First published online August 8, 2008
Journal of Experimental Biology 211, 2584-2599 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018663
Evidence for an apical Na–Cl cotransporter involved in ion uptake in a teleost fish
Junya Hiroi1,2,*,
Shigeki Yasumasu2,
Stephen D. McCormick3,4,
Pung-Pung Hwang5 and
Toyoji Kaneko6
1 Department of Anatomy, St Marianna University School of Medicine, Miyamae-ku,
Kawasaki 216-8511, Japan
2 Department of Materials and Life Sciences, Faculty of Science and Technology,
Sophia University, Chiyoda-ku, Tokyo 102-8554, Japan
3 USGS, Conte Anadromous Fish Research Center, Turners Falls, MA 01376,
USA
4 Department of Biology, University of Massachusetts, Amherst, MA 01003,
USA
5 Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei
11529, Taiwan, Republic of China
6 Department of Aquatic Bioscience, Graduate School of Agricultural and Life
Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
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Fig. 1. Western blot analysis of (A) NKCC1a and (B) NCC in the gills of Mozambique
tilapia acclimatized to freshwater (FW) or seawater (SW), and (C,D) a
preabsorption test for NCC immunofluorescence staining in the yolk-sac
membrane of freshwater-acclimatized tilapia embryos. The NCC-positive
immunoreactivity (C) is completely abolished by preabsorbing antibody against
NCC with the corresponding antigen peptide (D). Molecular mass standards are
indicated (kDa). Scale bar, 10 µm.
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Fig. 2. Multiple alignment of the deduced amino acid sequence of tilapia NKCC1a
(GenBank accession no. AY513737), tilapia NKCC1b (AY513738), tilapia NKCC2
(AY513739) and tilapia NCC (EU518934) cDNAs that were isolated from the gills
of Mozambique tilapia. Conserved and similar amino acids are shaded in black
and gray, respectively. Putative transmembrane segments are underlined
(TM1–TM12). The boxed amino-terminal sequences of tilapia NKCC1a and
tilapia NCC were used as antigens to produce antibodies against tilapia NKCC1a
and tilapia NCC.
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Fig. 3. A phylogenetic tree of full-length amino acid sequences of fish and human
cation–chloride cotransporters, constructed by the neighbor-joining
method. Tilapia NKCC1a, NKCC1b, NKCC2 and NCC are highlighted. Human
K+/Cl– cotransporter (KCC1) was used as an
outgroup to root the tree. The sequences were grouped into four clades (NKCC1
clade, NKCC2 clade, conventional NCC clade and fish-specific NCC clade).
Numbers at the nodes are the bootstrap values for 1000 replications, shown as
percentages. Bar, evolutionary distance of 0.05 amino acid substitution per
site. The GenBank accession numbers are as follows: tilapia (NKCC1a, AY513737;
NKCC1b, AY513738; NKCC2, AY513739; NCC, EU518934), eel (NKCC1a, AJ486858;
NKCC1b, AJ486859; NKCC2 , AJ564602; NKCC2β, AJ564603; NCC,
AJ564604; NCCβ, AJ564606), zebrafish (zNCCg, EF591989; NCCa,
NP_001038545; NCCb, XP_001342888; NCCc, XP_699464), medaka (NCCa, Ensembl
protein report for ENSORLP00000000509; NCCb, ENSORLP00000023616; NCCc,
scaffold1460 of golw_scaffold Hd-rR 200506), flounder (NCC, L11615), shark
(NKCC1, U05958; NKCC2, AF521915) and human (NKCC1, U30246; NKCC2, NM_000338;
NCC, X91220; KCC1, NM_005072).
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Fig. 4. Tissue distribution of mRNAs encoding (A) NKCC1a, (B) NKCC1b, (C) NKCC2,
(D) NCC, (E) NHE3 and (F) vacuolar-type H+-ATPase A-subunit in
Mozambique tilapia acclimatized to freshwater (FW) and seawater (SW),
quantified using real-time PCR. The yolk-sac membrane of embryos 4 days after
fertilization (YSM), adult gills, kidney, intestine and brain were examined.
Each value represents the mean ± s.e.m., N=7 (YSM) and
N=3 (adult tissues). The data were normalized to the
highest-expressing tissue for each gene, which were assigned an arbitrary
value of 1.
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Fig. 5. Changes in the levels of mRNA encoding NKCC1a, NCC, NHE3 and the
H+-ATPase A-subunit following transfer of Mozambique tilapia
embryos from (A) freshwater to seawater and (B) vice versa,
quantified using real-time PCR. Each value represents the mean ±
s.e.m., N=7. The data from both transfer experiments (which were
conducted simultaneously and assays run at the same time) were normalized to
the highest value for each gene (NKCC1a, at 6h of freshwater-to-seawater
transfer; NCC, at 72h of seawater-to-freshwater transfer; NHE3, at 24h of
seawater-to-freshwater transfer; H+-ATPase, at 72h of
freshwater-to-seawater transfer), which were assigned an arbitrary value of 1.
The data at 0h were derived from the same samples as used in
Fig. 4. Unfilled circles,
values in freshwater; filled circles, values in seawater. Values with
different lowercase letters are significantly different (e.g. a significant
difference is observed between `a' and `b', but not between `a' and `a,b', or
between `b' and `a,b'; P<0.0125, Tukey–Kramer test).
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Fig. 6. Classification of mitochondria-rich cells (MRCs) in the yolk-sac membrane
of Mozambique tilapia embryos into four types by means of quintuple-color
immunofluorescence staining: (A) type-I MRC (at 72h of seawater-to-freshwater
transfer), (B) type-II MRC (at 72h of seawater-to-freshwater transfer), (C)
type-III MRC (at 72h of seawater-to-freshwater transfer) and (D) type-IV MRC
(at 72h of freshwater-to-seawater transfer). The immunofluorescence signals
for Na+/K+-ATPase (red), NKCC1a (blue), NCC (cyan), NHE3
(yellow) and CFTR (green) are shown as separate channels, and the five
channels are merged in Merged X–Y and
X–Z planes. Merged X–Z plane,
the X–Z optical section cut transversely at the
horizontal lines indicated in Merged X–Y plane. Merged
X–Y plane, the X–Y optical
section cut at the lines indicated in Merged X–Z
plane. n, nucleus; ac, accessory cell. Scale bar, 10 µm.
Schematic diagrams of each of the four cell types are presented in the bottom
row, showing the apical or basolateral localization patterns of
Na+/K+-ATPase (red), NKCC1a (blue), NCC (cyan), NHE3
(yellow) and CFTR (green).
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Fig. 7. Variation in type-II mitochondria-rich cell morphology: (A–G) a cell
with a relatively small apical opening in an embryo in freshwater (at 0h of
freshwater-to-seawater transfer); (H–N) a remaining cell in an embryo
transferred from freshwater to seawater (at 72h of freshwater-to-seawater
transfer); (O–U) a newly appearing cell in an embryo transferred from
seawater to freshwater (at 24h of seawater-to-freshwater transfer); cells with
(V–AB) a deep apical opening and with (AC–AI) a wide apical
opening in an embryo transferred from seawater to freshwater (at 72h of
seawater-to-freshwater transfer). (A,H,O,V,AC) X–Z
optical sections, cut at the lines indicated in X–Y
optical sections. (B–D,I–K,P–R,W–Y,AD–AF)
X–Y optical sections, cut at three different lines
indicated in X–Z optical sections: the focus is placed
in the outer surface plane of the yolk-sac membrane (B,I,P,W,AD), in the plane
through the center of the NCC immunoreactivity (C,J,Q,X,AE) and in the plane
through the center of the nucleus (D,K,R,Y,AF). Channels for
Na+/K+-ATPase (red) and NCC (cyan) are merged in
A–D, H–K, O–R, V–Y and AC–AF. The merged
X–Y images are further merged with corresponding
differential-interference-contrast images
(E–G,L–N,S–U,Z–AB,AG–AI). n, nucleus.
Scale bar, 10 µm.
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Fig. 8. Quintuple-color immunofluorescence staining for
Na+/K+-ATPase (red), NKCC1a (blue), NCC (cyan), NHE3
(yellow) and CFTR (green), at (A–F) 0h and (G–L) 72h of the
freshwater-to-seawater transfer experiment, and at (M–R) 0h and
(S–X) 72h of the seawater-to-freshwater transfer experiment. Arrowheads,
arrows and double-lined arrows indicate the apical immunoreactivity for NCC,
NHE3 and CFTR, respectively. I, type-I mitochondria-rich cell (MRC);
II, type-II MRC; III, type-III MRC; IV, type-IV
MRC. Scale bar, 10 µm.
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Fig. 9. Changes in the density of the four mitochondria-rich cell (MRC) types
following transfer from (A) freshwater to seawater and (B) vice
versa. Each value represents the mean ± s.e.m., N=5.
Values with different lowercase letters are significantly different
(P<0.0125, Tukey–Kramer test). Red, type-I MRC; cyan,
type-II MRC; magenta, type-III MRC; green, type-IV MRC.
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