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Department of Molecular Biology and Pharmacology (Q.L., J.I.G.)
Washington University School of Medicine St. Louis, Missouri
63110
Department of Anatomy (S.M.K.) Faculty of
Medicine Kuwait University Safat 13110, Kuwait
Departments of Pathology, Molecular and Human Genetics, and Cell
Biology (K.A.C., M.M.M.) Baylor College of Medicine Houston,
Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-inhibin gene (inham1/m1) develop
gonadal sex cord stromal tumors that secrete large amounts of activins
A and B. Analysis of inham1/m1 mice, with
or without gonads, established that supraphysiological levels of
activins block differentiation of preparietal to acid-producing
parietal cells, differentiation of neck cells to pepsinogen-producing
zymogenic cells, and terminal differentiation of mucus-producing pit
cells. ActRII mRNA is normally present in pit, parietal, and zymogenic
cells.
inham1/m1actRIIm1/m1
compound homozygotes develop activin-secreting gonadal tumors but have
no abnormalities in their gastric epithelium, indicating that
persistent stimulation of ActRII-dependent signaling pathways produces
pleiotrophic effects on gastric epithelial differentiation. When a
lineage-specific promoter is used to ablate mature parietal cells
with an attenuated diphtheria toxin A fragment in transgenic mice,
there is increased proliferation of the multipotent gastric stem cell
and its committed daughters and subsequent development of gastric
neoplasia. Parietal cell loss in
inham1/m1mice is not associated with this
proliferative response. These different responses to parietal cell loss
suggest that stimulation of ActRII-dependent signaling pathways in
inham1/m1 animals affects the
proliferative activity of the stem cell and its immediate descendents.
This finding may have therapeutic significance. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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200 epithelial cells, representing five
lineages, all derived from a multipotent stem cell (1, 2). These
lineages give rise to pit, parietal, zymogenic, enteroendocrine, and
caveolated cells. A remarkable feature of the gastric unit is its
capacity to maintain an accurate steady state census of its component
cell types despite marked differences in their rates of differentation,
migration, and lifespan. In addition, the distinctive morphological
features of committed lineage precursors permit quantitative analysis
of their responses to changes in the census of their progeny, as well
as their responses to genetically engineered changes in expression of
potential mediators of epithelial cell proliferation, differentiation,
migration, or death programs.
Figure 1
outlines what is currently known about the
organization of gastric units in the corpus region. Proliferation is
largely confined to the centrally positioned isthmus. In
vivo pulse labeling with [3H]thymidine followed by
electron microscopic radioautography has identified a presumptive
multipotent isthmal stem cell with an estimated turnover time of 3
days (2). Three of the stem cell’s immediate descendants are known: a
granule-free pre-pit cell precursor, a granule-free pre-neck cell
precursor, and a granule-free pre-parietal cell precursor (2, 7).
Mucus-producing pit cells differentiate during a 3-day upward migration
through the upper portion of the gastric unit (the pit) to the surface
epithelium where they undergo an apoptotic or necrotic death (4).
Members of the zymogenic cell lineage differentiate during a
downward migration from the isthmus through the neck and base regions
of the gastric unit (5). Differentiation involves the
following sequence: granule-free pre-neck cell precursor
pre-neck
cellneck cellpre-zymogenic cellzymogenic cell. Zymogenic cells
have a lifespan of 190 days and die by necrosis or apoptosis. The
parietal cell lineage is the only one of the three principal gastric
epithelial lineages that completes its terminal differentiation within
the stem cell zone (isthmus). Preparietal cells are converted to mature
acid-producing parietal cells in 1 day (3, 8). Mature parietal cells
then undergo a bipolar migration away from the isthmus (3). Half
migrate up the pit where they are eliminated by necrosis, exfoliation,
or phagocytosis. An equal number of parietal cells move down through
the neck to the base were they are removed by apoptosis or phagocytosis
(3). Cellular lifespan averages 54 days (3).
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If parietal cells are important regulators of gastric epithelial
homeostasis, then it is important to identify factors that regulate
their differentiation and survival. Studies in knockout mice have
indicated that activins may play such a role (11). Activins are members
of the transforming growth factor-ß (TGFß) family. Family members
are known to modulate cellular proliferation and differentiation
programs in other organs of the developing and adult mouse (12, 13).
These disulfide-linked dimers initiate their signal transduction
cascades by interacting with two transmembrane serine/threonine
kinases, type I and type II receptors. Ligand binding to type II
receptors appears to function upstream of type I receptors in a
sequential kinase cascade (14, 15). Activins and inhibins share a
common ß-subunit. Inhibins are ß-heterodimers while activins are
composed of various combinations of closely related ß-subunits
[activin A = ßAßA homodimers; activin B = ßBßB
homodimers; activin AB = ßAßB heterodimers (14)]. Mice
homozygous for an activin ßA null allele or compound homozygotes for
activin ßA and activin ßB null alleles die of craniofacial
abnormalities within 1 day after birth (16, 17), well before completion
of gastric unit morphogenesis (7). Mice homozygous for a null
allele of the inhibin -subunit (inham1/m1)
develop gonadal sex cord-stromal tumors derived from
granulosa/Sertoli cells (11, 18). The tumors produce large quantities
of activins A and B, resulting in a wasting syndrome with anemia, loss
of hepatocytes due to massive apoptosis, and loss of parietal cells by
unknown mechanisms (11, 18). Removal of the gonads before development
of tumors prevents these changes (11).
Two type II activin receptors (ActR) have been identified: ActRII and ActRIIB (19, 20, 21). The majority of ActRII-deficient mice live to adulthood (22). Crossing ActRIIm1/m1 and inham1/m1 animals to generate compound homozygotes does not prevent development of activin-producing gonadal tumors but does prevent the weight loss, anemia, hepatocyte depletion, and parietal cell loss (23). This finding establishes a role for ActRII-mediated signaling in the pathogenesis of this syndrome.
In the current study, we have performed detailed analyses of epithelial proliferation and differentiation programs in the stomachs of inham1/m1 mice with and without gonads, actRIIm1/m1 mice, inham1/m1 actRIIm1/m1 compound homozygotes, actßAm1/m1 mice, actßBm1/m1mice, actßAm1/m1 actßBm1/m1 compound homozygotes, and actßBm1/m1actRIIm1/m1 compound homozygotes. The results, coupled with an analysis of the cellular patterns of expression of ActRII, indicate that activin stimulation of ActRII-mediated signaling pathways affects the differentiation programs of multiple gastric epithelial lineages and the proliferative status of the multipotent isthmal stem cell.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Inhibin-Deficient Mice with Gonadal Sex Cord-Stromal Tumors
Have Abnormalities in the Differentiation Programs of Multiple Gastric
Epithelial Lineages, A-D, shows the
distribution of pit, parietal, neck, and zymogenic cells in gastric
units located in the corpus region of the stomach of 8-week-old mice
homozygous for wild-type Inha and ActRII alleles.
Parietal cells can be readily identified by their distinctive
morphology (Fig. 2A), by their production of GalNAc-containing
glycoconjugates recognized by Dolicos biflorus agglutinin
(Fig. 2B), and by their synthesis of the noncatalytic ß-subunit of
H+/K+ ATPase (Fig. 2C). Parietal cells are
evident in the pit, isthmus, neck, and base compartments of these
normal gastric units. Pit cells elaborate fucosylated glycoconjugates
detectable with Anguilla anguilla and Ulex
europeaus agglutinins. They also produce glycoconjugates with
ganglioside GM1 epitopes that are recognized by cholera toxin B subunit
(Fig. 2C). Neck cells synthesize GlcNAcß1,4-containing
glycoconjugates that react with Griffonia simplifolica
agglutinin II (GSII) (Fig. 2D). As neck cells migrate through the neck
region they accumulate pepsinogen (Fig. 2D). Terminal differentiation
of neck to zymogenic cells is associated with loss of GSII reactivity,
continued production of pepsinogen, and induction of intrinsic factor
synthesis. Zymogenic cells are confined to the base of these units
(Fig. 2, B and D). Table 1 provides quantitative data on
the average number of lineage precursors and their fully differentiated
descendants per longitudinal section of a gastric unit in mice
homozygous for wild-type Inha and ActRII alleles.
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-inhibin
gene (inham1/m1) have developed
activin-secreting gonadal tumors and exhibit abnormalities in all three
principal gastric epithelial lineages. There is a block in the
differentiation of parietal cells: the number of mature parietal cells
is reduced 4-fold while preparietal cell number is not significantly
different from normal age- and gender-matched littermates (Table 1
and
Fig. 2F). There is also a complete and discrete block in the
differentiation program of the zymogenic cell lineage: pre-neck and
neck cell numbers are increased while their prezymogenic and zymogenic
descendants are entirely absent (Table 1 and Fig. 2, E, F, and H). The
loss of mature parietal and zymogenic cells is not due to an increased
apoptosis, or to increased necrosis, or to increased phagocytosis by
neighboring cells (Table 1 plus data not shown).
A statistically significant 2- to 2.5-fold increase in the steady state
number of pre-pit and pit cells per unit is also evident at 8 weeks of
age in inham1/m1 mice (Table 1). This can also
be viewed as an increase in their fractional representation: even
though parietal and zymogenic cells are lost, the total cellular census
of gastric units remains equivalent to that in age-matched wild-type
littermates (Table 1). While conversion of pre-pit to pit cells is not
blocked in inham1/m1 units, terminal
differentiation of this lineage is altered as judged by reduced
accumulation of -L-fucose-containing glycoconjugates
that react with Anguilla anguilla agglutinin and reduced
levels of ganglioside GM1 epitopes that react with cholera toxin B
subunit (compare panels C and G in Fig. 2).
As noted in the Introduction, the multipotent isthmal stem
cell also gives rise to the enteroendocrine and caveolated cell
lineages (6). Enteroendocrine and caveolated cells are much less
abundant than pit, parietal, or neck/zymogenic cells in wild-type
gastric units (Table 1). The two cell types have a similar long
lifespan [average = 100 days for enteroendocrine cells (6)]. The
function of caveolated cells is unknown although they appear to be a
source of nitric oxide (24).
While the total number of enteroendocrine cells is not increased in
8-week-old inham1/m1 mice, there is a marked
increase in precaveolated and mature caveolated cells (Fig. 2E and
Table 1). Like parietal cells, caveolated cells normally complete their
differentiation within the isthmus before undergoing a bipolar
migration (Ref. 6; cf Fig. 1). Immature caveolated cells are
readily apparent in inham1/m1 gastric units
(data not shown), suggesting partial inhibition of caveolated cell
differentiation.
More than 95% of male inham1/m1 mice die by 12
weeks of age while >95% of female mice succumb by 17 weeks (11, 18).
The changes in gastric epithelial cell biology noted at 8 weeks of age
in male and female inham1/m1 mice persist and,
in some cases, become more exaggerated in females that survive to 15
weeks of age. Mature parietal cells are no longer detectable. The
blockade in terminal differentiation of the zymogenic cell lineage is
manifested by an even greater augmentation of pre-neck cells (Table 1).
Caveolated cells are more numerous (Table 1). The steady state
population of isthmal-based lineage precursors is increased further to
3-fold above that of age-matched wild-type littermates (Table 1)
without a detectable change in proliferation (defined by the number of
5'-bromo-2'-deoxyuridine (BrdU)-positive (S-phase) or M-phase cells in
the isthmus; e.g. Table 1). These latter findings suggest
that gastric epithelial differentiation may be inhibited at very early
stages.
Two observations indicate that the alterations in parietal, zymogenic,
pit, and caveolated cell differentiation are related to the gonadal sex
cord-stromal tumors that develop in male and female
inham1/m1 mice. First, the evolution of these
gastric epithelial abnormalities corresponds to the time course of
progression of the gonadal tumors. For example, there are no
qualitative or quantitative abnormalities in the epithelial cell
lineages of 4-week-old male and female inham1/m1
animals (data not shown), a time when only microscopic foci of gonadal
neoplasia are present (11, 18). Second, when
inha-/- mice are gonadectomized at 4 weeks of
age and examined 8 weeks later, there are no detectable qualitative or
quantitative abnormalities in parietal, zymogenic, pit, or caveolated
cell differentiation (controls = age-matched
Inha+/+ and inha+/m1
littermates) (e.g. Fig. 2, I–K).
Signaling through ActRII Is Required to Produce the Gastric
Epithelial Abnormalities in inham1/m1
Mice with Gonadal Tumors
Autonomous production of activins by the gonadal tumors leads to
>10-fold elevations in the serum levels of activins A and B (11). The
observed rescue of gastric epithelial abnormalities by gonadectomy
raises two questions. What is the normal role of activins in regulating
gastric epithelial proliferation and differentiation programs in the
developing and adult stomach? Do the gastric epithelial lineage
abnormalities noted in adult -inhibin-deficient mice reflect
persistent stimulation of ActRII-mediated signaling pathways by high
circulating levels of gonadally derived activins?
Gastric Unit Morphogenesis and Epithelial Differentiation in Mice
that Lack Activin A, Activin B, and/or ActRII
Gastric unit morphogenesis in the mouse is not completed until the
third postnatal week (7). At embryonic day 12.5 (E12.5), the stomach is
lined with a simple undifferentiated epithelial monolayer. At E18,
nascent gastric units appear as short solid epithelial infoldings,
90% of whose cells have the morphological appearance of adult
isthmal stem cells, their granule-free committed daughters, plus more
differentiated pre-neck, pre-pit, and pre-parietal cell descendants
(7). From P1-P7, immature cells decrease to 20% of the total as
differentiated pit, neck, and parietal cells appear (7). Between P15
and P21, the multipotent stem cell and its descendants are assembled
into a distinct proliferative zone (the isthmus) and cellular
migration/differentiation programs become compartmentalized (7).
The cellular patterns of accumulation of mRNAs encoding the , ßA,
and ßB subunits of activins, the activin type IA and IB receptors,
and the activin type II and IIB receptors have been examined in the
developing (fetal) stomach by in situ hybridization (25, 26). - and ßA-subunit mRNAs are not detectable. ßB mRNA is
confined to the epithelium. ActRIA mRNA is restricted to the mesenchyme
while ActRIB mRNA is located in the epithelium. ActRII and ActRIIB
mRNAs are both present at low levels in the mesenchymal and epithelial
layers.
actßBm1/m1 mice survive to the adult stage (16). Our
single and multilabel immunohistochemical surveys of 3-, 4-, and
10-month-old mice failed to disclose any abnormalities in the
proliferative status of isthmal-based precursors or in the terminal
differentiation programs of any gastric epithelial lineage (Fig. 3, A–C, plus data not shown).
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, D and E, and data not
shown).
Northern blot studies of RNAs prepared from the corpus of adult
ActRII+/+ mouse stomachs revealed ActRII and
ActRIIB mRNAs (data not shown). Surveys of 15-week-old
actRIIm1/m1 mice disclosed no quantitative or
qualitative abnormalities in their gastric epithelium (Table 1 and Fig. 3F). As expected from these results, 3-month-old
actßBm1/m1 actRIIm1/m1
compound homozygotes had no detectable defects in gastric epithelial
proliferation or differentiation programs (data not shown). Together,
our findings establish that ActRII-mediated signaling is not essential
for normal gastric unit morphogenesis or for maintenance of normal
epithelial homeostasis. The data also indicate that two of the
receptor’s ligands, activins A and B, are dispensible.
Removal of a Functional ActRII Rescues the Gastric Epithelial
Abnormalities Associated with High Circulating Levels of Activins A and
B in inham1/m1 Animals
inham1/m1 actRII m1/m1
compound homozygotes develop bilateral gonadal sex cord-stromal tumors
and have serum levels of activins A and B equal to or greater than
those documented in inham1/m1 animals (23).
Nonetheless, the majority of these animals survive past 15 weeks
(males) or 18 weeks (females). Quantitative morphological and
multilabel immunohistochemical analysis of gastric units in 12-week-old
inha m1/m1 actRII m1/m1
compound homozygotes revealed no block in parietal or zymogenic cell
differentiation, no augmentation in the number of caveolated cells, and
no increase in isthmal lineage precursors (Table 1 and Fig. 3, G–I).
There was no increase in the fractional representation of pit cells
(Table 1), although terminally differentiated pit cells had reduced
production of ganglioside GM1-containing glycoconjugates (compare Fig. 3I with Fig. 2, C and J).
ActRII mRNA Is Present in Parietal, Zymogenic, and pit Cells
The results obtained from inha m1/m1actRII m1/m1 compound homozygotes suggested that
activins originating from the gonadal tumors of -inhibin- deficient
mice signal through an ActRII-dependent pathway to produce the observed
block in terminal differentiation of the parietal and zymogenic cell
lineages, as well as the other abnormalities enumerated above. At least
two mechanisms can be envisioned. 1) Members of the parietal,
zymogenic, pit, and caveolated cell lineages express ActRII and the
downstream effectors of the signaling pathway activated by its ligands.
Therefore, these lineages are equally vulnerable to the effects of
supraphysiological levels of activins. 2) Only the parietal cell
lineage expresses ActRII and members of its downstream signal
transduction pathway. In scenario 2, the primary effect of constitutive
ActRII-dependent signaling would be to block parietal cell
differentiation. The resulting loss of mature parietal cells would then
produce secondary effects on the zymogenic and pit cell lineages,
analogous to the situation that occurs when parietal cells are ablated
by an attenuated diphtheria toxin A fragment (tox176) in transgenic
animals (see Introduction).
To explore these possibilities, we defined the cellular distribution of
ActRII mRNA in the gastric epithelium of 15-week-old wild-type mice.
Northern blots of total cellular stomach RNA were probed with a cDNA
derived from the 3'-nontranslated region of ActRII mRNA. The results
confirmed that the probe reacted with a unique mRNA of the expected
size (data not shown). 33P-labeled antisense and sense
riboprobes, derived from the same 3'-nontranslated region, were
subsequently incubated with serial sections prepared from the corpus of
the stomach. ActRII mRNA is present in epithelial cells distributed
throughout the length of the gastric unit and is not detectable in the
muscularis layer (Fig. 4A). Pretreatment of the stomach
sections with RNAse A before application of the antisense probe reduced
the signal intensity to a level equivalent to that observed with the
sense strand probe (Fig. 4B). High-power views of sections
counterstained with hematoxyin and eosin show that ActRII mRNA is
present in terminally differentiated zymogenic, parietal, and pit cells
(Fig. 4, C–E).
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, A–C). This
proliferative response involves isthmal precursors that expand into the
‘former’ neck and base regions (Fig. 5C). Cells with the
morphological appearance of the gastric stem cell and its granule-free
committed daughters form adenomas and ultimately invade the muscularis
layer of the stomach between the sixth through twelfth months of life
(Q. Li, A. Syder, R. G. Lorenz, S. M. Karam, and J. I.
Gordon, manuscript in preparation). Neither augmented proliferation of
isthmal lineage precursors nor gastric neoplasia occur in
inham1/m1 mice during their 3- to 4-month
lifespan (n = 20). The lack of a proliferative response to
parietal cell loss in inham1/m1 mice suggests
that activation of ActRII pathways may serve to suppress cell division
among this precursor population. Second, complete loss of zymogenic
cells occurs in inham1/m1 mice even when there
is a small population of mature parietal cells present (Table 1),
whereas in the tox176 transgenic mice complete loss of parietal cells
is not associated with a complete loss of zymogenic cells. Third, there
was no increase in the number of caveolated cells in the transgenic
mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Conversion of pre-pit to pit cells is not blocked by persistent stimulation of activin type II receptors. Moreover, removal of ActRII from inhibin-deficient mice does not fully restore a normal terminal differentiation program to their pit cell lineage, in contrast to the complete rescue associated with removal of their activin-secreting gonadal tumors. The different responses of the pit, parietal, and zymogenic cell lineages to supraphysiological levels of activins A and B cannot be correlated with any discernible differences in their levels of ActRII mRNA. The different responses emphasize the importance of defining the downstream effectors of ActRII-dependent signaling in the various gastric epithelial lineages (e.g. Mad proteins; Ref.15).
The absence of any detectable abnormalities in gastric epithelial proliferation, differentiation, or death programs in actRIIm1/m1 mice with normal circulating levels of activins suggests that signal transduction pathways involving this receptor are not essential for completion of gastric unit morphogenesis or for maintaining normal epithelial homeostasis once these units are fully formed. It is possible that other receptors function in an analogous way to overcome loss of ActRII in these knockout mice. Alternatively, ActRII-dependent signaling pathways may normally be silent in the gastric epithelium or opposed by signals derived from other pathways. Since targeted disruption of ActRII blocks the gastric epithelial pathology produced by supraphysiological levels of activins A and B, there are apparently no other receptors in the gastric epithelium that are functionally equivalent to ActRII in terms of their ability to inhibit differentiation when persistently stimulated by these ligands.
Even though ActRII does not appear to be required for normal gastric epithelial morphogenesis and homeostasis, there are several reasons why this epithelium may be a good model for deciphering the contributions of (potential) components of ActRII-dependent signaling pathways to the regulation of cellular differentiation programs in adult mice. The response of the parietal and zymogenic cell lineages is pronounced and readily definable in inham1/m1 animals. The response appears to principally involve an alteration in differentiation. Such a response is quite distinct from the massive, activin-induced, p53-independent apoptotic response of hepatocytes (Refs. 11 and 27; and W. Shou and M. M. Matzuk, manuscript in preparation). Activins have been shown to impede differentiation in erythroid lineages [e.g. erythroid differentiation factor (28)]. However, unlike erythropoiesis, there is a physically well organized continuum of cellular proliferation and differentiation in normal and inham1/m1 gastric units that can facilitate an analysis of the interrelationships between execution of a given lineage’s differentiation program and accumulation of putative downstream components of ActRII-signaling cascades. The absence of a pathological response to high circulating levels of activins in the self-renewing intestinal epithelium is remarkable given the common themes shared by crypt-villus and gastric units: a multipotent stem cell, functionally anchored in a distinct proliferative compartment, giving rise to lineages that differentiate during an orderly migration (29). As such, the crypt-villus unit serves as a reference control that may help define factors required for activins A and B to produce their effects on epithelial differentiation programs.
Finally, the ActRII-mediated response to high levels of circulating activins appears to involve the multipotent stem cell and committed lineage progenitors located within the isthmus of gastric units. This conclusion is based largely on the fact that a proliferative response to loss of mature parietal cells is not manifested by the stem cell or its immediate daughters in inham1/m1 mice but is observed when parietal cells are ablated using an attenuated diphtheria toxin A fragment and a lineage-specific promoter. In the latter case, the eventual result is a gastric neoplasia composed of cells that resemble lineage precursors (Q. Li, A. Syder, R. G. Lorenz, S. M. Karam, and J. I. Gordon, manuscript in preparation). If persistent stimulation of ActRII-dependent signaling pathways affects the proliferative activity/potential of the multipotent isthmal stem cell and and its immediate daughters, such pathways may have therapeutic importance in the setting of gastric neoplasia where stimulation may prove useful in suppressing growth. Further evaluation of this possibility will require an analysis of the expression of ActRII and its downstream effectors in these neoplasms and whether progression of tumorigenesis is accompanied by mutations in this receptor and/or its effectors (30, 31, 32).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Single and Multilabel Light Microscopic Immunohistochemical
Studies
inham1/m1 mice were killed at 1, 2, 3, and 4 months
of age or were gonadectomized when they were 1 month old and then
killed 2 months later (n = 2–4 mice per time point).
actRIIm1/m1 mice were killed at 3 and 12 months
(n = 3). inham1/m1actRIIm1/m1 compound homozygotes were studied at 3 and
5 months (n = 4). actßAm1/m1 mice were
examined within 24 h after birth (n = 2).
actßBm1/m1 animals were killed at 3, 4, and 10
months of age (n = 1–2 mice per time point).
actßAm1/m1actßBm1/m1 compound homozygotes were surveyed at
postnatal day 1 (n = 2).
actßBm1/m1actRIIm1/m1 compound homozygotes were killed at 3
months (n = 3). Control littermates homozygous or heterozygous for
wild type Inha, ActßA, ActßB, and
ActRII alleles were killed at similar time points (at least
two animals per time point). ß-Subunit-l035 to
+24/tox176 transgenic mice and their normal littermates were
examined at 3 months (n = 5 per group). Some animals received an
intraperitoneal injection of 5'-bromo-2'-deoxyuridine (BrdU; 120 mg/kg)
and 5'-fluoro-2'-deoxyuridine (12 mg/kg) 90 min before death.
Stomachs were removed, opened along their cephalocaudal axis, and fixed in Bouin’s solution. Five micron-thick sections were cut from the middle of the stomach. Immunohistochemical analyses were conducted using methods described previously (8, 10). The following antisera were diluted in PBS-blocking buffer (8) and incubated overnight at 4 C with gastric sections: 1) rabbit anti-rat intrinsic factor [InF; specificity = zymogenic cells (9); final dilution = 1:1000]; 2) rabbit anti-rat pepsinogen [a generous gift of Michael Samloff, UCLA, Los Angeles, CA; subset of neck cells and zymogenic cells (9); 1:500]; 3) rabbit anti-ß-subunit of rat H+/K+ ATPase [kindly supplied by Michael Caplan, Yale University, New Haven, CT; parietal cells (9); 1:1000]; and 4) goat anti-BrdU (Ref. 9; 1:1000). Antigen-antibody complexes were detected using indocarbocyanine (Cy3)-labeled sheep or donkey anti-rabbit or anti-goat Igs (Jackson Immunoresearch, West Grove, PA; 1:500). No epithelial cell staining was observed when primary antibodies were omitted. Glycoconjugate production was assessed in various epithelial cell lineages using protocols described in Ref. 33 and the following fluorescein isothiocyanate (FITC)-tagged lectins: Dolicus biflorus agglutinin; Griffonia simplifolica II; cholera toxin B subunit;Anguilla anguilla agglutinin; and Ulex europeaus agglutinin type I (see Ref. 33 for sources).
Quantitative Light and Electron Microscopic Morphological Studies
of Epithelial Cell Populations Present in Gastric Units
inham1/m1 mice and their normal
littermates were killed at 1, 2, and 3–4 months of age;
actRIIm1/m1 mice and their normal littermates
were killed at 3–4 months; and
inham1/m1actRIIm1/m1
compound homozygotes were killed at 3 and 4 months (n = 2–3 mice
per genotype/time point). Tissue fragments (1 mm3) from
the corpus of each stomach were fixed in 0.1 M sodium
cacodylate containing 2% paraformaldehyde, 2.5% glutaraldehyde, and
0.2% tannic acid, washed in the cacodylate buffer (pH 7.4), postfixed
for 1 h at 4 C in 1% osmium tetroxide, dehydrated, and embedded
in Poly/Bed 812 (Polyscience, Niles, IL). Semithin sections (0.5 µm
thick) were stained with 0.1% toluidine blue for light microscopy.
Three tissue blocks from each mouse were used to prepare the 0.5 µm
thick sections. Fifteen to twenty longitudinally oriented gastric units
in a section were selected to identify epithelial cells having
morphological features similar to those described in adult C57BL/6 and
FVB/N mice (1, 8). At least one section from each of the three blocks
prepared per animal per time point per group was examined. All cells
per longitudinally sectioned unit were counted. Cellular identity was
confirmed by examining adjacent 0.1 µm thick sections with an
electron microscope after the sections had been stained with uranyl
acetate and lead nitrate.
In Situ Hybridization
A 640-bp ActRII cDNA, derived from the 3'-untranslated region of
ActRII mRNA (19), was subcloned into pBluescript SK+
(Stratagene, La Jolla, CA). The specificity of this cDNA for ActRII
mRNA has been established in previous RNA blot hybridization studies of
ActRII+/+ and actRIIm1/m1
mice (22, 23).[33P]UTP-labeled antisense and sense
riboprobes with identical specific activities were synthesized using
the plasmid as template plus T3 or T7 RNA polymerases, respectively.
In situ hybridizations were performed using
paraformaldehyde-fixed and protease K-digested sections prepared from
the stomachs of 15-week-old normal littermates and
33P-labeled riboprobes (5 x 106 dpm/100
µl hybridization solution) according to Ref. 34. Control tissue
sections were pretreated with RNAse A before application of the
antisense RNA probe (34). Experiments were repeated three times with
similar results (n = 2 animals per experiment).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by grants from the NIH (DK-33487, CA-60651, HD-32067) and the Kuwait Foundation for the Advancement of Sciences (KFAS 95–07-02). K.A.C. is a student in the Medical Scientist Training Program (NIH Grant EY07102).
1 Abbreviations used include:
ß-subunit-1035 to +24, nucleotides -1035 to +24 of the
mouse gene encoding the ß-subunit of H+/K+
ATPase; Inha, mouse gene encoding the -subunit of
inhibin; inham1/m1; mice homozygous for a
null allele (mutant allele number 1) of the Inha gene;
ActRII, mouse gene encoding the type II activin
receptor; InF, intrinsic factor. 
Received for publication September 12, 1997. Accepted for publication November 5, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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mice. Nature 360:313–319[CrossRef][Medline]
1-antitrypsin in human intestinal
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