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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
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Preparation of targeting construct. A lambda  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 beta -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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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.

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.

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.

                              
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Table 1.   Blood gases and plasma electrolytes


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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