Targeted disruption of the murine Nhe1 locus
induces ataxia, growth retardation, and seizures
Sheila M.
Bell1,
Claire M.
Schreiner1,
Patrick J.
Schultheis2,
Marian L.
Miller3,
Richard L.
Evans4,
Charles V.
Vorhees1,
Gary E.
Shull2, and
William J.
Scott1
1 Division of Developmental
Biology, Children's Hospital Research Foundation, Cincinnati 45229;
2 Department of Molecular
Genetics, Biochemistry, and Microbiology and
3 Department of Environmental
Health, University of Cincinnati College of Medicine, Cincinnati, Ohio
45267; and 4 Center for Oral
Biology, University of Rochester School of Medicine and
Dentistry, Rochester, New York 14642
 |
ABSTRACT |
In most cells, the
ubiquitously expressed
Na+/H+
exchanger isoform 1 (NHE1) is thought to be a primary regulator of pH
homeostasis, cell volume regulation, and the proliferative response to
growth factor stimulation. To study the function of NHE1 during
embryogenesis when these cellular processes are very active, we
targeted the Nhe1 gene by replacing
the sequence encoding transmembrane domains 6 and 7 with the neomycin
resistance gene. NHE activity assays on isolated acinar cells indicated
that the targeted allele is functionally null. Although the absence of
NHE1 is compatible with embryogenesis,
Nhe1 homozygous mutants
(
/
) exhibit a decreased rate of postnatal
growth that is first evident at 2 wk of age. At this time,
Nhe1
/
animals also
begin to exhibit ataxia and epileptic-like seizures. Approximately 67%
of the
/
mutants die before weaning. Postmortem
examinations frequently revealed an accumulation of a waxy particulate
material inside the ears, around the eyes and chin, and on the ventral
surface of the paws. Histological analysis of adult tissues revealed a
thickening of the lamina propria and a slightly atrophic glandular
mucosa in the stomach.
sodium/hydrogen exchanger; gene targeting; stomach; skin
 |
INTRODUCTION |
MEMBERS OF THE
Na+/H+
exchanger (NHE) family mediate the electroneutral exchange of
Na+ and
H+ across the plasma membrane.
Molecular cloning studies have led to the identification of five
distinct members of this gene family in a variety of mammalian species
(5, 8, 10, 18-20). Encoded by individual genes, NHE isoforms
1-5 (NHE1-NHE5) are distinguishable not only by differences
in amino acid sequence but also by their expression patterns,
functional characteristics, basolateral vs. apical localization in
polarized epithelial cells, and sensitivity to inhibition by amiloride
and its derivatives (reviewed in Ref. 7). Unlike isoforms
NHE2-NHE5, which exhibit a restricted tissue distribution (5, 8),
NHE1 is expressed to some extent in all mammalian tissues and cell
types (7). In situ hybridization analysis performed on sections of rat
brain (6) revealed that NHE1 is expressed at low levels throughout the
brain, with higher levels of expression evident in the hippocampus, in
the second and third layers of the periamygdaloid cortex, and in the
Purkinje and granule cells of the cerebellum. High levels of expression have also been observed in differentiating crypt and lower villus cells
of the small intestine (3) and in mucous neck cells and parietal cells
of the stomach (15).
Due to its ubiquitous expression pattern, NHE1 is considered the
"housekeeping" exchanger involved in the maintenance of pH homeostasis, cell volume regulation, and cellular proliferative responses to growth factors. Previous studies in our laboratory have
revealed that midgestational administration of teratogens known to
reduce embryonic intracellular pH
(pHi) (11, 13, 21) results in
abnormal murine embryonic development that is exacerbated by
coadministration of amiloride or one of its analogs (2). To further
study the role of pHi regulation
during embryogenesis, we used gene targeting to disrupt the murine
Nhe1 gene. We found that NHE1 activity
is dispensable for normal embryogenesis. However, postnatal growth of
homozygous mutants is adversely affected, mutants begin to exhibit
ataxia and seizures at 2 wk of age, and histological analysis reveals
an abnormal morphology of the glandular gastric mucosa. During the
course of these studies, Cox et al. (4) published a characterization of
the swe mouse line, which harbors a
spontaneous mutation in the Nhe1
allele. Similarities and differences between the phenotypes of the
swe and
Nhe1 gene-targeted animals are discussed.
 |
METHODS |
Preparation of targeting construct.
A
DASH II phage library containing genomic DNA of the
129SvJ mouse strain was screened with a partial mouse
Nhe1 cDNA. Identified clones were
characterized by restriction mapping and partial sequencing. The
targeting vector was MJK neo, a vector containing the neomycin resistance gene under control of the phosphoglycerate kinase promoter and the herpes simplex virus thymidine kinase gene. A 6.5-kb
BamH I fragment of an
Nhe1 genomic clone was used to isolate
a 1.8-kb Bgl
II/Bgl I fragment of the mouse
Nhe1 locus including intron sequences
and the coding sequence for amino acids 123-217. This fragment was
blunt-end ligated into the BamH I site
of the targeting vector MJK neo located 5' of the neomycin
resistance gene. To generate the 3' arm of homology, a 5.7-kb
Xho
I/Spe I fragment was subcloned into
the Xho I site flanking the neomycin
resistance gene.
Gene targeting.
Embryonic stem (ES) cells were maintained on a feeder layer of
embryonic fibroblasts in DMEM supplemented with 15% fetal bovine serum, 2 mM glutamine, and 0.1 mM
-mercaptoethanol. Before
electroporation, ES cells were trypsinized, washed, and resuspended in
PBS. This cell suspension was electroporated with targeting vector
previously linearized at a Not I site
in the vector located adjacent to the Nhe1 5' genomic sequences.
Electroporated cells were seeded onto fibroblast feeder layers and
allowed to equilibrate for 24 h before selection in the presence of
G418. One day later, secondary selection with gancyclovir was
initiated, and ES cell colonies resistant to both drugs were
subsequently isolated. DNA from each isolate was digested with
EcoR I and analyzed by Southern blot
hybridization using a 32P-labeled
BamH
I/EcoR I genomic fragment from the
region 5' to the fragments used to prepare the targeting
construct. Targeted clones were injected into C57BL/6 blastocysts for
the generation of chimeric animals. Male chimeras were bred with Black
Swiss females, and offspring containing ES cell-derived genetic
material were identified by the presence of the agouti coat color.
Southern blot analysis of isolated tail DNA confirmed the presence of
the Nhe1 null allele.
Animal husbandry.
All animals were housed in microisolator cages with food (Purina
Formulab 5008) and water ad libitum under a 12:12-h light-dark cycle.
The colony was maintained by interbreeding the heterozygous animals of
the F1 generation (129SvJ/Black
Swiss background). To generate the survival and growth curve data,
heterozygous matings were performed and 12 pregnant females were
monitored daily for delivery. All of the pups in each litter were
weighed weekly. Beginning on postnatal
day 14, cages were surveyed in the
morning and in the evening for dead animals. Gross examinations were
performed on dead animals. Genotypes were determined by Southern blot
analysis of isolated tail DNA.
Statistics.
Because of the high mortality, separate ANOVAs were performed on body
weights for each postnatal week. When significant body weight
differences were found, individual groups were compared by Duncan's
multiple range test.
Blood gases.
Animals were anesthetized with Nembutal before the collection of an
arterial blood sample in heparinized glass capillary tubes. Samples
were obtained from four animals representative of each genotype and
immediately loaded into a Radiometer model ABL5 blood gas machine for
the determination of whole blood pH,
PCO2, PO2, and
HCO
3. pH values were adjusted to the
internal body temperature of each animal at the time of sample collection.
Northern analysis.
Total RNA was isolated from the indicated tissues using Tri-Reagent
(Molecular Research Center, Cincinnati, OH) and analyzed by Northern
blot hybridization using a
32P-labeled 3.6-kb
Xba I fragment of the rat
Nhe1 cDNA that spanned the entire
coding sequence.
PCR analysis.
PCR analysis was used to determine the nature of the aberrant
transcripts identified by Northern blot analysis. Mutant and nonmutant
total RNA samples were treated with DNase, primed with oligo(dT), and
reverse transcribed with Superscript RT (GIBCO). For PCR analysis,
several combinations of primers were used. Primers on either side of
the targeted disruption, 5'-TTCCCAGTCCTGGACATTGACTAC-3' (3' end of coding exon 1) or
5'-ACGTCTTCTTCCTCTTCCTGCTG-3' (5' end of coding
exon 2), were used in combination with
5'-CATCACTACTCCTGAGGCGATGAG-3' (5' end of coding exon
4). To determine whether an
Nhe1-neomycin hybrid transcript had
been generated, the primer pair
5'-ACGTCTTCTTCCTCTTCCTGCTG-3' (from coding exon 2) and
5'-TGCAGTTCATTCAGGGCACC-3' (from the neomycin resistance
gene) was used. PCR products were characterized by DNA sequence analysis.
Measurement of NHE activity in isolated lacrimal gland acinar
cells.
All experiments were performed in a physiological saline solution (PSS)
containing (in mM) 135 NaCl, 5.4 KCl, 1.2 CaCl2, 0.8 MgSO4, 0.33 NaH2PO4,
0.4 KH2PO4,
10 glucose, 20 HEPES (pH 7.4 with NaOH), and 2 glutamine. Dispersed
acini were prepared from animals of each genotype (+/+, +/
, and
/
) by collagenase digestion using the method previously
described for parotid gland acini by Tanimura et al. (16) with
modifications. In brief, mice were anesthetized by inhalation of
CO2 and killed by cardiac
puncture. Lacrimal glands were removed, trimmed free of fat and
connective tissue, minced in a small volume of ice-cold digestion
medium [Earle's MEM (Biofluids, Rockville, MD) containing 0.075 U/ml collagenase P, 2 mM glutamine, and 0.1% BSA] and then
incubated in the same medium at 37°C for a total of 75 min.
Throughout this period, cells were kept continuously agitated, were
gassed with 95% O2-5%
CO2, and were periodically
dispersed at 30, 45, 60, and 75 min by trituration through a 10-ml
pipette fitted with a Rainin RT-96 yellow tip. The final cell
suspension was transferred to PSS containing 0.1% BSA and top gassed
with 100% O2.
Isolated lacrimal gland acini were loaded with the fluorescent pH
indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) by incubation with BCECF-AM (2 µM for 30 min at 30°C). After loading, cells were washed and resuspended in PSS plus 0.1% BSA.
Aliquots (150 µl) of acinar suspension were transferred to a
perfusion chamber, equipped with a coverslip glass base and gravimetric
perfusion system, that was fitted to the stage of a Nikon Diaphot
microscope. The acini were allowed to adhere to the base of the chamber
for 5 min and were then perfused with experimental PSS (warmed to
37°C) at a rate of 4 ml/min. The fluorescence from clumps of four
to ten acinar cells was monitored using a Spex ARCM spectrofluorometer
(Edison, NJ) interfaced to the microscope via a fiber-optic cable, by
alternating the excitation wavelength between 495 and 433 nm at 1-s
intervals and measuring emitted fluorescence at 530 nm. The ratio of
fluorescence at 495 nm to fluorescence at 433 nm was converted to
pHi using the
high-K+-nigericin calibration
technique as previously described (17).
NHE activity was determined by monitoring the ability of BCECF-loaded
mouse lacrimal gland acini to recover from an intracellular acid load
imposed by the addition of 30 mM sodium propionate (added as a 3.03 M
stock solution). In some experiments (tissue from +/+ and +/
animals), pHi recovery was also
monitored in the presence of ethylisopropyl amiloride (EIPA; 5 µM), a
specific inhibitor of the NHE. Because the absolute rate of
pHi recovery represents the
ability of the cells to respond to an acid load and because the time
course of the recovery conformed well to a linear response, we
quantitated the traces by calculating the slope of
pHi recovery.
Microscopy and morphometry.
Blocks 1-2 mm thick of cerebrum, cerebellum, stomach, kidney, and
skin were fixed in 4% paraformaldehyde in PBS. Tissues were postfixed
in 1% osmium tetroxide, dehydrated in an ascending series of ethanols
and propylene oxide, and embedded in Spurr's resin. Blocks were
oriented to produce transverse sections (1.5 µm), which were stained
with toluidine blue for light microscopy.
Light microscopic morphometry of the stomach was performed by measuring
the thickness of the gastric glands along the greater curvature of the
stomach in multiple ×1,250 fields. The thickness of the
epithelium of the gastric glands was determined by measuring a line
that began at and was perpendicular to the basement membrane and ended
at the lumen of the epithelium. Gastric gland epithelial measurements
were made in the region containing zymogen cells (base), parietal cells
(neck), and mucous cells (uppermost neck and surface of the gastric
gland). The thickness of the lamina propria between and beneath the
basal portion of the gastric glands was measured in micrometers. The
number of gastric glands per micrometer of basal lamina was determined
by measuring the length of basement membrane per low-magnification
field and counting the number of gastric glands for that distance.
Statistical significance was determined using a paired
t-test.
Isolated heart, lung, trachea, thyroid, thymus, spleen, liver,
pancreas, small and large intestine, colon, cerebrum, cerebellum, ear,
and testis were fixed in 10% neutral buffered Formalin, dehydrated through graded alcohols, and embedded. Sections 5 µm thick were stained with hematoxylin and eosin and examined by light microscopy.
 |
RESULTS |
Generation of Nhe1 null mutant mice.
As indicated in Fig.
1A,
the targeting construct
pNhe1neo-tk
was constructed so that recombination with the endogenous allele would
result in the replacement of the 3' end of exon 2 and part of the
adjacent intron with the neomycin resistance gene. The replaced
sequences from exon 2 encode amino acids 217-275, which encompass
the highly conserved sixth and seventh transmembrane-spanning domains.
After electroporation of the linearized construct into ES cells and
selection with gancyclovir and G418, Southern blot analysis identified
8 of 112 resistant clones as being correctly targeted. Four of the
targeted clonal ES cell isolates were injected into C57BL/6
blastocysts. Two of these clonal isolates produced chimeric progeny,
and one of them yielded germ line transmission of the targeted
Nhe1 allele. Heterozygous animals on
the 129SvJ/Black Swiss background of the
F1 generation were intercrossed to
produce homozygous progeny (Fig.
1B). Of the 226 pups born, 57 were
wild type, 105 were heterozygous, and 64 were homozygous for the
mutated Nhe1 allele, suggesting that
loss of NHE1 did not lead to prenatal lethality.

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Fig. 1.
Targeting of the Nhe1 locus.
A: diagram of targeting vector,
wild-type allele, and targeted allele.
B: Southern blot analysis of
EcoR I-digested tail DNA isolated from
a representative F1 litter. Blot
was hybridized with 5' BamH
I/EcoR I probe indicated in
A. Note that all 3 genotypes are
represented. E1, EcoR I; E5,
EcoR V; B,
BamH I; Bg1,
Bgl I; Bg2,
Bgl II; N,
Not I; S,
Spe I; X,
Xho I; neo, neomycin resistance gene;
tk, herpes simplex virus thymidine
kinase gene; +/+, wild type; +/ , heterozygous; / ,
homozygous mutant.
|
|
Expression of mutant Nhe1 allele.
To evaluate the expression of the mutated
Nhe1 allele, total RNA was isolated
from adult tissues and analyzed by Northern blot hybridization (Fig.
2A).
Consistent with the ubiquitous expression pattern observed in other
species, Nhe1 mRNA was detected in all of the tissue samples from wild-type and heterozygous animals. In all
of the samples analyzed, the heterozygous tissue expressed approximately one-half as much of the wild-type 4.8-kb message as
wild-type tissue. Two other mRNAs, ~4.5 and 3 kb in length, were
detected at varying levels in the tissues isolated from heterozygous and Nhe1
/
animals (Fig.
2A).

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Fig. 2.
Expression of wild-type and mutant
Nhe1 mRNAs.
A: Northern blot analysis of total RNA
from indicated tissues. Arrows indicate wild-type 4.8-kb transcript,
arrowheads point to upper and lower aberrant transcripts detected only
in tissues isolated from +/ and / animals.
B: RT-PCR analysis of brain total RNA
from each genotype with exon 2- and exon 4-specific primers. Note
absence of 613-bp wild-type product in / mutant sample
and 380-bp product present at low levels in +/ sample and as
only product in / mutant sample. Diagram at
right shows location of primers
(arrowheads) and aberrant alternative splicing that occurs in mutant
transcript. C: sequence analysis of
380-bp PCR product.
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|
To determine the structure of the aberrant transcripts, brain and lung
total RNA isolated from each of the three genotypes was analyzed by
RT-PCR. Using primers from the 5' end of the disrupted exon 2 and
the neomycin resistance gene, PCR products were generated from the
+/
and
/
samples (data not shown). These data
suggest that the smallest hybridizing transcript observed on the
Northern blot is likely to be a transcript containing the 5' end
of the Nhe1 mRNA that reads through to
the end of the neomycin resistance gene. RT-PCR with primers from the
5' end of coding exon 2 in combination with a primer from the
beginning of exon 4 resulted in a 613-bp PCR product in the wild-type
and heterozygous samples (Fig. 2B and
data not shown). A low level of a smaller 380-bp product was also
detected in the heterozygous samples; the 380-bp product was the only
product observed in the mutant samples. Sequence analysis of the 380-bp
product indicated that it was generated by aberrant splicing between a
cryptic splice donor site following codon 198 and the splice acceptor
at the beginning of exon 3 (Fig. 2, B
and C). This aberrantly spliced mRNA
probably corresponds to the transcript that appears slightly smaller
than the wild-type transcript in the Northern blot of heterozygous and
homozygous mutant tissues (Fig. 2A).
Although this mRNA remains in frame (Fig.
2C), it lacks 231 nucleotides
(encoding amino acids 199-275) that span 3 transmembrane domains.
To exclude the possibility that the aberrant
Nhe1 transcript encoded a mutant
protein that possessed NHE activity, we examined NHE activity in acinar
cells isolated from the mouse exorbital lacrimal glands of wild-type,
heterozygous, and homozygous mutant Nhe1 animals. In lacrimal acinar
cells, NHE plays a key role in buffering the intracellular acid load
that results from enhanced oxidative metabolism and membrane transport
processes during fluid secretion (9). Thus the ability of lacrimal
acinar cells to recover from a sodium propionate-induced acid load was
used as a measure of NHE activity. Representative traces are shown in Fig. 3A.
The addition of 30 mM sodium propionate to an acinus derived from a
wild-type animal induced a rapid initial decrease in BCECF fluorescence
(intracellular acidification, resulting from the permeation of
propionic acid into the cell and its subsequent dissociation into
propionate and protons) followed by a subsequent pHi recovery (alkalinization) to
original resting pHi values. However, in an acinus isolated from an
Nhe1
/
mutant animal, pHi recovery was completely
abolished. These data have been quantitated in Fig.
3B to show the absolute
pHi recovery rates in isolated acini. Acini from wild-type and heterozygous animals completely recover
their pHi after acid loading (+/+,
rate = 0.092 ± 0.009 pH units/min, half time to reach original
resting pHi = 263 ± 6 s;
+/
, rate = 0.068 ± 0.012, half time = 283 ± 9 s). In
contrast, recovery to the original resting
pHi was not observed in acini from
Nhe1
/
mutant mice (rate = 0.008 ± 0.002). Importantly, the NHE inhibitor EIPA (5 µM)
mimicked the effect of disrupting the
Nhe1 locus by blocking
pHi recovery in both wild-type
(rate = 0.011 ± 0.003) and heterozygous (data not shown) acini.
Taken together, these results indicate that NHE1 is the predominant regulator of pHi in mouse lacrimal
gland acinar cells and that the targeted
Nhe1 allele is functionally null.

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Fig. 3.
Lacrimal gland acini from Nhe1 mutants
lack NHE1 activity. A: effect of
Nhe1 mutation on ability of mouse
lacrimal acinus to recover to their resting intracellular pH
(pHi) following an acute acid
load. At time indicated by arrow, a single BCECF-loaded acinus isolated
from either a +/+ or a / animal was acid loaded by
exposure to 30 mM sodium propionate.
pHi was measured as described in
METHODS. To enhance clarity,
pHi trace for / acinus
is offset by 0.06 pH units. B:
absolute rates of pHi recovery of
+/+, +/ , and / lacrimal gland acini from an acute
acid load. Results are means ± SE for 4-7 separate experiments
made on acini prepared from 3 or 4 animals of each genotype.
pHi recovery rates were determined
by manually fitting a straight line to linear part of trace immediately
after peak acidification. In acini from +/+ and +/ animals, this
linear recovery period typically extended for at least 80 s over ~0.1
pH units. Because there was some variability between individual acini
in initial resting pHi and hence
in maximum peak acidification reached during sodium propionate
exposure, only cells with peak acidifications of 6.75 ± 0.1 pH
units were used in determination of recovery rates. Limitation of pH
range over which recovery rates were calculated minimized any effects
that variations in pHi or
cytosolic buffering capacity might have on NHE activity. Resting
pHi in +/+, +/ , and
/ acini were 7.15 ± 0.06 (n = 7), 7.13 ± 0.09 (n = 6), and 7.10 ± 0.04 (n = 7), respectively. Peak sodium
propionate-induced acidification values were 6.75 ± 0.02, 6.78 ± 0.07, and 6.73 ± 0.02, respectively. Ethylisopropyl amiloride
(EIPA; 5 µM) was added to +/+ cells 30 s before addition of
sodium propionate.
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Characterization of phenotype.
Through the first week of life, Nhe1
/
pups were indistinguishable from their littermates;
however, by 2 wk of age, the homozygous mutants exhibited a
statistically significant reduction in weight compared with either
heterozygous or wild-type littermates (Fig.
4A).
This difference persisted through adulthood, as evidenced by 10-wk-old
homozygotes weighing only ~18 g in contrast to the ~28 g
characteristic of wild-type animals. The growth rate of heterozygous
mice was indistinguishable from that of wild-type mice. Examination of
the life span of the Nhe1 null animals
indicated that, beginning on postnatal
days
16-18
and through postnatal day
29, ~68% of the
Nhe1
/
mice died (Fig.
4B). The
Nhe1
/
mice that reached
adulthood were fertile and capable of breeding with +/+ or +/
animals. No successful matings of
/
males with
/
females were observed.
Nhe1
/
females that were
mated with Nhe1 +/
males were
able to carry a litter to term, although the females that delivered
subsequently died several days postpartum. At death, the mutants were
poised with their forepaws in flexion suggestive of having had a
seizure.

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Fig. 4.
Nhe1 mutant growth and survival.
A: growth (means ± SE) for each
genotype. Pups from 12 different litters were weighed weekly. At 2 wk
of age, mutants are significantly lower in weight
(P < 0.05). * Statistically
significant different weights at P < 0.01. B: survival of
Nhe1 / mutants. No
+/ or +/+ progeny were found dead before weaning.
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During the second week of life, mutants began to exhibit an ataxic gait
that primarily involved an inability to efficiently move their
hindlimbs. When placed in a new environment, the mutants were also
hesitant to move and investigate their surroundings. The stress of
simply moving the cage could elicit a very excitable response in some
individuals, during which time they continually ran around the inside
of the cage, a well-known preconvulsant behavior. These episodes
frequently ended in a catatonic-like state resembling an absence
seizure, from which the animals usually recovered. This behavior was
most frequently observed in mutants before weaning but was also
observed in some adults.
Examination of mutants found within 16 h of death revealed varying
degrees of a waxy particulate material, especially on the ventral
surface of the paws, in the ears, and around the eyes and mouth and to
a lesser degree throughout the body fur (Fig 5). Morphometric analysis of the skin was
performed for signs of sebum and/or keratin overproduction. The
thickness of the whole skin, including the epidermis, dermis,
subcutaneous fat, and muscle was found to be significantly thinner
(P < 0.04) in
Nhe1
/
animals than in
wild-type animals, which is likely related to the differences in animal
body weights. In the mutants, the nonkeratinocytes in the epidermis
(Thy1 cells, Langerhans cells, and melanocytes) appeared
to be normal, as was the number of mast cells in the dermis. The
connective tissue also appeared to be normal, with no evidence of
inflammation. However, a slight increase in the accumulation of keratin
with small droplets of lipid on the surface of the cornified layer of
the epidermis was observed. These particles were about the same size as
the small particles observed on the skin of the animals. Notably,
accumulations of this waxy particulate material were not observed on
living mutants, suggesting that the postmortem observations of
particulate accumulation may be attributable to a lack of normal
grooming by the animals before death.

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Fig. 5.
Waxy particulate material. Mutants found within 16 h of death possessed
varying degrees of a particulate material prominently observed on
ventral surface of paws (A), inside
ear (B), on chin
(C), and within back fur
(D). Arrows indicate accumulations
of this waxlike material.
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A histological survey was performed on paraffin-embedded tissues
isolated from 12- to 13-wk-old animals of each genotype (+/+, +/
,
/
). The tissues examined included a variety of
organs (heart, lung, trachea, thyroid, thymus, spleen, liver, pancreas,
stomach, duodenum, jejunum, ileum, cecum, colon, cerebrum, cerebellum, testis, and ear) and revealed no abnormalities except in the stomach of
homozygous mutants (discussed below). Due to the high levels of NHE1
expression in the brain, additional morphometric analysis of
plastic-embedded brain sections was performed. No striking differences
in the appearance or number of Purkinje or granule cells of the folia
of the cerebellum were observed, nor was a difference in the thickness
of the molecular layer detected.
A morphometric evaluation of thin sections taken from the greater
curvature of the stomach revealed several differences between wild-type
and mutant animals. A trend was observed suggesting that the glandular
epithelium from muscularis mucosa to lumen was thinner in the
Nhe1 mutants than in wild type.
However, the thickness of the epithelial cells from the basement
membrane to the lumen of the gastric gland at the base of the glands
(primarily zymogen cells), at the neck (primarily parietal cells), and
at the surface (primarily surface mucous cells) was not different. As
depicted in Fig. 6, a considerable widening
of the interstitial space between gastric glands was observed. In
wild-type animals, the interstitial space was only 2.4 ± 2.8 µm wide compared with the 8.3 ± 3.4 µm in homozygous mutant
animals. Widening of the interstitial space in the mutants does not
appear to be attributable to an inflammatory response, since an
inflammatory infiltrate was not evident and since normal numbers of
tissue basophils were present. Consistent with the widening of the
interstitial space, the distance of basement membrane occupied by a
gastric gland and by the space between glands was found to be
significantly less in the wild-type animals than in the
Nhe1 homozygous mutants (33.7 ± 3.7 and 39.2 ± 3.5 µm, respectively;
P < 0.02).

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Fig. 6.
Sections through great curvature of stomach.
Top: montage of gastric glands from
Nhe1 / and +/+ mice
showing decrease in thickness of whole gastric epithelium. Widened
lamina propria between gastric glands and at base of gastric epithelium
is evident. Bottom: distance between
arrowheads is representative of regions measured to determine that
interstitial space is greater in Nhe1
/ mutant gastric tissue. lp, Lamina propria; m, muscle;
p, parietal cell region; z, zymogen cell region. Scale bars, 20 µm.
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|
Because NHE1 is considered one of the primary regulators of ionic
homeostasis, arterial blood samples collected from wild-type, heterozygous, and homozygous mutant animals were analyzed on a Radiometer ABL5 blood gas machine. The values obtained for whole blood
pH, PCO2,
PO2, and
HCO
3 were not different between the
genotypes (Table 1), indicating that the
lack of NHE1 activity caused no significant perturbations of systemic
acid-base homeostasis.
 |
DISCUSSION |
NHE1 recently was found to be coexpressed with NHE3 in the murine
oocyte and during the blastocyst stage of development (1). The
localization of NHE1 to the basolateral domain of the blastula trophectoderm cells suggests that NHE1 is not likely to be involved in
blastocoel cavity expansion. This conclusion is in agreement with the
observed Nhe1
/
targeted
progeny being observed in a normal Mendelian genetic ratio, indicating
a lack of embryo lethality. Thus the absence of NHE1 is compatible with
normal embryogenesis, suggesting that the embryo can rely on other
ionic regulatory systems such as other NHEs or
Na+-dependent
Cl
/HCO
3
exchangers to alleviate incurred acid loads. These findings are also
consistent with the recently reported spontaneous
Nhe1 mutant allele,
swe. The
swe allele is a point mutation within
the Nhe1 locus that introduces a
premature stop codon between the putative 11th and 12th transmembrane
domains. The swe allele is
functionally null, as determined by the inability of skin fibroblasts
isolated from swe mutants to
translocate Na+ in the presence or
absence of the NHE inhibitor EIPA (4). Like the
swe allele, the targeted allele also
disrupts the transmembrane-spanning domains of the protein by deleting
the sixth and seventh transmembrane domains. Analogous to the Northern
blot analysis of RNA isolated from swe
+/
tissues, tissues heterozygous for the targeted
Nhe1 allele also expressed
approximately one-half as much of the 4.8-kb mRNA as tissues from
wild-type animals. In contrast, the
Nhe1-targeted mice express an
aberrantly spliced in-frame Nhe1
transcript that lacks sequences encoding transmembrane domains five to
seven. However, the inability of lacrimal acini isolated from these
animals to recover from an acid load demonstrates that the targeted
gene is also a functionally null allele.
Phenotypic similarities observed between the targeted
Nhe1
/
mutant and the
swe
/
mutant include
1) an ataxic gait first evident at
~2 wk of age, 2) excitability in
neonates, followed by a brief period of total behavioral arrest,
3) an increase in mortality of
mutants before weaning, and 4) a
postmortem appearance suggestive of death by a convulsive seizure. The
swe allele has been transferred to
several genetic backgrounds with the finding that survival of
homozygous mutants past weaning is significantly related to the
background (4). The background reported herein for the targeted allele
is 50% 129SvJ and 50% Black Swiss. On this background, the mortality
rate is similar to that reported for the
swe animals on the SJL stock:
approximately two-thirds die before weaning. The targeted allele is
currently being backcrossed onto the C57BL/6 background.
Because high levels of Nhe1 mRNA
expression were observed in granule and Purkinje cells of rat brain, we
carefully examined the folia of the cerebellum; however, we found no
pathological differences between Nhe1
null mutants and either wild-type or heterozygous animals.
Abnormalities in these cell populations were also not observed in the
swe mutants. In the
swe mutants, dying neurons were
observed in the deep cerebellar nuclei. This population of neurons was
not examined in the brain sections of the gene-targeted
Nhe1 mutants.
Histological and morphometric analyses of stomachs from
Nhe1
/
mice revealed
abnormalities in the interstitial space of the gastric glands not
previously noted in the analysis of the swe phenotype. The relatively mild
stomach phenotype was in sharp contrast to the severe gastric
histopathology observed in Nhe2 null
mice (12). In both Nhe1 and
Nhe2 homozygous mutants, the lamina
propria at the base of the glands and the interstitial space was
significantly thicker. Within this region, numerous inflammatory cells
were evident in the Nhe2 mutant tissue
but were not detected in the Nhe1
mutant tissue. Nhe2
/
mice exhibit severe reductions in the number of gastric parietal and
chief cells, apparently due to reduced viability of the parietal cells (12); the loss of chief cells, however, may be secondary to the
reduction in parietal cells and loss of net acid secretion. In contrast
to the Nhe2 phenotype, there were no
apparent reductions in the number of parietal and chief cells in
Nhe1 null mice. The available data
indicate that the cell type and membrane distribution of NHE1 in
gastric mucosa are similar to those of NHE2. Both isoforms are abundant
in stomach (20) and are expressed in parietal, chief, and surface
mucous cells (14). NHE1 is localized on basolateral membranes of
gastric parietal cells (15), and a number of considerations (discussed
in Ref. 12) suggest that NHE2 is also a basolateral isoform in parietal
cells. Nevertheless, the sharp differences in phenotypes of
Nhe1 and
Nhe2 null mice indicate that the two exchangers serve different roles in gastric mucosa. Whether or not the
failure of Nhe1 null animals to grow
normally is related to the different functions between NHE1 and NHE2 in
the gastric mucosa is not known. However, it is noteworthy that, in
contrast to the Nhe1 null animals,
which are readily distinguishable by size from their littermates, the
Nhe2 null mice are indistinguishable from wild-type animals (12).
This characterization of the gene-targeted
Nhe1 null mouse line has revealed a
number of phenotypic similarities and differences between the two null
alleles of Nhe1 in the
swe and
Nhe1 knockout strains. Both exhibit an
ataxic gait, the occurrence of epileptic-like seizures, and diminished
levels of NHE1 expression in heterozygous individuals. Whether or not
the phenotypic differences observed, including growth retardation
[said to be slight in the swe
mice (4)], the abnormalities observed in the gastric mucosa, and the particulate material observed in the fur, are unique to the targeted mutation or are related to differences in the genetic backgrounds of the animals is not currently known.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by March of Dimes Grant 6-FY98-0648
(to S. M. Bell, C. M. Schreiner, and W. J. Scott) and National
Institutes of Health Grants DK-50594 (to P. J. Schultheis and G. E. Shull), ES-06096 (to M. L. Miller), and DE-08921 (to R. L. Evans).
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. M. Bell, Division of Developmental Biology, Children's Hospital
Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229 (E-mail: sheila.bell{at}chmcc.org).
Received 30 October 1998; accepted in final form 15 December 1998.
 |
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