* Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110;
and Onyx Pharmaceuticals, Richmond, California 94806
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Abstract |
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-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway and as an effector of cell-cell adhesion through its
association with cadherins. To explore the in vivo effects of
-catenin on proliferation, cell fate specification, adhesion, and migration in a mammalian epithelium, a human NH2-terminal truncation mutant
(
N89
-catenin) was expressed in the 129/Sv embryonic stem cell-derived component of the small intestine
of adult C57Bl/6-ROSA26
129/Sv chimeric mice.
N89
-Catenin was chosen because mutants of this
type are more stable than the wild-type protein, and
phenocopy activation of the Wnt/Wingless signaling pathway in Xenopus and Drosophila.
N89
-Catenin
had several effects. Cell division was stimulated fourfold in undifferentiated cells located in the proliferative
compartment of the intestine (crypts of Lieberkühn).
The proliferative response was not associated with any
discernible changes in cell fate specification but was accompanied by a three- to fourfold increase in crypt apoptosis. There was a marked augmentation of E-cadherin
at the adherens junctions and basolateral surfaces of
129/Sv (
N89
-catenin) intestinal epithelial cells and
an accompanying slowing of cellular migration along
crypt-villus units. 1-2% of 129/Sv (
N89
-catenin) villi exhibited an abnormal branched architecture. Forced
expression of
N89
-catenin expression did not perturb the level or intracellular distribution of the tumor
suppressor adenomatous polyposis coli (APC). The
ability of
N89
-catenin to interact with normal cellular pools of APC and/or augmented pools of E-cadherin may have helped prevent the 129/Sv gut epithelium from undergoing neoplastic transformation during
the 10-mo period that animals were studied. Together,
these in vivo studies emphasize the importance of
-catenin in regulating normal adhesive and signaling
functions within this epithelium.
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Introduction |
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-CATENIN plays important roles in cell adhesion and cell
signaling (for review see Miller and Moon, 1996
; Nusse,
1997
). The protein influences adhesion by providing a
functional bridge between cadherins and the actin cytoskeleton. Calcium-dependent homotypic interactions between the extracellular domains of cadherins result in the
formation of adhesion "zippers" between adjacent cells (Overduin et al., 1995
; Shapiro et al., 1995
). Although
these interactions help define the specificity of cellular interactions, they are not sufficient for productive adhesion.
Productive adhesion at the adherens junction is accomplished by the binding of
-catenin to the conserved cytoplasmic domains of cadherins and to the cytoplasmic protein
-catenin.
-Catenin, in turn, is linked to the cytoskeleton via its interactions with other proteins (e.g., actinin; Nagafuchi and Takeichi, 1988
; Ozawa et al., 1989
, 1990
; Aberle et al., 1994
; Hinck et al., 1994
; Jou et al., 1995
; Rimm
et al., 1995
).
-Catenin is also a critical downstream component of
the Wnt signal transduction pathway in vertebrates. In the
absence of a Wnt signal, serine/threonine phosphorylation
by glycogen synthase kinase-3 (GSK-3)1 leads to rapid
degradation of cytoplasmic pools of
-catenin through a
ubiquitin-proteosome pathway (Miller and Moon, 1996
; Munemitsu et al., 1996
; Yost et al., 1996
; Aberle et al.,
1997
; Cadigan and Nusse, 1997
). In contrast, stimulation of
the Wnt pathway leads to repression of GSK-3 (Noordermeer et al., 1994
; Cook et al., 1996
), decreased
-catenin
phosphorylation, and enhanced protein stability. The resulting augmentation of
-catenin pools facilitates formation of complexes between
-catenin and high mobility
group box transcription factors (T-cell factor [Tcf] and
lymphocyte enhancing factor-1 [LEF-1]); Behrens et al.,
1996
; Huber et al., 1996
). In the nucleus,
-catenin functions to coactivate transcription of largely unspecified
gene targets (Behrens et al., 1996
; Huber et al., 1996
; Molenaar et al., 1996
; Brunner et al., 1997
; Riese et al., 1997
;
van de Wetering et al., 1997
).
Studies in genetically manipulatable nonvertebrate species as well as nonmammalian vertebrate organisms have
shown that -catenin-mediated signaling affects axis formation and cell fate specification (McCrea et al., 1993
;
Heasman et al., 1994
; Funayama et al., 1995
; Cox et al.,
1996
; Molenaar et al., 1996
). However, attempts to test the
in vivo functions of
-catenin in mammals have been hampered by the fact that mice homozygous for a genetically
engineered null allele die during early embryogenesis
(Haegel et al., 1995
).
One function of -catenin in mammalian cell lineages
that has been recently explored is its role in oncogenic
transformation. Several reports have emphasized that the
tumor suppressor adenomatous polyposis coli (APC) functions to affect intestinal tumorigenesis through its regulation of
-catenin signaling. Mutations in APC lead to intestinal adenomas and adenocarcinoma in mice and humans (Su et al., 1992
; for review see Kinzler and Vogelstein,
1996
; Shibata et al., 1997
). The interaction between
-catenin and APC is enhanced when APC is phosphorylated by
GSK-3, thereby promoting
-catenin turnover (Su et al.,
1993
; Rubinfeld et al., 1993
, 1996
). When APC is absent,
or is mutated so that
-catenin binding is impaired, cytosolic pools of
-catenin are elevated, and
-catenin-Tcf signaling is induced (Munemitsu et al., 1995
; Korinek et al., 1997
; Morin et al., 1997
). Additional evidence for the importance of
-catenin signaling in tumorigenesis comes
from the observation that some human colorectal neoplasms
with wild-type APC genes have mutations in
-catenin (Ilyas
et al., 1997
; Morin et al., 1997
). Some of these mutations
affect potential sites for GSK-3 phosphorylation and
therefore are likely to lead to augmented pools of
-catenin.
In addition to its role in gut neoplasia, there are other
reasons why the self-renewing adult intestinal epithelium
represents an attractive mammalian model for studying
the in vivo functions of -catenin. Fundamental developmental processes such as cellular proliferation, lineage allocation, differentiation, migration/adhesion, and death
are continuously expressed in well-defined domains of the
small intestine's anatomically distinct crypt-villus units. Proliferation is confined to flask-shaped mucosal invaginations known as crypts of Lieberkühn. In the adult mouse,
each of the small intestinal crypts contains one or more active multipotent stem cells functionally anchored near its
base (Loeffler et al., 1993
; Potten et al., 1997
). These stem
cells give rise to daughters that undergo four to six rounds
of cell division in the midportion of the crypt and are allocated to four principal epithelial cell lineages. Each lineage completes its differentiation during a highly organized, rapid migration. Absorptive enterocytes (accounting for >80% of the epithelial population), mucus-producing
goblet cells, and enteroendocrine cells exit the crypt and
move up an adjacent finger-like structure (the villus) in
vertical coherent columns (Schmidt et al., 1985
; Hermiston
et al., 1996
). Cells are removed from the villus tip by apoptosis and/or extrusion into the lumen (Hall et al., 1994
).
The entire sequence is completed in 3-5 d (Cheng and Leblond, 1974a-c; Cheng, 1974a
). Members of the Paneth
cell lineage differentiate as they move downward to the
base of the crypt where they are removed after an ~20-d
residence (Cheng, 1974b
). E-Cadherin, the principal intestinal epithelial cadherin,
-catenin,
-catenin, and APC
are expressed in cells distributed along the length of crypt-villus units (Hermiston et al., 1996
; Näthke et al., 1996
;
Wong et al., 1996
). The importance of cadherins and their
related proteins to normal intestinal epithelial homeostasis was established when a dominant-negative cadherin was expressed in the 129/Sv component of C57Bl/6
129/
Sv chimeric mouse gut. Cell-cell and cell-substratum interactions were disrupted leading to alterations in migration rates, diminished cell survival, breakdown of mucosal
barrier function, inflammatory bowel disease, and adenoma formation (Hermiston et al., 1995a,b).
In the present report, we have examined the effects of
forced expression of a human -catenin lacking its NH2-terminal 89 residues (
N89
-catenin) on the small intestinal
epithelium of C57Bl/6
129/Sv chimeric mice.
N89
-Catenin was chosen for several reasons. Studies in cultured cell lines indicated that this mutant, which lacks putative GSK-3 phosphorylation sites (Yost et al., 1996
), has
a longer half-life than the wild-type protein, leading to its
accumulation both as a stable monomer and as a complex with APC (Munemitsu et al., 1996
; Barth et al., 1997
; Rubinfeld et al., 1997
). The NH2-terminal truncation does not
appear to affect binding to E-cadherin, does not involve
the domains involved in binding
-catenin or Tcf (Munemitsu et al., 1996
; Orsulic and Peifer, 1996
), and preserves
all of the protein's structurally and functionally important
armadillo repeats (Huber et al., 1997
). Finally, studies in
Drosophila and Xenopus had shown that NH2-terminal truncation mutants of
-catenin promote signaling (Yost
et al., 1996
; Pai et al., 1997
), whereas expression in cultured mammalian epithelial cells affects cell migration
(Pollack et al., 1997
). Thus, we anticipated that
N89
-catenin would allow us to examine the effects of
-catenin
on both signaling and adhesion/migration in the mouse intestine.
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Materials and Methods |
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Generation of Chimeric-Transgenic Mice
pL596hGHpNeoB2 (Hermiston et al., 1996
) contains nucleotides -596 to
+21 of the rat fatty acid binding protein gene (Fabpl) upstream of nucleotides +3 to +2150 of the human growth hormone gene (hGH). A phosphoglycerate kinase (pgk) neomycin resistance selection cassette is located just downstream of Fabpl-hGH. pL596hGHpNeo
B2 was cleaved at
its unique BamHI site located at nucleotide +3 of hGH, and a double-stranded oligonucleotide containing flanking BamHI sites and internal
KpnI and XbaI sites (5'-GATCCGGTACCATCGATGGGTCTAGAG-3') was inserted. The resulting DNA, pLFhGHpNeoK contains a unique KpnI
site for subcloning cDNAs into exon 1 of hGH. pCAN
-catenin
N90c.62 (provided by P. Polakis, Onyx Pharmaceuticals, Richmond, CA) contains a DNA insert encoding myc-tagged human
N89
-catenin. A 2.2-kb KpnI/XbaI fragment from pCAN
-catenin
N90c.62 was subcloned into KpnI/XbaI-digested pLFhGHpNeoK DNA, creating pLF
N89
cat. The 6.9-kb Fabpl-
N89
cat-hGH-pgkneo insert in pLF
N89
cat was excised with SacII, purified by gel electrophoresis, and then electroporated into
D3 129/Sv ES cells (Hermiston et al., 1995a). Stably transfected embryonic stem (ES) cell clones were injected into C57Bl/6 ROSA26+/
blastocysts (Wong et al., 1996
) to produce B6-ROSA26
129/Sv(
N89
-catenin) chimeric-transgenic mice. Chimeras generated from three of the cell
lines were analyzed at 6 wk, 6 mo, and 10 mo of life. Their 129/Sv contribution ranged from 20 to 80% based on coat color. The presence of ROSA26
in chimeras was established using a PCR protocol (The Jackson Laboratory, Bar Harbor, ME) and tail genomic DNA. Independent confirmation
was obtained by staining segments of tail with 5-bromo-4-chloro-3-inodyl
-D-galactoside (X-Gal) (Wong et al., 1996
). Nontransfected D3 ES cells
were used to generate B6-ROSA26
129/Sv chimeras (referred to as "normal control" chimeras in this study).
All animals were maintained in microisolator cages under a strict 12-h light cycle and fed a standard chow diet ad libitum (Pico Lab Rodent Diet 20; Purina Mills, Inc., St. Louis, MO). Routine screening of sentinel animals indicated that all chimeric mice were free of pathogens.
Assays for Transgene Expression
Reverse Transcriptase-PCR.
Total cellular RNA was isolated from the
proximal, middle, and distal thirds of the small intestine and the skeletal
muscle of 6-wk-old B6-ROSA26 129/Sv(
N89
-catenin) mice using
the RNeasy kit (QIAGEN Inc., Santa Clarita, CA). Oligo dT-primed
cDNA was generated from DNA-free intestinal RNA. Four separate
PCR reactions contained one of four primers that recognize human
-catenin sequences (5'-TTGATGGGCTGCCAGTACTG-3', 5'-CTACCAGTTGTGGTTAAGC-3', 5'-TGCACATCAGGATACCCAGC-3', or 5'-TATTGAAGCTGAGGGAGCCACAGC-3'), and a primer that
recognizes sequences derived from exon 2 of the hGH gene (5'-GGCAGAGCAGGCCAAAAGCC-3'). The following conditions were used for
PCR: denaturation at 95°C for 1 min; annealing at 65°C for 1 min; and extension at 72°C for 2 min using standard buffer and salt conditions (total = 30 cycles). Control reactions contained intestinal RNA prepared from normal control B6-ROSA26
129/Sv chimeras.
Immunoblots.
A segment was taken from the middle of the small intestine of B6-ROSA26 129/Sv(
N89
-catenin) and B6-ROSA26
129/Sv
mice and frozen immediately. After lyophilization, total cellular proteins were extracted by homogenizing the sample at 60°C in 1 ml of denaturation buffer (Laemmli, 1970
) supplemented with protease inhibitors (1 µg/ml leupeptin, 1 µg/ml pepstatin, 4 mM 4-[2-aminoethyl]-benzensulfonyl fluoride hydrochloride, 1 µg/ml aprotinin, 1 mM EDTA). Insoluble debris was removed by centrifugation at 12,000 g for 10 min. The protein
concentration in the resulting supernatant was defined (Hill and Straka,
1988
). Samples of protein (100 µg) were fractionated by SDS-PAGE and
electrophoretically transferred to polyvinylidene difluoride membranes.
The membranes were stained with Ponceau S to verify successful transfer,
preincubated for 1 h at 24°C with blocking buffer (PBS supplemented with
1% wt/vol gelatin and 0.5% vol/vol Tween-20) (Sigma Chemical Co., St.
Louis, MO), and then probed for 1 h at 24°C with one of the following antibody preparations: (a) affinity-purified rabbit anti-c-myc epitope (MEQKLISEEDLN, 1:50 in PBS blocking buffer; Upstate Biotechnology Inc., Lake Placid, NY); (b) rabbit antisera raised against the COOH-terminal 14 residues of human/mouse
-catenin (PGDSNQLAWFDTDL, 1:200; provided by J. Nelson, Stanford University, Stanford, CA); and (c)
rabbit anti-actin (1:5,000; Sigma Chemical Co.). Antigen-antibody complexes were detected using the reagents and protocols included in the
Western Light kit® from Tropix, Inc. (Bedford, MA).
Preparation and Staining of Intestinal Wholemounts
The small intestine was removed en bloc from mice immediately after killing and divided in half. Each half was gently flushed with ice-cold PBS,
followed by periodate-lysine-paraformaldehyde (PLP; McLean and Nakane, 1974). Each half was opened with a single longitudinal incision
along its mesenteric side and then subdivided into three equal parts. Each
part was pinned onto a wax substrate and then fixed in PLP for 1 h at
24°C. The samples were washed with PBS (three cycles of 5 min each), incubated in 20 mM DTT/20% ethanol per 150 mM Tris-HCl, pH 8.0, for 45 min to remove mucus, and then washed again with PBS (three cycles).
The epithelium was then genotyped by incubating all parts of the small intestine for 12 h at 4°C in PBS containing 2 mM X-Gal, 4 mM potassium
ferricyanide, 4 mM potassium ferrocyanide, and 2 mM MgCl2, final pH
7.6. The six regions of the small intestine were analyzed and photographed using a stereoscope (model SHZ10; Olympus, Tokyo, Japan), and then
placed in 2% agar/PBS. Care was taken to preserve the orientation of
crypt-villus units in each region while it was embedded in paraffin. Serial
sections (5-8-µm-thick) were prepared along the cephalocaudal axis of
each region (n = 300-500/region per animal).
Quantitation of Apoptotic and M-Phase Cells
Sections, prepared from the two middle regions of X-Gal-stained small intestine (jejunum), were counterstained with hematoxylin and eosin.
M-phase and apoptotic cells were identified in each of these regions by
their characteristic morphology (Hall et al., 1994; Coopersmith et al.,
1997
). Two 6-mo-old animals from each of three B6-ROSA26
129/
Sv(
N89
-catenin) lines and three 6-mo-old B6-ROSA26
129/Sv mice
were studied. A minimum of 200 well-oriented B6-ROSA26 and 200 well-oriented 129/Sv crypts were counted per mouse (well-oriented = crypt-sectioned parallel to the crypt-villus axis with Paneth cells represented at
the crypt base and an unbroken epithelial column extending from the
crypt base to the villus tip). A crypt had to be located within juxtaposed
patches of B6-ROSA26 and 129/Sv crypt-villus units to be tallied. Each
animal served as its own control; data from individual mice were expressed as the average number of M-phase (or apoptotic) cells per 129/Sv crypt divided by the average number of M-phase or apoptotic cells per B6-ROSA26 crypt. All slides were analyzed in a single-blinded fashion. Values obtained from animals from each line were averaged. Statistical analyses were conducted using unpaired Student's t-test.
Quantitation of Branched Villi
Sections obtained from the middle portions of the small intestine (see
above), were stained with nuclear fast red and scored for the presence of
branched villi. A minimum of 500-1000 villi in juxtaposed patches of B6-ROSA26 and 129/Sv crypt-villus units were scored per animal (n = two
6-mo-old mice/line of B6-ROSA26 129/Sv[
N89
-catenin] chimeric-transgenic animals; n = three age-matched B6-ROSA26
129/Sv normal
control chimeras). Branched villi were identified using the following criteria: (a) a villus had to have a continuous layer of epithelium over all of its
branches; (b) immunostaining of an adjacent serial section with rabbit
anti-laminin sera had to reveal a continuous ribbon of basement membrane underlying the uninterrupted epithelial sheet; and (c) the branched
villus had to be present in adjacent serial sections.
Single and Multilabel Immunohistochemistry
Mice were injected with 5'-bromo-2'deoxyuridine (BrdU, 120 mg/kg body weight; Sigma Chemical Co.) and 5'-fluoro-2'-deoxyuridine (12 mg/kg; Sigma Chemical Co.) 60 h before killing. The entire small intestine was removed en bloc and wholemounts were prepared, fixed in PLP, stained with X-Gal, embedded in paraffin as described above, and then cut into 5-µm-thick sections. Alternatively, the middle third of the small intestine was flushed with PBS, fixed in PLP for 1 h at 24°C, and then flushed and embedded with O.C.T.(Miles Inc., Kankakee, IL). The embedded tissue was then frozen in Cytocool (VWR, St. Louis, MO) and 6-8-µm-thick sections were prepared.
Protocols used for single- and multilabel immunohistochemical studies
have been described in our earlier publications (Hermiston et al., 1995a;
1996). PLP-fixed frozen sections of jejunum were stained with a 19-member panel of antibodies: (a) affinity-purified rabbit anti-Escherichia coli
-galactosidase (
-gal) (1:500; 5'
3' Inc., Boulder, CO); (b) rabbit anti-
-catenin sera (see above, final dilution in PBS/blocking buffer = 1:500);
(c) affinity-purified rabbit anti-c-myc (see above, 1:100); (d) affinity-purified rabbit antibodies raised against amino acids 1034-2130 of human
APC (APC2, a gift of P. Polakis; Näthke et al., 1996
; Wong et al., 1996
); (e) affinity-purified rabbit anti-
-catenin (1:500; gift of J. Nelson); (f) a
monoclonal rat antibody to E-cadherin (1:1,000; Sigma Chemical Co.;
Hermiston et al., 1995a); (g) rat anti-ZO-1 (polyclonal antibodies, 1:50;
Chemicon International, Inc., Temecula, CA); (h) rabbit anti-laminin (1:
1,000; Chemicon International Inc.); (i) rabbit anti-mouse fibronectin (1:
1,000; Chemicon International Inc.); (j) rabbit anti-mouse collagen type
IV (1:1,000; Chemicon International Inc.); (k) rat anti-mouse
1 integrin
(1:500; PharMingen, San Diego, CA); (l) rat anti-mouse
7 integrin (1:500;
PharMingen); (m) rat anti-mouse
4 integrin (1:500; PharMingen); (n) rat
anti-mouse
6 integrin (1:500; PharMingen); (o) goat anti-BrdU (1:1,000; Cohn et al., 1992
); (p) rabbit anti-serotonin (1:1,000, a marker of the predominant enteroendocrine subpopulation in the adult mouse intestine;
Incstar, Stillwater, MN); (q) rabbit anti-chromagranin A (1:1,000, a general marker of enteroendocrine cells; Incstar); and (r) rabbit anti-liver
fatty acid binding protein (1:1,000, an enterocyte lineage marker; Sweetser
et al., 1988
).
Antigen-antibody complexes were detected with indocarbocyanine (Cy3)- or FITC-conjugated donkey anti-rabbit, anti-rat, or anti-goat Ig (1:500; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
Analysis of the components of the diffuse gut-associated lymphoid tissue was performed on PLP-fixed frozen sections of jejunum. Antigen-
antibody complexes were visualized using tyramide signal amplification as
described previously (Garabedian et al., 1997).
PLP-fixed and X-Gal-stained sections of paraffin-embedded wholemounts were also incubated with a series of lectins (all used at a final concentration of 5 µg/ml PBS blocking buffer and detected according to Falk
et al., 1994): (a) Ulex europaeus agglutinin 1 (carbohydrate specificity = Fuc
1, 2Gal epitopes; lineage specificity = Paneth, goblet, and enteroendocrine cells; Sigma Chemical Co.); (b) Peanut (Arachis hypogaea) agglutinin (Gal
3GalNAc epitopes; all four epithelial lineages; Sigma Chemical
Co.); (c) Dolichos biflorus agglutinin (GalNAc
3GalNAc and GalNAc
3Gal
epitopes; Paneth and goblet cells plus enterocytes; Sigma Chemical Co.); (d) Helix pomentia agglutinin (
-GalNAc; GalNAc
4Gal epitopes; Paneth and goblet cells; Sigma Chemical Co.); (e) Jacalin-1 (Artrocarpus integrifolia agglutinin; Gal
6Gal; Gal
3GalNAc epitopes; enterocytes and goblet cells; E.Y. Laboratories, Inc., San Mateo, CA).
Stained sections were viewed with an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY) and/or model 2001 confocal microscope (Molecular Dynamics, Inc., Sunnyvale, CA). Scans in the confocal microscope were performed at 1-µm intervals.
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Results |
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Generation of B6-ROSA26 129/Sv(
N89
-Catenin)
Chimeric Mice
129/Sv ES cells were stably transfected with a recombinant
DNA consisting of nucleotides -596 to +21 of Fabpl
(Sweetser et al., 1988) positioned upstream of an open
reading frame encoding
N89
-catenin with an NH2-terminal myc tag. The Fabpl transcriptional regulatory elements were selected because previous studies in neonatal
and adult transgenic mice had shown that they could be used to direct expression of a variety of proteins to the region of small intestinal crypt containing the multipotent
stem cell, and to all four of the stem cell's descendant cell
lineages throughout the course of their differentiation
(Trahair et al., 1989
; Kim et al., 1993
; Simon et al., 1993
;
Hermiston et al., 1996
). Moreover, Fabpl-reporter transgene expression commences coincidently with initial cytodifferentiation of the intestinal epithelium in late fetal life
and is sustained throughout adulthood, with highest levels
of expression occurring in the middle third of the small bowel (jejunum).
Individual clones of stably transfected, Fabpl-N89
-catenin ES cells, or control nontransfected ES cells, were
injected into C57Bl/6-ROSA26 (B6-ROSA26) blastocysts
(Friedrich and Soriano, 1991
; Wong et al., 1996
). There
are several reasons why the resulting chimeras are well-
suited for studying the effects of transgene expression on
the gut. First, intestinal epithelial cells of each genotype are
separated into anatomically distinct units. By the time gut
morphogenesis is completed, all crypts in B6-ROSA26
129/
Sv chimeras are monoclonal; they contain either B6-ROSA26 or 129/Sv epithelial cells, but not a mixture of both (Wong
et al., 1996
). Several crypts surround the villus base and
supply epithelial cells to each villus. Thus, the chimeric
small intestine will contain patches of transgenic 129/Sv
crypt-villus units and adjacent patches of normal (nontransgenic) B6-ROSA26 crypt-villus units. A villus located
at the border of a patch of B6-ROSA26 crypts and a patch
of 129/Sv crypts will be polyclonal, containing a vertical
coherent column of transgenic epithelial cells emanating from a monoclonal 129/Sv crypt and an adjacent column of
normal epithelial cells emanating from a monoclonal B6
crypt (Fig. 1, A and B). Second, genotyping is simple. All
undifferentiated and differentiated epithelial cells in B6-ROSA26 patches produce E. coli
-gal (Wong et al., 1996
).
Therefore, the B6-ROSA26 component can be identified
by staining the opened chimeric small intestine with X-Gal
(see blue-stained villi in Fig. 1 A). The 129/Sv epithelium does not produce
-gal and can be identified, even in chimeras with a low percentage 129/Sv contribution, by its
white appearance. Third, regions of nontransgenic B6 epithelium serve as a critical internal control for defining the
effects of the transgene's product. Since the small intestine
is characterized by complex cephalocaudal variations in
cell production and differentiation, it is critical that the
control epithelial population be represented in a similar location as the genetically manipulated population. In the
case of a polyclonal villus, the effect of the transgene can
be defined by comparing 129/Sv and B6 cells located at a
given cell stratum of a single villus positioned at a unique
location along duodenal-ileal axis of an individual animal
(Fig. 1 B).
|
Four independent Fabpl-N89
-catenin ES cell lines
were injected into B6-ROSA26 blastocysts to generate
B6-ROSA26
129/Sv(
N89
-catenin) chimeric-transgenic
mice. Total RNA was isolated from the jejunum of 6-wk-old
animals with 20-80% 129/Sv contribution (defined by coat
color). Reverse transcriptase (RT)-PCR analysis revealed the expected sized product from the Fabpl-
N89
-catenin
transgene in all four chimeric-transgenic lines (Fig. 2 A).
The RT-PCR product was not present in RNA prepared
from the jejunum of normal control B6ROSA26
129/Sv
chimeras produced from nontransfected ES cells, or in
RNA isolated from the skeletal muscle of chimeric-transgenic animals (Fig. 2 A).
|
Transgene expression was independently confirmed by
immunoblot analysis of total jejunal proteins isolated from
6-wk-old chimeric-transgenic and normal control chimeric
mice with equivalent 129/Sv contributions. When the immunoblots were probed with antibodies that recognize the
myc tag, an 80-kD protein corresponding to the predicted
mass of myc-N89
-catenin was detected in the jejunums
of chimeric-transgenic but not control chimeric animals
(Fig. 2 B). Duplicate blots were probed with polyclonal
antibodies raised against a peptide derived from an absolutely conserved region of human and mouse
-catenin. The steady-state level of immunoreactive
-catenin species was severalfold higher in the jejunums of chimeric-transgenic mice compared with control chimeras (Fig. 2 B),
consistent with successful forced expression of
N89
-catenin.
A final confirmation of transgene expression was provided by immunohistochemistry. Serial sections of chimeric-transgenic and normal chimeric jejunums were stained
with antibodies raised against the myc tag and -gal. In
chimeric-transgenic mice, immunoreactive myc was present in
-gal-negative 129/Sv epithelial cells, but not in
-gal-positive B6-ROSA26 epithelial cells (Fig. 2 C). In normal control
chimeras, the myc tag was not detectable in either 129/Sv
or B6-ROSA26 epithelium (Fig. 2 D).
Villus Branching in
129/Sv(N89
-Catenin) Epithelium
Chimeric-transgenic animals generated from three of the four ES cell lines were characterized further. All had similar phenotypes. There were no statistically significant differences between the growth rates or adult body weights of chimeric-transgenic and normal chimeric mice (n = 72).
Surveys of X-Gal-stained intestinal wholemounts from
1.5-, 6-, and 10-mo-old chimeric-transgenic mice (n = 1-8
animals/line per time point) revealed that the -gal-negative 129/Sv(
N89
-catenin) epithelium contained branched
villi. Typically, there were two branches per villus, each of
equivalent height. Branched villi were similar in height to
surrounding unbranched villi (Fig. 3, A and B). When
polyclonal villi were encountered with branching, there was
always a wholly 129/Sv(
N89
-catenin) branch (Fig. 3 C).
|
The frequency of villus branching was quantitated by
examining serial sections of X-Gal-stained jejunal wholemounts from 6-mo-old chimeric-transgenic and normal
chimeric mice. An average of 3,400 villi were scored per
animal using the criteria described in Materials and Methods (n = two or three animals/line). The percentage of branched villi detected in 129/Sv(N89
-catenin) jejunal
epithelium ranged from 0.5 to 2%, depending upon the
Fabpl-
N89
-catenin ES cell line used to produce the chimeras (Fig. 4). In contrast, the frequency of villus branching in their B6-ROSA26 jejunal epithelium was <0.01%.
This difference was statistically significant (P < 0.05).
Branched villi were extremely rare (<0.01%) in both the 129/Sv and B6-ROSA26 components of jejunum harvested from aged-matched normal control chimeras (Fig.
4). Villus branching remained confined to the 129/Sv epithelium of 10-mo-old chimeric-transgenic mice (n = 20).
The frequency of branching was similar to that observed in
6-mo-old mice.
|
N89
-Catenin Expression Is Associated with
Alterations in Proliferation, Apoptosis, and Migration
but Not in Cell Fate Specification or Differentiation
Histochemical stains, plus a panel of antibodies and lectins
(refer to Materials and Methods), were used to determine
whether the epithelium overlying 129/Sv(N89
-catenin)
crypt-villus units exhibited any changes in differentiation.
Epithelial cells were compared in adjacent 129/Sv and B6-ROSA26 jejunal crypt-villus units, or within a single polyclonal villus, in both chimeric-transgenic and normal control chimeric mice. Expression of
N89
-catenin was not
associated with any detectable perturbations in cell fate specification or in the terminal differentiation programs of
the enterocytic, enteroendocrine, goblet, or Paneth cell
lineages. Cell polarity appeared unaffected in the enterocytic lineage, as judged by the intracellular distribution of
actin or by the distribution of apical- and Golgi membrane-associated glycoconjugates (Fig. 5 A, for example).
The subcellular and crypt-villus distributions of the tight
junction protein ZO-1 were similar in the 129/Sv and B6
components of polyclonal villi (Fig. 5 C). In addition, there
were no discernible changes in the levels or crypt-villus distributions of
6,
1,
4, or
7 integrin subunits, laminin, fibronectin, or type IV collagen (Fig. 5, B and D; data not
shown).
|
To determine the effects of N89
-catenin on cellular
proliferation, M-phase cells were scored in jejunal crypt-villus units of 6-mo-old chimeric-transgenic or normal chimeric mice. The ratio of M-phase cells in adjacent patches
of 129/Sv and B6-ROSA26 crypts was calculated for each
mouse in each line. Values obtained from all animals in a
given line were then averaged (n = 400-600 crypts scored/
mouse per line; n = two or three mice/line). Fig. 6 A shows
there was a statistically significant two- to fourfold increase in the mitotic ratio (index) in chimeric-transgenic
mice compared with age-matched normal chimeras (P < 0.05). This was due to an increase in the number of M-phase
cells per 129/Sv(
89
-catenin) crypt section. There were
no statistically significant differences in the number of
M-phase cells in the B6-ROSA26 crypts of chimeric-transgenic and normal chimeric mice (data not shown). The
proliferative abnormality produced by
N89
-catenin was
confined to the crypt epithelium; no M-phase cells were
noted on villi with normal morphology or with a branched
phenotype.
|
A crypt apoptotic index was also defined in these animals. Like the mitotic index, this index was expressed as
the ratio of apoptotic cells in adjacent patches of 129/Sv
and B6-ROSA26 crypts. An increase in apoptosis, equivalent to the increase in proliferation, was observed in 129/
Sv(N89
-catenin) jejunal crypts (Fig. 6 B). There were
no statistically significant differences in the number of apoptotic cells between the jejunal B6-ROSA26 crypts of chimeric-transgenic and normal control chimeric animals
(data not shown).
The effect of N89
-catenin expression on cell migration was also examined. 6-mo-old chimeric-transgenic and
normal control chimeric mice were injected with BrdU to
label crypt epithelial cells in S phase. Animals were killed
60 h later (n = two mice/line). Serial sections were prepared from their jejunums and then the sections were
stained with antibodies to
-gal and BrdU. Analysis of
polyclonal villi present in chimeric-transgenic mice revealed a marked difference in migration between 129/
Sv(
N89
-catenin) and B6-ROSA26 epithelial cells. 60 h
after pulse labeling, the leading edge of BrdU-positive B6
cells had moved from the crypt to the upper quarter of the
villus, whereas the leading edge of BrdU-positive 129/
Sv(
N89
-catenin) cells had only reached the middle portion of the villus (Fig. 7). This effect was related to forced
expression of
N89
-catenin: control studies revealed that
there were no detectable differences in migration between 129/Sv and B6-ROSA26 epithelial cells located in the
polyclonal villi of normal chimeras (for illustration see
Hermiston et al., 1996
). Based on previous determinations
of the time it takes BrdU-tagged cells to move from the
crypt to the villus tip (Hermiston et al., 1996
), the observed difference represents a slowing of 129/Sv(
N89
-catenin) cell migration by ~12-24 h.
|
Comparisons of polyclonal jejunal villi present in the
wholemount preparations of chimeric-transgenic and normal control chimeric mice (refer to Fig. 1 A and Fig. 3, A
and C) established that forced expression of N89
-catenin did not affect the orderliness of migration. The borders
between adjacent columns of B6 and 129/Sv(
N89
-catenin) epithelium were sharp. There was no sign of infiltration of 129/Sv(
N89
-catenin) epithelial cells into adjacent B6-ROSA26 cellular columns, as was seen when the
same Fabpl transcriptional regulatory elements were used
to force expression of wild-type human APC (Wong et al.,
1996
). Hematoxylin- and eosin-stained sections failed to disclose any piling up of cells at the junctions of 129/
Sv(
N89
-catenin) crypts and their associated villi. Similarly, there was no aberrant accumulation of cells at the
villus tip where cell extrusion normally occurs.
The slowing of migration was not accompanied by any
detectable perturbations in contacts between epithelial
cells. There was no evidence of disrupted mucosal barrier
function: (a) surveys of hematoxylin- and eosin-stained jejunal sections from 6-mo-old chimeric-transgenic mice
failed to show any signs of inflammatory bowel disease;
and (b) immunohistochemical analyses indicated that the
number and crypt-villus distributions of several components of the diffuse gut-associated lymphoid tissue were
unperturbed (e.g., 7-positive intraepithelial lymphocytes,
CD4+ T cells, and CD8+ T cells; refer to Fig. 5 D plus data
not shown).
Because aberrant -catenin signaling has been implicated in the pathogenesis of intestinal neoplasia (refer to
Introduction), we carefully surveyed X-Gal-stained wholemounts and serial sections of the small intestine from chimeric-transgenic animals for evidence of dysplasia, adenoma formation, or adenocarcinoma. None of the mice
had any evidence of these pathologic changes (n = 72;
ages 1.5-10 mo).
Mechanistic Analyses: N89
-Catenin Expression
Is Associated with an Augmentation in Cellular Pools
of E-Cadherin
Conventional light microscopic and confocal microscopic
surveys of jejunal sections, prepared from chimeric-transgenic mice and stained with antibodies against the myc tag
or -catenin, failed to disclose
N89
-catenin and endogenous
-catenin in the nuclei of transgenic 129/Sv or normal
B6-ROSA26 epithelial cells (refer to Fig. 2 C and see Fig.
8 A).
-Catenin was present at the adherens junctions and
basolateral surfaces of epithelial cells (Fig. 8 A).
|
We examined whether forced expression of N89
-catenin affected the intracellular distribution or levels of
its known partners APC, E-cadherin, or
-catenin. To do
so, jejunal sections were incubated with antibodies to each
protein. Surveys of adjacent 129/Sv(
N89
-catenin) and
B6-ROSA26 crypt-villus units, as well as polyclonal villi,
failed to disclose any appreciable differences in APC or
-catenin localization or levels.
-Catenin remained associated with adherens junctions and the basolateral surfaces of 129/Sv(
N89
-catenin) epithelial cells (data not shown),
whereas APC was prominent at the periphery of villus epithelial cells where it appeared to form small granular aggregates (Fig. 8 B).
In contrast, the steady-state level of E-cadherin was
markedly increased in N89
-catenin-producing epithelial cells (Fig. 8, C and D). The augmented concentration
of E-cadherin was not accompanied by detectable changes
in its intracellular location (adherens junctions and basolateral surfaces). Analysis of polyclonal jejunal villi from
normal chimeras established that the increased level of
E-cadherin was not simply due to genotypic differences between 129/Sv and B6-ROSA26 epithelium (Fig. 8, E and F).
Previous studies, using the same Fabpl transcriptional
regulatory elements used in this report, had shown that
forced expression of wild-type E-cadherin in the jejunal
crypt-villus units of chimeric-transgenic mice produced a
slowing of epithelial migration equivalent to that observed
with N89
-catenin (Hermiston et al., 1996
). To determine whether augmented levels of E-cadherin were responsible for the other phenotypic changes observed B6-ROSA26
129/Sv(
N89
-catenin) chimeras, we defined
the jejunal crypt mitotic and apoptotic indices in 6-mo-old
chimeric-transgenic mice generated using ES cells stably
transfected with Fabpl mouse E-cadherin DNA (Hermiston et al., 1996
). The increase in total M-phase cells/
crypt section associated with
N89
-catenin expression
was not evident in E-cadherin overexpressing crypts (refer
to Fig. 6 A), indicating that this feature was directly related to
N89
-catenin production rather than to a secondary effect of elevated E-cadherin concentrations. We
could not make such a statement in the case of the apoptotic response, because similar elevations in crypt apoptosis were associated with forced expression of wild-type
E-cadherin and
N89
-catenin (Fig. 6 B). Forced expression of E-cadherin did not produce villus branching (data
not shown).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of N89
-catenin, which lacks sites for GSK-3
phosphorylation, can be viewed as simulating at least one
effect of active Wnt signaling: generation of augmented
cellular pools of stable, hypophosphorylated
-catenin.
Comparable NH2-terminal truncation mutants are known
to phenocopy activation of the Wnt signaling pathway in
Xenopus and Drosophila. In these nonmammalian systems, the consequences include axis duplication and
changes in cell fate specification (Yost et al., 1996
; Pai et al.,
1997
). Moreover, components in the Wnt signaling pathway play critical roles in specifying endoderm and gut differentiation during early Caenorhabditis elegans development (Han, 1997
; Rocheleau et al., 1997
; Thorpe et al.,
1997
).
N89
-Catenin production in the small intestine of
chimeric-transgenic mice was not only designed as an in
vivo test of the role of Wnt signaling in establishing and maintaining a self-renewing mammalian epithelium, but
also as a test of the effects of
-catenin on a system with
complex adhesive requirements (i.e., preservation of cell-
cell contacts in a mucosal barrier, orderly yet rapid cell migration, and exfoliation at a defined point in the cellular
life cycle).
N89
-Catenin Expression Results in a Proliferative
Response Restricted to Crypts
Forced expression of Wnt-1 causes mammary epithelial
hyperplasia and tumor formation (Brown et al., 1986;
Tsukamoto et al., 1988
). In addition, ectopic expression of
Wnt-1 enhances proliferation in the developing mammalian central nervous system (Dickinson et al., 1994
). The
mitogenic response of crypt epithelial cells to
N89
-catenin is consistent with the observed mitogenic effects of active Wnt signaling in these other lineages. Surprisingly, this proliferative response was observed in the absence of
detectable myc-tagged
N89
-catenin (or endogenous
-catenin) within the nucleus of crypt epithelial cells. Forced
expression of wild-type
-catenin or
N89
-catenin in
Drosophila, Xenopus, two-cell mouse embryos, and cultured mammalian cells has been reported to result in redistribution of
-catenin to the cytoplasm and nucleus
(Funayama et al., 1995
; Huber et al., 1996
; Munemitsu et al.,
1996
; Yost et al., 1996
; Pai et al., 1997
). We assume that the
amount of immunoreactive
-catenin in the nuclei of crypt
epithelial cells was below the limits of detection of our
staining techniques.
The proliferative response did not involve villus epithelial cells despite sustained expression of N89
-catenin
from the crypt to the villus tip. Studies of nontransgenic
mice have shown that these short-lived postmitotic cells
retain many critical regulators that allow entry into S
phase (e.g., cyclin E and cdk4; Chandrasekaran et al.,
1996
). Moreover, the villus microenvironment does not
impose an absolute prohibition on crossing the G1/S
boundary: e.g., villus enterocytes can be induced to undergo a pRB-dependent reentry into the cell cycle by
forced expression of SV40 large T antigen (TAg; Chandrasekaran et al., 1996
; Coopersmith et al., 1997
). The
reason why
N89
-catenin fails to evoke a proliferative response in villus cells remains unresolved. However, terminal differentiation of these cells may change the availability or function of downstream effectors of the Wnt
pathway, such as high motility group box transcription factors.
Primary Versus Secondary Responses: Stimulation
of Apoptosis in N89
-Catenin-Producing Crypts and
Villus Branching
The intestinal epithelium has a great capacity to initiate
compensatory responses that preserve the steady-state cellular census when that census is threatened by changes in
proliferative status or cell survival (Hermiston et al.,
1995a; Coopersmith et al., 1997). As a consequence, it is
often difficult to distinguish between primary responses to
an applied stimulus and responses that compensate for an
effect produced by that stimulus. At this point, we cannot
determine whether the apoptosis reflects a direct effect of
augmented cytosolic
-catenin pools (and signaling), or a
secondary compensatory response to augmented crypt
proliferation. Whatever the underlying mechanism, given
the lack of discernible effects on villus height, we conclude
that the observed changes in cell death are generally able
to compensate for the observed changes in cell production.
A quandary exists regarding the interpretation of villus
branching. There is little information available about the
determinants of villus geometry (Totafurno et al., 1990).
For example, are the determinants of morphology primarily epithelial-based or do they also involve mesenchymal
signals? The normal variation in villus height observed
along the duodenal-ileal axis of the adult mouse intestine
can be correlated with the number of crypts that surround
the base of each villus (Wright and Irwin, 1982
). Totafurno et al. (1987)
examined rare branched villi in normal mice
and found that the number of crypts that surrounded their
base was approximately twice the normal number, suggesting that villus branching may be a compensatory response to increased epithelial cell input. Increased cell
production may not have to come from crypts: SV40 TAg
expression in villus enterocytes leads to reentry into the
cell cycle and branching in a subset of villi (Coopersmith et al., 1997
).
We do not view branching as analogous to the axis duplication observed in Xenopus when the Wnt pathway is
stimulated. It is unclear how a branched shape can be
maintained given the perpetual epithelial cell renewal that
occurs in crypt-villus units. In fact, the absence of a notable increase in the frequency of villus branching as B6-ROSA26 129/Sv(
N89
-catenin) mice age suggests that
branched villi have a limited life span. This is consistent with the view that normal intestine continually produces
new crypts and villi as a byproduct of its dynamic cell renewal (Totafurno et al., 1987
). Branching in our chimeric-transgenic mice may be a default response, reflecting the inability of some of their villi to adequately
compensate for a threatened or real increase in their
steady-state epithelial cell census beyond some critical threshold value. Such a threat may arise from the increased cell production observed in their crypts, from the
inability of crypt apoptosis to compensate for this proliferative response, and/or from the effects of slowed epithelial
cell migration. Branching could also represent a direct response to aberrant signals, originating from a genetically
manipulated epithelium and operating through interactions with the underlying mesenchyme. The significance of
branching is that it provides a potential inroad to deciphering how villus structure is preserved in the face of perpetual, rapid renewal of its principal cellular component.
N89
-Catenin Produces an Increase in Steady-State
Levels of E-Cadherin
Forced expression of N89
-catenin results in a marked
increase in E-cadherin levels at the adherens junction and
basolateral surfaces of intestinal epithelial cells. Several
recent studies suggest that this is due to activation of
E-cadherin gene expression. The mouse E-cadherin gene
contains a 7-bp binding sequence for Lef-1/Tcf (Huber et al.,
1996
). Yanagawa and co-workers (1997) used a Drosophila wing imaginal disc cell line to show that stimulation of
the Wingless pathway induces Drosophila E-cadherin
gene transcription and accumulation of DE-cadherin at
cellular junctions. They also found that forced expression
of Dishevelled, an inhibitor of Zeste-white 3 (the Drosophila GSK-3 homologue), led to accumulation of Armadillo (the
-catenin homologue) in the cytosol of these
cells, a marked increase in Drosophila E-cadherin mRNA,
and an elevation in junctional DE-cadherin. In addition,
they noted that forced expression of an NH2-terminal
truncation mutant of Armadillo produces elevated DE-cadherin mRNA and protein levels.
The N89
-catenin-mediated increase in E-cadherin
levels is likely to counteract the signaling activity of
N89
-catenin within the intestinal epithelium. Studies in
Xenopus have shown that the binding of
-catenin to cadherins opposes signaling by sequestering
-catenin (Heasman et al., 1994
; Fagotto et al., 1996
). The location of myc-tagged
N89
-catenin within intestinal epithelial cells provides evidence that a similar process occurs in a mammalian system.
The augmentation in cellular E-cadherin pools is also
likely to contribute to the slow migratory phenotype of
129/Sv(N89
-catenin) epithelium. When forced expression of E-cadherin was limited to villus enterocytes in
transgenic mice, enterocytic migration from the villus base
to villus tip was slowed (Hermiston et al., 1996
). In this
previous study, there was no augmentation of E-cadherin in the crypt epithelium, no perturbations in crypt proliferation or death, and therefore little likelihood that the observed effect on migration was due to a diminution in net
cellular output from the crypt.
APC and the Effects of N89
-Catenin on Intestinal
Epithelial Biology
N89
-Catenin production was not associated with any
detectable abnormalities in the intracellular distribution
or levels of APC within crypt-villus units. This may have
contributed to two phenotypic features of our chimeric-transgenic mice: (a) slowed migration and (b) lack of neoplastic transformation.
Studies in MDCK cells have indicated that -catenin affects APC's ability to organize microtubules (Näthke et al.,
1996
; Pollack et al., 1997
).
N89
-catenin-APC complexes are more stable than wild-type
-catenin-APC
complexes, leading to accumulation of
N89
-catenin at
clusters of APC positioned at the tips of MDCK cell membrane extensions (Näthke et al., 1996
; Barth et al., 1997
;
Pollack et al., 1997
). Based on these findings, Pollack and
co-workers (1997) proposed that this stabilization of
N89
-catenin-APC complexes inhibits (MDCK) migration by somehow perturbing APC function.
Regulation of -catenin degradation appears to play an
important role in colorectal tumorigenesis and in the formation of melanomas (Korinek et al., 1997
; Morin et al.,
1997
; Rubinfeld et al., 1997
). Levels of soluble
-catenin
are elevated in colon tumor cells (Munemitsu et al., 1995
).
In addition, mutations in the NH2 terminus of
-catenin
are oncogenic in cultured cells and are associated with intestinal neoplasms (Morin et al., 1997
). The stabilized
N89
-catenin mutant was produced in intestinal epithelial cells that were able to maintain normal levels of APC. This may account for the failure of these cells to undergo
neoplastic transformation during the 10-mo period that
animals were studied. In addition, the
-catenin mutations
that have been associated with human colorectal cancer
involve alterations in NH2-terminal serine residues. Although it may seem, superficially, that NH2-terminal truncations would have similar biochemical effects as serine
substitution (i.e., protein stabilization), NH2-terminal phosphorylation may have other roles, including affects on protein-protein interactions that modulate signaling.
![]() |
Prospectus |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The self-renewing intestinal epithelium is able to invoke a
variety of compensatory responses when its census is
threatened. This capacity to compensate makes sense
given the enormous energetic investments required to sustain normal self-renewal. Compensation provides an experimental challenge when trying to decipher the function
of molecules such as -catenin, since dramatic or even noticeable phenotypes may be difficult to elicit. Further analysis of the effects of active
-catenin-mediated signaling
on epithelial homeostasis may require forced expression
of
-catenin-Lef-1 (or Tcf) fusion proteins or dominant-negative Tcf/Lef-1 mutants (Molenaar et al., 1996
). The
chimeric-transgenic system described above provides a
way of assaying the effects of these mutants in vivo under
well-controlled conditions.
![]() |
Footnotes |
---|
Received for publication 4 February 1998 and in revised form 11 March 1998.
Address all correspondence to Jeffrey I. Gordon, Department of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: (314) 362-7243. Fax: (314) 362-7047. E-mail: jgordon{at}pharmdec.wustl.eduWe thank D. O'Donnell (Washington University School of Medicine) for expert technical assistance. We are grateful to J. Nelson and P. Polakis for supplying reagents and for their many valuable suggestions during the course of this work.
These studies were supported in part by grants from the National Institutes of Health (DK37960, DK30292, and T32 HlO7275).
![]() |
Abbreviations used in this paper |
---|
-gal,
-galactosidase;
N89
-catenin, NH2-terminal truncation mutant of human
-catenin lacking amino acid
residues 1-89;
APC, adenomatous polyposis coli protein or gene;
BrdU, 5'-bromo-2'deoxyuridine;
Cy3, indocarbocyanine;
DBA, Dolichos biflorus
agglutinin;
ES cell, embryonic stem cell;
Fabpl, fatty acid binding protein
gene;
GSK-3, glycogen synthase kinase-3;
hGH, human growth hormone
gene;
LEF-1, lymphocyte enhancing factor-1;
PLP, periodate-lysine-paraformaldehyde;
RT, reverse transcriptase;
TAg, T antigen;
Tcf, T-cell
factor;
X-Gal, 5-bromo-4-chloro-3-indolyl
-D-galactoside.
![]() |
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