Department of Anatomy and Cell Biology, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8
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ABSTRACT |
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Previous studies of chimeric animals demonstrate that multipotential stem cells play a role in the development of the gastric epithelium; however, despite much effort, it is not clear whether they persist into adulthood. Here, chemical mutagenesis was used to label random epithelial cells by loss of transgene function in adult hemizygous ROSA26 mice, a mouse strain expressing the transgene lacZ in all tissues. Many clones derived from such cells contained all the major epithelial cell types, thereby demonstrating existence of functional multipotential stem cells in adult mouse gastric epithelium. We also observed clones containing only a single mature cell type, indicating the presence of long-lived committed progenitors in the gastric epithelium. Similar results were obtained in duodenum and colon, showing that this mouse model is suitable for lineage tracing in all regions of the gastrointestinal tract and likely useful for cell lineage studies in other adult renewing tissues.
parietal cells; zymogen cells; mucous cells; committed progenitors; cell lineage tracing; small intestine; colon; ROSA26 mice
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INTRODUCTION |
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THE GASTRIC EPITHELIUM is organized into numerous gastric units. Each unit is composed of three main structural elements: a planar surface epithelium, tubular invaginations of the surface epithelium called pits, and tubular extensions of the pits called glands. The simple epithelium forming gastric units is continuously renewed. Most cell production occurs near the junction between the pit and its associated gland, in a region called the isthmus. The epithelium is composed of several cell types derived, it is proposed, from a multipotential stem cell population found in the isthmus, possibly among the immature-looking, granule-free cells of the isthmus (15, 17, 18, 22, 25, 29). Different cell types follow different migratory paths from the isthmus. In the corpus of the stomach, the mucus-secreting pit cells migrate up to the pit and surface, whereas cells in the zymogenic lineage migrate down into the glands. Parietal, enteroendocrine, and caveolated cells migrate in both directions (15, 17, 20, 22-25, 29). In the antrum, units are similarly organized but consist mostly of mucous cells, although parietal and enteroendocrine cells are also found in some antrum units (18, 27, 30).
A lineage model has been proposed both for corpus and for antrum units based on circumstantial morphological and cell kinetic evidence. Some of the granule-free cells located in the isthmus are proposed to act as multipotential stem cells giving rise to prepit, preparietal, and preneck cells, which then act as progenitors committed to their respective cell lineages (22, 25, 30). However, there is no direct evidence in the adult stomach for multipotential stem cells. Nor is it known whether any of the committed progenitors are long lived, as is the case in the small intestinal epithelium (6).
Studies of chimeric mammals (chimeric either as a result of random x-chromosome inactivation during development or as a result of mixing blastomeres from different mouse strains) show that most gastric units are monoclonal by adulthood, although they need not have started out that way (33, 35, 36, 44, 45). Thus, at some stage during development, there are single cells capable of generating all epithelial lineages. However, we cannot conclude from such results that multipotential stem cells exist in the adult gastric epithelium, because it remains plausible, for example, that the multipotential cells are lost during development but leave behind a series of committed progenitors to maintain the adult epithelium. Similarly, although transgenic mouse studies of the effects of parietal cell ablation (10, 31, 41) or of signal-transduction pathways (7, 26, 32) indicate regulation of gastric progenitor populations, they provide only suggestive evidence for adult multipotential stem cells and various committed progenitor populations. A direct demonstration of the persistence of a common stem cell population into adulthood is still lacking.
In the adult small intestine and colon, direct evidence for the existence of multipotential stem cells has been obtained by inducing somatic mutations in one of a variety of endogenous genes and then examining the clones of cells derived from such mutated cells (6, 14, 38, 48). The dlb-1 locus works well for small intestine (6, 48) but suffers from normally variegated expression in stomach and parts of the colon (unpublished observations; see also Ref. 45), making it unsuitable for lineage tracing in these tissues. The enzyme glucose-6-phosphatase has been used for similar studies both in colon and in small intestine (14, 38), but these mice are not commercially available and have not been applied to the stomach. Here, we introduced the use of a well-characterized strain of transgenic mice for lineage tracing and used it to establish the presence of multipotential stem cells and committed long-lived progenitors in the gastric epithelium.
We used chemical mutagenesis of a transgene to label a cell and its
progeny. Transgenic mice expressing the bacterial gene for
-galactosidase (lacZ) are available from Jackson
Laboratories as the ROSA26 strain. They were originally constructed by
infecting embryonic stem cells with a retrovirus promotor trap
construct (13). One of the resulting lines has one copy of
lacZ inserted into the genome (13, 49). The
associated mouse promotor drives
-galactosidase expression in all
adult tissues tested, including the gastrointestinal tract. These mice
were originally generated as a byproduct of a program to look for genes
with interesting expression patterns but have since been used
principally in transplantation or chimeric mouse experiments as markers
for tissue of specific origin. To apply these mice to lineage tracing,
we took advantage of the fact that mice hemizygous for lacZ
contain only one transgene allele. Thus cells incurring inactivating
mutations in the single lacZ allele (for example, a point
mutation introducing a stop codon) will lose
-galactosidase activity
and will not stain for the enzyme. If the mutated cell is a progenitor,
all of its progeny will inherit the nonfunctional allele and hence will
also be unstained and thus distinguished from neighboring stained
cells. This allows clones of related cells to be identified.
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MATERIALS AND METHODS |
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Isolated epithelium from stomach, duodenum, and descending colon
obtained from male C57BL/6J mice was stained overnight at 37°C for
-galactosidase (40) to determine the background level of intrinsic galactosidase activity in these tissues. The background level of intrinsic galactosidase activity was negligible using this
procedure (data not shown).
N-ethyl-N-nitrosourea dosing and animals.
N-ethyl-N-nitrosourea (NEU) was used as the
chemical mutagen in this study. NEU (250 mg/kg) was administered
intraperitoneally (6) to 7-wk-old male hemizygous ROSA26
mice. Groups of two animals each were killed 5, 10, 15 (3 animals), 20, 30, and 48 wk after NEU administration. Control animals (injected with
vehicle only) were killed 10, 15, 25, and 42 wk after injection. Under anesthesia, the animals were perfused with 30 mM EDTA in PBS, and the
epithelium from duodenum, descending colon, and stomach was isolated by
vibration (5) into 0.25% glutaraldehyde in PBS and fixed
for 10 min. The isolated epithelium was stained overnight at 37°C for
-galactosidase (40).
Identification of the four main mature gastric cell types with differential interference contrast microscopy. All mutant units were microdissected and mounted on slides to determine the clone composition and distribution using differential interference contrast (DIC) microscopy. Mature parietal and zymogen cells were easily recognized with DIC microscopy. Recognition of mucous and enteroendocrine cells required experience. Our ability to recognize these cells with DIC microscopy was initially confirmed using specific staining. Enteroendocrine cells were stained with antibodies specific for chromogranin A (DAKO Diagnostics Canada, Missisauga, Canada), whereas mucous cells were stained with Kasten's fluorescent periodic acid Schiff (PAS) method (12).
Cell identification using specific gastric epithelial cell
markers.
Multiple labeling with specific gastric epithelial cell markers was
used to confirm the presence of the four main mature gastric epithelial
cell types in homogeneous units (units in which all cells are unstained
and hence represent a clone derived from a single stem cell, see
RESULTS). Rabbit primary antibodies specific for
chromogranin A, intrinsic factor, and
H+,K+-ATPase -subunit were used sequentially
to label enteroendocrine, zymogen, and parietal cells, respectively.
Peroxidase-conjugated donkey anti-rabbit secondary antibodies were used
with a different enzyme substrate after each primary to differentiate
between the three cell types (the deposit formed by the reaction
product masks the antibody complexes, thereby minimizing interaction
with subsequent antibodies). Mucous cells were demonstrated with alcian
blue staining.
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RESULTS |
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lacZ expression in gastric units from ROSA26 mice.
lacZ transgene product staining was observed in all cells in
the vast majority of corpus and antrum units (Fig.
1). The staining density appeared to
decrease slightly with cell maturity, due perhaps to increased numbers
of intracellular organelles in the mature cell types (Fig. 1). Also
contributing to a nonuniform appearance is the fact that units have a
cylindrical geometry. Thus there is a double layer of cytoplasm in the
more central portions of the unit, making it appear more densely
stained. This effect is enhanced by the protrusion of parietal cells
from the lateral surface of the unit (Fig. 1, E-G). There
was little variation in staining intensity between units in a given
animal; however, we did notice variation in staining intensity between
animals (perhaps due to slight variation in duration of fixation).
Clones of unstained cells are readily seen against this background of stained cells (see below).
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Identification of the four main mature gastric cell types with DIC
microscopy.
Mature parietal and zymogen cells are easily recognized with DIC
microscopy. Parietal cells are characterized by the presence of the
distinctive circumnuclear cannaliculi and the numerous surrounding
mitochondria (21). Both organelles are evident under DIC
microscopy (Fig. 2, A and
B). Zymogen cells contain many characteristic supranuclear
secretory granules that can easily be seen (Fig. 2,C and
D) (21). Morphological recognition of mucous
cells with DIC microscopy is more subtle, because individual mucous
granules are not discernible; however, collectively, the mucous
granules appear as a distinctive aggregate visible in the lumenal
aspect of the cells, consistent with description based on electron
microscopy (21). This means of identification was
initially confirmed with Kasten's fluorescent PAS method to stain
mucin (Fig. 2, E-H). Enteroendocrine cells are recognized by
their prominent nucleus and basal collection of small granules (21;
Fig. 2, I and K), as initially confirmed by
staining with antibodies specific for chromogranin A (Fig. 2,
J and L).
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Classification of mutant clones in gastric units.
Most mutant units contained a mixture of stained and unstained cells
and are referred to as mixed units (Fig.
3A). Some mutant units
contained only mutant cells and are referred to as homogeneous units
(Fig. 3B). Clones could be variously categorized. The
following scheme proved useful for both corpus and antrum:
1) spanning clone; mutant cells distributed throughout
the length of a unit, although not necessarily occupying the entire
unit; thus all homogeneous units and many mixed units contained
spanning clones (Fig. 3, A,B,F, G,K,M,O); 2)
partial clone; mutant cells distributed in the top one-third of a unit,
and possibly lower, but not throughout the full length of a unit (Fig.
3, C-E); 3) restricted clone; mutant cell
distribution does not include the top one-third of the unit or clones
containing only a single mature cell type (i.e., mucous, parietal, or
zymogen cells; Fig. 3, H-J).
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Frequency of mutant units.
In the gastric epithelium, NEU induced a significant increase in the
frequency of mutant units compared with the background control
frequency (Fig. 4). The average frequency
of mutant units in control mice was 0.000217, whereas the average
frequency in NEU-treated mice was 0.00136. There was no significant
variation in clone frequency with time. The fraction of mutant units in control animals that were mixed was 0.89, whereas the remaining 0.11 were homogenous. About 0.56 of mutant units in controls contained spanning clones, whereas 0.22 contained partial and 0.22 contained restricted clones. There was no statistical evidence of change during
the experimental period (from 10 to 42 wk of age) in the relative
distribution of the types of clones in control animals, in contrast to
the significant changes observed in the NEU-treated animals we now
describe.
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Dynamics of spanning and partial clones in NEU-treated animals.
Most mutant units in the corpus contained either a spanning or a
partial clone (Fig. 5A). The
proportion of mutant units with spanning clones increased significantly
with time (1.32% per week; P = 0.0000232), whereas the
proportion of units containing partial clones decreased (0.867% per
week; P = 0.0009). The proportion of mutant units
containing restricted clones seemed to slowly decrease with time (Fig.
5A), but the slope was not significantly different from 0 (
0.0045% per week; P = 0.06). Clonal expansion occurred more rapidly in the antrum than in the corpus. From 20 wk
onward, only spanning clones were observed in antrum (Fig. 5B) in contrast to the corpus, where partial clones were
still observed at 48 wk. Restricted antrum clones were occasionally observed, but their numbers were so sparse that we refrain from making
detailed comments on them.
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Dynamics of mixed and homogeneous units in NEU-treated animals. With time the proportion of mixed units decreased while that of homogeneous units increased significantly (0.7% per week; P = 0.0001; Fig. 5C), although even at 48 wk, mixed units made up the majority of mutant units. It is important to note, especially at the later time points, that in a substantial fraction of mixed units, the only stained cells were mature cells (usually parietal or zymogen cells; Fig. 3, K and L). This suggests that in such units, all cells were clonally related with the exception of residual long-lived mature cells, which were not derived from the mutant stem cell and hence were stained. Figure 5E shows the result of combining into a single "homogeneous" category the homogeneous units and the mixed units whose stained cells were all mature cells. Note that by 48 wk, the majority of mutant units are of the homogeneous type. A similar pattern was observed in the antrum (Fig. 5, D and F).
Homogeneous units contain the four main epithelial cell lineages.
Homogeneous units containing the four main cell lineages (parietal,
zymogen, enteroendocrine, and mucous cells) were observed using DIC,
indicating that all four lineages were derived from a single common
stem cell in the adult. The presence of the four lineages in
homogeneous units was subsequently confirmed by simultaneous lineage-specific staining (parietal, zymogen, enteroendocrine, and
mucous cells were demonstrated by presence of
H+,K+-ATPase -subunit, intrinsic factor,
chromogranin A, and mucin, respectively; Fig.
6).
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Branching mutant units.
Branching mutant units (Fig. 3A, E, F,
and M-P) were observed at all time points both in corpus and
in antrum (Fig. 5, G and H). The branching rate
of mutant units we observed is similar to the branching rate reported
by others in normal mouse gastric units (35), indicating
that mutation of the transgene does not affect the behavior of the
population. The proportion of branching mutant units appeared to
decrease with time in the corpus (0.2% per week; P = 0.048), but the decrease in the antrum was not significant (
0.4% per
week; P = 0.096). The decrease is probably due to
aging, because decrease in the rate of branching with age has been
reported in normal mouse gastric units (35).
Mutant crypts in the intestine. Mutant crypts were readily detected in duodenum and colon of NEU-treated mice (Fig. 3, Q and R). The process of clonal purification is much more rapid in duodenum and colon than in stomach, so a majority of mutant crypts (an average of 94% of mutant duodenum and 95% of mutant colon crypts) was homogeneously mutant. As a result, little of interest occurred in colon and duodenum during the course of our observations. Our purpose in reporting the results is to establish the applicability of this mouse model to the entire gastrointestinal tract. In duodenum, the average fraction of crypts containing a mutant clone was 0.006 ± 0.00064 (mean ± SE) in NEU-treated vs. 0.00017 ± 0.00014 in control mice. In colon, the average fraction of crypts containing a mutant clone was 0.004 ± 0.00036 in NEU-treated mice vs. 0.00014 ± 0.00012 in control mice.
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DISCUSSION |
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We show that ROSA26 (13, 49) is a simple and broadly applicable model for lineage tracing in all regions of the adult mouse gastrointestinal tract. We have used it specifically to establish the presence of both multipotential stem cells and committed long-lived progenitors in the adult mouse gastric epithelium.
Adult gastric epithelium has multipotential stem cells. We report the generation in adult mice of long-lived mutant clones containing the four main epithelial cell types (parietal, zymogen, enteroendocrine, and mucus; Figs. 3 and 6). These clones frequently expand to occupy entire gastric units. These observations directly demonstrate the existence of multipotential stem cells in the adult gastric epithelium. It might be argued that such units could have resulted from simultaneous mutations in two or more long-lived committed progenitor cells in the same unit and hence, that we have not demonstrated a common stem cell. However, given that transgene inactivation by mutagenesis in a given cell is random and independent of events in neighboring cells, the probability that the transgene is inactivated in two or more long-lived progenitor cells in the same unit should be very small compared with the probability of mutation of a single progenitor in the unit. This makes simultaneous mutation untenable as an explanation for our observations, because we observed far more long-lived clones containing multiple cell types (spanning or partial clones) than clones containing a single cell type (restricted clones; Fig. 5A).
With time, partial clones seem to convert into spanning clones. This conclusion is derived from the similar composition of the two clone types and the fact that their frequencies follow inverse time courses (units containing partial clones decrease in frequency with a corresponding increase in frequency of spanning clones; Fig. 5A). Thus it is likely that partial clones gradually expand to become spanning clones, probably through the slow downward migration of cells in the parietal and zymogen lineages. This suggests that the multipotential stem cells are located in the upper portions of the unit, a conclusion consistent with previous morphological observations localizing the putative stem cells, the granule-free cells, to the isthmus (22, 29).Adult gastric epithelium contains long-lived committed progenitors. Multipotential stem cells are not the only long-lived progenitor population in the stomach. We observed clones containing only parietal, zymogen, or mucous cells (Fig. 3, H-J) as late as 48 wk after NEU administration, suggesting the presence of long-lived committed progenitors for each of these lineages in the stomach. Because mature gastric mucous cells normally live for only a few days (22, 23, 28, 29), our finding of persistent mucous-only clones indicates the existence of long-lived mucous progenitors. The situation regarding zymogen and parietal clones is less clear, however, because mature zymogen and parietal cells may live for hundreds of days (20, 24) making it difficult to specify bounds on the lifespan of the committed progenitor. However, the large number of cells contained in many of these clones, their extensive distribution along the axis of the unit, and the slow migration rate of these cell types (20, 22) suggest the existence of committed progenitors with prolonged proliferative capacity. There is precedence for committed long-lived progenitors in the gastrointestinal tract. In the small intestine, long-lived committed columnar and mucous cell progenitors have been documented (6).
Multistep accumulation of mutations by clones of long-lived progenitor cells is thought to be a mechanism leading to many cancers (4, 34, 37, 46). It is usually assumed that the multipotential stem cells are the only long-lived progenitor in the gastrointestinal epithelium and hence are the only population at significant risk of cancer. However, the long-lived committed progenitors we have observed could also be at risk of accumulating the mutations leading to cancer. Therefore, a characterization of the various long-lived committed progenitors might aid our understanding of specific forms of gastrointestinal cancer (1-3, 9, 11, 19, 39, 43, 47). Committed long-lived progenitors might also be targets, in addition to stem cells, for long-term gastrointestinal gene therapy (8). Finally, development of better means to protect the gut from the damaging effects of radiation and chemotherapy may benefit from an understanding of the subtleties of the various progenitor populations and their regulation.Clonal segregation in adult units. The majority of mutant units contained a mixture of stained and unstained cells at all time points, but with time, the proportion of mixed units decreased, whereas that of homogeneous units increased (Fig. 5, C and D), suggesting a transformation from mixed to homogeneous units. The lacZ-positive cells in a substantial number of the mixed units, especially at the later time points, were mature parietal or zymogen cells (Fig. 3, K and L). This suggests that in such units, the stem cell population is clonally homogeneous but residual long-lived mature normal cells persist. A plot of the proportion of mutant units containing either no stained cells or only mature stained cells indicates the impact of the long-lived mature cell populations on the homogenization process (compare Fig. 5, C and E).
The processes leading to homogenization of mixed units are important but poorly understood. Clonal purification may result from clonal partitioning after unit branching, from clonal extinction, or from a combination of both processes. New units are thought to result from a process of unit branching (16, 27) that presumably is accompanied by a distribution of the progenitor population among the resulting units. Nomura et al. (35) report that "monoclonal units do not emerge immediately following splitting of a polyclonal gland." However, we observed numerous examples of mutant units, which, on completion of the branching process, would likely yield at least one homogeneous unit from a mixed parental unit (Fig. 3, N and P). Also, clusters of neighboring units were observed in which some units were mixed, whereas neighbors were homogeneously mutant, suggestive of branching events that resulted in one mixed and one homogeneous unit (Fig. 3, B, G, and O). Clonal extinction is also clearly important for the homogenization process. This includes the extinction of stem cells, committed progenitors, and all of their extant mature progeny. It is important to realize that the long life of some of the mature cell populations in gastric units slows down the homogenization of a mutant unit. For example, it may take hundreds of days for all of the original population of stained zymogen and parietal cells to die off. Furthermore, the presence of long-lived committed progenitors and any potential competition among various stem cells inhabiting units undoubtedly add subtleties to the process.Adult colon and small intestine have multipotential stem cells. Although not entirely novel, it is worth reporting our observation of homogeneously mutant crypts both in the small intestine and in colon using this model system. First, it confirms the applicability of the hemizygous ROSA26 mice to lineage tracing in these regions of the gastrointestinal tract and confirms the presence of multipotential stem cells. Second, it is important to note that our use of a transgene marker serves to complement previous studies using mutation of physiological genes (dlb-1 or glucose-6-phosphatase; see also Refs. 6, 14, 38, and 48). It was plausible that loss of endogenous gene function altered cellular behavior in some way that either gave the mutant cells a competitive advantage or changed adhesive characteristics causing the cells to segregate. Transgene mutation is more likely to be neutral. Therefore, we have confirmed that clonal purification is a normal process in small intestine and colon, not an artifact resulting from loss of function of an endogenous gene.
A potential issue with the use of a transgene marker, such as the bacterial gene lacZ, is the generation of clones by epigenetic mechanisms such as suppression of gene expression by methylation. A comparison of the frequency of lacZ-negative clones in small intestine from control mice reported here with that of clones from control mice marked by an endogenous gene and scored similarly in isolated epithelium (6) indicates comparable clone frequencies (~10 ![]() |
ACKNOWLEDGEMENTS |
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This research was funded by a grant from the Canadian Institutes of Health Research.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Bjerknes, Dept. of Anatomy and Cell Biology, Medical Sciences Bldg., Univ. of Toronto, Toronto, Ontario, Canada M5S 1A8 (E-mail address: matthew.bjerknes{at}utoronto.ca).
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. Section 1734 solely to indicate this fact.
May 10, 2002;10.1152/ajpgi.00415.2001
Received 27 September 2001; accepted in final form 1 May 2002.
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