A Possible Role for the High Mobility Group Box Transcription
Factor Tcf-4 in Vertebrate Gut Epithelial Cell Differentiation*
YoungJoo
Lee
,
Bethany
Swencki
,
Sarah
Shoichet
, and
Ramesh
A.
Shivdasani
§¶
From the
Department of Adult Oncology, Dana-Farber
Cancer Institute and the § Department of Medicine, Harvard
Medical School, Boston, Massachusetts 02115
 |
ABSTRACT |
The Wingless (Wg)/Wnt signaling pathway activates
High Mobility Group (HMG)-box transcription factors of the T-cell
Factor (Tcf)/Lymphoid Enhancer Factor (LEF) subfamily and mediates
diverse functions in development, possibly including endoderm and gut differentiation. Determinants of tissue specificity in the response to
Wg/Wnt signaling remain unknown. We have identified Tcf-4 as the
predominant Tcf/LEF factor in the developing mouse gut. During fetal
development, Tcf-4 mRNA expression is restricted to gut epithelium
and specific regions of the brain, the thalamus and roof of the
midbrain. In adults, expression is widespread, with highest levels
observed in the liver, an endodermally derived organ, and persists in
the gastrointestinal tract. Murine Tcf-4 has multiple RNA splice
variants with consequently significant heterogeneity in sequences 3' to
the HMG box. Microinjection of mRNA or plasmid DNA encoding Tcf-4
into Xenopus embryos results in ectopic expression of
molecular markers of endoderm and differentiated gut epithelium in
isolated animal cap explants. Taken together, these findings point to a
potentially important function for Tcf-4 in development of the
vertebrate gastrointestinal tract.
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INTRODUCTION |
The Wingless (Wg)1 /Wnt
signaling pathway mediates essential aspects of early development and
has been elucidated through a combination of genetic and biochemical
studies in several species (1). One critical component of this
signaling pathway is the cytoplasmic protein Armadillo/
-catenin,
which is maintained at a low concentration in the free form in the
cytoplasm. Wg/Wnt signaling raises the free
-catenin concentration
to permit association with High Mobility Group (HMG)-box proteins of
the T-cell Factor (Tcf)/Lymphoid Enhancer Factor (LEF) sub-family and
translocation to the nucleus (2, 3), where the complex is presumed to effect a Wg/Wnt-responsive program of gene expression (4). Transcriptional targets of this signaling pathway include the developmentally regulated genes engrailed,
siamois, labial, and ultrabithorax
(5-7), although many other genes are undoubtedly controlled through
Wg/Wnt signaling in diverse cell types.
Besides the established role of Wg/Wnt signaling in vertebrate
mesodermal differentiation and axis formation and in development of the
larval cuticle in Drosophila, several lines of evidence point to a role for this pathway in the differentiation of endodermal derivatives. First, genetic and biochemical studies in
Drosophila suggest that larval midgut development depends on
the Wg signal (8, 9). Second, recent genetic evidence implicates
homologs of Wg/Wnt signaling pathway components in gut development in
Caenorhabditis elegans (10, 11). Finally, latency of
cytoplasmic
-catenin may be maintained in part through the function
of the product of the adenomatous polyposis coli
(APC) gene (12, 13), a frequent target of mutation in
human colorectal and other gastrointestinal epithelial malignancies
(14). This potential role of APC in the Wg/Wnt signaling cascade likely
reflects a critical function in maintaining gastrointestinal epithelial
cell homeostasis. Indeed, a fraction of colorectal tumors with intact
APC harbor activating mutations in the
-catenin gene (15), and at
least one Tcf/LEF protein, human (h) Tcf-4, is commonly expressed in
colon cancer cell lines and mediates transcriptional activation therein
(16). The sum of these observations strongly implicates
-catenin and Tcf/LEF family proteins in normal gut development and in the
pathogenesis of gastrointestinal tumors.
The important question of how Wg/Wnt signaling achieves
lineage-specific outcomes in diverse cell types remains unresolved and
relies in part on a better understanding of the transcriptional effectors of the signaling pathway. In Drosophila, mutations
in dTCF (also known as pangolin) result in
phenotypes that are identical to those seen in wg mutants
(9, 17), implying that Pangolin functions exclusively within this
pathway. The correspondence may, however, be more complicated in
vertebrates, which have multiple Tcf/LEF-related proteins with varying
patterns of expression in embryos and adults. Both Tcf-1 and LEF-1 were
originally identified through studies in lymphocytes, where their
expression is restricted in adult mice (18-20); during fetal
development, their expression is wide and largely overlapping (21, 22).
Mice lacking Tcf-1 develop normally (23), whereas
LEF-1
/
mice manifest developmental abnormalities
consistent with a role for LEF-1 in inductive interactions between
mesenchymal and epithelial cells (22). Development of the gut has long
been recognized to depend upon such inductive interactions but Tcf-1
and LEF-1 are not expressed in this organ and absence of either gene
does not lead to obvious gut anomalies. Characterization of other
members of this HMG-box protein subfamily has been less detailed, and the full extent of the subfamily is unknown.
We sought to identify Tcf/LEF proteins that are expressed in the
developing vertebrate gut and to examine their function in differentiation of the epithelium. Using degenerate polymerase chain
reaction (PCR) cloning, we isolated a single Tcf/LEF family member as
the dominant protein of this class in the developing mouse gut. The
mRNA and predicted amino acid sequence of this clone are most
closely related to those of hTcf-4, previously identified through near
uniform expression in colon cancer cell lines (16); during preparation
of this report, Korinek et al. also reported the cloning of
murine (m) Tcf-4 (24). In mouse embryos, expression of Tcf-4 mRNA
is restricted to the gut epithelium and specific regions of the
developing brain; in adults, expression is widespread, with highest
levels observed in the liver, an embryonic midgut derivative. mTcf-4
mRNA possesses multiple alternative splice forms, the significance
of which is presently unclear. Ectopic expression of one of these
mTcf-4 mRNA isoforms in Xenopus embryos induces
expression of gastrointestinal epithelial markers in isolated animal
cap explants. These observations point to a possibly important function
for Tcf-4 in differentiation of the gastrointestinal epithelium and
vertebrate gut development.
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EXPERIMENTAL PROCEDURES |
Construction of Mouse Embryonic Gut cDNA
Library--
Poly(A)+ RNA isolated from the fore- and
mid-guts of 250 ICR strain mouse fetuses at post-coital day 13.5 was
reverse transcribed, size selected over 1 kb by column chromatography,
and cloned directly into a modified pCS2 vector (pCS105; kindly
provided by Dr. R. Harland, Berkeley, CA) using the SuperScript Plasmid
System (Life Technologies, Inc., Bethesda, MD). The library contained
>5 × 105 transformants, of which 105
clones were arrayed robotically onto high density membrane filters (Genome Systems, St. Louis, MO).
PCR Cloning--
Total RNA from the fore- and midguts of
embryonic day (ED) 14.5 mouse fetuses was reverse transcribed and PCR
amplified using degenerate oligonucleotide primers spanning nucleotides
encoding the HMG-box domains of mTcf-1 and LEF-1
(5'-AAYGCNTTYATGCTNTAYATGAARGARATG-3' and
5'-CCANCCNGGMTANAGYTGCATRTGNAGYTG-3', where Y = C/T, N = A/C/G/T, R = A/G, and M = A/C). The resulting
180-base pair amplified fragments were cloned into pCR2.1 (Invitrogen,
Carlsbad, CA), sequenced, radiolabeled, and used to probe the above
high density membrane filters to obtain full-length cDNA clones.
For cloning splice variants, PCR primers were
5'-AAGCCCCACATAAAGAAGCCCCT-3' and 5'-GCGAACCAAGAACCAGAAGGAA-3'.
In Situ Hybridization--
C57BL/6 mouse embryos were harvested
at various stages of gestation, fixed in 4% paraformaldehyde overnight
at 4 °C, dehydrated in ethanol, and embedded in paraffin. Serial
5-µm sagittal and parasagittal sections were mounted on Superfrost
Plus glass slides (Fisher). mTcf-4B plasmid DNA was linearized, and
sense and antisense digoxigenin-UTP-labeled RNA probes were synthesized
using a commercial kit (Boehringer Mannheim). The slides were
deparaffinized, rehydrated, treated with proteinase K, refixed with 4%
paraformaldehyde, and treated for 5 min with 0.1 M
triethanolamine, 0.5% acetic anhydride before overnight hybridization
with the probes at 60 °C. After washing at final stringency of 0.2×
SSC, the slides were incubated with alkaline phosphatase-conjugated
anti-digoxigenin antibody (Boehringer Mannheim) and visualized with
nitro blue tetrazolium/BCIP chromogenic substrates. Sections were
post-fixed in 4% paraformaldehyde, counterstained with 1% nuclear
fast red solution (Rowley Biochemical Institute, Danvers, MA), and
mounted in Permount (Fisher).
Northern Hybridization--
1 µg of poly(A)+ RNA
was isolated from mouse embryonic brain and gut or adult brain and
stomach, resolved on a 1% formaldehyde-agarose gel, and transferred to
Hybond-N+ membranes (Amersham Pharmacia Biotech); for other adult
tissues, a multiple tissue Northern blot (CLONTECH,
Palo Alto, CA) was used. The full-length mTcf-4B probe was radiolabeled
by random hexamer priming, and hybridizations were conducted in
ExpressHyb Solution (CLONTECH). The blots were washed in a final solution of 0.1× SSC and 0.1% SDS and exposed to autoradiography.
Xenopus Embryo Microinjection and Animal Cap
Dissection--
Xenopus embryos were fertilized in vitro,
dejellied with 3% cysteine, and cultured in 0.1× modified Marc's
Ringer's solution prior to microinjection of 4.6 nl of DNA or
synthetic capped mRNA (Ambion, Austin, TX) in 3% Ficoll solution
into the animal pole. Animal caps were dissected between
Nieuwkoop-Faber stages 9 and 10.5 and cultured overnight at 23 °C
with or without 50 ng/ml recombinant activin A (provided by the
National Hormone and Pituitary Program). The medium was changed on the
following day, and the animal cap explants were cultured further and
harvested when sister tadpoles reached stages 38-40.
RT-PCR Analysis--
Total RNA from animal caps was isolated
using RNAzol B (Tel-Test, Friendswood, TX) and reverse-transcribed
using oligo(dT) primer. Aliquots of the reverse transcriptase reactions
were used for PCR in the presence of 0.2 µCi
[
-32P]dCTP as tracer. PCR reactions were run for
20-22 cycles for EF1
(primers 5'-cctgaatcacccaggccagattaa-3' and
5'-gagggtagtctgagaagctctccacg-3'), 24 cycles for endodermin
(5'-tattctgactcctgaaggtgttgga-3' and 5'-gagaactgcccatgtgcctcttg-3'),
and 30-32 cycles for Xlhbox8 (5'-gcagtcatgctgaacctgacagagag-3' and
5'-atagaaggaacttgattggactggga-3') and intestinal fatty acid binding
protein (IFABP) (5'-caagtttacccttgcacaaccc-3' and
5'-atggcccgtcaggtcaataatg-3') at an annealing temperature of 60 °C.
Reaction products were resolved on 4% polyacrylamide gels and exposed
for autoradiography.
 |
RESULTS |
Isolation of a Tcf/LEF Subfamily Member Expressed in the Developing
Mouse Gut--
The epithelial lining of the murine gastrointestinal
tract begins to differentiate in mid-gestation. To study the early
development of this epithelium, we prepared cDNA from foregut and
midgut tissues harvested from fetuses at ED13.5. Using degenerate
oligonucleotides complementary to the ends of the highly conserved HMG
box of Tcf/LEF proteins, we performed PCR with this cDNA as the
template and cloned the amplified products. DNA sequencing of ten PCR
clones revealed nine identical inserts with sequence most closely
related to hTcf-4 and a single clone encoding the HMG box of mTcf-1.
The latter transcript is expressed at varying levels in many parts of
the developing mouse embryo but has not been detected previously in the
gut, and its amplification in this experiment is of unclear significance. In contrast, hTcf-4 is known to be expressed in the
epithelial lining of the adult colon and in many colon cancer cell
lines (16).
We used the PCR-amplified HMG-box of mTcf-4 as a probe to screen
105 plasmid clones from an embryonic gut cDNA library
and recovered a single strongly hybridizing clone whose nucleotide
sequence is >80% homologous to that of hTcf-4 and whose predicted
amino acid sequence bears >95% identity with hTcf-4 up to and
including the HMG box. The sequence of this clone through amino acid
position 405 is identical to that reported recently (24), except that the space between amino acids designated 268 and 269 has the residues SFLSS; this difference increases the homology with Tcf-3 (3). Additional PCR on ~5 × 105 clones using
mTcf-4-specific primers corresponding to sequences in the 3' end of the
gene resulted in isolation of three additional clones encoding two
distinct C termini (Fig. 1); one of these (here designated mTcf-4(K)) is identical to the recently reported cDNA sequence of mTcf-4 (24). Thus, Tcf-4 appears to be the predominant Tcf/LEF-subfamily HMG-box protein expressed in the gut of
the mid-gestation mouse embryo.

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Fig. 1.
Amino acid sequence C-terminal to the HMG box
of mTcf-4, deduced from independent cDNA clones and compared with
corresponding sequences from the human gene. Multiple splice
variants and greater interspecies sequence divergence are seen in this
region, similar to the closely related gene Tcf-1; additional splice
variants may also exist in the gut and other sites. Nomenclature and
numbering are according to the scheme adopted by Clevers and colleagues
(16, 24), and the mTcf-4 cDNA isolated by these investigators is
designated mTcf-4(K). Dashes indicate amino acid identity;
parentheses mark regions not reported among hTcf-4, or
missing from known mTcf-4, splice variants; and the dots
designate sites of alternative RNA splicing based on cDNA
isolates.
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mTcf-4 is closely related to other members of the Tcf/LEF subfamily and
particularly to Tcf-1 in the extensive alternative mRNA splicing
that leads to a heterogeneous pool of mRNAs differing at their 3'
ends; presumptive intron-exon junctions toward the 3' terminus also
appear to be conserved between both mouse and man (Fig. 1). In
addition, there is a remarkable 79% identity of nucleotide sequences
within the 5'-untranslated regions (UTRs) of the human and mouse genes
(Fig. 2), raising the possibility that
important regulatory information is encoded by this sequence.

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Fig. 2.
Comparison of the 5'-untranslated regions of
mouse and human Tcf-4, indicating remarkable conservation of nucleotide
sequence (79% identity) over the entire region. Numbering is
relative to the initiation codon at +1, dashes represent
nucleotide identity, and spaces are positioned for the best
sequence fit.
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Domain of Tcf-4 mRNA Expression During Development--
The
expression profiles of the two best characterized Tcf/LEF proteins
follow a strikingly similar theme. Both Tcf-1 and Lef-1 are expressed
only in lymphocytes in adult mice but exhibit wide and partially
overlapping domains of expression in the fetus (21, 22). Whereas mice
lacking Tcf-1 have an isolated T-cell defect (23),
LEF-1
/
mice die as a result of multiple developmental
abnormalities that correlate with dominant sites of fetal expression
(22). To acquire insight into the potential functions of Tcf-4, we
examined its pattern of expression.
mTcf-4 mRNA is first detected between ED11.5 and 13.5, when it is
localized to the thalamus in the developing central nervous system
(Fig. 3). Extension of the staining into
the roof of the midbrain is detected as early as ED13.5; expression in
both the di- and mesencephalon becomes more prominent by ED15.5 (Fig.
3), when it assumes its characteristic pattern in the posterior portion of the mesencephalic roof, spatially separated from predominant expression within the thalamus. This pattern of central nervous system
expression persists at least until ED18 (Fig. 3) and probably beyond
(see below). Although brain expression dominates the in situ
hybridization studies, lower levels of Tcf-4 mRNA are clearly detected in the embryonic gut by Northern analysis (Fig.
4A); by in situ
hybridization, this expression is confined to the epithelial lining of
the developing gastrointestinal tract (Fig. 4B). This is
consistent both with our original isolation of mTcf-4 from ED13.5 gut
cDNA and with the apparently low (
1/105 clones)
representation of mTcf-4 mRNA within this source. Notably, mTcf-4
is expressed both in the developing stomach (Fig. 4C) and intestine (Fig. 4, B and D) and in many sections
displays a patchy distribution among epithelial cells, with some areas
staining more prominently than others (Fig. 4, C and
D). However, the stratified and incompletely differentiated
epithelium of the mouse fetal gut precludes better characterization of
the strongly positive cells. We do not detect mTcf-4 mRNA
expression outside of the central nervous system and gut during fetal
development, and particularly note its absence in other major
endodermal derivatives, the liver and bronchial epithelium.

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Fig. 3.
Expression of mTcf-4 during fetal
development. In situ hybridization of mouse fetuses
between ED11.5 and ED18 with full-length mTcf-4B antisense RNA probe,
showing the most prominent sites of expression to be the diencephalon
(thalamus, t) and roof of the mesencephalon (rm)
in the developing brain. The only other site of expression, the gut, is
not easily appreciated at this resolution. v = ventricle.
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Fig. 4.
Expression of mTcf-4 in the developing
gastrointestinal tract. A, Northern analysis of
mRNA isolated from the whole embryo (reflecting mostly brain
expression, as judged by in situ hybridization; see Fig. 3)
or from isolated fore- and midgut at ED15.5 and probed sequentially
with full-length mTcf-4B and Actin cDNA probes. B and
D, in situ hybridization of separate transverse
sections of ED15.5 fetal intestine (panels B and
D) and ED17.5 fetal stomach (C) probed with
full-length mTcf-4B antisense RNA probe, indicating patchy (panel
D and arrow in panel C) but definite
expression of mTcf-4 mRNA in epithelial cells. B and
C, no counterstain; D, counterstained with
nuclear fast red. In each case, hybridization with either sense or
irrelevant RNA probes yielded no signal (data not shown).
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mTcf-4 Expression in the Adult--
Unlike Tcf-1 and LEF-1, whose
expression becomes remarkably restricted after birth, mTcf-4 is
expressed widely in adult tissues. Although the brain remains a major
site of expression, high levels of Tcf-4 mRNA are also observed in
the liver; lower but detectable levels are present in the heart, lungs,
kidneys, and testis, with lowest levels in muscle and spleen (Fig.
5A). This expression pattern
is distinct from that reported by Korinek et al. (24) who
failed to detect appreciable mTcf-4 levels outside of the adult brain.
In our hands also, mTcf-4 is difficult to detect on Northern blots with
total RNA and requires a high specific activity probe against 1-2 µg
of poly(A)+ RNA to generate a signal. Furthermore, Northern
analysis consistently reveals two distinct RNA species in embryos as
well as adults, corresponding to transcript lengths of 4.7 and 4.0 kilobases; the smaller species is expressed principally in the fetal
and adult brain, whereas the larger species predominates in all other sites, including the liver. Both species react with full-length mTcf-4
as well as with a probe encompassing ~400 nucleotides in the 3'-UTR
at high stringency (Fig. 5A). This finding virtually excludes the possibility of a cross-reacting transcript and suggests that the distinct mRNA species reflect heterogeneity outside the 3'-UTR. As predicted, mTcf-4 is also expressed in the adult
gastrointestinal tract. Korinek et al. have reported a small
increase in mRNA levels along the rostro-caudal axis of the
intestine (24). Here we show that expression also extends rostrally to
the stomach (Fig. 5B), the most proximal portion of the gut
that is lined by a glandular epithelium, but at a much lower level than
in the brain.

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Fig. 5.
Expression of mTcf-4 in adult tissues.
Northern analysis of mRNA isolated from various tissues
(panel A, commercial blot) or brain and stomach (panel
B) from 8-12-week adult mice and probed sequentially with DNA
probes corresponding to full-length mTcf-4B, mouse -Actin, and a
400-base pair region in the 3'-UTR of mTcf-4. Autoradiographic exposure
times: mTcf-4, 2 days; Actin, 6 h; 3'-UTR, 12 h.
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Function of mTcf-4 in Endodermal Differentiation--
Protein
functions in cell differentiation are best assessed in experimental
systems that approximate normal tissue development. The animal cap of
Xenopus blastulas is normally fated to develop into ectoderm
but retains the capacity for both mesodermal and endodermal
differentiation in the presence of the TGF-
-related ligand activin
or selected ectopically expressed genes (25-28). To examine the
potential role of Tcf-4 in the differentiation of endoderm and its
tissue derivatives, we expressed mTcf-4B mRNA in one-cell stage
embryos and followed the expression of three molecular markers in
animal cap explants: endodermin, a pan-endodermal marker (29), Xlhbox8
(also known as Pdx1, IPF1, or STF-1), a marker of foregut derivatives
including the duodenum and pancreas (30), and the IFABP (31).
Expression of these markers is weakly but reproducibly induced by
full-length mTcf-4B mRNA and by the known inducer chordin but not
by a 5'-truncated and frameshifted mTcf-4 control (Fig.
6A). Notably, however, the
magnitude of induction is considerably smaller than that by activin
(Fig. 6A) or by the recently described homeobox gene
Mixer (28), a potent early activator of endoderm
differentiation in Xenopus embryos (data not shown).

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Fig. 6.
Endodermal differentiation in Xenopus
animal cap explants following embryonic microinjection of mTcf-4B
mRNA (A) or plasmid DNA (B) or human LEF-1
plasmid DNA (C). RT-PCR detects expression of the
pan-endodermal marker endodermin (Edd), foregut-specific marker
Xlhbox8, small intestine-specific marker IFABP, and loading control
EF1 . Marker induction is compared with embryos injected with chordin
mRNA (A) and with uninjected caps cultured in 50 ng/ml
recombinant activin A (A and B). ctl
designates injection of water (C) or RNA (A) or
plasmid DNA (B) corresponding to an out-of-frame 5'
truncation of the first 76 nucleotides of the mTcf-4B coding sequence.
These results are representative of three independent experiments with
DNA and RNA injections, respectively.
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Injection of mRNA into Xenopus embryos results in
potentially rapid gene expression that persists through many cell
divisions; in contrast, injected plasmid DNA undergoes zygotic
transcription only after the mid-blastula transition. Injection of
mTcf-4B plasmid DNA also induced the gastrointestinal markers in
explanted Xenopus animal caps (Fig. 6B),
suggesting that the ectopic expression can influence endodermal
differentiation relatively late. Injection of plasmid DNA encoding
hLEF-1 had a similar effect (Fig. 6C), suggesting that other
Tcf/LEF subfamily proteins can substitute for this function. We
conclude that Tcf-4 participates in a biochemical pathway of
differentiation that leads to gastrointestinal derivatives of the endoderm.
 |
DISCUSSION |
The molecular mechanisms by which the endoderm-derived epithelial
cells lining the aerodigestive tract differentiate are largely unknown.
Several lines of evidence point to a role for Wg/Wnt signaling in
differentiation of the gastrointestinal epithelium, including genetic
studies in invertebrates (6, 10) and biochemical analysis of colon
carcinomas (16). This raises the intriguing possibility that there are
gut-specific components or modifiers of the Wnt signaling pathway that
mediate tissue-specific responses. However, many of the components of
this signaling pathway in gut epithelial cells are largely presumed on
the basis of biochemical studies in other developmental systems. We
have, therefore, focused on characterizing gut epithelium-specific
aspects of Wnt signaling. Here we report that the predominant Tcf/LEF
subfamily member present in the developing vertebrate gut is Tcf-4,
which is only expressed here and in selected regions of the central
nervous system during fetal development.
mTcf-4 Structure and Expression--
Mouse and human Tcf-4 are
virtually identical at the amino acid level, at least in sequences
N-terminal to the HMG box. The relationship of Tcf-4 to Tcf-1 extends
to a remarkable similarity in splice variants and of a second reading
frame in one of the terminal exons that encodes the isoform designated
Tcf-4B. This potentially leads to considerable heterogeneity in the C
termini of both proteins (32), and its evolutionary conservation
suggests that this may be of functional importance. Although the
various C termini of Tcf-1 do not reveal detectable differences in
transactivation properties in transient transfection assays (32), the
Tcf-4 isoforms may well harbor functional heterogeneity that is
relevant in vivo. Tcf-1 is also heterogeneous at the N
terminus, in part reflecting use of alternate promoters (32). Although
we have not observed the same structure in mTcf-4 cDNAs isolated
from the fetal gut, the major mRNA isoform present in the brain is distinct from that in other tissues (Fig. 5A) and probably
reflects 5' heterogeneity; the precedent with Tcf-1 suggests that this also may represent dual promoter usage. The roughly equal frequency with which we isolated alternatively spliced clones suggests, but does
not prove, that these splice variants are expressed in low but equal
proportions in the fetal gut; our data do not address the existence or
relative abundance of mTcf-4 splice variants in the brain.
Recently, Korinek et al. (24) reported cloning the mouse
Tcf-4 gene, and our in situ hybridization studies confirm
localization of fetal expression to the gut epithelium and
di/mesencephalon. Whereas these investigators failed to detect
appreciable mTcf-4 RNA levels in most adult tissues, however, we note
that the gene is widely expressed postnatally, with highest levels in
the liver, an endoderm-derived organ, and brain. The same pattern is
seen in Northern analysis with either a full-length cDNA probe or
one corresponding to a fragment of the 3'-UTR, which argues strongly in
favor of specificity over cross-reactivity with related species. This
apparent discrepancy is best explained by our use of high specific
activity probes against Northern blots of poly(A)+ rather
than total RNA. Indeed, Korinek et al. (24) readily detected
mTcf-4 transcripts in poly(A)+ RNA isolated from various
segments of the adult intestine, and we were also repeatedly frustrated
in efforts to demonstrate expression outside the brain using total RNA.
Thus, the expression pattern of Tcf-4 departs significantly from that
of either Tcf-1 or LEF-1, both of which are expressed broadly during
fetal development but restricted to lymphocytes in adult mice.
The restricted fetal expression of Tcf-4 might suggest that it mediates
essential aspects of signaling by Wnts or related molecules during
development of the gut epithelium and, especially, of the diencephalon
(thalamus), where expression levels are highest. Notably, at least 7 of
the 16 known mammalian Wnt genes are expressed in various
regions of the central nervous system and at least one of these, Wnt-1,
is required for mid- and hind-brain development (33, 34). The lack of
brain abnormalities in mouse embryos lacking Tcf-1 or LEF-1 further
hints at a possible requirement for Tcf-4 in central nervous system
development or function.
Tcf-4 Function--
Our most important finding pertains to the
potential role of Tcf-4 in differentiation of endodermally derived
tissues. Injection of mTcf-4 in the early Xenopus embryo
leads to ectopic expression of endodermal and gut markers in animal cap
explants; at sibling tadpole stages beyond 35-36, endodermin
specifically marks endodermal derivatives in the gut (29), whereas
Xlhbox8 and IFABP are specific markers of the duodenum and pancreas
(30) and small intestine (31), respectively. This implicates Tcf-4 as
functioning within a biochemical pathway that promotes gastrointestinal
epithelial cell differentiation.
Several aspects of this finding merit further discussion. First, the
assay does not directly address the extent of cytodifferentiation promoted by Tcf-4; indeed, complete differentiation of endodermal derivatives in vivo is highly dependent on inductive
interactions with mesenchyme (35) and probably does not occur in
isolated animal caps (26). However, induction of Xlhbox8 mRNA, a
specific marker of differentiated foregut derivatives (30), suggests that Tcf-4 might promote relatively advanced differentiation and may be
particularly relevant to development of the pancreas. Second, the
mechanism of cellular changes induced by Tcf-4 in the animal cap
remains uncertain. Untreated animal caps differentiate into atypical,
ciliated epidermis in isolation but retain considerable developmental
plasticity early on. Ectopic expression of gastrointestinal markers in
this tissue may reflect selective expansion of rare progenitor cells
with intrinsic endodermal potential, realization of such potential in
naive embryonic cells, or perhaps some combination of these
possibilities; this consideration applies to all experiments with
Xenopus animal caps. Finally, loss-of-function studies,
including gene targeting in mice, are necessary to establish a
functional requirement for Tcf-4 in development of the endoderm and its
derivatives in vivo. During the review of this manuscript,
Korinek et al. reported that Tcf-4 knockout mice die at
birth and their only detectable histopathologic abnormality is a
reduced number of cells in small intestinal crypts, suggestive of a
depleted stem cell compartment (36). Together with the restricted fetal
expression in mice, this observation and our gain-of-function studies
in Xenopus implicate Tcf-4 as a regulator of the vertebrate
gut epithelium. The similar results with either mRNA or DNA
injection in Xenopus embryos further suggest that Tcf-4 can
function in this capacity relatively late, i.e. after the
mid-blastula transition, in embryonic development.
Although genetic experiments in Drosophila suggest that
dTcf/Pangolin exclusively subserves Wg signaling (9, 17), it is
entirely possible that in the vertebrate gut ligands other than Wnt
signal through Tcf-4 to influence epithelial cell differentiation. The
full complement of Wnt-related proteins expressed in the developing gut
remains unknown, and several proteins without a known connection to Wnt
signaling have also been implicated in endodermal and gut epithelial
differentiation (37-39). The required inductive effect of adjacent
mesenchyme for complete maturation of the gut epithelium (35) raises
the further possibility that Tcf-4 is an effector for signals delivered
by cell surface ligands. The interactions between the biochemical
pathways involved in these processes and in gastrointestinal
tumorigenesis remain to be determined. Identification of Tcf-4 as the
predominant Tcf/LEF protein expressed in the developing and adult gut
epithelium and its potential role in cytodifferentiation should
facilitate molecular approaches to these questions.
 |
ACKNOWLEDGEMENTS |
We are grateful to Lydia Otalora and David
Smoller for assistance with the mouse embryonic gut cDNA plasmid
library; to Leonard Zon, Jeremy Green, and members of their
laboratories for assistance with Xenopus methodology; to
Eddy De Robertis, Randall Moon, and Paul Mead for providing chordin,
hLEF-1, and Mix.3/Mixer constructs, respectively; and to Massimo Loda
for assistance with in situ hybridization.
 |
FOOTNOTES |
*
This work was supported by fellowships from the Harcourt
General Trust, the Dolphin Trust, and the Cancer Research Fund of the
Damon Runyon-Walter Winchell Foundation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF107298 and AF107299.
¶
To whom correspondence should be addressed: Dana-Farber Cancer
Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-5746; Fax:
617-632-5739; E-mail: ramesh_shivdasani{at}dfci.harvard.edu.
The abbreviations used are:
Wg, Wingless; HMG, High Mobility Group; Tcf, T-cell factor; LEF, Lymphoid Enhancer Factor; IFABP, intestinal fatty acid binding protein; PCR, polymerase chain
reaction; ED, embryonic day; UTR, untranslated region; BCIP, 5-bromo-4-chloro-3-indolyl phosphate.
 |
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