(Received for publication, November 21, 1995)
From the
The self-renewing epithelial populations present in the gastric
units of the mouse stomach are descended from a multipotent stem cell
and undergo an orderly migration-associated differentiation followed by
apoptosis. The steady state census of the three principal cell types
(acid-producing parietal cells, mucus-producing pit cells, and
pepsinogen and intrinsic factor-producing zymogenic cells) is
accurately controlled, despite marked differences in the rates of
migration of each lineage. A transgenic mouse model has been created to
define functional interrelationships between the proliferation,
differentiation, and death programs of these lineages. Nucleotides
-1035 to +24 of the noncatalytic subunit gene of mouse
H
/K
-ATPase were used to direct
expression of an attenuated diphtheria toxin A subunit in the parietal
cell lineage. These transcriptional regulatory elements are not active
in members of the pit and zymogenic lineages. Stomachs, prepared from
postnatal day 28-80 transgenic mice and their normal littermates,
were subjected to single- and multilabel immunohistochemical studies as
well as qualitative and quantitative light and electron microscopic
morphologic analyses. The toxin produced complete ablation of
differentiated parietal cells. Loss of parietal cells was accompanied
by a 5-fold increase in the number of undifferentiated granule-free
cells located in the proliferative compartment of gastric units. This
amplified population of granule-free cells included the multipotent
stem cell as well as committed precursors of the pit and zymogenic
lineages. Loss of mature parietal cells was also associated with (i) a
block in the differentiation program of the zymogenic lineage with an
accumulation of pre-neck cells and a depletion of their neck and mature
zymogenic cell descendants, and (ii) an
2-fold amplification of
pit cells. These findings are consistent with the notion that
epithelial homeostasis within gastric units is maintained by
instructive interactions between their different cell lineages. Unlike
pit and zymogenic cells, parietal cells complete their differentiation
in the gastric unit's proliferative compartment before undergoing
a bipolar migration along the unit. Thus, the mature parietal cell is
in a strategic position to influence decision-making among gastric
epithelial cell precursors and to modulate the migration-associated
terminal differentiation programs of the pit and zymogenic lineages.
The glandular epithelium of the mouse stomach is self-renewing. Self-renewal occurs in tube-shaped mucosal invaginations known as gastric units(1) . The gastric unit represents a model system for studying how the proliferation, differentiation, and death programs of several distinct lineages are coordinated to maintain a steady state population of cells and whether these programs are controlled in a cell autonomous or nonautonomous fashion.
Each gastric unit located in
the corpus of the adult mouse stomach contains an average of 200
cells(1) . The apical third of the gastric unit, known as the
pit region, is populated with 40 mucus-producing pit
cells(2) . A centrally positioned isthmus contains one or more
active multipotent stem cells and their undifferentiated yet committed
descendants(3) . The neck region is located just below the
isthmus and contains
12 mucus-producing neck cells(4) .
The base of the gastric unit is inhabited by
70 pepsinogen- and
intrinsic factor-positive zymogenic cells(4) . The gastric
unit's
25 acid-producing parietal cells are distributed in
all four of its regions(5) , as are members of the
enteroendocrine lineage(6) .
Tritiated thymidine
labeling-electron microscopic (EM) ()autoradiographic
studies have identified the presumptive stem cell and its committed,
undifferentiated descendants(3) . These studies have also
provided morphologic descriptions of the differentiation programs of
each of the principal epithelial
lineages(2, 3, 4, 5, 6) . A
pit cell precursor devoid of secretory granules gives rise to pre-pit
cells within the isthmus. Pre-pit cells become pit cells when they
enter the pit region. They migrate up the pit and upon reaching the
surface epithelium degenerate and die. The entire sequence is completed
in 3 days(2) . Members of the zymogenic lineage differentiate
during a downward migration from the isthmus. A granule-free pre-neck
cell precursor produces pre-neck cells, which are transformed to neck
cells as they migrate from the isthmus to the neck. Neck cells complete
their journey through the neck region in 14 days(4) . Upon
arrival at the upper portion of the base of gastric units, they become
pre-zymogenic cells. Terminal differentiation to zymogenic cells occurs
during a continued downward descent to the lower portion of the base of
gastric units. Zymogenic cells die by necrosis or apoptosis. The
sequence is completed in
190 days(4) . Conversion of
undifferentiated granule-free cells to pre-parietal cells occurs in the
isthmus and takes
1 day(5) . Differentiation of
pre-parietal to parietal cells also takes place in the isthmus.
Parietal cells subsequently undergo a bipolar migration to both the pit
and base. Death ensues and cells are disposed of by extrusion or
phagocytosis. The overall turnover time for parietal cells is 54
days(5) .
A steady state cellular census is maintained among these different epithelial lineages (1) despite differences in their rates and direction of migration. How is homeostasis achieved? Are instructive interactions between cells critical for controlling decisions about proliferation, differentiation, and death, or is regulation entirely cell autonomous? In a self-renewing system, an entirely cell autonomous mechanism of control poses the risk that perturbations in the rate of proliferation, or migration, or death could result in unopposed changes in cellular census.
The parietal
cell lineage represents an attractive starting point for examining the
general issue of whether one lineage influences the proliferation,
differentiation, or death programs of the gastric unit's other
lineages. Parietal cells are derived from three precursors(3) .
Although the majority (95%) of parietal cells originate from an
undifferentiated granule-free cell, thymidine labeling-EM studies
indicate that the daughters of pre-pit cell precursors and the
daughters of pre-neck cell precursors can each adopt two fates in the
adult mouse(3) . Ninety-nine percent of the progeny of a
pre-pit cell precursor become pre-pit cells, but 1% become pre-parietal
cells. Likewise, a small fraction (
2%) of the daughters of the
pre-neck cell precursor become pre-parietal cells, while the remainder
adopt a pre-neck cell fate.
These observations raise a question
about whether perturbations in parietal cell differentiation could
modify decisions concerning the fate of daughters of the pre-neck and
pre-pit cell precursors. One recent experiment addressed this
issue(7) . Functional mapping studies of transcriptional
regulatory elements in the mouse
H/K
-ATPase
subunit gene,
performed in transgenic animals using light and EM immunohistochemical
techniques, have shown that its nucleotides -1035 to +24 are
only active in pre-parietal cells and their differentiated parietal
cell descendants (7, 8) . When
H
/K
-ATPase
subunit
was used to direct expression
of Simian virus 40 T antigen, a 50-70-fold amplification of
pre-parietal cells occurred so that they became the predominant cell
type in the gastric units of adult (postnatal day 28-80)
transgenic animals. Differentiation to mature parietal cells was
blocked. Remarkably, even though the transgene was not expressed in
members of the pit or zymogenic lineages, there was a block in
conversion of pre-zymogenic to zymogenic cells. The effect was quite
specific; conversion of pre-neck to neck cells and neck to
pre-zymogenic cells was not perturbed(7) . There was also a
2-3-fold increase in mature pit cells.
Although this
experiment revealed an apparent interrelationship between the
differentiation programs of the parietal, zymogenic, and pit cell
lineages, it was not possible to determine whether the phenotype
reflected the consequences of amplifying the normally rare isthmal
pre-parietal cell and distributing these cells to ``ectopic
sites'' along the length of gastric units or whether the phenotype
was due to depletion of mature parietal cells. To distinguish between
these possibilities, we have used
H/K
-ATPase
subunit
to direct expression of an
attenuated diphtheria toxin A fragment (DT-A) to pre-parietal and
parietal cells so that they would be destroyed.
Eighty-seven live-born mice were screened using the polymerase chain reaction, tail DNA, and two primers that span intron 2 of the hGH gene. Primer 1 (5`-AGGTGGCCTTTGACACCTACCAGG-3`) anneals to the antisense strand in exon 1 of hGH. Primer 2 (5`-TCTGTTGTGTTTCCTCCCTGTTGG-3`) hybridizes to the sense strand in exon 3. The following thermocycling conditions were used to generate a 360-base pair polymerase chain reaction product: denaturation = 94 °C for 90 s, annealing = 55 °C for 90 s, and extension = 72 °C for 90 s for a total of 30 cycles, followed by 72 °C for 10 min. Six transgenic founders were identified. Pedigrees were established from 5 of these founders by crosses to normal FVB/N littermates. One founder was sterile.
Mice were maintained in a specific pathogen-free state in microisolator cages under a 12 h light cycle. Animals were given a standard irradiated chow diet (Pico Lab Rodent Diet 20; PMI Feeds) ad libitum.
The
following panel of antibodies and lectins were used: rabbit anti-rat
intrinsic factor (InF; 1:1000; (8) ); rabbit anti-pepsinogen
sera (1:500; (8) ); rabbit anti- subunit of rat
H
/K
-ATPase (amino acids 2-23;
dilution = 1:500; (8) ), goat anti-BrdUrd sera (1:1000; (14) ); Dolicus biflorus agglutinin (DBA; carbohydrate
specificity =
GalNAc epitopes; cellular specificity
= FVB/N parietal cells; Refs. 7, 8, and 15); Griffonia
simplifolica II (GSII, GlcNAc
1, 4; FVB/N neck cells; Refs. 8,
15, and 16), cholera toxin B subunit (ganglioside G
; FVB/N
pit and surface mucous cells; (15) and (17) ) and Anguilla anguilla agglutinin (AAA,
-L-fucose;
FVB/N pit and surface mucous cells; (15) ). All lectins
contained a fluorescein isothiocyanate (FITC) tag and were diluted to 5
µg/ml with PBS-blocking buffer (minus the powdered skim milk).
Figure 1: Quantitation of epithelial lineages that populate gastric units in normal P1, P15, and P28 FVB/N mice. Cells were identified in toluidine blue-stained 0.5-µm-thick sections of gastric units located middle third of the stomach(1) . The mean ± S.E. are plotted. Isthmal cells = pre-pit, pre-neck, and granule-free cells.
Fragment A of diphtheria
toxin (DT-A) functions as an ADP-ribosyl transferase. When introduced
into cells, it ADP-ribosylates and inactivates elongation factor 2,
causing inhibition of protein synthesis and cell death(10) .
Because of its extreme cytotoxicity, even very low basal levels of
H/K
-ATPase
subunit
/wild type DT-A
expression could, in theory, result in the death of nontarget cell
populations. Therefore, we used an attenuated DT-A, tox176,
which is 15-30-fold less cytotoxic than wild type
DT-A(10) , to ensure that the ablation would be limited to
members of the parietal cell lineage.
Finally, since previous EM
immunohistochemical studies of
H/K
-ATPase
subunit
expression had been conducted
using the hGH gene as a reporter(7) , tox176 was
inserted into exon 1 of hGH, upstream of hGH's initiator Met
codon. This served two purposes. It provided introns and a
polyadenylation signal to enhance the chances of efficiently processing
the transgene's DT-A containing mRNA transcript. It also reduced
the chances of perturbing the pre-parietal cell-specific pattern of
H
/K
-ATPase
subunit
transcriptional activation.
The forestomach, zymogenic,
mucoparietal, and pure mucous zones of transgenic mice and their normal
littermates were compared. Hematoxylin- and eosin-stained sections of
the forestomach disclosed no histopathologic changes in transgenic
animals. In normal littermates, differentiated parietal cells were
readily detected in the zymogenic and mucoparietal zones based on their
reaction with an antibody to the noncatalytic subunit of
H
/K
-ATPase and with D. biflorus agglutinin (Fig. 2, A and B). In
contrast, neither of these reagents reacted with cells present in the
zymogenic or mucoparietal (or pure mucous) zones of transgenic mice (Fig. 2, C and D).
Figure 2:
Single- and multilabel immunohistochemical
studies of H/K
-ATPase
subunit
/DT-A (tox176)
transgenic mice. Five-µm sections were prepared from the zymogenic
zone of the stomachs of P35 transgenic mice and their age-matched
normal FVB/N littermates. Panel A, section from a normal mouse
incubated with rabbit anti-rat
subunit of
H
/K
-ATPase sera. Antigen-antibody
complexes were visualized with Cy-3 labeled donkey anti-rabbit Ig.
Parietal cells appear red. Panel B, section from a
normal mouse incubated with FITC-labeled DBA. Parietal cells that react
with the lectin appear green. Multilabel studies indicated
that >98% of normal P35 FVB/N parietal cells coexpress the
subunit of H
/K
-ATPase and fucosylated
glycoconjugates recognized by DBA (data not shown). Panels C and D, sections prepared from a transgenic littermate and
processed as in panels A and B, respectively. No
specific cellular staining for the
subunit (C) or
glycoconjugates recognized by DBA (D) are evident, indicating
the absence of parietal cells. Panel E, section from a normal
mouse incubated with FITC-labeled AAA and goat anti-BrdUrd.
Antigen-antibody complexes were detected with Cy-3-labeled donkey
anti-goat Ig. Pit and surface epithelial cells that produce
glycoconjugates recognized by AAA appear green. S phase cells
in the isthmus appear orange-red. Panel F, section
from a transgenic littermate, processed as in E. The number of
S phase (BrdUrd-positive orange) cells is greater when
compared to the age- and gender-matched normal cagemate shown in E. Panels G and H, low and high power views
of a hematoxylin- and eosin-stained section prepared from a transgenic
mouse. The high power view provides evidence of increased proliferation (e.g. open arrow pointing to M phase cell) and increased
apoptosis (solid arrows). Panel I, section of a
normal FVB/N stomach incubated with FITC-labeled GSII, goat
anti-BrdUrd, Cy3-labeled donkey anti-goat Ig, and bis-benzidine.
GSII-positive neck (and pre-neck) cells appear aqua, while S
phase cells appear red. Bis-benzidine stains nuclei purple. Panel J, section from a transgenic littermate
processed as in I. Note the increased number of
BrdUrd-positive cells. Panel K, section from a transgenic
mouse incubated with FITC-labeled GSII, goat anti-BrdUrd, and
Cy3-labeled donkey anti-goat Ig. This high power scanning confocal
microscopic view of the isthmus shows BrdUrd-positive (red)
pre-neck cells that produce glycoconjugates recognized by GSII (green). Panel L, double exposure of the section
shown in panel B after incubation with FITC-labeled DBA,
rabbit InF, and Cy-3-labeled donkey anti-rabbit Ig. Differentiated,
InF-positive zymogenic cells (orange) are located in the base
of these normal mouse gastric units. Panel M, double exposure
of the section shown in panel C after treatment with the
reagents described in panel L. Panels N and O, sections from normal (N) and transgenic (O) littermates were incubated with rabbit anti-rat pepsinogen
and Cy-3-labeled donkey anti-rabbit Ig. Comparison of the two panels
indicates that the number of pepsinogen-positive cells is reduced in
transgenic gastric units and that they are confined to the base. Bars = 25 µm.
Comparisons of toluidine blue stained thin (0.5 µm) sections prepared from the zymogenic and mucoparietal zones provided independent confirmation of the loss of parietal cells in transgenic animals (Fig. 3, A-D; Table 1).
Figure 3:
Toluidine blue-stained sections of gastric
units present in a postnatal day 35
H/K
-ATPase
subunit
/DT-A (tox176)
transgenic mouse and its normal littermate. Panel A, base of a
normal FVB/N gastric unit. Differentiated parietal cells are evident (closed arrows). Zymogenic cells (e.g. open arrow)
contain numerous dense secretory granules. Panel B, pit region
of a normal gastric unit. Parietal cells are indicated by closed
arrows. Pit cells with apical granules are denoted by open
arrows. Panel C, lower power view of a section taken from
the zymogenic zone (corpus) of a transgenic littermate's stomach. Panel D, high power view of the section in panel C,
showing a dilated central lumen in the gastric unit with a degenerated,
extruded cell (open arrow), apoptotic bodies (e.g. closed
arrow), a pre-zymogenic cell (closed arrowhead), and the
absence of parietal cells. Panel E, another high power view of panel C. Comparison with panel B emphasizes the
increase in pit cell number associated with ablation of parietal cells. Bars = 25 µm.
Previous
[H]thymidine labeling-EM autoradiographic studies
had demonstrated that degeneration and apoptosis of parietal cells is
normally confined to the pit and base regions of gastric units (5) . Dead cells are either extruded into the lumen of gastric
units, phagocytosed by neighboring cells, or engulfed by macrophages
that invade from the surrounding mesenchyme(5) . These
processes were increased as early as P28-P35 in transgenic
gastric units (e.g.Fig. 3D and 4A).
Pre-parietal cells were identified by viewing serial 0.5- and
0.1-µm sections of gastric units under light and electron
microscopes, respectively. Pre-parietal cells were largely confined to
the isthmus region of gastric units in both transgenic and normal mice (Fig. 4A). Steady state levels of pre-parietal cells
were 7-fold elevated in transgenic animals (from an average of
0.2-1.4 cells/longitudinally sectioned gastric unit; cf.Table 1). Many of the pre-parietal cells present in
transgenic gastric units were in various states of degeneration (Fig. 4, A and C). These results contrast with
those obtained from identically aged FVB/N
H/K
-ATPase
subunit
/SV40 TAg transgenics raised
in the same barrier facility. Their pre-parietal census ranged from 50
to 70 cells per longitudinally sectioned of gastric unit (i.e. a 250-350-fold amplification; cf.(7) ).
Moreover, the pre-parietal cells were distributed in coherent vertical
bands or clusters from the pit to the base rather than being limited to
the isthmus.
Figure 4:
Transmission electron microscopic view of
gastric units from a postnatal day 42
H/K
-ATPase
subunit
/DT-A (tox176) mouse. Panel A, longitudinal section of a gastric unit. The
midportion of this unit contains a granule-free precursor (gf), a dividing pre-pit cell (pp), a pre-neck cell (pn), and a pre-parietal cell (ppar). The
pre-parietal cell shows signs of degeneration with partial dissolution
of cytosol, dense lysosomal bodies, and a dilated nuclear envelope. The
remnants of two extruded cells are seen in the central lumen (L). g, secretory granules; G, Golgi
apparatus. Magnification,
5000. Panel B, montage of the
midportion of a gastric unit showing amplified population of
granule-free (gf) precursors. A pre-neck (pn) and
neck (n) cell are also evident. Magnification,
3000. Panel C, the pre-parietal cell (ppar) in the mid-left portion of the panel appears normal and contains
apical microvilli, numerous mitochondria, and a characteristic
secretory canaliculus (c). The pre-parietal cell in the mid-right portion of the panel contains mitochondria with an
abnormal pale matrix, indicative of early degeneration. Two pre-neck
cells (pn) are present as is an enteroendocrine cell (ee). Panel D, existing populations of pre-neck,
neck, and zymogenic cells have normal morphology. The pre-neck cell (pn) contains scattered secretory granules with electron dense
cores. The number of these secretory granules is markedly increased in
a neck cell (nc). A fully differentiated zymogenic cell (zc) contains large (900-1300 nm) granules with uniform
electron density. Magnification,
6000.
Surveys of P28-P80 transgenic mice revealed a
persistent absence of parietal cells as defined by immunohistochemical
and morphologic criteria. Not surprisingly,
H/K
-ATPase
subunit
/DT-A (tox176) mice
were unable to acidify their gastric contents. Luminal pH ranged from 6
to 7 in nonfasting transgenic animals and from 1 to 2 in normal
littermates sacrificed at the same time of day (n = 6).
Members of the enlarged proliferating cell population were identified using light and electron microscopy. Ablation of mature parietal cells was associated with a 4-5-fold increase in the number of (proliferating) granule-free cell precursors in the isthmus (Fig. 4, A-C; Table 1). These isthmal precursors became the third most populous cell type in transgenic gastric units, in contrast to the situation in normal gastric units where their fractional representation was considerably lower (Table 1).
This report describes a transgenic mouse model where diphtheria toxin-mediated ablation of mature parietal cells is associated with a perturbation in the proliferation and differentiation programs of the pit and zymogenic lineages. The results provide direct evidence that loss of mature parietal cells reduces the efficiency of zymogenic cell differentiation from pre-neck cell precursors, while at the same time producing an amplification of pit cells. These findings are consistent with the notion that epithelial homeostasis within gastric units is maintained by instructive interactions that occur between different lineages.
The responses to the ablation of mature parietal cells suggest that both positive and negative regulatory cascades operating within gastric units derive from, and/or are dependent upon, this cell type. It is not yet clear whether instructive interactions involving parietal cells require direct cell-cell contacts or whether they involve short or long range paracrine loops. Our data underscore the fact that the mature parietal cell is in a strategic position to influence decision making. Parietal cells complete their differentiation in the isthmus, unlike pit and zymogenic cells. Thus, DT-A-induced degeneration of mature isthmal parietal cells could have marked effects on decisions concerning the proliferative status or fate of isthmal-based precursors of the pit and zymogenic lineages (e.g. through the release of positively and/or negatively acting factors from these dying cells or as a consequence of the loss of their normal instructive functions).
As noted in the Introduction, one fascinating feature of the self-renewing gastric unit is its ability to maintain an accurate steady state census of its component cell types even though these cells migrate at different rates along the base-pit axis. The bipolar migration of mature parietal cells through the pit and neck/base regions of gastric units could provide a series of extra-isthmal instructive interactions that serve to modulate progression through the pit and zymogenic cells' terminal differentiation programs.
The regulatory significance of cell-cell
contacts involving parietal cells can now be assessed using a strategy
analogous to that recently employed in the small intestine of
chimeric-transgenic mice, namely disruption of endogenous
cadherin-mediated cell adhesion by employing a lineage-specific
promoter such as H/K
-ATPase
subunit
to direct expression of a
dominant negative cadherin(19, 20) . The ability to
readily identify the committed precursor cells of the gastric
unit's principal epithelial lineages using morphologic criteria
permits a more complete description of the effects of such
perturbations on decision making than is achievable in other
self-renewing systems, including the small intestine's
crypt-villus unit.