|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online December 12, 2006
doi: 10.1242/10.1242/dev.02700
1 Department of Pathology and Immunology, Washington University School of
Medicine, St Louis, MO 63110, USA.
2 Department of Molecular Biology and Pharmacology, Washington University School
of Medicine, St Louis, MO 63110, USA.
3 Department of Biological Sciences and the Purdue Cancer Center, Purdue
University, West Lafayette, Indiana 47907-2064, USA.
Author for correspondence (e-mail:
jmills{at}pathology.wustl.edu)
Accepted 18 October 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Bhlhb8, Mucous neck cell, Laser-capture microdissection, Microarray, Mouse
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Karam and Leblond, 1993d;
Karam and Leblond, 1993c;
Karam and Leblond, 1993b;
Karam et al., 2003) have
revealed that the gastric unit in the stomach corpus (body) can be organized
by anatomy and cell function into: the pit zone, which opens into the gastric
lumen and is lined by mucus-secreting pit cells; the isthmus, which houses the
stem cell; the neck zone, which contains mucus-secreting neck cells located
among acid-secreting parietal cells; and the base zone, which contains
digestive enzyme-secreting zymogenic cells (ZCs)
(Fig. 1). The developmental
pathways characteristic of the adult gastric epithelium are established
postnatally: the ZC lineage, the last to appear, emerges at 
3 weeks of
age in mice (Li et al.,
1996).
Gastric epithelial differentiation is strongly correlated with migration
(Fig. 1B). Specific cell
lineages must execute the proper spatial (i.e. toward the lumen or base) and
temporal program. The mucus-secreting cells of the neck, for example, mature
as they descend over 14 days toward the base of the gastric unit. By
contrast, pit cells mature along a more short-lived (3 days in mice)
gradient climbing toward the gastric lumen. Acid-secreting parietal cells
arise in the isthmus, and most thereafter migrate into the neck zone, where
they live 54 days in mice (Karam et
al., 1997).
The most poorly understood differentiation pathway in the gastric unit is
that of the ZC, which is found only in the base zone but, like all gastric
epithelial cells, continuously turns over (half-life, 194 days) and must be
replenished by the stem cell in the isthmus. Morphological studies
(Suzuki et al., 1983;
Karam and Leblond, 1993c;
Ge et al., 1998) suggest that
ZCs arise from mucous neck cells that migrate into the base zone. However,
this neck-to-ZC transition hypothesis has been recently called into question
(Hanby et al., 1999). The
reason for the skepticism is that if neck cells transition into ZCs, they must
first form an elaborate mucussecretory apparatus that they then must
dismantle, shed or convert, as they subsequently construct the equally
elaborate serous secretory apparatus characteristic of the mature ZC
(Fig. 1C). The cell and
molecular processes that could convert a postmitotic, fully functional
mucus-secreting cell into a zymogen-secreting cell with a different function
have not previously been described.
In this report, we analyze ZC differentiation at the molecular level to
identify potential factors that might regulate the neck-to-ZC transition. We
developed an RNA-preserving approach for laser-capture microdissection
(LCM)-based purification of individual gastric epithelial cell lineages,
generated gene expression profiles from each lineage, and performed a
bioinformatic screen that targeted one high probability candidate regulator of
ZC differentiation. The candidate, Mist1 (Bhlhb8 - Mouse
Genome Informatics), encodes a class B basic helix-loop-helix transcription
factor previously shown to be expressed in only a handful of specialized
secretory cell lineages, such as acinar cells of the salivary gland and
pancreas (Pin et al., 2000;
Johnson et al., 2004).
Molecular and genetic studies have shown that formation of Mist1 homodimers is
crucial for Ca2+-regulated exocytosis and intercellular
communication in acinar tissues (Pin et
al., 2001; Rukstalis et al.,
2003; Zhu et al.,
2004; Luo et al.,
2005).
Therefore, we hypothesized a regulatory role for Mist1 in ZC differentiation and, thus, performed quantitative and qualitative molecular and ultrastructural analyses of gastric unit development in Mist1-/- mice. We find that normal, mature ZCs do not form in the bases of Mist1-/- gastric units. Rather, basal Mist1-/- ZCs have markedly underdeveloped apical cytoplasms with multiple ultrastructural defects. In the absence of normal ZCs, there is accumulation - at the transition between neck and base zones - of cells with features of both neck and zymogenic cells. Although increased in abundance, these transitional cells are otherwise normal. Thus, our results provide molecular and cellular evidence that ZCs do in fact arise from neck cells. Furthermore, we identify Mist1 as the first gene necessary for this maturation process and present evidence that Mist1 specifically regulates genes involved in extension of the apical cytoplasm, which we identify as a key feature of normal ZC differentiation from neck cells.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Tissue for the LCM/GeneChip studies was dissected from
stomachs of wild-type FVB/N mice (The Jackson Laboratory, Bar Harbor, ME,
USA).
RNA-preserving triple channel immunolabeling for LCM
Stomachs for LCM were excised immediately following sacrifice, quickly
flushed with RT PBS, inflated by duodenal injection of O.C.T. (Sakura Finetek,
Torrance, CA), and frozen in Cytocool II (Richard-Allen Scientific, Kalamazoo,
MI). Serial 7 µm-thick cryosections were cut onto Superfrost slides (Fisher
Scientific), fixed in 70% ETOH, rehydrated in nuclease-free water
(nuclease-free solutions from Ambion, Austin, TX) and then incubated in
immunolabeling buffer. Several buffers and incubation times for multichannel,
fluorescent immunolabeling cell lineages were tested. Although labeling was
optimal in nuclease-free PBS, these conditions led to activation of endogenous
gastric RNases and complete degradation of RNA within 5 minutes (RNA integrity
assessed by Agilent 2100 Bioanalyzer, Palo Alto, CA). RNA preservation was
enhanced in buffers with lower pH (e.g. Sodium Acetate, pH 4.3, prepared in
nuclease-free water) and/or lower ionic strength (e.g. 10 mmol/l nuclease-free
Tris-HCl, multiple pHs from 5-7). However, the optimal balance of preservation
and labeling occurred by diluting all immunolabeling reagents in unbuffered,
nuclease-free water with total incubation time no longer than 30 minutes. For
the current studies, reagents for triple-labeling were: (1) to identify
parietal cells, 20 µg/ml AlexaFluor532-labeled (Molecular Probes, Eugene,
OR) Dolichos biflorus agglutinin (DBA) lectin (E-Y Laboratories, San
Mateo, CA); (2) to label neck cells (Falk
et al., 1994), 20 µg/ml Alexafluor488-labeled Griffonia
simplicifolia II (Molecular Probes); (3) to identify pit cells and ZCs,
10 µg/ml mouse anti-E-cadherin (BD Biosciences), prelabeled using the
Alexafluor647 IgG2a mouse Zenon primary antibody labeling kit (Molecular
Probes). After labeling, slides were alcohol or xylene dehydrated and stored
in desiccation chambers (no more than 3 hours) until LCM was performed.
LCM and GeneChips
Individual parietal cells (visualized by DBA-positivity and
autofluorescence) from well-oriented gastric units were dissected (PixCell II
LCM apparatus, 7.5 µm spot diameter; CapSure HS LCM caps, Arcturus,
Mountain View, CA) one at a time from the pit zone and collected for
GeneChips. Pit cells (E-cadherin-positive, DBA-negative) were then collected
from the same gastric units. Only the pit cells in a two- to three-cell-thick
region at the apex of the gastric unit - but not yet upon the gastric surface
- were taken. ZCs (E-cadherin-positive, GSII/DBA-negative cells in the base
zones) were collected from corpus gastric units after potentially
contaminating basal parietal cells had first been dissected and discarded.
Between 3000 and 5000 individual cells from each cell lineage were isolated
from four to five individual mice. RNA was purified by PicoPure kit
(Arcturus). RNA integrity was verified, and RNAs for each lineage were pooled
from multiple mice, and 10 ng total RNA was then amplified, labeled and
fragmented (by the Arcturus RiboAmp HS kit followed by the RNA Amplification
and Labeling Kit from Enzo Life Sciences). The resulting biotinylated cRNA
probes were hybridized to Affymetrix (Santa Clara, CA) MOE430v2 GeneChips.
Bioinformatic analysis
Chip quality control and GeneChip to GeneChip comparisons to generate
expression profiles were performed using dChip
(Li and Wong, 2001;
Zhong et al., 2003). Cell
lineage-specific profiles were generated by extracting those genes whose
expression was increased in the given cell lineage relative to the other two
cell lineages (parameters: lower bound of 90% confidence of fold-change
1.3, expression intensity difference 50). The length (in number of
probesets) of each expression profile was as follows: LCM ZC (1066), LCM
parietal cell (665), LCM Pit (469), Previous ZC (1121), Previous parietal cell
(285), Previous Pit (701). To compare these expression profiles across
different versions of Affymetrix GeneChips and different microarray platforms,
each expression profile (i.e. the list of genes with expression that was
increased in each cell population) was re-expressed as a function of the Gene
Ontology (GO) terms represented in each list, and then compared to all others
on the basis of overall GO term distributions using the GOurmet software
(Doherty et al., 2006).
Multi-organ GeneChip comparisons
To identify which of the 41 candidate regulators of ZC differentiation were
preferentially expressed in salivary gland, we performed head-to-head
comparisons (parameters: fold-change lower bound 1.2, intensity difference
100) between the salivary gland and all other organs (excluding stomach)
from a panel of 15 mouse organs (RC Lindsley and KM Murphy, contributors; GEO
Series GSE1986;
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1986).
Only two of the 41 genes were expressed preferentially in salivary gland: one
probe set for Mist1 and three independent probe sets all representing
Xbp1.
Antibodies and immunostaining
Primary antibodies used for the non-LCM experiments in this study: rabbit
(1:10,000) and goat (1:2000) anti-human gastric intrinsic factor (gifts of Dr
David Alpers, Washington University), monoclonal
mouse-anti-ß-galactosidase (1:50), monoclonal mouse anti-Tff2 IgM (1:10,
gift of Sir Nicholas Wright), sheep anti-PepsinogenC (Pgc 1:10,000, from
Abcam), rabbit anti-Mist1 (1:200, described previously in Pin et al.
(Pin et al., 2001), goat
anti-Brdu (1:20,000, gift of Dr Jeff Gordon). Secondary antibodies for non-LCM
immunofluorescence were: AlexaFluor (488, 594 or 647) conjugated donkey
anti-goat, anti-rabbit, anti-sheep or anti-mouse (1:200-1:500, Molecular
Probes).
Stomachs for immunofluorescent analysis were excised immediately, flushed with RT PBS via the duodenal stub and then inflated with freshly prepared methacarn fixative. The stub was clamped by hemostat, and the stomach suspended in fixative in a 150 ml Erlenmeyer flask for 15-30 minutes at RT, followed by multiple rinses in 70% ETOH, arrangement in 2% agar in a tissue cassette, and routine paraffin processing. Sections (5 µm) were cut, deparaffinized and rehydrated with graded xylenes, alcohols and water, then antigen-retrieved by boiling in 50 mmol/l Tris-HCl, pH 8.0 (9.0 for Mist1). Slides were blocked in 1% BSA, 0.3% Triton-X100, in PBS, then incubated overnight at 4°C in primary antibodies, rinsed in PBS, incubated 1 hour at RT in secondary antibodies and/or 1 µg/ml fluorescently labeled GSII lectin (Alexafluor488, 594 from Molecular Probes, Alexafluor647 made by directly conjugating E-Y Labs lectin to Molecular Probes Alexauor647), rinsed in PBS, incubated 5 minutes in 1 µg/ml bisbenzimide (Molecular Probes), and mounted in glycerol:PBS.
Immunofluorescence quantification
Stomachs from seven Mist1-/- and seven
Mist1+/- littermates were fluorescently stained with
bisbenzimide, and the following combinations of neck and ZC markers:
GSII+anti-GIF, GSII+anti-Pgc, Tff2+anti-GIF. Photomicrographs (output as TIFF
files from a Zeiss Axiovert 200 microscope with Axiocam MRM camera and with
Apotome optical sectioning filter) from 10-13 well-oriented gastric units from
each genotype and staining combination were randomly selected. All cells
positive for neck and/or ZC markers were numbered as a function of increasing
distance from the stem cell zone. The mean fluorescent intensity (MFI) was
determined by manually outlining the cytoplasm of each cell and measuring mean
8 bit grayscale pixel intensity in the neck cell marker channel (e.g. GSII)
and then the ZC marker channel (e.g. GIF) channel across the lassoed area.
Values were blanked by subtracting MFIs in each channel for parietal cells
(which do not label with either marker). Distribution of Mist1
expression was quantified in wild-type mouse stomachs by staining with
anti-Mist1, GSII and either anti-GIF or anti-Pgc. Every well-oriented gastric
unit across the entire circumference of a random section through the
mid-section of the corpus of each mouse was scored for GSII, GIF or Pgc, and
Mist1. For the Brdu studies, four Mist1-/- and three
age-matched Mist1+/- mice were injected intraperitoneally
with a 15 µl/g live weight solution of 8 mg/ml bromodeoxyuridine and 0.8
mg/ml fluorodeoxyruidine (both from Sigma, St Louis, MO). Stains and counts
were performed as for determination of Mist1 distribution.
|
|
1 mm-thick, transverse stomach rings
were cut from the mid-corpus region, and then 1 x 1 mm-thick
sections perpendicular to the ring axis were taken from the mid-corpus region;
both greater and lesser curvature were sampled. Post-fixation was in 2%
OsO4 (in 0.1 mmol/l cacodylate buffer). Tissues were embedded in
PolyBed 812 (Polysciences Inc., Warrington, PA); 90-100 nm sections were cut
with a Diatome diamond knife and stained with uranyl acetate in 50% MeOH and
Venable's lead citrate. Transmission EM was performed on a JEOL JEM 1200-EX
microscope with AMT Advantage HR (Advanced Microscopy Techniques Corp.,
Danvers MA) high-speed, wide-angle 1.3 megapixel TEM digital camera. Granule
area was calculated by measuring two perpendicular diameters for every vesicle
from size-calibrated photomicrographs of well-oriented gastric units. At least
100 vesicles per cell differentiation state were scored.
|
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To increase purity of the ZC isolation, we first individually captured and
removed the potentially contaminating parietal cells that sporadically occur
in the bases of gastric units before retrieving ZCs. For a reference control,
we microdissected and pooled another population of parietal cells: those from
the region above the neck zone of the gastric unit, where neither neck cells
nor ZCs could contaminate the dissection. For a second reference population,
we microdissected pit cells in the most apical portion of the pit zone, above
the isthmus and neck of the gastric unit but below the gastric surface to
exclude all neck and parietal cells. RNA from each cell lineage (ZCs, pit
cells and parietal cells, 10 ng/cell lineage/mouse) was purified,
linearly amplified and hybridized to Affymetrix MOE430v2 GeneChips.
|
), to annotate the genes preferentially expressed by a
given cell type with the corresponding GO terms for each gene
(Harris et al., 2004) and then
compute the relative representation of each GO term in the list of genes as a
whole. Because GO terms describe the functional properties of genes, this
translation of expression profiles from a list of genes into a distribution of
GO terms allowed us to quantitatively compare entire expression profiles at
the level of the functions and properties of their component genes. Hence,
comparisons were rendered essentially independent of methods used to generate
the expression profile. Using this approach, we determined that our
LCM-derived expression profiles paralleled the corresponding previously
generated profiles (Fig. 2A,C),
and the types of genes that were enriched in each cell lineage profile were
consistent with the function of that cell
(Fig. 2B).
Prior gene expression profiles of ZCs
(Mills et al., 2003;
Mueller et al., 2004),
although informative about key functional aspects of these long-lived highly
secretory cells (Fig. 2B), did
not yield much insight about factors regulating ZC differentiation. By
contrast, GOurmet analysis of the LCM-based expression profile of ZCs
identified 41 genes that could be classified as having a known or putative
role in transcription regulation (Fig.
2D). To narrow this list of possible ZC differentiation mediators
for subsequent functional studies, we performed a bioinformatic metanalysis.
We reasoned that, because ZCs are acinar secretory cells, transcription
factors regulating their differentiation should also be enriched in serous
salivary glands, in which the tissue parenchyma is almost exclusively composed
of similar acinar cells. Thus, we screened a publicly available panel of adult
mouse organ expression profiles (GEO Series GSE1986;
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1986)
for salivary gland-enriched mRNAs. Of our 41 ZC-enriched transcripts, only
Xbp1 and Mist1 showed this pattern. As Xbp1 is
involved in the protein unfolding response and general ER maintenance in
diverse tissues (Brewer and Hendershot,
2005), we excluded it from further analysis. Thus, we focused on
Mist1 as a potential key regulator of ZC differentiation and
undertook a detailed analysis of its possible functional role in ZC
development and maturation.
|
), and the effects of loss of Mist1 function are confined to
the cells in which Mist1 is normally expressed, making the
Mist1 mice an excellent tool for analysis of differentiation pathways
in the adult gastric epithelium. To determine the cellular origins and
development of ZCs in the presence or absence of Mist1, we first
established a system for quantitative analysis of neck cell and ZC
differentiation in the adult gastric unit. The lectin GSII (Griffonia
simplicifolia-II) labels the mucous granules of neck cells intensely, and
antibody against gastric intrinsic factor (GIF) labels the zymogenic granules
of murine ZCs with corresponding intensity
(Fig. 3A). In wild-type or
Mist1 heterozygous mice, the mucus-containing cells in the neck zone
labeled exclusively with GSII, and the mature ZCs in the base zone labeled
exclusively with GIF. We noted, however, a transition zone between the end of
the neck zone and the beginning of the base that harbored a small number of
cells with cytoplasm that contained both mucus and zymogens
(Fig. 3A,B), consistent with
the hypothesis that ZCs arise from neck cells that exchange mucous secretion
for digestive enzyme secretion as they enter the base of the gastric unit. As
ZCs migrated away from the neck, their GIF expression tended to increase,
indicating a developmental gradient from the progenitor neck zone to the base
of the gastric unit (Fig.
3A,B,C).
Loss of Mist1 leads to quantitative increase in neck-to-ZC transitional cells
Analysis of Mist1 null mice showed that the transition cells at
the end of the neck zone were markedly increased in abundance. Furthermore,
the highest levels of GIF, the ZC marker, were seen within cells in the early
base, immediately adjacent to the neck zone; GIF levels in cells that had
migrated farther into the base were considerably lower
(Fig. 3A,B,C). Thus, our data
indicated that, although neck cell differentiation appeared normal,
Mist1-/- mice had a defect in ZC maturation.
To determine the extent of the increased abundance of transitional cells in
Mist1-/- mice, we quantified ZC differentiation by
measuring mean cytoplasmic fluorescent intensity in every cell expressing
above background levels of neck cell markers and/or zymogenic cell markers in
33 gastric units in seven Mist1-/- and seven
Mist1+/- mice. The data summed across 1100 cells from
each genotype using three combinations of neck and ZC markers (GSII and
anti-GIF, GSII and anti-Pgc, anti-Tff2 and anti-GIF) quantified the
substantially increased abundance of transitional cells coexpressing neck cell
and ZC markers in Mist1-/- gastric units
(Fig. 3C shows scatter plot for
GSII-GIF; summed data for all markers in
Fig. 3D). These transitional
cells could be gated into two populations. The first was delineated by
coexpression of approximately equal levels of neck cell and ZC markers (i.e.
levels within 2 s.d. of mean ZC and mean neck cell marker levels). These cells
(the GSII+GIF+ pattern in
Fig. 3A) showed a twofold
increase in abundance (3.8 to 8.2% with GSII and GIF; 3.2 to 12.0% with Tff2
and GIF; 11.4 to 19.3% with GSII and Pgc; P-value by paired
two-tailed t-test <0.033) in Mist1-/- mice
(Fig. 3C, red dashed boxes).
The second subset of transitional cells was characterized by high ZC marker
expression (levels 1 s.d. above the mean intensity for the ZC marker) with
neck cell marker levels significantly above background
(GSII+GIFHi pattern in
Fig. 3A, blue dashed box in
Fig. 3C). These
GSII+GIFHi-type cells were nearly tenfold more abundant
in Mist1-/- mice versus wild-type mice, no matter the
combination of neck and ZC marker used
(Fig. 3D,
P<0.017).
|
If Mist1 is necessary for maturation of all ZCs from the neck cell zone,
then all mature ZCs should express Mist1 normally, and expression
should be restricted to ZCs and perhaps transitional cells. To verify this, we
quantified distribution of Mist1 in randomly selected gastric units from
multiple mice using a polyclonal antibody against Mist1 (see, for example,
Fig. 4A). Mist1 was seen in 97%
of ZCs (1168 cells counted) and 47% of the cells coexpressing GSII and a ZC
marker (123 cells counted). We have never observed Mist1 in neck cells;
accordingly, 0 of 450 neck cells were Mist1-positive in this experiment. We
also determined distribution of Mist1 promoter activity in
Mist1-/- mice, taking advantage of the nuclearlocalized
lacZ coding region that had been targeted to the Mist1 null
allele (Pin et al., 2001). In
Mist1 nulls, expression from the Mist1 locus began in a
subset of TZ2 cells (GSII+/GIFHi TZ2 cells;
Fig. 4B) abutting - but no
longer within - the neck zone and was uniformly present in all ZCs that had
migrated away from the neck and into the base. Mist1 expression was
rarely found in TZ1 cells (Fig.
4B).
|
There are alternative explanations of our results: (1) that the apparent
transitional cells with coexpression of neck and ZC markers may represent a
common progenitor cell from which both the neck and ZC arise; and/or (2) that
these cells may be increased in Mist1-/- mice because loss
of Mist1 leads to non-specifically increased proliferation rates that alter
the differentiation pattern of the neck and ZC lineages. To address these
possibilities, we performed pulse-chase labeling experiments with
bromodeoxyuridine (Brdu), a thymidine analog that is permanently incorporated
into replicating DNA strands of dividing cells. The results
(Fig. 4C,D) indicate no
difference in active sites of proliferation (as measured by distribution of
Brdu-labeled cells at 90 minutes after injection) or migration of progenitor
cells (i.e. distribution of Brdu label after chases of 2 and 5 days) between
Mist1-/- and Mist1+/- mice (Pearson's
correlation of the fractional distribution of Brdu label between the two
genotypes was 0.999, where perfect correlation=1.0). Furthermore, total
incorporation of label was almost identical at each time point between
genotypes, (mean for Mist1+/- mice of 1.6±0.4
Brdu-positive cells/gastric unit and 1.3±0.4 for
Mist1-/- mice), arguing strongly against a generalized
increase in proliferation in Mist1-/- mice and confirming
no experimental bias in Brdu levels injected or subsequent immunostaining. No
label was identified within the neck-to-ZC transitional zone at any time
during the experiment, and ZCs never constituted more than 1% of the labeled
cells in either genotype at the first three time points. However, in a single
Mist1-/- mouse followed for 9 days after Brdu injection, a
small fraction (4.5%) of the aberrant, basal ZCs (see below) that formed in
these mice began to show label incorporation, and, although they were not
identified in the specific sections and counting parameters established for
the multi-timepoint experiment in Fig.
4D, occasional labeled transitional cells could be found
incidentally at this timepoint. Thus, the pattern of Brdu labeling in both
genotypes of mice was consistent with published reports of normal gastric unit
differentiation (Karam and Leblond,
1993a; Li et al.,
1996), indicating that proliferative activity in the gastric unit
occurs in the isthmus and pit zones, with a somewhat smaller fraction of neck
cells also showing proliferative activity. Our results are also consistent
with the hypothesis that ZCs arise only after a differentiation timecourse
that takes many days following cessation of mitosis. Furthermore, the data
indicate that the population of apparent neck-to-ZC transitional cells are not
sites of active proliferation in Mist1-/- mice or controls
and so they are unlikely to be a common progenitor for both the neck cells,
which are themselves actively proliferating, and the ZCs, which do not appear
to proliferate.
TZ2 (immature zymogenic) cells in Mist1-/- stomachs have normal ultrastructure but do not become mature basal ZCs
Overall, the data argue that Mist1 does not have a role in initial
specification of the ZC fate, because the Mist1-/- TZ2
cells express abundant markers of ZC differentiation (GIF, Pgc). In addition,
cells of the ZC lineage can eventually migrate into the base and turn off
expression of neck cell markers even in the absence of Mist1 (e.g.
Fig. 3A,B; and note appearance
of Brdu in ZC-marker-positive, neck-cell marker negative cells at day 9 in
Fig. 4D). However, ZC marker
expression is clearly also lost as TZ2 cells migrate away from the neck
(Fig. 3A,D,E), indicating that
following the TZ2 stage, ZC differentiation is abnormal. Earlier published
experiments in other tissues had suggested that Mist1 was necessary
specifically for formation of the secretory apparatus in highly secretory
exocrine cells (Pin et al.,
2001; Johnson et al.,
2004); however, the TZ2 cell phenotype, characterized by abundant
expression of secretory zymogenic markers, also supports the argument against
a role in secretory apparatus elaboration in ZCs. To help address these
issues, we studied the pattern of ZC differentiation in
Mist1-/- mice at the ultrastructural level using
transmission electron microscopy (TEM). The development of zymogenic granules
in representative differentiating wild-type ZCs is shown in
Fig. 5C, and in Fig. S1 in the
supplementary material. Note that the most immature ZC exiting the neck zone
(Fig. 5C, the ZC adjacent to
the parietal cell in the neck zone) contains abundant, large zymogenic
granules, and subsequent more mature cells (to the right) maintain this level
of granularity. Whereas immature ZCs also contained abundant, large granules
in Mist1-/- mice, it is striking that, as ZCs migrated
from the neck zone, they contained fewer and smaller granules
(Fig. 5A,B; note that EM
results are representative of nine blocks examined from three different
Mist1-/- mice). The immature ZCs with abundant granules
corresponded in morphology and location (i.e. nearest the neck zone) to the
GSII+GIFHi TZ2 population detailed in
Fig. 3 (see left inset,
Fig. 5A, where the cell
contains abundant large secretory granules, some of which contain remnants of
electron-lucent material, consistent with neck cell mucins). In addition, the
TEM results showed that the pattern of decreased GIF staining as a function of
ZC distance from the neck zone (Fig.
3A,B,E) accurately reflected the differentiation-related loss of
secretory granules as a whole in Mist1-/- ZCs.
Thus, the results indicated uniform defects in Mist1-/- ZCs as they migrate away from the neck zone, and the ultrastructural defects correlate spatiotemporally with Mist1 gene expression in wild-type mice (and expression of lacZ from the Mist1 promoter in Mist1-/- ZCs; Fig. 4A,B). To assess in more detail how loss of Mist1 affected the final stage of ZC differentiation, we analyzed ZCs in the bases of Mist1-/- and wild-type mice in our TEM sections. We noted that normal ZCs elaborated their supranuclear cytoplasm in a coordinated extension of their apices with neighboring cells, such that ZCs at the base of a gastric unit were pyramidal or Erlenmeyer flask shaped with minimal luminal surface area (Fig. 1C,D).
However, examination of multiple Mist1-/- mice showed
that the bases of their gastric units were devoid of mature ZCs. Basal
Mist1-/- ZCs were cuboidal or cuboidocolumnar, with
stunted supranuclear cytoplasm and extensive apical luminal surfaces (see
cells at right in Fig. 5A or
cell in Fig. 6A), indicating
that neighboring ZCs had failed to coordinate extension of their apical
cytoplasm. Without coordinated apical expansion, the lumens in the bases of
gastric units as a whole were wider and resembled ducts more than the acinar,
pouch-like structures of normal mice. Mist1-/- ZCs had
significantly smaller (P<0.001,
Fig. 6D), less abundant
vesicles and their supranuclear rough endoplasmic reticulum (rER) network was
sparse. Furthermore, they exhibited a reproducible, 500 nm region of
amorphous, organelle-free cytoplasm immediately subjacent to the plasma
membrane (Fig. 6A,B), and their
apical plasma membrane generally had a tufted, dynamic appearance that was in
marked contrast with the smooth apical membranes of wild-type ZCs
(Fig. 1D,
Fig. 6C). Interestingly, the
subnuclear and basolateral cytoplasm of Mist1-/- ZCs was
indistinguishable from wild type, showing abundant lamellar rER and even
occasional zymogenic granules of normal size
(Fig. 6A). Junctional complexes
also appeared normal (Fig.
6A).
Thus, our TEM observations confirmed our immunofluorescent data, indicating that immature TZ2 ZCs in Mist1-/- mice contained abundant, zymogenic granules of normal size and shape, further arguing against a role, at least in the stomach, of Mist1 in generation of secretory granules or establishment of the core cellular secretion machinery (e.g. an elaborate rER network). Rather, the results support the argument that Mist1 regulates genes needed for immature ZCs to undergo the apical morphological changes required for terminal differentiation into the mature, acinar cells at the base of the gastric unit.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Karam and Leblond,
1993c; Ge et al.,
1998). However, before the current study, no gene required for any
step in the differentiation of neck cells into mature, basal ZCs had been
identified. Furthermore, it had been posited that the conversion of a mostly
postmitotic, long-lived, terminally differentiated mucus-secreting cell into a
serous secretory cell was highly unusual
(Hanby et al., 1999). Our
results show for the first time that deletion of a single gene,
Mist1, blocks normal terminal ZC differentiation with all basal
zymogen-secreting cells in Mist1-/- mice showing multiple
structural defects. The block in terminal differentiation is accompanied by
accumulation of cells at the interface between the neck and the base zones of
the gastric unit. Those interface cells show coexpression of neck cell and ZC
markers and clearly represent stages in the transition between the neck cell
and ZC, thereby providing the first molecular-genetic evidence that ZCs arise
from neck cells.
The mechanism of the Mist1-/- phenotype
Mist1-/- mice form cells with abundant zymogenic
granules (TZ2 cells). They also still form aberrant ZCs in bases of
Mist1-/- gastric units with basal cells expressing GIF and
Pgc in the absence of neck cell markers. Thus, Mist1 is not necessary for
initial conversion of mucus granules to zymogenic granules, and it is
necessary neither for migration of nascent ZCs into the base nor for
downregulation of neck cell markers. But in Mist1-/- mice
there are no mature ZCs with abundant, large secretory granules, elaborate
supranuclear cytoplasms and pyramidal, acinar cell shapes. Thus,
Mist1 is a developmentally regulated transcription factor that must
be necessary for the terminal functional and structural maturation of ZCs (our
interpretation of both normal ZC development and the aberrant development are
modeled in Fig. 7).
The molecular mechanism of the defective differentiation phenotype in Mist1-/- mice will undoubtedly be complex: Mist1 is a transcription factor and may concomitantly activate multiple cellular pathways. A starting point for molecular analysis of the role of Mist1 might be to examine differences in cytoskeletal organization between Mist1-/- and wild-type ZCs. The tufted apical plasma membrane and organelle-free region of sub-apical cytoplasm in the defective Mist1-/- ZCs suggest that their apical cytoskeleton is hyperdynamic or hypercontractile. Abnormalities in the normal cytoskeletal lattice that regulates secretory granule release could explain both the loss of granules as Mist1-/- ZCs leave the neck zone and the failure in coordinated extension of the apical cytoplasm that these cells normally undertake as they mature.
Gastric epithelial niche-related regulation of Mist1 expression
Our results illustrate some of the advantages of the stomach as a model
system for studying differentiation processes in a mammalian system with
relevance to human disease. The quantitative ZC differentiation analysis
system that we developed allowed us to detect a normally rare precursor
population (i.e. the immature TZ2 ZC) and to tease out differences between
those cells and the mature ZCs of the base. The analysis was greatly
facilitated by the spatiotemporal organization of development in the stomach,
where, unlike mouse pancreas (the other organ where Mist1 has
primarily been studied) or the Drosophila nervous system [in which
the fly homolog dimm has been studied
(Hewes et al., 2003)],
cell-type differentiation can be followed in a stage-by-stage fashion in
tissue sections even in adults.
Eventually, it will be important to establish whether Mist1 regulates
identical genes in different organ systems (e.g. pancreas versus stomach). For
example, it has been reported that Mist1-/- acinar cells
of the pancreas undergo an age-related switch in differentiation state, in
which serous, acinar cells begin to resemble pancreatic ductal cells in older
mice (Zhu et al., 2004).
Ductal cells have cuboidal morphology with scant supra-apical cytoplasm,
similar to the morphology of the basal ZCs we observe in
Mist1-/- mice, and because of the failure of ZCs to extend
apical cytoplasms, the base zones of Mist1-/- mice
resemble ducts more than acini. It will be interesting to determine whether
basal ZCs in Mist1-/- mice aberrantly express duct markers
and whether the abnormal duct-like cells in Mist1-/-
pancreases show defects in cytostructural organization like those of ZCs in
Mist1-/- stomachs.
In the stomach, the pattern of defective differentiation and the timing of
Mist1 expression both suggest that induction of Mist1 occurs
as transitional cells begin to exit from the neck. Migration from the neck
involves migration away from direct cell-cell contact with parietal cells and
from the factors they secrete, such as the morphogen Shh
(Lees et al., 2005;
Merchant, 2005). Parietal
cells occupy most of the basement membrane surface in the neck zone (see, for
example, Fig. 1C), so ZCs
entering the base must rapidly expand their basal contact with the basement
membrane. Either loss of parietal cell-mediated factors and/or increased
exposure to factors in extracellular matrix (or possibly subjacent
capillaries) could regulate induction of Mist1 expression.
Relevance for human disease
An exigency for studying neck-to-ZC differentiation is that this
developmental pathway is aberrant in the precancerous state of chronic gastric
atrophy (Houghton et al., 2004;
Katz et al., 2005;
Nomura et al., 2005;
Shiotani et al., 2005), and
mucous neck-like cells form the bulk of tumor cells in diffuse-type, signet
ring adenocarcinomas (Yamashina,
1986; Charlton et al.,
2004). Despite the abundant evidence for aberrant ZC
differentiation in carcinogenesis, little is known about the molecular and
cellular details of the defects. Our current results should allow us to
examine differentiation during carcinogenesis with greater molecular
resolution. For example, Mist1 expression could be used as a marker
of definitive ZC differentiation and also might be lost as an early event in
carcinogenesis.
Conclusion
Our data show how an approach that combines in situ cell purification with
bioinformatics and detailed morphological analysis can take advantage of the
spatiotemporal organization of the gastric unit to elucidate the
molecular-genetic underpinnings of the development of a long-lived, slowly
differentiating, secretory cell lineage. We argue that the organization of the
stomach makes it a useful model system for studies of fundamental
developmental processes in an adult mammal. Furthermore, because the stomach
is an all too frequent site for pathophysiological differentiation processes
(e.g. cancer), such studies may also often have the additional advantage of
directly furthering our understanding of human disease.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/02700/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Brewer, J. W. and Hendershot, L. M. (2005). Building an antibody factory: a job for the unfolded protein response. Nat. Immunol. 6,23 -29.[CrossRef][Medline]
Charlton, A., Blair, V., Shaw, D., Parry, S., Guilford, P. and
Martin, I. G. (2004). Hereditary diffuse gastric cancer:
predominance of multiple foci of signet ring cell carcinoma in distal stomach
and transitional zone. Gut
53,814
-820.
Doherty, J. M., Carmichael, L. K. and Mills, J. C. (2006). GOurmet: A tool for quantitative comparison and visualization of gene expression profiles based on gene ontology (GO) distributions. BMC Bioinformatics 7, 151.[CrossRef][Medline]
Falk, P., Roth, K. A. and Gordon, J. I. (1994). Lectins are sensitive tools for defining the differentiation programs of mouse gut epithelial cell lineages. Am. J. Physiol. 266,G987 -G1003.
Ge, Y. B., Ohmori, J., Tsuyama, S., Yang, D. H., Kato, K., Miyauchi, M. and Murata, F. (1998). Immunocytochemistry and in situ hybridization studies of pepsinogen C-producing cells in developing rat fundic glands. Cell Tissue Res. 293,121 -131.[CrossRef][Medline]
Hanby, A. M., Poulsom, R., Playford, R. J. and Wright, N. A. (1999). The mucous neck cell in the human gastric corpus: a distinctive, functional cell lineage. J. Pathol. 187,331 -337.[CrossRef][Medline]
Harris, M. A., Clark, J., Ireland, A., Lomax, J., Ashburner, M.,
Foulger, R., Eilbeck, K., Lewis, S., Marshall, B., Mungall, C. et al.
(2004). The Gene Ontology (GO) database and informatics resource.
Nucleic Acids Res. 32Database issue, D258-D261.
Hewes, R. S., Park, D., Gauthier, S. A., Schaefer, A. M. and
Taghert, P. H. (2003). The bHLH protein Dimmed controls
neuroendocrine cell differentiation in Drosophila.
Development 130,1771
-1781.
Houghton, J., Stoicov, C., Nomura, S., Rogers, A. B., Carlson,
J., Li, H., Cai, X., Fox, J. G., Goldenring, J. R. and Wang, T. C.
(2004). Gastric cancer originating from bone marrow-derived
cells. Science 306,1568
-1571.
Johnson, C. L., Kowalik, A. S., Rajakumar, N. and Pin, C. L. (2004). Mist1 is necessary for the establishment of granule organization in serous exocrine cells of the gastrointestinal tract. Mech. Dev. 121,261 -272.[CrossRef][Medline]
Karam, S. M. (1993). Dynamics of epithelial cells in the corpus of the mouse stomach. IV. Bidirectional migration of parietal cells ending in their gradual degeneration and loss. Anat. Rec. 236,314 -332.[CrossRef][Medline]
Karam, S. M. and Leblond, C. P. (1993a). Dynamics of epithelial cells in the corpus of the mouse stomach. I. Identification of proliferative cell types and pinpointing of the stem cell. Anat. Rec. 236,259 -279.[CrossRef][Medline]
Karam, S. M. and Leblond, C. P. (1993b). Dynamics of epithelial cells in the corpus of the mouse stomach. II. Outward migration of pit cells. Anat. Rec. 236,280 -296.[CrossRef][Medline]
Karam, S. M. and Leblond, C. P. (1993c). Dynamics of epithelial cells in the corpus of the mouse stomach. III. Inward migration of neck cells followed by progressive transformation into zymogenic cells. Anat. Rec. 236,297 -313.[CrossRef][Medline]
Karam, S. M. and Leblond, C. P. (1993d). Dynamics of epithelial cells in the corpus of the mouse stomach. V. Behavior of entero-endocrine and caveolated cells: general conclusions on cell kinetics in the oxyntic epithelium. Anat. Rec. 236,333 -340.[CrossRef][Medline]
Karam, S. M., Yao, X. and Forte, J. G. (1997). Functional heterogeneity of parietal cells along the pit-gland axis. Am. J. Physiol. 272,G161 -G171.
Karam, S. M., Straiton, T., Hassan, W. M. and Leblond, C. P.
(2003). Defining epithelial cell progenitors in the human oxyntic
mucosa. Stem Cells 21,322
-336.
Katz, J. P., Perreault, N., Goldstein, B. G., Actman, L., McNally, S. R., Silberg, D. G., Furth, E. E. and Kaestner, K. H. (2005). Loss of Klf4 in mice causes altered proliferation and differentiation and precancerous changes in the adult stomach. Gastroenterology 128,935 -945.[CrossRef][Medline]
Lees, C., Howie, S., Sartor, R. B. and Satsangi, J. (2005). The hedgehog signalling pathway in the gastrointestinal tract: implications for development, homeostasis, and disease. Gastroenterology 129,1696 -1710.[CrossRef][Medline]
Li, C. and Wong, W. H. (2001). Model-based
analysis of oligonucleotide arrays: expression index computation and outlier
detection. Proc. Natl. Acad. Sci. USA
98, 31-36.
Li, Q., Karam, S. M. and Gordon, J. I. (1996).
Diphtheria toxin-mediated ablation of parietal cells in the stomach of
transgenic mice. J. Biol. Chem.
271,3671
-3676.
Luo, X., Shin, D. M., Wang, X., Konieczny, S. F. and Muallem,
S. (2005). Aberrant localization of intracellular organelles,
Ca2+ signaling, and exocytosis in Mist1 null mice. J.
Biol. Chem. 280,12668
-12675.
Merchant, J. L. (2005). Inflammation, atrophy, gastric cancer: connecting the molecular dots. Gastroenterology 129,1079 -1082.[CrossRef][Medline]
Mills, J. C., Andersson, N., Stappenbeck, T. S., Chen, C. C. and
Gordon, J. I. (2003). Molecular characterization of mouse
gastric zymogenic cells. J. Biol. Chem.
278,46138
-46145.
Mueller, A., Merrell, D. S., Grimm, J. and Falkow, S. (2004). Profiling of microdissected gastric epithelial cells reveals a cell type-specific response to Helicobacter pylori infection. Gastroenterology 127,1446 -1462.[CrossRef][Medline]
Nomura, S., Yamaguchi, H., Ogawa, M., Wang, T. C., Lee, J. R.
and Goldenring, J. R. (2005). Alterations in gastric mucosal
lineages induced by acute oxyntic atrophy in wild-type and gastrin-deficient
mice. Am. J. Physiol. Gastrointest. Liver Physiol.
288,G362
-G375.
Pin, C. L., Bonvissuto, A. C. and Konieczny, S. F. (2000). Mist1 expression is a common link among serous exocrine cells exhibiting regulated exocytosis. Anat. Rec. 259,157 -167.[CrossRef][Medline]
Pin, C. L., Rukstalis, J. M., Johnson, C. and Konieczny, S.
F. (2001). The bHLH transcription factor Mist1 is required to
maintain exocrine pancreas cell organization and acinar cell identity.
J. Cell Biol. 155,519
-530.
Rukstalis, J. M., Kowalik, A., Zhu, L., Lidington, D., Pin, C.
L. and Konieczny, S. F. (2003). Exocrine specific expression
of Connexin32 is dependent on the basic helix-loop-helix transcription factor
Mist1. J. Cell Sci. 116,3315
-3325.
Shiotani, A., Iishi, H., Uedo, N., Ishiguro, S., Tatsuta, M., Nakae, Y., Kumamoto, M. and Merchant, J. L. (2005). Evidence that loss of sonic hedgehog is an indicator of Helicobater pylori-induced atrophic gastritis progressing to gastric cancer. Am. J. Gastroenterol. 100,581 -587.[CrossRef][Medline]
Suzuki, S., Tsuyama, S. and Murata, F. (1983). Cells intermediate between mucous neck cells and chief cells in rat stomach. Cell Tissue Res. 233,475 -484.[Medline]
Yamashina, M. (1986). A variant of early gastric carcinoma. Histologic and histochemical studies of early signet ring cell carcinomas discovered beneath preserved surface epithelium. Cancer 58,1333 -1339.[CrossRef][Medline]
Zhong, S., Li, C. and Wong, W. H. (2003).
ChipInfo: Software for extracting gene annotation and gene ontology
information for microarray analysis. Nucleic Acids
Res. 31,3483
-3486.
Zhu, L., Tran, T., Rukstalis, J. M., Sun, P., Damsz, B. and
Konieczny, S. F. (2004). Inhibition of Mist1 homodimer
formation induces pancreatic acinar-to-ductal metaplasia. Mol.
Cell. Biol. 24,2673
-2681.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  K. Nozaki, V. Weis, T. C. Wang, A. Falus, and J. R. Goldenring Altered gastric chief cell lineage differentiation in histamine-deficient mice Am J Physiol Gastrointest Liver Physiol, June 1, 2009; 296(6): G1211 - G1220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Capoccia, W. J. Huh, and J. C. Mills How form follows functional genomics: gene expression profiling gastric epithelial cells with a particular discourse on the parietal cell Physiol Genomics, April 10, 2009; 37(2): 67 - 78. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Y. Liu, H. Seno, A. V. Miletic, J. C. Mills, W. Swat, and T. S. Stappenbeck Vav proteins are necessary for correct differentiation of mouse cecal and colonic enterocytes J. Cell Sci., February 1, 2009; 122(3): 324 - 334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tashima, S. Zhang, R. Ragasa, E. Nakamura, J. H. Seo, A. Muvaffak, and S. J. Hagen Hepatocyte growth factor regulates the development of highly pure cultured chief cells from rat stomach by stimulating chief cell proliferation in vitro Am J Physiol Gastrointest Liver Physiol, February 1, 2009; 296(2): G319 - G329. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhu, J. Hatakeyama, B. Zhang, J. Makdisi, C. Ender, and J. G. Forte Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin Am J Physiol Gastrointest Liver Physiol, February 1, 2009; 296(2): G185 - G195. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Doherty, M. J. Geske, T. S. Stappenbeck, and J. C. Mills Diverse Adult Stem Cells Share Specific Higher-Order Patterns of Gene Expression Stem Cells, August 1, 2008; 26(8): 2124 - 2130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Y. Choi, A. I. Romer, Y. Wang, M. P. Wu, S. Ito, A. B. Leiter, and R. A. Shivdasani Requirement of the Tissue-Restricted Homeodomain Transcription Factor Nkx6.3 in Differentiation of Gastrin-Producing G Cells in the Stomach Antrum Mol. Cell. Biol., May 15, 2008; 28(10): 3208 - 3218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Park, O. T. Shafer, S. P. Shepherd, H. Suh, J. S. Trigg, and P. H. Taghert The Drosophila Basic Helix-Loop-Helix Protein DIMMED Directly Activates PHM, a Gene Encoding a Neuropeptide-Amidating Enzyme Mol. Cell. Biol., January 1, 2008; 28(1): 410 - 421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L Pin, C. L Johnson, B. Rade, A. S Kowalik, V. C Garside, and M. E Everest Identification of a Transcription Factor, BHLHB8, Involved in Mouse Seminal Vesicle Epithelium Differentiation and Function Biol Reprod, January 1, 2008; 78(1): 91 - 100. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. F. Kim Paving the road for lung stem cell biology: bronchioalveolar stem cells and other putative distal lung stem cells Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1092 - L1098. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
