First published online August 10, 2007
doi: 10.1242/10.1242/dev.006544
Development 134, 3177-3190 (2007)
Published by The Company of Biologists 2007
ß-catenin/TCF/Lef controls a differentiation-associated transcriptional program in renal epithelial progenitors
Kai M. Schmidt-Ott1,2,*,
T. Nestor H. Masckauchan3,
,
Xia Chen1,,
Benjamin J. Hirsh1,
Abby Sarkar1,
Jun Yang1,
Neal Paragas1,
Valerie A. Wallace4,
Daniel Dufort5,
Paul Pavlidis6,
,
Bernd Jagla6,
Jan Kitajewski3 and
Jonathan Barasch1,*
1 Department of Medicine, Columbia University College of Physicians and
Surgeons, 630 West 168th Street, New York, NY 10032, USA.
2 Max-Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10,
D-13125 Berlin, Germany.
3 Department of Obstetrics and Gynecology, Columbia University College of
Physicians and Surgeons, New York, NY 10032, USA.
4 Molecular Medicine Program, Ottawa Health Research Institute and University of
Ottawa Eye Institute, Ottawa, ON, Canada.
5 Department of Obstetrics and Gynecology, Division of Experimental Medicine,
McGill University Health Center, Royal Victoria Hospital, Montreal, QC,
Canada.
6 Department of Biomedical Informatics, Columbia University College of
Physicians and Surgeons, New York, NY 10032, USA.
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Fig. 1. Rat metanephric mesenchymes recapitulate differentiation of kidney
epithelia in vivo under defined culture conditions. (A) Metanephric
mesenchymes cultured in basal media undergo apoptosis. (B) Addition of
Fgf2 and Tgf to the culture media induces survival of clusters of
progenitors. (C) These aggregates express Wnt4 as detected by
in situ hybridization. (D) After continued culture with Fgf2 and
Tgf, mesenchymes degenerate without differentiating into epithelia.
(E-J) NHBF, Lif or NGAL, when combined with Fgf2 and Tgf, each
induced continued expansion of metanephric mesenchymes and their
differentiation into organotypic epithelia within 7 days of organ culture.
Tubules stain positive for Cdh1 (E-cadherin; F), whereas glomerular-like
structures express podocalyxin-like (Podxl) (G). Histologically, these
structures resemble kidney epithelia at and beyond the S-shaped body stage
(H-J). (K) The sequence of metanephric mesenchymal differentiation in
organ culture recapitulates epithelial differentiation in vivo (in this case
in the presence of Lif). Arrows delineate a mesenchymal aggregate, after 3-4
days of differentiation in vitro, reminiscent of pretubular aggregates in
vivo. UB, ureteric bud; CM, condensed mesenchyme; PA, pretubular aggregate;
SB, S-shaped body; Tb, tubule; Gl, glomerular-like structure.
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Fig. 2. Microarray analysis of gene expression during epithelial differentiation
in rat metanephric mesenchyme. (A) Heatmap representation of all
genes upregulated independently by Lif, NGAL and NHBF in a temporal sequence
from day 0 (freshly isolated mesenchyme) to day 7 (differentiated renal
epithelia). Average profiles (right) were deduced arithmetically and grouped
into three classes based on the time point of peak expression (class A, day
1-2; class B, day 3-5; class C, day 7). Note that only genes upregulated by
all three inducers were included in this analysis. (B) Correlation
matrices comparing global gene expression at individual time points (day 0 to
day 7) were calculated and hierarchical clustering identified time points of
major shifts in gene expression (tree diagram). Pearson correlation
coefficients R of neighboring time points (blue) illustrate the
degree of change in global gene expression. Epithelial differentiation is
depicted at the times of peak expression of each class of genes.
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Fig. 3. Prediction of TCF/Lef target genes in differentiating epithelial
progenitors. (A) Real-time RT-PCR confirmation of an upregulation
of putative TCF/Lef target genes during epithelial differentiation induced by
Lif. Expression levels peak on either day 5 (class B genes) or day 7 (class C
genes) of organ culture. Maximal expression levels were set to 100%.
Prediction is based on either published evidence or computational
identification of conserved TCF/Lef consensus sites in the promoter region
(see below). *, P<0.05 versus Lif 2 days
(n=3). (B) Example of the prediction of a TCF/Lef binding site
99 bp upstream of the transcriptional start site of the cyclin D1
(Ccnd1) gene. Mouse, rat and human sequences are aligned and reveal a
high degree of conservation in the region surrounding the TCF/Lef consensus
motif (red). (C) Overrepresentation of conserved TCF/Lef binding sites
in promoters of genes induced during epithelial differentiation.
Identification of genes containing at least one conserved TCF/Lef binding site
in different intervals preceding the transcriptional start site consistently
reveals a statistically significant overrepresentation in classes B and C
compared with control genes ( 2 test, P values for
different intervals are indicated in orange). (D) Specific
overrepresentation of the TCF/Lef core motif as measured by an
overrepresentation index (see Materials and methods) is demonstrated by
comparison with 4096 control motifs of equal dimension. The TCF/Lef matrix
(red) scores in the 97.1st percentile of all examined matrices.
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Fig. 4. TCF/Lef-dependent induction of predicted ß-catenin
targets. (A) Adenoviruses expressing wild-type
(Ad-CTNNBWT) or stabilized (Ad-CTNNBS37A) ß-catenin
induce TCF/Lef-dependent Topflash reporter activity in human embryonic kidney
(HEK)293 cells at moderate and high levels, respectively. The effect of
Ad-CTNNBS37A is effectively blocked by co-infection of Ad-DN-TCF.
(B) As determined by real-time RT-PCR, Ad-CTNNBS37A
significantly induces expression of 10/15 (66%) predicted TCF/Lef target genes
in metanephric mesenchymes after 14 hours of culture when compared with Ad-GFP
only. This induction is consistently blocked by co-infection of
Ad-CTNNBS37A and Ad-DN-TCF (n=3, values are represented as
mean±s.e.m.). Expression levels under control conditions (Ad-GFP only)
were set to 1. Conversely, stabilization of ß-catenin in metanephric
mesenchyme does not induce expression of genes associated with epithelial
differentiation that lack predicted TCF/Lef sites (Frzb, Lama1,
Tcfap2b), or of Myc, a TCF/Lef target gene in other cell types,
which is not significantly regulated during epithelial differentiation
(Controls). *, Significantly upregulated versus Ad-GFP only
(P<0.05); #, significantly downregulated versus
Ad-CTNNBS37A+Ad-GFP (P<0.05).
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Fig. 5. Conserved co-expression of ß-catenin/TCF/Lef targets in the
renal epithelial lineage in vivo. (A) Ccnd1, Pax8 and
Emx2 are expressed in epithelia deriving from the metanephric
mesenchyme in vivo as determined by in situ hybridization on rat and mouse
E15.5 kidneys. Ccnd1 and Pax8 are first activated in
pretubular aggregates and maintained in nascent early epithelia. Emx2
is activated slightly later and appears first in early epithelia. Expression
patterns are strictly conserved in the two species. (B)
TCF/Lef-lacZ activity as determined by X-Gal staining is strong in
emerging epithelia overlapping with the expression domain of target genes.
Note that Pax8, Emx2 and the TCF/Lef reporter are also detected in
the ureteric bud. SB, S-shaped body; PA, pretubular aggregate; UB, ureteric
bud.
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Fig. 6. ß-catenin associates with chromatin in promoters of TCF/Lef target
genes in vivo. ChIP was performed on rat E15.5 kidneys. (A)
Chromatin was cross-linked and DNA was fragmented to  400 bp. (B)
Subsequently, ß-catenin-containing DNA-protein complexes were
immunoprecipitated as verified by immunoblotting. (C) Following
reversal of cross-links, a strong enrichment of promoter sequences containing
TCF/Lef sites (targets, indicated in diagrams on the left) was observed in
ß-catenin immunoprecipitates (ß-catenin IP) but not control IP (no
antibody). Off-target control sites were not enriched, demonstrating
specificity.
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Fig. 7. ß-catenin signaling triggers survival and proliferation of
epithelial progenitors, but not tubulogenesis. (A) Introduction of
stabilized ß-catenin (Ad-CTNNBS37A) into epithelial
progenitors marked by Pax2 (red) prevents apoptosis determined by
immunostaining for activated caspase 3 (a-CASP3, blue) observed after 3 days
of culture under control conditions (Ad-GFP only). This anti-apoptotic
response is blocked by dominant-negative TCF (Ad-DN-TCF). (B)
Proliferative activity measured by phospho-histone H3 staining (blue) is
observed in Pax2-expressing (red) progenitors following infection with
Ad-CTNNBS37A, but not Ad-GFP (arrows). Note the presence of a small
number of phospho-histone-positive cells in the Pax2-negative cell compartment
in both conditions (arrowheads). (C) Only scattered Pax2-positive
progenitors (red) are initially infected by Ad-CTNNBS37A, as
detected after 16 hours of culture following infection and staining for the HA
epitope on the adenovirally coded protein (green). Infected cells display a
strongly positive membrane pool of HA-CTNNBS37A and a nuclear pool
of variable intensity, which colocalizes with Pax2 (yellow areas). After 3
days of culture, HA-negative progenitors have largely disappeared suggesting
positive selection of cells infected by Ad-CTNNBS37A, which
indicates that the effect of the adenovirus is cell-autonomous. (D)
Metanephric mesenchymes infected with Ad-CTNNBS37A develop into
cell aggregates, but do not display evidence of tubulogenesis or segmentation.
Conversely, tubular and glomerular-like structures are abundantly observed
after treatment with a low dose of the Gsk3ß inhibitor BIO. (E)
Aggregates induced by Ad-CTNNBS37A stain negative for Cdh1 (green),
a marker of polarized epithelial cells. By contrast, metanephric mesenchymes
induced with the Gsk3ß inhibitors lithium chloride or BIO for the same
time period display Cdh1-positive tubules. Note that treatment with BIO also
induces glomerular-like structures marked by podocalyxin expression (Podxl,
red). (F) As detected by real-time RT-PCR, markers of polarized
epithelial cells are strongly induced after epithelial induction with BIO for
4 days (d4 BIO) or Lif for 7 days (d7 Lif) (time points of robust epithelial
differentiation), but remain negative 4 days (d4) and 7 days (d7) after
infection with Ad-CTNNBS37A (S37A). Values are compared with
freshly isolated metanephric mesenchymes (d0), expression levels of which were
set to 1. *, P<0.05 versus metanephric mesenchyme. Gl,
glomerular-like structure; Tb, tubule.
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Fig. 8. Progenitors with compromised TCF/Lef signaling are depleted from the
epithelial lineage. (A) Metanephric mesenchymes are infected with
Ad-GFP or GFP-tagged dominant-negative TCF (Ad-DN-TCF) followed by culture in
the presence of inductive media containing Tgf, Fgf2 and Lif and virus
withdrawal after 14 hours. Efficiency of infection of Pax2-positive epithelial
progenitors (red) with Ad-GFP (green, cytoplasmic staining) or Ad-DN-TCF
(green, nuclear staining) is similar as determined after 14 hours of culture.
Conversely, after 48 hours, cells expressing DN-TCF are observed at a
decreased frequency in the Pax2-positive cell population as compared with
cells expressing GFP only. Occasionally, staining for activated caspase 3
(blue) is observed in Pax2-positive cells infected by Ad-DN-TCF (arrowheads),
indicating ongoing apoptosis in this population (note occasional apoptosis
also in the Pax2-negative cell population with both viruses). After 6 days of
culture, DN-TCF-expressing cells (green) are rarely observed in Pax2-positive
Cdh1-negative (Pax2+ Cdh1-) epithelial progenitors or
arising Pax2+ Cdh1+ epithelial clusters, whereas cells
infected with Ad-GFP (green) readily contribute to either of these pools
(asterisks). (B) Quantitative evaluation of percentage of
green-fluorescent cells in either Pax2+ progenitors or
Pax2+ Cdh1+ epithelia demonstrates progressive depletion
from the differentiating epithelial lineage of cells expressing GFP-tagged
DN-TCF, but not GFP only.
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© The Company of Biologists Ltd 2007
