bright and punctate CFTR staining was frequently
observed at 48 h and 72 h (Fig.
5K). Type-I and type-II cells were observed throughout 72 h of the
transfer experiment. Type-III cells decreased in number and disappeared by 72
h.
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The ratios of the four cell types at 0 h were: type I, 14%; type II, 0%; type III, 13%; type IV, 73%. Those at 72 h were: type I, 15%; type II, 25%; type III, 60%; type IV, 0. The temporal changes in the number of cells (Fig. 6B) were significant for type II (F=6.73, P=0.0026), type III (F=23.34, P<0.0001) and type IV (F=75.61, P<0.0001), but were not significant for type I (F=0.62, P=0.65). The mean number of type-I cells ranged between 52 and 75 cells mm-2. Type-II cells were not found at 0 h, first appeared at 12 h (14 cells mm-2), and reached 99 cells mm-2 at 72 h (P<0.0125 between 0 h and 48-72 h). From 0-12 h to 24-72 h, the mean number of type-III cells increased sharply from 77 cells mm-2 (12 h) to 285 cells mm-2 (24 h), whereas that of type-IV cells decreased from 240 cells mm-2 (12 h) to 31 cells mm-2 (24 h) (P<0.0125 between 0-12 h and 24-72 h for both cell types).
Size/frequency distributions of the cell types existing at 0 h and 72 h (type I, type III and type IV for 0 h; type I, type II and type III for 72 h) are shown in Fig. 7B. The size distributions of type-I and type-II cells were narrower than for type-III and type-IV cells which had distinctive bimodal distributions (peak: 200-250 µm2 and 450-500 µm2 for type III+type IV at 0 h; 200-250 µm2 and 500-550 µm2 for type III at 72 h).
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Discussion |
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Seawater-type ion secretory cell
Type-IV cells were the only type showing immunoreactivity for all three ion
transport proteins examined, Na+/K+-ATPase, NKCC and
CFTR (Fig. 4). The
intracellular distributions of both Na+/K+-ATPase and
NKCC were throughout the cell except for the nucleus and the apical region.
Previous electron microscopic studies have demonstrated that
Na+/K+-ATPase was present on both the basolateral
membrane and the extensive tubular system, which was continuous with the
basolateral membrane of MRCs (Evans et al.,
2005). Therefore, the presence of
Na+/K+-ATPase and NKCC within MRCs represents their
basolateral distributions. The basolateral distributions of
Na+/K+-ATPase and NKCC and a distinct apical CFTR
distribution in type-IV cells are completely consistent with the current
accepted model for ion secretion by MRCs, indicating that type-IV cells are
seawater-type ion secretory cells. This is also supported by our finding that
type-IV cells were purely seawater specific: they were not observable in
freshwater, rapidly appeared following transfer from freshwater to seawater
(Fig. 6A), and disappeared
following transfer from seawater to freshwater
(Fig. 6B).
Type-III cells showed basolateral staining of Na+/K+-ATPase and NKCC, but lacked apical CFTR staining. Following transfer from freshwater to seawater, the number of type-III cells decreased whereas type-IV cells increased, and the size/frequency distribution of type-III cells at 0 h and that of type IV cells at 72 h showed similar unimodal, positively skewed distributions. Based on these observations, we propose that after seawater exposure type-III cells synthesize CFTR de novo and arrange it at the apical membrane following transfer from freshwater to seawater, and consequently the cells are counted as type-IV cells. Transfer from seawater to freshwater showed these changes in reverse: the numbers of type-III cells increased whereas type-IV cells decreased, and the size of type III+type IV at 0 h and that of type III at 72 h had similar bimodal distributions. Therefore, it is most likely that type-IV cells lose apical CFTR and stop their ion secretory function to change into type-III cells following transfer from seawater to freshwater. From these observations, we conclude that type-III cells are a proto- or dormant-type of type-IV cells that only becomes active in ion secretion after placement of CFTR in the apical membrane.
Our previous in vivo sequential observations revealed that 75% of
pre-existing MRCs in tilapia embryos survived for 96 h following transfer from
freshwater to seawater (Hiroi et al.,
1999). After transfer from freshwater to seawater in the present
study, the number of type-III cells at 0 h and that of type-IV cells at 72 h
corresponded to 50% (233 cells mm-2) and 75% (345 cells
mm-2) of total pre-existing cells at 0 h of freshwater-to-seawater
transfer (452 cells mm-2), respectively (stacked graph in
Fig. 6A). Therefore, almost all
type-III cells are expected to survive to be transformed into type-IV cells,
while the remaining type-IV cells would be differentiated from type-I cells
(presumptive immature MRCs; see below). The in vivo sequential
observations also demonstrated that each surviving cell enlarged following
seawater transfer, similar to the present results in which the size of type-IV
cells at 72 h exceeded that of type-III cells at 0 h
(Fig. 7A). Therefore, the
transformation from type III to type IV would include not only the appearance
of apical CFTR but also the increase in cell size, which may imply the
enlargement of the basolateral/tubular system in order to localize more
Na+/K+-ATPase and NKCC for active NaCl secretion in
seawater.
The transition between types III and IV reflects the appearance and
disappearance of apical CFTR immunoreactivity in MRCs, and these changes
started within 12 h and occurred sharply between 12 h and 24 h after transfer
from freshwater to seawater and vice versa
(Fig. 6). The similar rapid
CFTR response to salinity change was observed in killifish Fundulus
heteroclitus: following transfer from freshwater to seawater, CFTR mRNA
started to increase at 8 h and showed a ninefold increase at 24 h
(Singer et al., 1998), and
apical CFTR immunoreactivity in MRCs increased between 24 h and 48 h
(Marshall et al., 2002
);
following transfer from seawater to freshwater, apical CFTR immunoreactivity
was reduced at 12 h and disappeared at 24 h
(Katoh et al., 2003
).
Therefore, tilapia and killifish, the two typical experimental models for
euryhaline fish, seem to share a similar on-off mechanism of active ion
secretion, which is achieved by the rapid appearance and disappearance of CFTR
at the apical membrane of MRCs. It has also been reported that MRCs close the
apical openings quickly (about 30 min or a few hours, which seem likely to be
quicker than the response of CFTR) in response to abrupt salinity changes or
acid-base disturbances (Goss et al.,
1995
; Sakamoto et al.,
2000
; Daborn et al.,
2001
; Lin and Hwang,
2004
). Although we confirmed in DIC observations that all of the
type-III cells that appeared following transfer from seawater to freshwater
possessed a distinct apical opening, it is possible that the apical opening of
the MRCs was closed within 12 h, in advance of the disappearance of the apical
CFTR distribution.
The noticeable characteristics of type-IV cells were clear discontinuous,
punctate CFTR staining at the apical region
(Fig. 4F,H,M) and indentation
by accessory cells (Fig.
4G,H,J,M,O). A similar punctate distribution of apical CFTR was
observed in seawater-adapted Hawaiian goby
(McCormick et al., 2003).
Shiraishi et al. (1997
)
presented transmission electron micrographs of a horizontal section cut
through an apical crypt of a seawater-type MRC in the yolk-sac membrane of the
same species. In the micrographs, an accessory cell interdigitated with a main
MRC, and extended cytoplasmic processes to the apex, so that cytoplasmic
processes of the MRCs and accessory cells appeared to be arranged alternately.
Supposing that CFTR is present only in the MRC itself and not in the
interdigitating accessory cell, the apical CFTR staining may result in a
discontinuous pattern associated with the alternate arrangement of the
interdigitation. Thus, the discontinuous CFTR staining in the present study
would reflect the complex architecture between chloride and accessory cells.
In the current model for NaCl secretion by MRCs, while Cl- is
considered to be secreted through MRCs, Na+ secretion occurs
via a paracellular pathway formed between chloride and accessory
cells. Therefore, the discontinuous CFTR staining might indirectly imply
type-IV cells as the site not only for Cl- secretion but also that
they are involved in paracellular Na+ secretion. Previous
transmission electron microscopic studies revealed that the interdigitation
and leaky junction between chloride and accessory cells appeared within 1-3 h
after transfer of larval Ayu Plecoglossus altivelis from freshwater
to seawater, and disappeared within 3 h after transfer of juvenile stone
flounder Kareius bicoloratus from seawater to freshwater
(Hwang and Hirano, 1985
;
Hwang, 1990
). We consider it
likely that the development and degeneration of the interdigitation between
chloride and accessory cells occurs reversibly in parallel with the appearance
and disappearance of CFTR at the apical membrane of MRCs.
Freshwater-type ion absorptive cell
Type-II cells possessed apical NKCC, basolateral
Na+/K+-ATPase and showed freshwater-specific changes in
number during the transfer experiments: type-II cells were absent in seawater,
appeared following transfer from seawater to freshwater
(Fig. 6B), and tended to
decrease following transfer from freshwater to seawater
(Fig. 6A). These changes in
response to salinity changes provide indirect evidence that type-II cells
possess an ion transport function favorable for freshwater acclimation,
presumably ion absorption.
The apical localization of NKCC in MRCs is surprising. A number of previous
reviews on ion transport mechanisms of teleost gills have suggested
Na+ absorption by H+-ATPase and Na+ channel,
or by a Na+/H+-exchanger, and Cl- absorption
by Cl-/HCO3- exchanger, but did not refer to
apical NKCC (e.g. Marshall,
2002; Perry et al.,
2003
; Evans et al.,
2005
). An active NaCl uptake mechanism with apical NKCC has been
proposed in crabs Carcinus maenas and Chasmagnathus
granulatus in brackish water
(Riestenpatt et al., 1996
;
Onken et al., 2003
), and only
Kirschner (2004
) has proposed
that the mechanism may exist in estuarine fish. In other euryhaline teleost
species examined so far, the presence of apical NKCC has not been reported: in
Atlantic salmon Salmo salar and Hawaiian goby, NKCC has a basolateral
localization in MRCs in both freshwater and seawater conditions
(Pelis et al., 2001
;
McCormick et al., 2003
); in
killifish, NKCC is condensed and localized in lateral parts of MRC complexes
in freshwater, and apparently not in the apical membrane area of MRCs
(Marshall et al., 2002
).
Apical NKCC staining is currently detectable only in MRCs in the gills
(Wu et al., 2003
) and in the
embryonic yolk-sac membrane (present study) of freshwater-acclimated
Mozambique tilapia.
In mammals, the Na+/K+/2Cl- cotransporter
occurs in two major isoforms: a secretory isoform, termed NKCC1, and an
absorptive isoform, termed NKCC2 (Xu et
al., 1994; Payne and Forbush,
1994
). NKCC1 is widely distributed in mammalian tissues and is
especially prominent in secretory epithelial cells, in which NaCl is secreted
in the same manner as seawater-type MRCs. By contrast, NKCC2 is found only in
the apical membrane of epithelial cells in the thick ascending limb of the
loop of Henle (TAL) (Lytle et al.,
1995
; Kaplan et al.,
1996
; Nielsen et al.,
1998
). The localization and stoichiometry of the NKCC2 appears to
be conserved among vertebrates, though alternative splicing results in altered
affinities (Gagnon et al.,
2003
). In the TAL, NKCC and K+ channel (ROMK,
Xu et al., 1997
) are present
at the apical membrane, whereas Na+/K+-ATPase and
Cl- channel (CLC-K2, Uchida,
2000
) are in the basolateral membrane. In the present study, the
presence of two out of four proteins, apical NKCC and basolateral
Na+/K+-ATPase defined type-II cells. Therefore, it is
possible that type-II cells are involved in NaCl absorption by the same manner
as mammalian TAL epithelial cells. It should be noted, that the luminal
Na+ concentration in the TAL is higher than the intracellular
Na+, providing a driving force for the NKCC to transport
Na+, K+ and 2Cl- into the cell, whereas this
would be absent under most freshwater conditions (Na+
concentrations of freshwater are lower than the Na+ concentration
in MRCs). It is possible that this uptake mechanism may only function when
ambient Na+ is relatively high (e.g. >10 mmol l-1),
or that intracellular Na+ near the apical membrane is kept at very
low levels in these cells by basolateral Na+/K+-ATPase.
If the type-II cell is indeed involved in uptake, it seems likely that the
observed apical staining is an NKCC2-like absorptive isoform, since the
anti-NKCC (T4) used in this study is known to react with both NKCC isoforms
(Lytle et al., 1995
). We have
recently succeeded in isolating two homologs of NKCC1 (GenBank accession No.
AY513737, AY513738) and one homolog of NKCC2 (AY513739) from the gills of
adult Mozambique tilapia. Ascertaining the cellular distribution of these NKCC
genes and the immunolocalization of their proteins will help clarify their
involvement in ion absorption and secretory processes.
The apical NKCC staining of type-II cells showed some variations during the transfer experiments. Type-II cells with a cup-like apical NKCC staining (Fig. 2A-J) were frequently observed in embryos fully acclimated to freshwater, at 0 h of freshwater-to-seawater transfer and at 48 and 72 h of seawater-to-freshwater transfer. On the other hand, type-II cells with pinhole-like or hairline-like apical NKCC staining, which corresponded with the boundaries between pavement cells (Fig. 2K-O), appeared at 12 h and 24 h of seawater-to-freshwater transfer. These cells seem to be an earlier developmental stage of type-II cells that have just differentiated from type-I cells, and are expected to enlarge the apical opening and place more NKCC at the apical membrane during freshwater acclimation.
Previous morphological observations on the same species identified three
types of MRCs with different apical surfaces: wavy convex, shallow basin and
deep hole (Lee et al., 1996;
Lin and Hwang, 2001
,
2004
). The wavy-convex-type
cells, which possessed a wide apical opening and a rough apical surface
appearance, increased in number in low Cl- water, suggesting this
cell type is involved in active Cl- uptake. We also recognized a
small number of type-II cells (approximately less than 5%) possessed a wide
apical opening and a rough apical surface appearance 72 h after transfer from
seawater to freshwater (Fig.
2P-S). Although the apical surface of the cells looked concave,
not convex, this may have been due to the removal of the yolk-sac membrane in
the present study, which may change a convex apical surface into a concave
one. Therefore, these type-II cells seem likely to be identical to the
wavy-convex-type cells, and it is of great interest to determine whether they
proliferate in low Cl- conditions.
Although type-III cells are unlikely to be involved in ion secretion, the
possibility that they are involved in ion absorption in freshwater condition
cannot completely ruled out. Wilson et al.
(2000b) reported that
H+-ATPase and Na+ channel immunoreactivity were
co-localized to pavement cells, and apical
Cl-/HCO3- exchanger immunoreactivity was
found in MRCs in the gills of the same species, proposing that the absorption
of Na+ and Cl- occurs separately in pavement cells and
MRCs, respectively. H+-ATPase immunoreactivity was also found in
pavement cells in the yolk-sac membrane of tilapia larvae
(Hiroi et al., 1998b
).
Therefore, if Cl-/HCO3- exchanger is
localized in the apical membrane of type-III cells, the cell type could be
involved in Cl- absorption. There is evidence that there are
different ion uptake mechanisms in zebrafish (Danio rerio) acclimated
to soft water and hard water (Boisen et
al., 2003
), and it is also possible that different ion uptake
mechanisms by different cell types exist concurrently and are regulated by
ambient ion levels. Future studies on the localization of the
Cl-/HCO3- exchanger as well as other ion transport
proteins in relation to ambient ion changes are warranted. Moreover, some
type-III cells appearing after transfer from seawater to freshwater showed
relatively strong NKCC staining at the apical region, in which
Na+/K+-ATPase staining was absent
(Fig. 3K-U). We counted these
cells as type-III cells rather than type-II cells, because of their distinct
basolateral NKCC staining (type-II cells were defined as showing apical NKCC
and basolateral Na+/K+-ATPase, without basolateral
NKCC). These type-III cells might synthesize absorptive type NKCC de
novo, arrange it at the apical membrane and function as the ion
absorptive site in the same manner as type-II cells. The development of
antibodies that can distinguish absorptive and secretory isoforms of NKCC is
expected to specify the function of these cells further.
|
In conclusion, the characteristics of the four MRC types and their presumptive interrelationships are summarized in Fig. 8. Immature type-I cells are considered to develop into type-II ion absorptive cells (arrow A in Fig. 8) and type-III cells (arrow B) in freshwater, and into type-IV ion secretory cells in seawater (arrow C). Following transfer from freshwater to seawater, it seems likely that most type-III cells are transformed into type-IV cells (arrow D), while a certain number of type-I cells may also develop into type-IV cells (arrow C). When transferred from seawater back to freshwater, type-IV cells seem to change into type-III cells to stop ion secretion (arrow E), and type-I cells develop into type-II cells to function as in ion absorption (arrow A). The euryhalinity of tilapia embryos seems to depend on the rapid and reversible changes between type-III cells and type-IV cells, as well as the continual presence of immature type-I cells, which can develop into the other cell types. Finally, we would like to emphasize that MRCs of euryhaline teleosts are not only important in understanding iono- and osmo-regulation of fishes, but also serve as a unique and excellent model for the ion transport properties of epithelial cells in general.
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Acknowledgments |
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