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The Journal of Neuroscience, November 15, 1998, 18(22):9192-9203
Na,K-ATPase Subunit 
1 knock-in Prevents Lethality
of 2 Deficiency in Mice
Philipp
Weber1,
Udo
Bartsch1,
Melitta
Schachner1, 2, and
Dirk
Montag1, 3
1 Department of Neurobiology, Swiss Federal Institute
of Technology, CH-8093 Zürich, Switzerland, 2 Zentrum
für Molekulare Neurobiologie, Universität Hamburg, D-20246
Hamburg, Germany and 3 Research Group Neurogenetics,
Leibniz Institute for Neurobiology, D-39118 Magdeburg, Germany
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ABSTRACT |
The 
2 subunit of the Na,K-ATPase displays functional properties
of both an integral constituent of an ion pump and an adhesion and
neurite outgrowth-promoting molecule in vitro. To
investigate whether the 1 subunit of the Na,K-ATPase can
functionally substitute for the 2 isoform in vivo, we
have generated 2/1 knock-in mice by homologous
recombination in embryonic stem cells. In 2/1 knock-in mice, expression of 2 was abolished, whereas
1 mRNA expression from the mutated gene amounted to ~15% of the
normal expression of 2 in the adult mouse brain and prevented the
juvenile lethality observed for 2 null mutant mice. In contrast to
2 null mutant mice, the overall morphological structure of all
analyzed brain regions was normal. By immunohistochemical analysis,
1 expression was detected in photoreceptor cells in the retina of knock-in mice at an age when expression of 1 and
2, respectively, is downregulated and persisting in the wild-type
mice. Morphological analysis by light and electron microscopy revealed
a progressive degeneration of photoreceptor cells. Apoptotic death of
photoreceptor cells determined quantitatively by terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis
increased in 2/1 knock-in mice with age. These
observations suggest that the 1 subunit of the Na,K-ATPase can
substitute sufficiently, at least in certain cell types, for the role
of the 2 subunit as a component of a functional Na,K-ATPase, but
they do not allow us to determine the possible role of the 2 subunit
as an adhesion molecule in vivo.
Key words:
Na,K-ATPase; knock-in; retinitis pigmentosa; photoreceptor cells; adhesion molecule on glia; AMOG; mouse;
subunit; ionic homeostasis
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INTRODUCTION |
The Na,K-ATPase is an ubiquitously
expressed ion pump located in the plasma membrane. The pump maintains
the flux of sodium and potassium ions across membranes and thus
regulates, by directly influencing ion gradients, cellular activities
such as cell volume and size, action potentials, and secondary active
transport systems. The functional Na,K-ATPase is a heterodimeric ion
pump which consists of a 
subunit and a subunit. Three subunits (1, 2, and 3) and three subunits (1, 2, and
3) have been identified (Mercer et al., 1986
; Shull et al., 1986;
Hara et al., 1987; Herrera et al., 1987; Gloor, 1989; Malik et al.,
1996). The subunit comprises the catalytic and transport activities
of the Na,K-ATPase (Jørgensen and Andersen, 1988; Skou, 1990; Blanco
et al., 1994). The functional role of the subunit is less well
understood, but it appears to be involved in the structural maturation,
correct routing of the functional heterodimeric Na,K-ATPase to the
plasma membrane, and localization of the subunit in the plasma
membrane (Geering et al., 1989, 1996; McDonough et al., 1990; Geering, 1991). Combinations of different subunits (1, 2, and 3)
with subunits (1, 2, 3) by recombinant expression in
Xenopus oocytes show that different and subunits can
associate with each other to form functionally active pumps
(Horisberger et al., 1991; Schmalzing et al., 1991, 1992, 1997; Jaisser
et al., 1992; Munzer et al., 1994; Blanco et al., 1995a,b; Therien et
al., 1996).
The subunits of the Na,K-ATPase show distinct expression patterns. The
1 subunit is expressed in all tissues. 2 is expressed mainly in
skeletal muscle and also in brain and heart, and 3 is expressed only
in brain and heart (Emanuel et al., 1987; Orlowski and Lingrel, 1988).
Expression of the 1 subunit is detected in most neural cells, being
predominantly located in neurons and astrocytes (Lecuona et al., 1996;
Peng et al., 1997). During the second postnatal week, expression of the
1 subunit by glial cells and photoreceptor cells in the optic nerve
and the retina, respectively, is downregulated (Lecuona et al., 1996).
The 2 subunit of the Na,K-ATPase is predominantly expressed by glial
cells in the CNS and additionally by distinct neuronal cell types,
including, for instance, granule cells in the cerebellar cortex and
photoreceptor cells in the retina (Magyar et al., 1994). Expression of
2 is first detectable in the brain at late embryonic stages,
increases during the first 2 postnatal weeks, and reaches highest
levels in the adult (Pagliusi et al., 1990; Lecuona et al., 1996),
whereas it is hardly detectable outside the CNS (Antonicek et al.,
1987; Antonicek and Schachner, 1988; Gloor et al., 1990; Pagliusi et al., 1990). 3 subunit expression has been detected in human placenta and various rat tissues, including skeletal muscle and lung of 7-d-old
animals and the developing and adult brain (Malik et al., 1996;
Arystarkhova and Sweadner, 1997).
The 2 subunit of the Na,K-ATPase was originally identified as an
adhesion molecule on glia (AMOG) mediating adhesion between neurons and
astrocytes (Antonicek et al., 1987; Antonicek and Schachner, 1988).
Sequence analysis of AMOG identified it as a homolog of the 1
subunit of the Na,K-ATPase (Gloor et al., 1990). Here, we refer to AMOG
as the 2 subunit of the Na,K-ATPase. The 2 subunit, but not the
1 subunit of the Na,K-ATPase, promotes neurite outgrowth in
vitro (Müller-Husmann et al., 1993). A monoclonal antibody
to 2 that blocks adhesion increases Na,K-ATPase activity of cultured
astrocytes (Gloor et al., 1990). The dual function of the 2 subunit
in cell recognition and ion transport has been hypothesized to couple
cell recognition with regulation of the ionic milieu (Gloor et al.,
1990). Mice deficient in 2 exhibit lack of motor coordination
at 15 d of age and subsequent tremor and paralysis of extremities,
and they die at 17-18 d after birth (Magyar et al., 1994).
Morphological analysis of the CNS of 17-d-old 2-deficient mice
revealed enlarged ventricles, swollen astrocytic end feet in the brain
stem, thalamus, and spinal cord, and apoptotic photoreceptor cell death
in the retina during the second postnatal week (Magyar et al., 1994;
Molthagen et al., 1996).
Analysis of the phenotype of 2-deficient mice led to the
interpretation that the morphological abnormalities could be caused by
the absence of pump activity or the absence of adhesion molecule function or both. In the hope of distinguishing between these possibilities, we generated 2/1 knock-in mutant mice
via homologous recombination in embryonic stem cells. In these animals,
the 1 subunit cDNA is placed into the 2 gene, yielding the
replacement of 2 expression by 1 expression under the regulatory
elements of the 2 gene. Here we show that in contrast to
2-deficient animals, 2/1 knock-in mutants have a
normal life span. Moreover, swollen and enlarged astrocytic end feet
were not detectable in the brain stem of knock-in mutants.
Degeneration of photoreceptor cells was reduced in 2/1
knock-in mutants when compared with 2 null mutants, but
it was significantly increased when compared with wild-type animals.
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MATERIALS AND METHODS |
2/1 targeting construct. The targeting
construct consisted of a 1 kb 5' region of the mouse 2 gene and the
mouse cDNA coding for the 1 subunit of the Na,K-ATPase inserted in
frame into the unique XmnI site in exon I of the 2 gene, followed by
a 4.7 kb 3' region of the 2 gene containing exons II to VII (Magyar
et al., 1994) (see Fig. 1A-C). By use of the
conserved XmnI site in the 2 genomic sequence and in the 1 cDNA
sequence, a fusion between the sequence coding for 18 amino acids of
the N-terminal part of 2 and the cDNA sequence coding for amino
acids 14 to 304 of the 1 isoform (Gloor, 1989) (see Fig.
1D) was obtained. The herpes simplex virus (HSV)
thymidine kinase gene (tk) at the 3' end of the construct
allowed for selection against random integration (Mansour et al.,
1988). For positive selection, the neomycin resistance gene driven by
the PGK promoter (Soriano et al., 1991) and flanked by loxP sites and
polyadenylation sites (loxpAPGKneopAlox) was inserted 3' to
the 1 cDNA sequence, resulting in the targeting construct designated
2/1loxpAneoloxtk (see Fig.
1B).
Cell culture. The embryonic stem cell line E14.1 (Hooper et
al., 1987) was cultured on irradiated primary mouse embryonic fibroblasts (MEF). Embryonic stem cells (2 × 107) were transfected by electroporation (Bio-Rad
Gene Pulser; 230V, 500 µF) with 20 µg of SalI linearized
targeting construct, cultured on irradiated MEFneoR
feeder cells (gift of Dr. H. Blüthmann, F. Hofmann-LaRoche, Basel, Switzerland), and selected with 0.2 µM 1-(2-deoxy,
2-fluoro--D-arabinofuranosyl)-5-iodouracil (FIAU) (Bristol-Myers,
New York, NY) and 300 µg/ml G418 (Life Technologies-BRL, Rockville,
MD) for 3 and 6 d, respectively. Single colonies were expanded,
and aliquots of clones were frozen as described (Chan and Evans, 1991)
or cultured in medium containing 60% buffalo rat liver
cell-conditioned medium without feeder cells for DNA isolation.
Screening of recombinant clones and Southern blot analysis.
Embryonic stem cells were lysed and DNA was isolated as described (Ramirez-Solis et al., 1992). DNA of individual embryonic stem cell
clones was digested with BamHI and analyzed by Southern
blotting as described (Montag et al., 1994) using the probe 5'EXT (416 bp StyI-SnaBI fragment of the 2 gene 5' of the
construct) (see Fig. 1C). The probe was labeled to
108 cpm/µg according to Feinberg and Vogelstein
(1983). Genomic DNA from positive embryonic stem cells was further
characterized after restriction with appropriate enzymes by Southern
blot analysis as described above using probe 3'INT (1690 bp fragment
from XmnI exon I to EcoRV intron I) (see Fig.
1C).
Blastocyst injection and mating of mice. Blastocyst
injections were performed by Dr. J. P. Julien and his coworkers
(McGill University, Montreal, Canada) on a commercial basis. Male
chimeras were mated with C57BL/6J females. Heterozygous offspring were crossed to obtain homozygous mice. The genotype of mice was determined by Southern blot analysis of DNA isolated from tail biopsies.
RNA preparation and Northern blot analysis. Total RNA from
brains of 5-week-old wild-type (2/1+/+),
heterozygous (2/1+/ki), and homozygous
(2/1ki/ki) 2/1 knock-in mice
was isolated using the RNeasy Kit (QIAGEN, Santa Clarita, CA). RNA was
electrophoresed in a 1.5% agarose gel containing 7% formaldehyde and
transferred onto Hybond-N membranes (Amersham, Uppsala, Sweden).
Hybridization was performed with the following random-primed probes
(cDNA probes labeled to 108 cpm/µg): 1079 bp
EcoRI fragment of construct
2/1loxpAneoloxtk coding for the 1 cDNA
(probe 1), 686 bp PstI-EcoRV fragment of
pBSKS+AMOG2 encoding exons II to VII of 2 (probe 2), and 625 bp
ApaI-SacII fragment of BlueKS+/AMOG (probe
2-5'UT), representing 556 bp of 5' untranslated and 75 bp
translated sequence of the 2 gene. Relative mRNA expression levels
were estimated by visual comparison of band intensities.
Antibodies. Polyclonal antibody to the mouse 2 subunit,
monoclonal antibodies 426 and BSP/3 to the mouse 2 and 1
subunits, respectively, and polyclonal antibodies to mouse L1 have been described (Gorvel et al., 1984; Rathjen and Schachner, 1984; Antonicek et al., 1987; Schmalzing et al., 1991). For indirect
immunofluorescence, polyclonal and monoclonal antibodies were
visualized by fluorescein isothiocyanate (FITC)-conjugated antibodies
to rat or rabbit IgG (diluted 1:100) (Dako, Hamburg, Germany).
Protein analysis of brain extracts. For analysis of
proteins, retinae of 17-d-old or brains of 5-week-old wild-type
(2/1+/+) and 2/1 knock-in
mice (2/1ki/ki) were homogenized in buffer H
(1 mM NaHCO3, 0.2 mM
CaCl2, 0.2 mM MgCl2,
1 mM spermidine, pH 7.9) complemented with protease inhibitors (10 µg/ml soybean trypsin inhibitor, 10 µg/ml turkey egg-white trypsin inhibitor, 1 mM phenylmethylsulfonyl
fluoride, 0.5 mM iodoacetamide). The homogenate was
centrifuged at 4°C and 30,000 × g for 30 min. The
pellet was solubilized for 2 hr at 4°C in buffer S (20 mM
Tris, 1 mM EDTA, 1 mM EGTA, 0.15 M
NaCl, 0.5% Triton X-100, pH 7.2) complemented with protease inhibitors as detailed above. The solubilized fraction was centrifuged at 4°C
and 100,000 × g for 45 min. The protein concentrations
of supernatants of crude membrane fractions were determined using the
BCA-assay (Pierce, Rockford, IL). After addition of 2× loading buffer
and heat denaturation, samples were analyzed under reducing (L1) or
nonreducing conditions (polyclonal anti-2 antibody, BSP/3) by
SDS-PAGE (Laemmli, 1970) and Western blotting (Towbin et al., 1979).
Primary antibodies were visualized using horse radish
peroxidase-coupled antibodies to rat or rabbit IgG (diluted 1:10,000)
(Dianova, Hamburg, Germany) and detection by enhanced chemiluminescence
(ECL kit; Amersham). Relative protein expression levels were estimated
by visual comparison of band intensities.
For deglycosylation of 1 in tissue homogenates of retinae, membrane
fractions (10 µg of protein) from 17-d-old wild-type (2/1+/+) and 2/1 knock-in
mice (2/1ki/ki) were incubated with
N-glycosidase F (PNGase F) and/or O-glycosidase as described (Holm et al., 1996). The proteins were resolved and subjected to immunoblot analysis as described above.
Light and electron microscopy. For light and electron
microscopy, mice were deeply anesthetized and perfused through the left ventricle with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Tissue was removed and
post-fixed in the same fixative for 2 hr at room temperature. Vibratome
sections of eyes dissected through central regions of the retina and of brains, 200-500 µm in thickness, were incubated in 2%
OsO4 for 2 hr, dehydrated in an ascending series of
methanol, and embedded in Epon resin as described (Bartsch et al.,
1989; Montag et al., 1994). For light microscopy, 3-µm-thick sections
were stained with Toluidine blue and examined with a Zeiss Axiophot
microscope. For electron microscopy, ultrathin sections were
counterstained with lead citrate and examined with a Zeiss EM 10C
electron microscope.
Immunohistochemistry. Indirect immunofluorescence on
sections of fresh-frozen retinae was performed as described including the negative controls with secondary antibodies only (Bartsch et al.,
1989; Wintergerst et al., 1993).
Visualization of apoptotic cell death. To visualize
degenerating cells in the retina, fragmented DNA of apoptotic cells was detected using the terminal deoxynucleotidyl transferase-mediated dUTP
nick end labeling (TUNEL) technique (Gavrieli et al., 1992). Briefly,
cryosections through central regions of the retinae from 17-d-old,
4-month-old, and 9-month-old wild-type
(2/1+/+) and 2/1 knock-in
mice (2/1ki/ki) were mounted onto silan-coated
coverslips and processed as described (Molthagen et al., 1996).
Sections were finally mounted onto slides and analyzed with a
fluorescence microscope (Axiophot, Zeiss). Labeled cells in the outer
nuclear layer were counted at a final magnification of 200×.
Subsequently, sections were counterstained with Toluidine blue, and the
area of the outer nuclear layers was determined using an image analysis
system (Neurolucida V2.1i, MicroBrightFields). At least three animals
were analyzed for each genotype and age. Statistical analysis of data
was performed using ANOVA and the Fischer's protected least
significant difference test (Fischer's PLSD).
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RESULTS |
Generation of 2/1 knock-in mice
After electroporation of the linearized targeting vector into
strain 129Ola-derived embryonic stem cells and double selection with
FIAU and G418, 1 in 20 clones carried the expected mutation as
determined by Southern blot analysis with the external probe 5'EXT
(Fig. 1). In addition to the wild-type
band of 8.4 kb, the appearance of a 2.9 kb band was detected because of
the presence of a new BamHI site introduced by insertion of
the 1 cDNA sequence into exon I of the 2 gene (Fig.
2A). Further analysis
with the 3' internal probe 3'INT confirmed the pattern expected
after homologous recombination.
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Figure 1.
2 gene, 2/1 knock-in
targeting construct, and structure of the 2/1
knock-in gene. A, Restriction map of the
mouse 2 gene. Translated and nontranslated exons are represented by
closed and open boxes, respectively, and
are numbered with Roman numerals. E, B, Sn,
X, and RV represent cleavage sites for
EcoRI, BamHI, SnaBI,
XhoI, and EcoRV (not all sites
indicated), respectively. Arrow indicates the
translation initiation codon. B, Restriction map of the
2/1 knock-in targeting construct
2/1loxpAneoloxtk, containing 1.0 and 4.7 kb of homologous sequences on the 5' and 3' site flanking the
1cDNAloxpAneopAlox insertion and thus interrupting
2 gene in exon I. LoxP sites are indicated by
triangles, the 1 cDNA, the PGKneobpA
cassette, the HSVtk cassette, and the Bluescript (KS-)
vector are indicated by open boxes. S
represents cleavage sites for SalI. C,
Expected and observed structure of the 2/1
knock-in gene after homologous recombination and
localization of probes. Horizontal bars indicate the
localization of hybridization probes 5'EXT and 3'INT. D,
Alignment of N-terminal amino acid sequences of 1 and 2 subunits
and the 2/1 fusion protein. Residues of the 1 and 2 subunit
contributing to the fusion protein are underlined. The
vertical bar indicates the fusion site.
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Figure 2.
Southern blot analysis of
2/1+/+ and 2/1+/ki
targeted embryonic stem cells, and Southern, Northern, and Western blot
analysis of 2/1+/+,
2/1+/ki, and 2/1ki/ki
mice. A, Southern blot analysis. DNA from
2/1+/+ (lane 1) and
2/1+/ki targeted embryonic stem cells
(lane 2) and DNA from 2/1+/+
(lanes 3 and 6),
2/1+/ki (lanes 4 and
7), and 2/1ki/ki
(lanes 5 and 8) mice digested with
BamHI (lanes 1, 2, 6-8) or
EcoRI (lanes 3-5) was hybridized with
probes 5'EXT (lanes 1-5) or 3'INT (lanes
6-8). The size of DNA fragments in kilobases is indicated at
the left margin. B, Northern blot
analysis. RNA from brains of 2/1+/+
(lanes 1, 4, and 7),
2/1+/ki (lanes 3, 6, and
9), and 2/1ki/ki (lanes
2, 5, and 8) mice was hybridized with probe 2
(exon II to exon VII; lanes 1-3), probe 1
(lanes 4-6), or probe 2-5'UT specific for
the 5' untranslated region of the 2 mRNA also present in the
2/1 knock-in fusion mRNA (lanes
7-9). The size of RNA fragments in kilobases is indicated at
the left margin. C, Western blot analysis with 10 µg
of protein per lane of detergent extracts from crude membrane fractions
from brains of 5-week-old 2/1+/+ (lanes
1, 3, and 5) and
2/1ki/ki (lanes 2, 4, and
6) mice using polyclonal antibodies against 2
(lanes 1 and 2), and monoclonal
antibodies BSP/3 against 1 (lanes 3 and
4). Polyclonal antibodies against L1 were used to
confirm equal loading of proteins (lanes 5 and
6). 2 and 1 are clearly detectable as broad
bands at 47-53 and 43 kDa, respectively (lanes 1, 3,
and 4). No signal with polyclonal 2 antibodies
is obtained in 2/1ki/ki (lane
2), whereas an additional band of ~40 kDa is observed with
monoclonal antibodies BSP/3 (lane 4). The
molecular mass is indicated at the left margin. D,
Western blot analysis of deglycosylated proteins. Ten micrograms of
soluble fractions of detergent lysates of crude membrane fractions from
retinae of 17-d-old 2/1+/+ (wt)
and 2/1ki/ki (ki) mice were
incubated with N-glycosidase F
(N), O-glycosidase
(O), both enzymes (N + O), or
without enzyme ( ), subjected to SDS-gel electrophoresis, and reacted
with monoclonal antibody BSP/3 after Western blotting. Molecular mass
standards are indicated in kilodaltons at the left margin.
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Highly chimeric mice were obtained after injection of targeted
embryonic stem cells into blastocysts. Chimeric males showed germline
transmission of the integrated 1 cDNA sequence as confirmed by
Southern blot analysis. Crossing of heterozygous offspring yielded
homozygous 2/1 knock-in mice with Mendelian
frequencies. Southern blot analysis of these mice with probes 5'EXT and
3'INT showed the pattern expected for a single integration by
homologous recombination (Fig. 2A). Neither
heterozygous nor homozygous 2/1 knock-in mice showed
any obviously abnormal behavioral phenotype. In contrast to
2-deficient mice, 2/1 knock-in mice had a life span
not different from wild-type mice (data not shown).
Total RNA from brains of 5-week-old wild-type, heterozygous, and
homozygous 2/1 knock-in mice was subjected to Northern blot analysis to determine whether the mutated 2 gene was
transcribed (Fig. 2B). After hybridization with probe
2, no signal was detectable with RNA from 2/1
knock-in mice. In contrast, 2 mRNA of ~3.0 kb was
easily detectable in wild-type and heterozygous animals (Fig.
2B). After hybridization with probe 1, similar
amounts of 1 mRNA in the range of 1.5 to 2.5 kb were detectable in
wild-type, heterozygous, and homozygous mice (Fig.
2B). To distinguish between endogenous and
knock-in-derived 1 mRNA, the Northern blot was hybridized
with probe 2-5'UT. A signal corresponding to 2 mRNA of ~3.0 kb
was detectable in wild-type and heterozygous mice, whereas no signal of
this size was detectable with RNA from 2/1 knock-in
mice. However, an additional band at ~2.1 kb corresponding to
the transcript of the knock-in 1 gene was detected in
heterozygous and homozygous 2/1 knock-in mice with an
intensity corresponding to ~10-20% of wild-type 2 mRNA
expression (Fig. 2B).
To confirm that the mutation generated a null allele for 2, proteins
from membrane fractions of brains of 5-week-old wild-type and 2/1
knock-in mice were subjected to immunoblot analysis. The
2 subunit of the Na,K-ATPase was detectable in 10 µg of protein from brains of wild-type mice using a polyclonal antibody to the mouse
2 subunit, whereas no signal could be detected in the same amount of
protein from brains of 2/1 knock-in mice (Fig.
2C). To determine the amount of 1 protein, 10 µg of
protein from brains of 5-week-old wild-type and 2/1
knock-in mice were subjected to Western blot analysis using
monoclonal antibody BSP/3. The 1 subunit of the Na,K-ATPase was
detectable with a molecular mass of ~43 kDa in wild-type mice and in
higher amounts in 2/1 knock-in mice. This increase in
1 expression in 2/1 knock-in mice amounted to
20-30% of that found in wild-type animals. In addition, a band of
~40 kDa was observed by immunoblot analysis in 2/1
knock-in mice (Fig. 2C).
To determine whether this additional band is caused by a different
glycosylation pattern of the 1 subunit, the carbohydrate contribution to the molecular mass and the type of carbohydrate modification were analyzed. Proteins (10 µg) from membrane fractions of retinae from 17-d-old wild-type and 2/1 knock-in
mice were subjected to enzymatic deglycosylation. After PNGase F
treatment, the molecular masses of all 1-immunoreactive proteins
from retinae of wild-type and 2/1 knock-in mice were
reduced. The band at 43 kDa in wild-type and 2/1
knock-in mice and the additional band at 40 kDa in 2/1
knock-in mice shifted to a single band at ~33 kDa. No
change in the molecular mass of BSP/3-immunoreactive proteins was
observed after treatment with O-glycosidase (Fig. 2D).
Analysis of 2/1 knock-in mice
by immunohistochemistry
In retinae of 17-d-old and 4-month-old wild-type mice, the
plexiform layers and the ganglion cell layer showed immunoreactivity for the 1 subunit (Fig.
3a,e), whereas strongest
immunoreactivity for the 2 subunit was detected in association with
the inner segments of photoreceptor cells (Fig.
3b,f). In retinae of 17-d-old and 4-month-old
2/1 knock-in mice, immunoreactivity for the 1
subunit was detected not only in the plexiform layers and the ganglion
cell layer but also in the inner segments of photoreceptor cells (Fig.
3c,g). Immunoreactivity for the 2 subunit was not observed in retinae of 2/1 knock-in mice (Fig.
3d).
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Figure 3.
Immunohistological analysis of
2/1+/+ and 2/1ki/ki
mice. Immunohistological localization of 1 (a, c) and
2 (b) in sections of 17-d-old
2/1+/+ (a, b) or
2/1ki/ki (c) retinae
using monoclonal antibodies BSP/3 (a, c) and 426 (b) recognizing 1 and 2 isoforms,
respectively. Note the intense 1 immunoreactivity of inner segments
of photoreceptor cells of 2/1ki/ki mice. No
2 immunoreactivity is detectable on sections from
2/1ki/ki mice incubated with monoclonal
antibody 426 (d). The immunohistological
localization of 1 (e, g) and 2
(f) in sections of 4-month-old
2/1+/+ (e, f) or
2/1ki/ki (g) retinae
using monoclonal antibody BSP/3 (e, g) or 426 (f). Note the expression of 1 by
photoreceptor cells of 2/1ki/ki mice
(g). 1, Ganglion cell layer and
nerve fiber layer; 2, inner plexiform layer;
3, inner nuclear layer; 4, outer
plexiform layer; 5, outer nuclear layer;
6, inner and outer segments of photoreceptor cells.
Scale bar (shown in a for a-g): 100 µm.
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Morphological analysis of retinae of 2/1
knock-in mice
At the light microscopic level, the overall morphology of
the brain was similar between wild-type and 2/1
knock-in littermates at the ages of 17 d, 4 months, and
9 months. In contrast to 2-deficient mice, neither enlarged
ventricles nor swollen astrocytic end feet were observed in the brain
stem, thalamus, or spinal cord of 2/1 knock-in mice
(data not shown). Furthermore, the cytoarchitecture of the cerebellar
cortex of 2/1 knock-in mice appeared normal and the
thickness of different cortical layers was similar to that of wild-type
littermates (data not shown).
Semithin sections through central regions of the retinae of 17-d-old,
4-month-old, and 9-month-old wild-type and 2/1
knock-in littermates revealed a progressing degeneration of
photoreceptor cells in the mutants. In retinae of 17-d-old 2/1
knock-in mice, the thickness of the outer nuclear layer
appeared similar to that of age-matched wild-type animals (Fig.
4a,b), whereas the thickness of the outer nuclear layer in 2 knock-out mice was
reduced in thickness (Fig. 4c). In retinae of 4-month-old
2/1 knock-in mice, a reduction in the thickness of the
outer nuclear layer was observed when compared with wild-type animals
(Fig. 4d,e). In retinae of 9-month-old 2/1
knock-in mice, the outer nuclear layer was either absent
(data not shown) or reduced to a few rows or a single row of
photoreceptor cells (Fig. 4g). In the mutant, the lengths of
inner and outer segments of photoreceptor cells were significantly
reduced when compared with wild-type littermates (Fig. 4, compare
f, g). Analysis of retinae from wild-type and 2/1 knock-in mice by electron microscopy confirmed a
progressing degeneration of photoreceptor cells in retinae of 17-d-old,
4-month-old, and 9-month-old mutant mice (for 4-month-old wild-type and
mutant animals, see Fig. 5, a
and b, respectively).
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Figure 4.
Light microscopic analysis of retinae of
2/1+/+ and 2/1ki/ki
mice. Semithin sections through retinae of 17-d-old
(a-c), 4-month-old (d, e), and
9-month-old (f, g)
2/1+/+ (a, d, f),
2 / (c), and
2/1ki/ki (b, e, g) mice. Note
that the thickness of the outer nuclear layer and the length of the
inner and outer segments of photoreceptor cells is significantly
reduced in 17-d-old 2/
(c) and 4-month-old
2/1ki/ki mice (e), and
dramatically reduced in 9-month-old 2/1ki/ki
mutants when compared with age-matched wild-types (a, d,
f). 1, Ganglion cell layer and nerve
fiber layer; 2, inner plexiform layer; 3,
inner nuclear layer; 4, outer plexiform layer;
5, outer nuclear layer; 6, inner and
outer segments of photoreceptor cells. Scale bar (shown in
c for a-g): 100 µm.
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Figure 5.
Electron microscopic analysis of photoreceptor
cells of 2/1+/+ and
2/1ki/ki mice. Ultrathin sections through
retinae of 4-month-old 2/1+/+
(a) and 2/1ki/ki
(b) mice. Note that the length of inner and outer
segments of photoreceptor cells in 2/1ki/ki
animals (b) is significantly reduced when
compared with 2/1+/+ littermates
(a). ELM, External limiting
membrane; IS, inner segments; ONL, outer
nuclear layer; OS, outer segments; P,
pigment epithelium. Scale bar (shown in b for
a, b): 5 µm.
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Detection of apoptotic cell death in the retina of 2/1
knock-in mice
Degeneration of photoreceptor cells in central regions of the
retinae of 17-d-old, 4-month-old, and 9-month-old wild-type and
2/1 knock-in mice was visualized using a modified
TUNEL method (Molthagen et al., 1996). In retinae of 17-d-old wild-type mice, only a few degenerating cells were visible in the outer nuclear
layer (Fig. 6a). In
comparison, a significantly increased number of apoptotic photoreceptor
cells was detectable in retinae of 17-d-old 2/1
knock-in mice (Fig. 6b), but it was still below the number of apoptotic cells observed in 17-d-old retinae of 2
knock-out mice (Fig. 6c). In retinae of
4-month-old (Fig. 7a) or
9-month-old wild-type mice, hardly any degenerating cells were visible,
whereas in age-matched 2/1 knock-in mice (Fig.
7b) numerous apoptotic photoreceptor cells were
detectable.
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Figure 6.
Apoptotic cell death of photoreceptor cells in
17-d-old 2/1+/+,
2/1ki/ki, and 2/
mice. Visualization of apoptotic cell death in the retina of 17-d-old
2/1+/+ (a),
2/1ki/ki (b), and
2/ (c) mice using the
TUNEL method. In the retina of 17-d-old 2/1+/+
animals, only a few degenerating photoreceptor cells are detectable
(a). In contrast, in retinae of 17-d-old
2/1ki/ki mice (b),
apoptotic cell death is increased when compared with wild-type mice. In
comparison, massive apoptotic cell death is visible in the outer
nuclear layer of 17-d-old 2-deficient mice (c)
(also see Molthagen et al., 1996). As a positive control, sections were
incubated with DNaseI before the TUNEL method was applied, and all
retinal cells are labeled (d).
e-h represent the phase-contrast photomicrographs of
a-d, respectively. 1, Ganglion cell
layer and nerve fiber layer; 2, inner plexiform layer;
3, inner nuclear layer; 4, outer
plexiform layer; 5, outer nuclear layer;
6, inner and outer segments of photoreceptor cells.
Scale bar (shown in e for a-h): 100 µm.
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Figure 7.
Apoptotic cell death of photoreceptor cells in
4-month-old 2/1+/+ and
2/1ki/ki mice. Visualization of apoptotic cell
death in the retina of 4-month-old 2/1+/+
(a) and 2/1ki/ki
(b) mice using the TUNEL method. In the retina of
4-month-old 2/1+/+ animals, apoptotic
photoreceptor cells are virtually absent (a),
whereas in retinae of 4-month-old 2/1ki/ki
mice (b), apoptotic cell death of photoreceptor
cells is frequently observed. c and d
represent the phase-contrast photomicrographs of a and
b, respectively. 1, Ganglion cell layer
and nerve fiber layer; 2, inner plexiform layer;
3, inner nuclear layer; 4, outer
plexiform layer; 5, outer nuclear layer;
6, inner and outer segments of photoreceptor cells.
Scale bar (shown in c for a-d): 100 µm.
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Quantitative determination of the density of TUNEL-labeled cells
revealed that at postnatal day 17 the number of apoptotic photoreceptor
cells was increased in mutant 2/1 knock-in mice (132.3 ± 24.0 TUNEL-labeled cells per mm2;
mean ± SEM) when compared with wild-type mice (33.8 ± 3.2 TUNEL-labeled cells per mm2; p < 0.0001) (Fig. 8). In 4-month-old
wild-type mice, the number of degenerating photoreceptor cells was
reduced to only very few cells (7.0 ± 1.0 TUNEL-labeled cells per
mm2), whereas in age-matched 2/1
knock-in mice the number of apoptotic cells increased
significantly (175.2 ± 7.2 TUNEL-labeled cells per
mm2; p < 0.0001) (Fig. 8). The
highest density of degenerating photoreceptor cells was found in the
outer nuclear layer of 9-month-old 2/1 knock-in mice
(265.3 ± 28.2 TUNEL-labeled cells per mm2).
Retinae of age-matched wild-type mice contained 5.3 ± 0.8 apoptotic cells per mm2 (p < 0.0001) (Fig. 8).
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Figure 8.
Density of TUNEL-labeled cells in the outer
nuclear layer of 2/1+/+ (open
bars) and 2/1ki/ki
(filled bars) mice at different ages. Animals
were analyzed at postnatal day 17 (P17) and at 4 months (4M) and 9 months
(9M) of age. Compared with wild-type mice, the
density of apoptotic cells was significantly increased in the outer
nuclear layer of 2/1ki/ki mice at all ages
analyzed (***p < 0.0001; according to Fischer's
PLSD). Bars represent mean numbers of apoptotic photoreceptor
cells/mm2 ± SEM from at least three animals of each
genotype and age.
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DISCUSSION |
We have generated knock-in mice expressing the 1
isoform instead of the 2 isoform of the Na,K-ATPase via homologous
recombination in embryonic stem cells. The cDNA sequence coding for the
1 subunit was inserted in frame into the first exon of the 2
gene, thereby abolishing 2 gene expression. The deduced fusion
protein contains 18 residues of the N-terminal part of 2, followed
by residues 14 to 304 of 1. Southern blot analysis with 5' external
and 3' internal 2 probes showed the hybridization pattern expected
after homologous recombination. The absence of 2 gene expression in the mutant was confirmed by Northern blot, Western blot, and
immunohistochemical analysis.
Northern blot analysis revealed transcription of the inserted 1 cDNA
in the knock-in mice but to a lower extent than 2 gene transcription in the wild-type, possibly because of reduced stability of the primary transcript. This 1 transcript amounted to only 10-20% of the 2 transcript in the wild-type mice. The level of expression of the introduced 1 cDNA was below detection for in situ hybridization analysis using digoxigenin-labeled
1-specific cRNA probes (our unpublished observations). By Western
blot analysis, expression of 1 subunit protein from the
knock-in cDNA in mutant mice was revealed and amounted in
retina extracts to 20-30% more 1 in comparison with 1
expression in the wild-type mice. Detection of an additional smaller
band in tissue extracts from mutant mice recognized by an antibody
against the 1 subunit suggested an additional 1 isoform caused by
altered glycosylation. After deglycosylation with PNGase F, all
1-immunoreactive bands shifted to one single band at ~33 kDa,
indicating a different glycosylation pattern of the
knock-in-derived 1 protein compared with the endogenous 1 subunit in at least some cell types. Expression of the
knock-in 1 subunit by cells normally expressing the 2
subunit may explain this change in the glycosylation of the 1
subunit. The -isoforms of the Na,K-ATPase are only
N-glycosylated with three or nine glycosylation sites
predicted from the 1 and 2 sequences, respectively (Antonicek et
al., 1987; Fahrig et al., 1990). As described previously, O-linked glycosylation was not observed for any of the
BSP/3-positive components detected in either genotype.
Expression of the 1 subunit from the knock-in gene was
further confirmed by immunohistochemical analysis, which revealed expression of the 1 subunit instead of 2 by photoreceptor cells in the retina of 2/1 knock-in mice. The different
reactivities of the antibodies recognizing either 2 or 1
subunits, however, did not permit a quantitative comparison between
endogenous 2 and knock-in 1 protein expression levels.
The use of antibodies raised against the first 18 residues of the 2
subunit and also contained in the 2/1 knock-in protein
may clarify this problem, under the assumption that these are not
cleaved by proteases.
In contrast to 2-deficient mice, 2/1 knock-in mice
have a normal life span. Thus, expression of the 1 subunit in place of 2 abolishes the lethal phenotype reported for 2-deficient mice
(Magyar et al., 1994). Deficits in motor coordination, tremors, or
paralysis of extremities were not observed. Moreover, the abnormal histological phenotype in some brain regions of 2-deficient mice (Magyar et al., 1994) was not observed, and the general morphology of
ventricles and other brain structures in 2/1 knock-in
mice appeared normal. Spongiform encephalopathy characterized by
intracellular vacuoles in the brain tissue of 2-deficient mice
(Magyar et al., 1994) was not observed in 2/1 knock-in
mice. At least to some extent, the 1 subunit of the Na,K-ATPase can
functionally substitute for the 2 subunit, and the absence of an
abnormal histological phenotype as described for some brain regions of
2-deficient mice implies a functional compensation for the absence
of the 2 subunit by the knock-in 1 isoform. Our
results support the interpretation that the basis of the phenotype of
2-deficient mice is caused by altered Na,K-ATPase pump activity
(Magyar et al., 1994). It was shown that all six possible isozymes
between 1, 2, 3 and 1 and 2 can be formed in
vitro, supporting the assumption that different isozymes exist
in vivo (Lemas et al., 1994; Schmalzing et al., 1997).
Although different kinetic properties of functional --isozymes
of the Na,K-ATPase have been described (Blanco et al., 1995a,b),
sufficient ionic homeostasis seems to be achieved in many cells in
which expression of the 2 subunit is substituted by 1 expression,
resulting in an apparently normal phenotype in the knock-in
animals regarding the spongiform encephalopathy and enlarged ventricles
detected in 2-deficient mice. The complex temporal and spatial
regulation of expression of the different and subunits by
distinct cell types thus may provide a system for the optimal
regulation of Na,K-ATPase pump activity.
In support of this view, we detected in the knock-in mutant
animals a higher level of photoreceptor cell death than in wild-type animals. In 2-deficient mice, apoptotic death of photoreceptor cells
in the retina was observed during the last days of the mutant's life
(Molthagen et al., 1996), whereas in wild-type animals, apoptotic cell
death in the retina occurs predominantly until the second postnatal
week and after this time only sporadic cell loss is observed (Chang et
al., 1993; Portera-Cailliau et al., 1994). Using the TUNEL method to
analyze apoptotic cell death in retinae of 2/1
knock-in mice, we observed a progressive degeneration of
photoreceptor cells, although it was less than in 2-deficient mice
[this study and Molthagen et al. (1996)]. Thus, apoptotic cell death
of photoreceptor cells in the retina was delayed considerably in the
knock-in mice compared with 2-deficient mice. The
progressive loss of photoreceptor cells in the retina of 2/1
knock-in mice leads to a reduction in the thickness of the
outer nuclear layer. Furthermore, inner and outer segments of
photoreceptor cells in retinae of 9-month-old 2/1
knock-in mice were hardly detectable. The progressive cell
death of photoreceptor cells in 2/1 knock-in mice may
be indicative of a suboptimal or insufficient Na,K-ATPase activity
needed for the highly active photoreceptor cells. The -1-isozyme
of the Na,K-ATPase may possess kinetic properties different from those
of the -2 isozyme causing the degeneration of these particular
cells. Alternatively, photoreceptor cells may depend on a particularly
high Na,K-ATPase activity for which the level of 1 subunit
expression may be insufficient in the 2/1 knock-in
mice. On the other hand, the phenotype of the knock-in mutant may be explained by the absence of the adhesive properties of
the 2 subunit. Because the RNA expression level from the
knock-in gene was lower than from the 2 gene in wild-type
animals and the amount of protein cannot be compared directly, we
cannot rule out either possibility. Using the knock-in mice
to dissect the two functions of the AMOG/2 molecule as a pump, on
the one hand, and as an adhesion molecule, on the other hand, was thus
possible only for cells in which sufficient ionic homeostasis was reached.
The selective loss of photoreceptor cells in 2/1
knock-in mice resembles the human disease retinitis
pigmentosa (RP). In photoreceptor-specific forms of human RP, night
blindness and loss of peripheral vision are the initial symptoms,
reflecting degeneration of rod photoreceptors. Mutations in the genes
for rhodopsin, peripherin, and cGMP phosphodiesterase have been
identified in mouse models for some forms of RP (Dryja et al., 1990;
Farrar et al., 1991; McLaughlin et al., 1993). It was shown that
apoptotic cell death of photoreceptor cells occurs in these three mouse models (Chang et al., 1993; Portera-Cailliau et al., 1994).
Photoreceptor cells undergo apoptosis not only during development for
fine tuning the number of cells in the retina and their
interconnections but also in maturity as a response to aberrant stimuli
(Finlay, 1992). Internucleosomal DNA fragmentation that occurs during
apoptotic cell death is thought to be mediated by a nuclear
endonuclease that can be triggered by a rise in calcium concentration
(Duke et al., 1983; Cohen and Duke, 1984; McConkey et al., 1989, 1990; Schwartzmann and Cidlowski, 1993). This mechanism of apoptosis was
discussed for the retinal degeneration mouse, in which an increase in intracellular cGMP concentration initiates among other events a rise in calcium concentration (Chang et al., 1993). The Na,K-ATPase can directly influence the intracellular calcium
concentration via the Na,Ca exchanger. In the 2/1
knock-in mice, a malfunction of the Na,K-ATPase may also
lead to altered intracellular concentrations of ions other than
potassium and/or sodium and therefore to a similar induction of nuclear
endonuclease activity, with the consequence of cell death. Studies are
under way to investigate this possibility and to use the 2/1
knock-in mice as a model for retinitis pigmentosa.
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Abstract
Introduction
