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J Biol Chem, Vol. 273, Issue 39, 25310-25319, September 25, 1998
From the Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, Missouri 63110
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Ids are dominant-negative helix-loop-helix (HLH)
proteins that play overlapping yet distinct roles in antagonizing basic
HLH transcription factors. Although Ids affect myogenesis,
neurogenesis, and B-cell development, little is known about their
in vivo functions in epithelia. We have examined the
effects of forced expression of Id-1 in the small intestinal epithelium
of adult chimeric mice. 129/Sv embryonic stem cells, transfected with
DNA containing Id-1 under the control of transcriptional regulatory
elements that function in all intestinal epithelial cell lineages, were
introduced into C57Bl/6 (B6) blastocysts heterozygous for the
ROSA26 marker. The B6 ROSA26/+ intestinal
epithelium of the resulting adult chimeras produces Escherichia
coli 
-galactosidase, allowing identification of this internal
control cell population. Chimeras produced from nontransfected
embryonic stem cells served as additional controls. Immunohistochemical
studies of the control chimeras indicated that the small intestinal
epithelium supports a complex pattern of endogenous Id expression. Id-1
is restricted to the cytoplasm; levels do not decrease as descendants
of multipotent intestinal stem cells differentiate. Id-2 and Id-3 are
only detectable in nuclei; levels increase markedly as epithelial cells
differentiate. Forced expression of Id-1 in the 129/Sv epithelium
results in a decline in Id-2 and Id-3 to below the limits of
immunodetection. A subset of chimeric-transgenic mice lacked growth
factor- and defensin-producing Paneth cells in their 129/Sv epithelium
and also developed intestinal adenomas. These changes were not present in normal control chimeras. Adenomas were composed of proliferating -Gal-positive and -negative epithelial cells, suggesting that they
arose through cooperative interactions between 129/Sv(Id-1) and B6
ROSA26/+ cells. These chimeras provide a model for studying how perturbations in Id expression affect tumorigenesis.
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INTRODUCTION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Dimeric basic helix-loop-helix (bHLH)1 transcription factors play important roles in cellular development. Their HLH domain promotes dimer formation, while their basic domain mediates binding to an E-box sequence (CANNTG) present in target genes. Homodimers of class B, tissue-specific bHLHs activate transcription of "master regulatory" bHLHs that allow cells to commit to a particular fate. Tissue-specific bHLHs can also form heterodimers with ubiquitously expressed class A bHLHs (also known as E-proteins). These heterodimers activate transcription of genes associated with differentiation (reviewed in Refs. 1-4).
The four known Ids (inhibitors of DNA binding; Ref. 5) are naturally occurring dominant-negative HLH proteins that lack the basic domain (1, 2, 4). Each Id forms high affinity heterodimers with E-proteins (6). These heterodimers cannot bind to DNA and therefore inhibit transcription of differentiation-associated genes by sequestering E-proteins (5-9). Forced expression of Ids has been used to show that tissue-specific bHLHs regulate differentiation in cultured cells and in specified cell lineages in vivo (10-19). Individual Ids have distinct affinities for tissue-specific bHLH proteins (6), suggesting that each Id could regulate expression of different subsets of bHLH target genes. Individual Ids also interact in distinct ways with non-bHLH proteins (20-23).
Ids and their affiliated proteins also affect proliferation. For
example, Ids appear to be required by human diploid fibroblasts and NIH
3T3 cells for progression through G1/S (24, 25).
Interactions between Id-2 (but not Id-1 or Id-3) and pRB are associated
with enhanced proliferation in cultured cells (21, 26). Yan et al. (27) recently reported that E2A
/
mice that escape neonatal lethality develop T-cell lymphomas. In
situ hybridization studies of developing normal mouse embryos revealed high levels of Id expression in proliferating undifferentiated cells and decreased expression in committed differentiating cells (28,
29).
Although considerable attention has been paid to the roles of Ids in myogenesis, neurogenesis, and B-cell development (1-4), there have been few in vivo studies of their functions in, and effects on, epithelia. The mouse intestinal epithelium has two features that make it attractive for examining the effects of Id expression. First, it renews itself rapidly and continuously throughout life. Proliferation, lineage allocation, differentiation, and death are confined to readily identifiable regions in each of the adult intestine's crypt-villus units: i.e. cells at all developmental stages are represented. Second, this epithelium is known to produce several (b)HLH proteins (28, 30-32).
The self-renewing adult mouse small intestinal epithelium contains ~1 million flask-shaped structures known as crypts of Lieberkühn (33). One or more active multipotent stem cells are functionally anchored near the base of each long lived crypt (34-39). Stem cells give rise to daughters that undergo 4-6 rounds of cell division in the mid-portion of the crypt (40). Epithelial cells are allocated to four principal lineages through mechanisms that are poorly understood. Committed cells belonging to each lineage complete their differentiation during a highly organized, rapid migration. Differentiating absorptive enterocytes, mucus-producing goblet cells, and enteroendocrine cells exit the crypt and move up an adjacent finger-like structure (the villus) in coherent columns (41, 42). Cells are removed at the villus tip by apoptosis and/or by extrusion into the lumen (43). The entire sequence is completed in 3-5d (44-47). Paneth cells differentiate as they move down to the crypt base, where they secrete antimicrobial peptides and growth factors (34, 48-52). Their life span at the crypt base is ~20 days (48). One of the intestine's known bHLH proteins, BETA2/NeuroD, regulates expression of secretin, a peptide produced during terminal differentiation of a subset of enteroendocrine cells (32). Another bHLH protein, MATH-1, is related to atonal, a master regulator of neurogenesis and is expressed almost exclusively in the intestinal epithelium of adult mice (31). Its function is unknown.
There are several reasons why we decided to assess the effects of forced expression of Id-1 on intestinal epithelial homeostasis. Id-1 is normally expressed at a critical point in gut development, embryonic day 16, when the endoderm begins to undergo cytodifferentiation to an epithelial monolayer. At this time, the intestinal epithelium is one of the few sites where Id-1 mRNA levels are high (28). Moreover, forced expression of Id-1 in many cultured cell lines has been shown to inhibit bHLH-regulated differentiation (10-15). Finally, Id-1 has been used in transgenic mice to disrupt bHLH functions in nonepithelial cell lineages: B-cells (16) and skeletal muscle (53). In this report, we describe the use of chimeric mice to assess effects of Id-1 on the accumulation of other Ids and on intestinal epithelial homeostasis. Surprisingly, a subset of these mice developed intestinal adenomas.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Generation of Chimeric Mice--
A DNA fragment derived from
nucleotides 4-558 of mouse Id-1 mRNA (5) was produced by reverse
transcriptase (RT)-PCR using small intestinal RNA from a 3-month-old
C57Bl/6 (B6) animal. Each PCR primer contained a BamHI
restriction site at its 5' end (5'-GAATTCGGATCCGTACAACCTTTCTCCAAC-3' and 5'-GAATTCGGATCCATCTGGTCCCTCAGTGC-3'). The PCR product was purified
and subcloned into the T/A vector (InVitrogen). Sequence analysis
confirmed that no errors had been introduced during the amplification.
A 576-base pair BamHI fragment, containing the mouse Id-1
open reading frame and stop codon, was excised from the T/A vector and
inserted into pL596hGHpNeo
B2 (42) at its unique
BamHI site to produce pLF/Id-1. This resulted in placement of the Id-1 open reading frame at nucleotide +3 of the human growth hormone (hGH) gene, and under the control of nucleotides 
596 to +21
of a rat fatty acid-binding protein gene (Fabpl; Refs. 54
and 55). Id-1 contains the first in-frame Met codon in this recombinant
DNA so that Id-1, but not hGH, is produced from the mRNA
transcript. A pgk-neomyocin resistance selection cassette is
located just downstream of hGH in pLF/Id-1.
129/Sv(Id-1) chimeric-transgenic mice. Control
"normal" B6 ROSA26/+ 129/Sv chimeras were generated
using nontransfected D3 ES cells.
All animals were maintained in microisolator cages in a barrier
facility and given Pico Lab Rodent Diet 20 (Purina Mills) ad
libitum. Routine screens of sentinel mice for hepatitis, minute, lymphocytic choriomeningitis, ectromelia, polyoma, Sendai, pneumonia, and adenoviruses plus enteric bacterial pathogens and parasites were
negative.
Preparation of Intestinal Whole Mounts-- Mice were given an intraperitoneal injection of an aqueous solution of 5'-bromo-2'-deoxyuridine (BrdUrd) and 5'-fluoro-2'-deoxyuridine (12 mg and 1.2 mg/g body weight, respectively) in order to label cells in S phase. Animals were sacrificed 90 min later. The entire small intestine was removed en bloc. The proximal third was snap frozen in liquid nitrogen and used as a source of total cellular RNA to verify transgene expression (see below).
The remaining distal two-thirds of the small intestine was flushed with ice-cold phosphate-buffered saline (PBS, pH 7.4), followed by periodate/lysine/paraformaldehyde (PLP; Ref. 59). The segments were then opened with a longitudinal incision along their cephalocaudal axis and pinned, serosal side down, on dissecting wax. Following fixation with PLP for 1 h at 4 °C, the whole mount preparation was washed with PBS, incubated with gentle shaking in 20 mM dithiothreitol, 20% ethanol, 150 mM Tris-HCl, pH 8, for 45 min at room temperature to remove mucus and then washed again in PBS. To stain-galactosidase (-Gal)-producing B6 ROSA26/+ cells, whole mounts were placed in a solution containing PBS, 2 mM 5-bromo-4-chloro-3-indolyl -D-galactoside
(X-gal), 4 mM potassium ferricyanide, 4 mM
potassium ferrocyanide, and 2 mM MgCl2 (final
pH=7.6) for 36 h at 40 °C. Some whole mounts were stained with
Bluo-Gal instead of X-Gal using the protocol described by its
manufacturer (Life Technologies, Inc.). Following staining, whole
mounts were postfixed in 10% phosphate-buffered formalin (Fisher) for
4-6 h at room temperature and then stored in 70% ethanol at
4 °C.
Histochemistry--
Segments of X-Gal- or Bluo-Gal-stained
intestinal whole mounts were embedded in paraffin, and 5-µm sections
were cut and collected on glass slides. The sections were
deparaffinized using xylene (one cycle of 10 min followed by two cycles
of 5 min each), incubated in isopropyl alcohol (three cycles, 5 min
each), and then rehydrated in distilled water (5 min). Typically, 300 serial sections were prepared from both the middle and distal thirds of
the small intestine of each animal. These regions are referred to as
jejunum and ileum, respectively. Sections were cut parallel to the
cephalocaudal axis of the intestine: every 30th section was stained
with nuclear fast red or hematoxylin and eosin (60) to assess cellular
morphology and crypt-villus architecture. Members of specific cell
lineages were visualized in adjacent serial sections using the
following histochemical stains (60): (i) Alcian Blue (detects acidic
mucins in goblet cells); (ii) phloxine/tartrazine (visualizes the
apical secretory granules of Paneth cells); and (iii) periodate acid Schiff (PAS; detects neutral mucins present in goblet cell mucus globules and Paneth cell granules, as well as glycoconjugates associated with the apical brush-border membrane of enterocytes). Intracellular X-Gal and Bluo-Gal precipitates were not removed during
these staining procedures, allowing us to distinguish -Gal-positive B6 ROSA26/+ epithelium from -Gal-negative 129/Sv
epithelium.
RT-PCR Assays--
Frozen portions of the proximal third of the
small intestine from a normal B6 ROSA26/+ 129/Sv chimera
or a B6 ROSA26/+ 129/Sv(Id-1) chimeric-transgenic mouse
were added to RNAzol (Tel-Test; 1 ml of RNAzol/30 mg of tissue) and
homogenized with a TissueMizer (Tekmar), and total cellular RNA was
isolated according to the manufacturer's protocol. cDNA was
synthesized using Avian Myeloblastosis Virus reverse transcriptase
(Promega) and oligo(dT)12-18 primers according to the
supplier's protocol. An aliquot of each reaction was then subjected to
PCR as follows. RNA transcripts from the Fabpl-Id-1-hGH
transgene were amplified using two different sets of primers. In one
set, the upstream and downstream primers (5'-TGAACTCGGAGTCTGAAGTC-3'
and 5'-TTGGCGAAGACACTCCTGAG-3', respectively) spanned the Id-1/hGH
boundary. In the other set, the upstream and downstream primers
(5'-CTGCTGCTCATCCAGTCGTG-3' and 5'-GCAGCTAGAAGCCACAGCTG-3', respectively) spanned intron 4 of hGH. PCR conditions were as follows:
annealing at 60 °C for 30 s; extension at 72 °C for 2 min;
denaturation at 95 °C for 30 s, for a total of 34 cycles. DNAs
representing mRNAs derived from endogenous mouse Id and -actin genes were also amplified, using primers and cycling conditions described by Riechmann et al. (9).
Immunohistochemistry-- A panel of previously characterized antibodies was used to define expression of various differentiation markers in each of the four principal small intestinal epithelial cell lineages. In addition, a set of anti-peptide antibodies specific for HLH proteins were used to characterize their cellular and intracellular patterns of accumulation. Staining was performed on paraffin-embedded sections prepared from X-Gal-stained whole mounts. Sections were deparaffinized, rehydrated, placed in blocking buffer (PBS containing 1% bovine serum albumin and 0.05% Tween 20) for at least 30 min at room temperature, and then incubated overnight at 4 °C with one of the following antibodies: (i) rabbit anti-mouse Id-1 (generated against residues 129-148 of the protein; final concentration of 0.7 µg/ml PBS-blocking buffer; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); (ii) rabbit anti-mouse Id-2 (generated against residues 167-186; 0.7 µg/ml; Santa Cruz Biotechnology); (iii) rabbit anti-human Id-3 (residues 98-117; 0.7 µg/ml; Santa Cruz Biotechnology); (iv) rabbit anti-human E12/E47 (residues 632-649; 2 µg/ml; Santa Cruz Biotechnology); (v) goat anti-rat apolipoprotein B (1:500; specific for enterocytes in the small intestine; kindly provided by Patrick Tso, University of Cincinnati School of Medicine, Cincinnati, OH); (vi) goat anti-rat apolipoprotein AIV (1:1000; enterocytes; P. Tso); (vii) rabbit anti-serotonin (1:1000, specific for subpopulation of enteroendocrine cells (61); Incstar); (viii) rabbit anti-cryptdin (1:500, reacts with Paneth cell cryptdins 1, 2, 3, and 6 (49, 52, 62); kindly provided by Michael Selsted, University of California, Irvine); (ix) rabbit antibodies to the secreted phospholipase A2 encoded by Pla2 g2a (1:40,000; Paneth cells (52, 63, 64); kindly provided by Rita Mulherkar, Cancer Research Institute, Bombay, India)); (x) rabbit anti-human lysozyme (1:1000; Paneth cells (49); Dako); and (xi) goat anti-BrdUrd (1:2000; cells in S-phase (54, 65); kindly provided by Steve Cohn, Washington University).
Antibody-antigen complexes were visualized with indocarbocyanine (Cy3)-conjugated donkey anti-goat IgG or anti-rabbit IgG (1:1000; Jackson ImmunoResearch). Nuclei were counterstained with bis-benzimide (100 ng/ml PBS; Sigma) for 10 min at room temperature. Blocking controls were performed for the Id-1, Id-2, Id-3, and E12/E47 antibodies by incubating them with the appropriate peptide antigen (10 µg of peptide/µg of antibody; Santa Cruz Biotechnology) for 4-6 h at room temperature before they were added to tissue sections.Lectin Staining--
Glycoconjugate production is a sensitive
marker of the differentiation programs of mouse intestinal epithelial
cell lineages (57, 66). Therefore, PLP-fixed and X-gal-stained sections of whole mounts were deparaffinized, rehydrated, and stained with horseradish peroxidase-conjugated lectins as described in an earlier report (66). Each lectin was used at a final concentration of 5 µg/ml
PBS blocking buffer. Members of the lectin panel included (i)
Ulex europaeus agglutinin 1 (Sigma; recognizes Fuc
1,2Gal containing glycoconjugates synthesized by Paneth, goblet, and enteroendocrine cells); (ii) Anguilla anguilla agglutinin
(EY Labs; reacts with -L-Fuc-containing glycoconjugates
produced by Paneth, goblet, and enteroendocrine cells); and (iii)
Maackia amurensis agglutinin (Boehringer Mannheim;
recognizes Neu5Ac2,3Gal4Glc/GlcNAc-containing glycoconjugates
present in Paneth cells, goblet cells, and enterocytes).
Light Microscopy-- Stained sections were viewed with a Zeiss Axiophot microscope and/or a Molecular Dynamics model 2001 confocal microscope. Scans in the confocal microscope were performed at 1-µm intervals.
Electron Microscopy-- Intestinal whole mounts were prepared exactly as described above except that PBS, 2% paraformaldehyde, 0.4% glutaraldehyde was used as the initial fixative. Following incubation with Bluo-Gal, intestines were washed with PBS, and regions containing adjacent 129/Sv and B6 ROSA26/+ patches were isolated by dissection. Pieces of tissue were then post-fixed overnight at 4 °C in 2% paraformaldehyde, 2% glutaraldehyde, washed in PBS, treated for 1 h in 2% osmium tetroxide at room temperature, dehydrated in graded alcohols, and embedded in Poly/Bed 812 (Electron Microscopy Sciences). Semithin (0.5-µm) sections were cut with a glass knife and examined using bright field microscopy until regions containing juxtaposed patches of B6 ROSA26/+ and 129/Sv crypt-villus units were located. Thin sections (0.1 µm) were then prepared and viewed with a JOEL model 100C electron microscope. The Bluo-Gal precipitate was retained in B6 ROSA26/+ epithelial cells after fixation and embedding, allowing the epithelium to be genotyped under the electron microscope.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Description of the Chimeric-Transgenic System Used to Assess the
Effects of Id-1 on Intestinal Epithelial Homeostasis--
129/Sv ES
cells were stably transfected with a recombinant DNA consisting of Id-1
under the control of nucleotides 596 to +21 of a fatty acid-binding
protein gene (Fabpl). These transcriptional regulatory
elements are well suited to assess the effects of Id-1 on
proliferation, commitment, and differentiation in the small intestinal
epithelium. They have been used to express a variety of proteins in the
region of crypt containing the multipotent stem cell. Expression is
maintained in members of all four of the stem cell's descendant
lineages as they proliferate, differentiate, and then complete their
life on the villus (e.g. see Refs. 42, 55, 58, 67-69).
Fabpl-reporter transgenes are expressed from embryonic day
15 through the period that crypt-villus units form (postnatal weeks
1-3) and during the subsequent 1.5-2 years of life. Highest levels of
reporter mRNA and protein occur in the distal half of the small
intestine (jejunum/proximal ileum).
-galactosidase. Therefore, the B6
component can be readily identified by staining whole mount preparations of the small intestine with X-Gal or Bluo-Gal. The 129/Sv
epithelium does not produce -Gal and is identifiable, even in
chimeras with a low 129/Sv contribution, by its white appearance (Fig.
1A).
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Id-1 Levels Are Elevated in Transgenic Epithelium--
Seventeen
3-12-month-old B6 ROSA26/+ 129/Sv(Id-1)
chimeric-transgenic mice were studied. They were produced from two
different clones of stably transfected ES cells. Twenty-seven
age-matched normal chimeras were generated using nontransfected 129/Sv
ES cells and were employed as external controls to identify
strain-specific differences between B6 ROSA26/+ and 129/Sv
gut epithelium.
129/Sv chimeras (Fig. 2A).
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Forced Expression of Id-1 Results in Marked Reductions in Cellular Id-2 and Id-3 Levels-- RT-PCR assays of RNA isolated from the proximal and distal halves of the small intestines of normal adult mice revealed that the endogenous Id-2, Id-3, and Id-4 genes are also expressed in this organ (data not shown). Moreover, immunohistochemical studies of normal control chimeras disclosed that Id-2 and Id-3 concentrations increase abruptly as B6 ROSA26/+ and 129/Sv epithelial cells exit the cell cycle and arrive at the crypt-villus junction. Id-2 and Id-3 levels do not change appreciably as cells complete their migration from the base to tips of villi (Fig. 3, A and B, and data not shown). Unlike Id-1, Ids 2 and 3 are located in the nucleus (Fig. 3B). Despite the positive RT-PCR result, we were unable to detect Id-4 in the 129/Sv or B6 intestinal epithelium of normal chimeras using commercially available antibodies and sensitive immunohistochemical detection techniques.
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A Subset of Chimeric-Transgenic Mice Develop Paneth Cell Depletion
and Adenomas--
Analysis of sections prepared from the distal
two-thirds of the small intestine from 14 of the 17 chimeric-transgenic
mice (age 3-10 months; 129/Sv contribution of <1 to 30%) failed to reveal any abnormalities in epithelial homeostasis (external controls were normal, age-matched chimeras). Transmission electron microscopic analysis of 129/Sv crypts indicated that forced expression of Id-1 did
not produce detectable morphologic changes in their epithelial cell
populations. This includes the region near the crypt base where stem
cells reside (Fig. 4, A and
B). Forced expression of Id-1 in these 14 mice did not
result in any significant quantitative changes in
proliferation within jejunal and ileal crypts, whether defined by
pulse-labeling mice with BrdUrd 90 min prior to sacrifice and noting
the number of S-phase cells/crypt section (Fig. 4C) or by
surveying the number of M-phase cells per crypt section after
hematoxylin and eosin staining (data not shown). In addition, there
were no qualitative changes in proliferation: epithelial cells did not re-enter the cell cycle as they migrated up the villus
(Fig. 4C). Paneth cell number and differentiation appeared normal, as judged by transmission electron microscopy (Fig. 4, A and B) and by their reaction with two
histochemical stains (phloxine/tartrazine and PAS), three lectins that
recognize fucosylated- or Neu5Ac2,3Gal4Glc/GlcNAc-containing glycoconjugates, and three antibodies that recognize gene products produced by this lineage (cryptdins, lysozyme, and a secreted phospholipase A2) (Fig. 4D). The number and
differentiation of goblet cells, as defined by their reaction with
three lectins and two histochemical stains, were unaffected (Fig.
4E). The crypt-villus distributions of terminal
differentiation markers in enterocytes and enteroendocrine cells were
also unperturbed when cellular pools of Id-1 were increased (Fig. 4,
F and G, and data not shown). Finally, forced
expression of Id-1 did not affect the orderliness of cell migration:
(i) polyclonal villi contained coherent vertical columns of wholly B6
ROSA26/+ or wholly 129/Sv cells; (ii) the borders between
the cellular columns were well demarcated; and (iii) there was no
"wandering" of cells of one genotype into columns of the opposite
genotype (Fig. 4H). Orderly migration was also observed in
the follicle-associated epithelium overlying Peyer's patches; the
submucosal lymphoid aggregates that can serve as inductive sites for
initiation of immune responses (Fig. 4H).
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129/Sv(Id-1) mice was
composed of -Gal-positive and -Gal-negative cells
(e.g. Fig. 5,
A-C). 5'-Bromo-2'-deoxyuridine labeling 90 min prior to sacrifice revealed numerous S-phase cells in both the -Gal-positive and -negative components of these neoplasms (Fig. 5, C and
D). -Gal-positive and -negative cells appeared to be
poorly differentiated based on their morphologic appearance after a
variety of histochemical stains. They also had similar patterns of Id
expression. For example, Id-3 levels in both -Gal-positive and
-negative cells were markedly diminished or undetectable (Fig.
5E), just as they were in the 129/Sv(Id-1) epithelium of
normal appearing crypt-villus units located elsewhere in these animals
or in the 14 chimeric-transgenic mice without neoplasms.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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We have found that the normal adult mouse small intestinal epithelium supports a complex pattern of expression of three Id genes. Cellular Id-1 levels do not change appreciably as the descendants of the multipotent intestinal stem cell proliferate and then undergo terminal differentiation. In contrast, Id-2 and Id-3 increase markedly as postmitotic cells exit the crypt and differentiate. Id-2 and Id-3 are targeted to the nucleus, while Id-1 appears to be restricted to the cytoplasm. The cytoplasmic location of immunoreactive Id-1 and E12/E47 indicates that nuclear import of these transcription factors is normally highly restricted in this epithelium. Transient transfection studies of COS cells indicate that E-proteins function as nuclear chaperons for Ids (75). This raises the possibility that Id-1 lacks a functional chaperon within the cytoplasm of small intestinal epithelial cells and/or that Id-1 and E12/E47 are somehow prevented from interacting with one another within this cellular environment.
Forced expression of Id-1 produces a global change in Id accumulation: Id-1 levels increase, but the protein remains restricted to the cytoplasm while cellular Id-2 and Id-3 levels decline to below the limits of detection. These findings not only disclose the interrelated nature of Id accumulation within this epithelium but also indicate that forced expression of Id-1 in the mouse intestine should be viewed as producing both loss-of-function (abolition of Ids 2 and 3) as well as a "gain" of Id-1.
Modest changes in the activities of bHLH proteins can have a dramatic effect on cellular function. E2A regulation of B cell development provides one such example: a 2-fold reduction in bHLH activity is associated with an equivalent reduction in the likelihood of adopting this cellular fate (73). As noted in the Introduction, forced expression of Id-1 inhibits the differentiation of many cultured cell lines. In contrast, forced expression of Id-1 does not produce a detectable effect on intestinal epithelial cell differentiation in the majority of mice we examined. The cytoplasmic location of both Id-1 and E12/E47 could account for the general insensitivity of small intestinal epithelium to augmented Id-1 levels.
Since the loss of Id-2 and Id-3 was generally not accompanied by detectable perturbations in proliferation, lineage allocation, or differentiation, it appears that neither Id is absolutely required to control these processes or that their loss can be functionally complemented. Moreover, the fact that Id-2 and Id-3 are confined to the villus epithelium further suggests that their role is not to inhibit terminal differentiation of villus-associated lineages.
A recent report mentioned that mice homozygous for a null allele of
Id-1 had no phenotype, based on a histologic survey that included the gastrointestinal tract (27). The same group noted that
Id-3/ mice were phenotypically normal, while
compound homozygotes were not viable (27). These results led them to
conclude that Id-1 and Id-3 have overlapping functions. Based on our
findings, it would be interesting to explore whether loss of the one Id
resulted in an increase in the other Id(s), thereby maintaining a net
level of Id function in these Id-1/ or
Id-3/ mice. The effects of knocking out
Id-2 have not been reported, and thus we do not know the
contribution of this Id to establishing and maintaining
intestinal epithelial homeostasis.
A subset of our chimeric-transgenic mice exhibited Paneth cell loss in their 129/Sv epithelium plus adenomas. It is not clear why only a subset of mice exhibited these abnormalities or why the long-lived Paneth cell lineage was more responsive than other lineages to the effects of forced expression of Id-1. The Paneth cell depletion/adenoma phenotype is unlikely to be due to insertion of the Fabpl-Id-1 transgene at a particular site within the mouse genome, since it was evident in chimeras generated from two independent, stably transfected ES cell clones. Our analysis of 50 normal control chimeras indicated that they do not spontaneously lose Paneth cells or develop adenomas. Thus, we believe that the presence of this phenotype in 3 of 17 chimeric-transgenic mice with relatively low 129/Sv contribution in their ileum/jejunum (5, 7, and 15%) is significant. If forced expression of Id-1 was sufficient in itself to produce tumorigenesis, we would have expected adenomas to be present in mice with the highest fractional representation of 129/Sv(Id-1) epithelium. Such a correlation was not apparent, although the number of animals with adenomas was small. Thus, our findings suggest that elevated levels of cytoplasmic Id-1 and/or reduced levels of nuclear Id-2 and Id-3 are not sufficient to directly initiate tumorigenesis and that other factors are required.
Transgenic mice with a genetically engineered diphtheria toxin A fragment-mediated ablation of the Paneth cell lineage do not have detectable proliferative abnormalities in their crypts and do not develop intestinal adenomas (52). Although this result indicates that loss of Paneth cells per se is not sufficient to initiate tumorigenesis, the loss of at least one Paneth cell product has been shown to help promote tumorigenesis in mice with a predilection to initiation. Animals heterozygous for a mutant allele of the Adenomatous polyposis coli gene, ApcMin, develop multiple intestinal adenomas (76). Mom1 is a semidominant modifier of tumor multiplicity in Min/+ animals. Genetic studies indicate that the secreted phospholipase A2 encoded by Pla2g2a is a very likely candidate for Mom1 (77-80). Two Pla2g2a alleles are known (79). One contains a frameshift mutation that prevents production of the intact protein and is associated with a significantly greater number of intestinal adenomas (Mom-1S). The other allele, Mom1R, does not contain the mutation. A transgene that permits forced expression of a functional Pla2 g2a product reduces tumor multiplicity and size in Min/+ mice to the level associated with a single copy of Mom-1R (80). The mechanism by which this secreted phospholipase A2 influences tumorigenesis is unclear at present. Studies in intestinal isografts and aggregation chimeras indicate that Apc and Mom1 act locally to affect the (crypt) cell lineage from which adenomas arise (81, 82). B6 ROSA26/+ epithelium contains Mom1S (79), and immunoreactive enzyme was not detectable in adenomas or in the 129/Sv(Id-1) crypts that lacked Paneth cells. Nonetheless, we have no direct evidence that loss of this phospholipase contributes to tumorigenesis in our chimeric-transgenic animals. However, the combination of (i) loss of this and other Paneth cell products, (ii) the effects of dysregulated Id expression on the crypt epithelium, and (iii) other as yet unspecified (environmental) factors may play a key role in determining whether tumorigenesis can proceed in some crypts.
Each of the solitary adenomas present in B6-ROSA26/+ 129/Sv(Id-1) chimeras was composed of proliferating populations of both
-Gal-positive and -Gal-negative epithelial cells. One possible explanation for this phenotype is that adenoma formation was initiated in the B6 ROSA26/+ (crypt) epithelium and that at some point
during progression there was silencing or deletion of the
ROSA26 locus. In other words, these tumors were not composed
of B6-ROSA26 and 129/Sv cells but rather were amplified
clones of B6-ROSA26+/ and B6-ROSA26/
cells. Studies by Dove and co-workers (82, 83) argue against this
scenario. They never found mixed populations of -Gal-positive and
-negative cells in adenomas arising in nonchimeric ROSA26/+, Min/+ mice or in aggregation chimeras that contain
ApcMin on ROSA26/+ lineages (Ref. 82;
cf. Ref. 83). A more likely and intriguing possibility is
that these adenomas arise through cooperative interactions between B6
ROSA26/+ and 129/Sv(Id-1) cells. Two recent
studies of humans suggest that during early stages of adenoma
formation, tumors are polyclonal. Patients with familial adenomatous
polyposis inherit a mutant APC allele, predisposing them to
develop hundreds to thousands of intestinal adenomas during their first
several decades of life (84). Analysis of a XYXO mosaic patient with
familial adenomatous polyposis revealed that this individual's small
intestine and colon crypts were monoclonal but that three quarters of
his microadenomas were polyclonal (85). Bjerknes and co-workers
(86) also studied individuals with familial adenomatous polyposis.
Immunohistochemical analyses of their crypts and early adenomas using
APC antibodies, coupled with microdissection and APC
genotyping also revealed polyclonality. Conversion to monoclonality at
later stages of tumor progression was attributed to a proliferative
advantage conferred on a subclone as a consequence of additional
mutations.
Our findings suggest that in the chimeric mouse system used for this study, perturbations in Id gene expression are associated with juxtacrine or paracrine signaling between initiated 129/Sv(Id-1) cells and normal B6 ROSA26/+ cells and that such signaling may facilitate recruitment and subsequent retention of the B6 ROSA26/+ population in an evolving tumor. If this hypothesis is correct, then this chimeric system provides a model for studying the recruitment process. At a minimum, the system provides an opportunity to examine how perturbations in Id expression might affect vulnerability to tumorigenesis.
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ACKNOWLEDGEMENTS |
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We thank David O'Donnell, Maria Karlsson, Melissa Wong, Lisa Roberts, Elvie Taylor, Bill Coleman, and Marlene Green for technical help and suggestions. B. M. W. thanks Dwight Towler and members of our laboratory for helpful discussions and encouragement.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK 37960.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.
To whom correspondence should be addressed: Dept. of Molecular
Biology and Pharmacology, Box 8103, Washington University School of
Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-7243; Fax: 314-362-7047; E-mail: jgordon{at}pharmdec.wustl.edu.
The abbreviations used are:
bHLH, basic
helix-loop-helix; HLH, helix-loop-helix; hGH, human growth hormone; ES
cell, embryonic stem cell; B6, C57Bl/6 strain of mice; BrdUrd, 5'-bromo-2'-deoxyuridine; PAS, Periodic Acid Schiff stain; PLP, periodate/lysine/paraformaldehyde; -Gal, -galactosidase; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactosideCy3, indocarbocyaninePCR, polymerase chain reactionRT, reverse
transcriptasePBS, phosphate-buffered saline.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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