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First published online August 10, 2007
doi: 10.1242/10.1242/dev.006544
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.
* Authors for correspondence (e-mails: kms2115{at}columbia.edu; jmb4{at}columbia.edu)
Accepted 18 June 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Metanephric mesenchyme, Epithelial differentiation, ß-catenin, TCF/Lef-type transcription factors, Neutrophil gelatinase-associated lipocalin, Leukemia inhibitory factor, Wnt4, Emx2, Pax8, Cyclin D1 (Ccnd1), Frzb
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Initially,
metanephric mesenchymal progenitors organize at the tips of the ureteric bud
forming the `condensed mesenchyme'. This is followed by translocation relative
to the elongating ureteric bud stalk to generate a `pretubular aggregate'
characterized by expression of Wnt4 and Pax8
(Stark et al., 1994).
Establishment of apical-basal polarity in this aggregate produces the renal
vesicle, a structure characterized by the onset of expression of E-cadherin
(also known as cadherin 1, Cdh1), a crucial component of epithelial adherens
junctions (Barasch et al.,
1999; Cho et al.,
1998). Segmentation events lead to the formation of the `S-shaped'
body, the direct precursor to mature nephrons, which includes a glomerular
pole proximally and a tubular pole distally that connects to the tip of the
ureteric bud (Dressler,
2006).
In vivo, epithelial differentiation of mesenchymal progenitors is dependent
on the presence of the ureteric bud. This sequence can be modeled in vitro by
application of defined combinations of growth factors to rat metanephric
mesenchyme, which we and others have previously identified by protein
chromatography from a ureteric bud cell line
(Barasch et al., 1996;
Barasch et al., 1999;
Karavanova et al., 1996;
Plisov et al., 2001;
Yang et al., 2002a;
Yang et al., 2002b).
Application of these growth factors results in the highly reproducible and
synchronized appearance of segmented tubules that include glomerular-like
structures, proximal tubules and distal tubules
(Barasch et al., 1999).
The molecular events that drive stage transitions in the epithelial lineage
are subject to ongoing research. Although extracellular factors from the WNT,
transforming growth factor ß (TGFß), fibroblast growth factor (FGF)
and interleukin 6 (IL6) families have been implicated as regulators of
different aspects of epithelial differentiation
(Barasch et al., 1999;
Carroll et al., 2005;
Kispert et al., 1998;
Oxburgh et al., 2004;
Perantoni et al., 1995;
Stark et al., 1994), little is
known about the transcriptional events involved in these processes. In the
current paper, we analyzed genome-wide transcriptional profiles associated
with epithelial differentiation using microarray analysis. Activated
transcripts included multiple putative target genes of transcription factors
of the TCF/Lef family, suggesting that these factors might participate in the
control of epithelial differentiation.
TCF/Lef transcriptional activity is principally regulated through
alteration of ß-catenin levels in the nucleus
(Brembeck et al., 2006;
Clevers, 2006). ß-catenin,
a bifunctional protein that comprises a structural component of adherens
junctions and a transcriptional co-activator, converts TCF/Lef from a
transcriptional repressor into an activator of specific target genes and
thereby regulates crucial transcriptional programs throughout development.
Nuclear ß-catenin levels are predominantly regulated by a multiprotein
complex that targets glycogen synthetase kinase Gsk3ß and casein kinase I
(CKI; also known as Csnk1) to mediate amino-terminal serine-threonine
phosphorylation of ß-catenin and, thereby, promotes proteasomal
degradation of ß-catenin. This process is inhibited by ligands of the WNT
family, which results in stabilization of intracellular ß-catenin.
However, the process is not specific for WNT proteins as it can be triggered
by additional ligands (Brembeck et al.,
2006; He, 2006)
and, conversely, in many cases the cellular actions of WNT proteins are
independent of ß-catenin and mediated through alternative pathways,
including c-Jun N-terminal kinase (also known as Mapk8 - Mouse Genome
Informatics) or calcium signaling (Veeman
et al., 2003). Although multiple lines of evidence have
established an involvement of WNT signaling in the differentiation of renal
epithelial progenitors in both mice and rats
(Carroll et al., 2005;
Herzlinger et al., 1994;
Kispert et al., 1998;
Osafune et al., 2006;
Plisov et al., 2001;
Stark et al., 1994), little is
known about TCF/Lef-mediated events as a potential downstream signal.
In the current paper, we show that even in the absence of stimulation by exogenous WNT ligands, epithelial differentiation of metanephric mesenchyme is characterized by the activation of multiple TCF/Lef-dependent targets of ß-catenin. We further demonstrate that ß-catenin/TCF/Lef signaling is involved in the regulation of survival and proliferation of epithelial progenitors and induces stage progression characterized by the induction of a subset of the tubulogenic transcriptional program. Importantly, cells with impaired TCF/Lef-dependent transcription are progressively depleted during epithelial differentiation, suggesting that this signaling axis controls cellularity in the renal epithelial lineage.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Gsk3ß inhibitors used were lithium chloride (Sigma-Aldrich, St Louis, MO)
and 6-bromoindirubin-3'-oxime (BIO; EMD Biosciences, San Diego, CA).
Isolation of non-heparin-binding fraction (NHBF)
Approximately 200 l of serum-free media, conditioned by monolayers of
ureteric bud cells (Barasch et al.,
1996), were concentrated, desalted and applied to
heparin-Sepharose (GE Healthcare, Piscataway, NJ) in 10 mM
Na2PO4 (pH 7.0). Flow-through was reapplied to a fresh
heparin-Sepharose column twice to ensure removal of all heparin-binding
substances. Following concentration and desalting, the flow-through was
applied to ANX Sepharose 4FF columns (GE Healthcare) in 20 mM bis-Tris buffer
(pH 9.0) and eluted with a NaCl gradient. The active non-heparin-binding
fraction was concentrated, desalted and 100 µg of total protein was
subjected to immunoblotting for Lif (polyclonal anti-mouse Lif; R&D) and
NGAL (Lcn2) (Yang et al.,
2002b), confirming the absence of these heparin-binding inductive
activities.
RNA extraction, reverse transcription and real-time PCR
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) as
described previously (Schmidt-Ott et al.,
2005). cDNA was synthesized using Omniscript Reverse Transcriptase
(Qiagen). Real-time PCR was carried out using iQ SYBR Green Super Mix and a
MyiQ Single-Color Real-Time PCR Detection System (Biorad, Hercules, CA).
Primer sequences are available upon request. Specificity of the amplification
was checked by melting curve analysis, agarose gel electrophoresis and
sequencing of PCR products. Relative levels of mRNA expression were normalized
to ß-actin mRNA and calculated according to the
CT method as described
(Schmidt-Ott et al.,
2006).
Microarray analysis
Metanephric mesenchymes were cultured in basal media supplemented with Fgf2
(50 ng/ml; R&D Systems) and Tgf
(20 ng/ml; R&D Systems) and one
of the following inducers: Lif (50 ng/ml; R&D Systems), siderophore-loaded
NGAL (100 µg/ml) (Yang et al.,
2002b) or NHBF (100 µg total protein per ml). Labeled cRNA was
prepared from 20 mesenchymes per condition or from freshly dissected E15.5 rat
kidneys for two biological replicates per condition as described
(Schmidt-Ott et al., 2005) and
hybridized to Rat Genome 230 2.0 Arrays (Affymetrix). Raw data are available
at Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo/,
GEO accession GSE5478).
Statistical analysis and classification of microarray data
Image files of microarray data were analyzed by robust multichip analysis
as previously described (Schmidt-Ott et
al., 2005). To test for significant regulation of gene expression
over time, we utilized a recently developed method for significance analysis
of time-course microarray experiments
(Storey et al., 2005). An
average time curve for all inducers was modeled using the software package
EDGE. A gene was considered significantly regulated if its false discovery
rate (FDR) of differential regulation over time was less than 10% and its
expression level was either upregulated or downregulated more than 2-fold by
each inducer compared with the baseline. The lists of genes identified thereby
are provided in Tables S1 and S2 in the supplementary material. To identify
global shifts in gene expression, hierarchical clustering was carried out
based on Pearson correlation coefficients between average expression levels at
different time points (Montaner et al.,
2006).
Identification of putative TCF/Lef binding sites
To identify putative TCF/Lef binding sites in genes activated during
epithelial differentiation (classes B and C), sequences 10 kb upstream of the
transcriptional start site were extracted from published rat genomic sequences
and aligned with the corresponding promoter sequences from the mouse and human
genome using ClustalW (Thompson et al.,
1994). Alignments were then subjected to an automated search for
the TCF/Lef consensus recognition sequence [5'-(A/T)(A/T)CAAAG-3']
on both strands. Only sites conserved between all three species were
considered. To test for significant overrepresentation of TCF/Lef consensus
sites in classes B and C, the frequency of occurrence of at least one TCF/Lef
site in various promoter intervals was compared against a set of 2591 control
genes (least significant probe sets in the statistical analysis above). A
2 test was used to test for overrepresentation and
P<0.05 was assumed to indicate statistical significance. To
confirm specificity of overrepresentation of the TCF/Lef consensus sequence in
the promoter region of class B and C genes, 4096 nucleotide matrices, equal in
dimension but different in sequence to the TCF/Lef matrix, were used to repeat
the same type of analysis. An overrepresentation index i was calculated for
the TCF/Lef matrix and all control matrices according to the formula
i=fBC2/fcon, where fBC is the
percentage of 10 kb promoters from class B and C genes that contain at least
one occurrence of the nucleotide matrix and fcon is the
corresponding percentage of promoters from control genes. The calculated value
for i for the TCF/Lef matrix was 22.31, which represented the 97.1st
percentile of all assayed matrices.
Adenoviral gene transfer
Recombinant adenovirus vectors Ad-GFP, Ad-CTNNBWT and
Ad-CTNNBS37A were described previously
(Masckauchan et al., 2005).
Ad-DN-TCF was prepared from a pEGFP-DN-Tcf4 construct (a kind gift of P.
Petzelbauer and W. Holnthoner) (Holnthoner
et al., 2002) according to standard methods
(Masckauchan et al., 2005).
Five freshly dissected metanephric mesenchymes per condition were exposed to
1.5x108 plaque forming units (PFU) of the appropriate
adenovirus in 150 µl basal culture media for 1 hour at 37°C and then
plated onto Transwell filters and cultured in media supplemented as
indicated.
Reporter assays
HEK293T cells were transfected in 12-well plates using Lipofectamine 2000
(Invitrogen, Carlsbad, CA) using 190 ng of the TCF/Lef reporter construct
Topflash or the mutated control plasmid Fopflash
(Korinek et al., 1997) and 10
ng of a plasmid containing Renilla luciferase for normalization. A
total of 1.5x107 PFU of adenovirus was added to the culture
media. Cells were harvested after 24 hours of culture and reporter activity
was assayed using the Dual Luciferase Assay System (Promega, Madison, WI).
Histology, X-Gal staining, immunofluorescence and in situ hybridization
For histology, metanephric mesenchymes were embedded in Epon, sectioned and
stained with Toluidine Blue as described previously
(Barasch et al., 1999).
TCF/Lef-lacZ embryos (Mohamed
et al., 2004) were fixed with 2% paraformaldehyde,
cryostat-sectioned and stained in lacZ staining solution
(1xPBS, 2 mM MgCl2, 5 mM potassium ferricyanide, 5 mM
potassium ferrocyanide and 1 mg/ml X-Gal) at 37°C.
For immunofluorescence staining, whole-mount metanephric mesenchymes were
fixed in 4% paraformaldehyde and stained using primary antibodies for Pax2
(Invitrogen), goat anti-Cdh1 (R&D Systems), mouse anti-Cdh1 (BD
Biosciences, Franklin Lakes, NJ), podocalyxin (R&D Systems), ACTIVE
caspase 3 (Promega), phospho-histone H3 (Ser10) (Cell Signaling, Danvers, MA),
the HA epitope (Clone 3F10, Roche Applied Sciences, Indianapolis, IN) and the
appropriate secondary antibodies labeled with either Cy2, Cy3 or Cy5 (Jackson
ImmunoResearch Laboratories, West Grove, PA). For double-staining using Pax2,
ACTIVE caspase 3 and phospho-histone H3 antibodies, which are all raised in
rabbits, we biotinylated the Pax2 antibody using the EZ-link Solid Phase
Biotinylation Kit (Pierce, Rockford, IL) and performed immunostaining for
ACTIVE caspase 3 or phospho-histone H3 followed by Pax2, where
cross-reactivity of the biotinylated Pax2 antibody was prevented by
preincubation with rabbit gamma globulins (Jackson ImmunoResearch)
(Wurden and Homberg, 1993).
All fluorescent imaging was performed on a Zeiss LSM 510 META scanning
confocal microscope.
In situ hybridization on frozen sections was performed using DIG-labeled
probes as described previously
(Schmidt-Ott et al., 2006). To
produce riboprobes, PCR fragments of genes of interest were generated from
embryonic kidney cDNA using reverse primers containing a 5' T7
polymerase promoter sequence (primer sequences available upon request). The
resulting fragments were purified and sequence-verified, and
digoxigenin-labeled cRNA was produced using T7 polymerase.
Chromatin immunoprecipitation (ChIP) assays
In vivo ChIP was performed essentially as described
(Chamorro et al., 2005;
Lowry et al., 2005), but using
cells isolated from kidneys of rat E15.5 embryos. Chromatin was cross-linked
for 15 minutes in 1% paraformaldehyde in PBS. Cells were lysed in SDS lysis
buffer (ChIP Assay Kit, Upstate USA, Charlottesville, VA), homogenized, and
sonicated four times for 15 seconds at an output power of 4 Watts so as to
fragment the DNA (`input DNA'). Subsequently, immunoprecipitation was carried
out using rabbit anti-ß-catenin antibody (H-102, Santa Cruz) and immune
complexes pulled down using protein A-agarose beads blocked with salmon sperm
DNA (Upstate). Following washes according to manufacturer instructions
(Upstate), complexes were eluted in 1% SDS/100 mM NaHCO3 and
cross-links were reversed by adding NaCl to 200 mM and heating to 65°C for
14 hours. DNA was extracted and subjected to PCR using primers flanking the
conserved TCF/Lef sites identified by our in silico analysis or off-target
control sequences (primer sequences available upon request). DNA fragmentation
was monitored by agarose gel electrophoresis of input DNA. Successful
immunoprecipitation of ß-catenin-containing protein-DNA complexes was
confirmed by immunoblotting using a mouse anti-ß-catenin antibody (BD
Transduction Laboratories, San Jose, CA).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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permitted survival of these metanephric mesenchymal explants under defined
conditions in organ culture (Fig.
1A-C) (Barasch et al.,
1997; Karavanova et al.,
1996; Perantoni et al.,
1995). These factors induced the formation of clusters of
epithelial progenitor cells expressing the characteristic markers Pax2, Wt1
and Wnt4, which were competent to undergo expansion and epithelial
differentiation upon addition of an inducer
(Fig. 1B-D)
(Barasch et al., 1997;
Barasch et al., 1999;
Karavanova et al., 1996). We
and others have previously isolated two such inducers from ureteric bud cell
lines by protein chromatography, and identified them as leukemia inhibitory
factor (Lif) and neutrophil gelatinase-associated lipocalin (NGAL; also known
as Lcn2 - Mouse Genome Informatics)
(Barasch et al., 1999;
Plisov et al., 2001;
Yang et al., 2002b). While
these two factors were isolated based on their heparin-binding properties, we
also noted an additional, distinct ureteric bud cell-derived inductive
activity, which was not retained on heparin-Sepharose columns. We partially
purified this activity and provisionally designated it `non-heparin-binding
fraction' (NHBF). In its presence, as in the presence of Lif and NGAL, rat
metanephric mesenchymes underwent expansion and converted into organotypic
nephron epithelia within 7 days (n=40) in stages reminiscent of the
morphological sequence in vivo (Barasch et
al., 1996; Karavanova et al.,
1996) (Fig.
1E-K).
These molecular tools provided the methodological basis of this study and
enabled us to monitor transcriptional events in differentiating renal
epithelial progenitors in a synchronized organ culture setting
(Barasch et al., 1999;
Plisov et al., 2001;
Yang et al., 2002b), which
contrasts with the variety of different stages of epithelial differentiation
co-existing at a given time point in vivo. We obtained transcriptional
profiles during the course of metanephric mesenchymal differentiation in the
presence of either Lif or NGAL or NHBF as indicated in
Fig. 2 using Rat Genome 230 2.0
Arrays (Affymetrix), which contain 31,099 probe sets representing a large
proportion of the rat transcriptome. In a preliminary analysis, we identified
probe sets that were upregulated more than 8-fold by at least one inducer at
one or more time points, revealing that a substantial proportion (24.4%) of
upregulated probe sets was common to all three inducers (see Fig. S1 in the
supplementary material). To relate this finding to gene expression in the
embryonic kidney in vivo, we analyzed expression of all upregulated probe sets
on a separate set of microarrays prepared from freshly dissected rat E15.5
kidneys. These kidneys display robust epithelial differentiation up the
S-shaped body stage. When compared with freshly isolated metanephric
mesenchyme, these developing kidneys displayed an enrichment (1.5-fold or
more) of 71.2% of the probe sets upregulated by all three inducers. By
contrast, only 41.3% of the probe sets that were upregulated by only one
individual inducer displayed such enrichment in the developing kidney. Thus,
we reasoned that selection of probe sets common to all three inducers would
focus the analysis on genes specific to epithelial differentiation in the
developing kidney and subtract out inducer-specific effects. To obtain a
robust set of genes upregulated by all three inducers, we utilized a
statistical approach specifically designed for time-course microarray
experiments (Storey et al.,
2005) at a 10% false discovery level and selected genes with (1)
significantly modulated expression levels and (2) an upregulation of at least
2-fold during the course of differentiation when compared with freshly
isolated mesenchyme. This analysis identified 854 probe sets upregulated by
each of the three inducers, for which we derived average temporal expression
profiles to deduce a common temporal sequence of gene activation
(Fig. 2A, see Table S1 in the
supplementary material).
|
; Stark et al.,
1994). In situ hybridization confirmed that these genes and a
third class B gene with previously unknown expression pattern, Frzb,
were expressed in pretubular aggregates in the developing rat kidney (see Fig.
S3 in the supplementary material). Class C genes contained multiple
transcripts associated with mature polarized kidney epithelia, including those
encoding Cdh1, Cdh16 (cadherin 16) and Lama1 (laminin 1) (see Fig. S2
in the supplementary material). In addition, class C genes included
transcripts known to be expressed in specific segments of the nephron, such as
early glomeruli [podoplanin (Pdpn), CD2ap, Mafb], proximal
tubules [jagged 1 (Jag1), megalin (also known as Lrp2 -
Mouse Genome Informatics)], the thick ascending limbs of Henle
[Na+-K+-2Cl- cotransporter (Nkcc2;
also known as Slc12a1)] and distal tubules (Hnf1ß,
AP-2ß and Brn1 - also known as Tcf2, Tcfap2b
and Pou3f3, respectively) (see Fig. S2 in the supplementary
material). Real-time reverse transcriptase (RT)-PCR was used to validate these
temporal gene expression patterns in independent biological samples (see Fig.
S2 in the supplementary material).
|
;
Yu et al., 2004). In a search
for potential key regulators of this transcriptional program associated with
epithelial differentiation in our model, we manually screened class B and C
genes for characteristic target genes of known developmentally regulated
pathways. We noted the presence of multiple transcripts that represent known
targets of TCF/Lef signaling in other biological settings, including cyclin D1
(Ccnd1) (Tetsu and McCormick,
1999), osteopontin (also known as Spp1 - Mouse Genome
Informatics) (El-Tanani et al.,
2004), ecto-5'-nucleotidase (Nt5e)
(Spychala and Kitajewski,
2004) and Emx2 (Theil
et al., 2002). Real-time RT-PCR confirmed that these transcripts
were induced in close temporal correlation with the establishment of segmented
epithelia (Fig. 3A).
Given the previous reports of the crucial involvement of WNT proteins in
kidney organogenesis (Carroll et al.,
2005; Stark et al.,
1994) and in epithelial differentiation of the metanephric
mesenchyme in response to Lif (Plisov et
al., 2001), we hypothesized that ß-catenin-induced
TCF/Lef-mediated transcriptional programs might constitute a central
downstream signal in differentiating epithelial progenitors. To obtain a more
comprehensive picture of this putative transcriptional program activated by
TCF/Lef, we performed an in silico analysis on transcripts in classes B and C
to detect conserved consensus TCF/Lef binding sites in the promoter region of
these genes. For this purpose, genomic sequences 10 kb upstream of the
transcriptional start were extracted for rat, mouse and human homologs,
aligned and analyzed for conserved occurrences of core TCF/Lef binding sites
[5'-(A/T)(A/T)CAAAG-3'] of either orientation (for an example, see
Fig. 3B). Genomic sequences and
ortholog annotations were sufficient to complete this analysis for 71 genes
from class B and 171 genes from class C. This approach identified at least one
conserved TCF/Lef binding site in the predicted promoter region of 11 genes in
class B (15.5%) and 27 genes (15.8%) in class C. Analysis of various intervals
of promoter sequences consistently showed that TCF/Lef binding sites were
present significantly more frequently in promoters of genes from classes B and
C than in a set of 2869 control genes, the expression of which was unchanged
during epithelial differentiation (Fig.
3C). Furthermore, when we carried out the same type of analysis
using a set of 4096 control nucleotide matrices, equal in dimension but
different in sequence to the TCF/Lef matrix, only 2.9% of these sequences
displayed an equal or higher degree of overrepresentation in class B/C genes
compared with the TCF motif (Fig.
3D). This suggested that TCF/Lef binding sites were specifically
enriched in the promoter regions of genes associated with the stages directly
preceding and coinciding with epithelial differentiation. The analysis
identified conserved TCF/Lef sites in the promoter region of Ccnd1
and Emx2, genes that have already been identified as target genes of
TCF/Lef signaling in other biological settings. Importantly, the analysis
predicted 36 putative, but as yet unconfirmed TCF/Lef targets, including the
transcription factor Pax8, which is known to be a crucial regulator of
specification, survival and proliferation in the nephric lineage
(Bouchard et al., 2002).
ß-Catenin activates a TCF/Lef-dependent transcriptional program in epithelial progenitors
To test the hypothesis that these putative TCF/Lef targets were activated
by ß-catenin signaling and to determine the physiological consequences of
an activation of ß-catenin/TCF/Lef signaling in the metanephric
mesenchyme, we used adenoviral gene transfer of genetic constructs that
activate or antagonize the ß-catenin/TCF/Lef signaling axis. Although
replication-deficient adenovirus has previously been used to achieve efficient
gene transfer in cultured kidneys
(Leung-Hagesteijn et al.,
2005), we initially set out to verify that metanephric mesenchymal
progenitors were efficiently targeted by adenovirus without significant
pathogenicity. Initial experiments using a replication-deficient adenovirus
expressing green fluorescent protein (Ad-GFP) revealed that 14 hours after
infection of freshly isolated metanephric mesenchyme with Ad-GFP, followed by
organ culture, 30-40% of Pax2-positive epithelial progenitors displayed GFP
fluorescence (see Fig. S4 in the supplementary material, also see
Fig. 8). GFP fluorescence was
maintained at continuously high levels for up to 8 days in organ culture, the
longest interval tested. Similar to uninfected metanephric mesenchymes, those
infected with Ad-GFP and cultured without addition of growth factors underwent
global apoptosis within 3 days of culture (see
Fig. 7), indicating that the
adenoviral infection itself had no effect on the survival of epithelial
progenitors. Conversely, in the presence of inductive growth factors, these
GFP-positive cells participated in the epithelialization process, indicating
that the adenoviral infection was not inhibitory to epithelial differentiation
(see Fig. 8). These
observations verified that adenoviral gene transfer could achieve efficient
and long-lasting expression of a gene of interest in epithelial progenitor
cells without non-specifically perturbing the biological processes relevant to
our experimental model.
|
|
).
Conversely, to block ß-catenin/TCF/Lef-dependent transcription of target
genes, we generated an adenovirus expressing a fusion protein of GFP and an
N-terminally truncated, dominant-negative Tcf4 protein (Ad-DN-TCF), which
lacks the ß-catenin interaction domain and acts as a constitutive
repressor of TCF/Lef target genes (Korinek
et al., 1997). To verify the effectiveness of these adenoviruses,
we assayed their effect on the expression of a TCF/Lef-dependent reporter,
Topflash, in HEK293 cells. As predicted, cells infected with
Ad-CTNNBS37A displayed a marked (174-fold) increase in
TCF/Lef-dependent reporter activity compared with control conditions (Ad-GFP)
(Fig. 4A). Conversely,
co-infection of Ad-DN-TCF reduced Ad-CTNNBS37A-induced Topflash
activity by approximately 20-fold (Fig.
4A). These initial studies indicated that these adenoviruses
provided efficient tools to activate and antagonize
ß-catenin/TCF/Lef-mediated signaling. We used Ad-CTNNBS37A, and Ad-GFP as a control, to assay the effect of stabilized ß-catenin on the expression level of predicted TCF/Lef target genes in the metanephric mesenchyme. We examined the tissue 14 hours after infection, when epithelial progenitors had not yet initiated the default apoptotic program and when cultured explants were morphologically identical under both conditions (see Fig. 7A). We determined by real-time RT-PCR that 10 out of 15 of the predicted target genes were upregulated significantly in response to infection with Ad-CTNNBS37A (Fig. 4B). To test if this response was mediated by TCF/Lef-type transcription factors, we co-infected adenovirus expressing dominant-negative TCF. This consistently antagonized the stimulation of target gene expression by stabilized ß-catenin (Fig. 4B). These results established that the majority of putative ß-catenin target genes predicted by our analysis were indeed induced by ß-catenin in a TCF/Lef-dependent fashion.
Three of these target genes, Pax8, Emx2 and Ccnd1,
particularly attracted our attention because they displayed high levels of
induction and had previously been implicated in processes relevant to our
model. Transcription factors of the Pax2/8 family are required for the
survival and differentiation of progenitor cells in the intermediate mesoderm
(Bouchard et al., 2002).
Emx2 is a homeobox gene that is required for normal urogenital tract
development (Miyamoto et al.,
1997), and in the brain cooperates with ß-catenin to promote
expansion of progenitor cells (Muzio et
al., 2005). Ccnd1, although uncharacterized in the
context of kidney development, is a crucial regulator of cell cycle
progression in multiple systems (Stacey,
2003).
|
;
Stark et al., 1994)
(Fig. 5A) and, also,
Ccnd1 expression was strictly conserved in mouse and rat kidneys
(Fig. 5A) indicating that this
transcriptional program is conserved in differentiating kidney epithelia in
rodents.
To further substantiate that these transcripts were controlled by
ß-catenin/TCF/Lef in vivo, we used two independent approaches. First, we
employed TCF/Lef-lacZ transgenic mice that express
ß-galactosidase under the control of six TCF/Lef response elements,
thereby enabling spatial resolution of TCF/Lef-mediated transcription in vivo
(Mohamed et al., 2004). In the
developing kidneys of these mice, ß-galactosidase activity was detected
primarily in nascent epithelial tubules shortly after the establishment of
epithelial polarity (Fig. 5B).
Thus, consistent with the concept that TCF/Lef control expression of Emx2,
Pax8 and Ccnd1, the TCF/Lef reporter displayed a close overlap
with the expression of these genes. The fact that Pax8 and
Ccnd1 preceded the expression of Emx2 and the activation of
the TCF/Lef reporter might indicate that additional pathways participate in
the transcriptional control of these genes. In the case of the TCF/Lef
reporter, the delayed onset of expression might also reflect a limited
sensitivity of the construct, which has been previously suggested
(Mohamed et al., 2004).
Nevertheless, the sensitivity of the TCF/Lef reporter used in this study seems
to be improved compared with alternative versions, which display lacZ
staining only in the ureteric bud, but not in metanephric mesenchymal
derivatives (Maretto et al.,
2003; Schwab et al.,
2007).
In a second approach, we aimed to confirm that ß-catenin-containing
transcriptional complexes associate with the predicted TCF/Lef binding sites
in the promoter region of these genes in vivo. For this purpose, we conducted
in vivo chromatin immunoprecipitation (ChIP) assays using an
anti-ß-catenin antibody that had recently been shown to function in
immunoprecipitating chromatin associated with several TCF/Lef target genes
(Chamorro et al., 2005;
Lowry et al., 2005). We used
freshly isolated kidneys from E15.5 rat embryos, cross-linked the protein-DNA
complexes, fragmented the DNA to an average size of 400 bp, and then
immunoprecipitated ß-catenin-containing complexes
(Fig. 6A,B). In samples
immunoprecipitated with anti-ß-catenin antibody, we detected `target
sequences' by PCR using primers that encompassed one of the putative TCF/Lef
binding sites in the promoters of Pax8, Ccnd1 and Emx2, and
these displayed strong enrichment compared with control immunoprecipitates (no
antibody) (Fig. 6C). We also
found enrichment of a conserved TCF/Lef motif in intron 1 of Pax8 and
of a previously published TCF/Lef site 3' to the Emx2 gene
(Theil et al., 2002)
(Fig. 6C). Conversely, when
using primers amplifying `off-target' sequences that lacked conserved TCF/Lef
sites within the same genes, we did not observe such enrichment
(Fig. 6C). These results
confirmed that ß-catenin-containing transcriptional complexes
specifically associated with those promoter regions containing TCF/Lef binding
sites and, to our knowledge, provide the first in vivo ChIP data on embryonic
kidneys. Overall, these results revealed a good correlation between the in
vivo expression pattern of predicted ß-catenin/TCF/Lef target genes, the
binding of ß-catenin-containing protein complexes to their predicted
TCF/Lef consensus binding motifs, and the expression of TCF/Lef reporter
activity.
|
; Kispert et al.,
1998). To determine whether ß-catenin signaling was
sufficient to phenocopy these responses, we induced expression of stabilized
ß-catenin in the metanephric mesenchyme by adenoviral gene transfer. As
expected, metanephric mesenchyme infected with the control virus Ad-GFP and
cultured in basal media in the absence of growth factors underwent apoptosis
(Fig. 7A). After 3 days of
culture, the entire Pax2-expressing pool of epithelial progenitors stained
positive for activated caspase 3, a marker of apoptotic cells
(Fig. 7A). By contrast,
metanephric mesenchymes infected with Ad-CTNNBS37A were salvaged
from apoptosis. This was evident after 2-3 days of culture, when aggregates of
progenitor cells expanded and formed clusters (100% of infected metanephric
mesenchymes, n>20, Fig.
7A). These cell aggregates were positive for Pax2 indicating that
they represented the nephric epithelial lineage (n=5,
Fig. 7A). The absence of
activated caspase 3 in these nodules demonstrated that these Pax2-positive
cells were selectively spared from apoptosis (n=5,
Fig. 7A). By contrast, cells
outside of the Pax2-positive cell population, despite efficient infection (see
Fig. 7C), were not rescued and
underwent apoptosis (Fig. 7A).
These data indicated that an activation of ß-catenin signaling exerted
anti-apoptotic effects specifically in epithelial progenitors. To test whether an activation of ß-catenin signaling in Pax2-positive epithelial progenitors promoted proliferation in addition to this anti-apoptotic effect, we assayed the number of proliferating cells in the progenitor compartment. Co-staining for Pax2 and phospho-histone H3, a marker of cells in metaphase, revealed that approximately 10% of epithelial progenitors were undergoing cell division 24 hours after infection with Ad-CTNNBS37A (n=4, Fig. 7B). Conversely, under control conditions (Ad-GFP), Pax2-positive epithelial progenitors showed no evidence of proliferation, although a few phospho-histone H3-positive cells were observed in the Pax2-negative cell population (Fig. 7B). This result indicated that ß-catenin signaling induced proliferation in epithelial progenitor cells.
|
|
Finally, we tested whether ß-catenin signaling alone was sufficient to induce epithelial differentiation of the metanephric mesenchyme. For this purpose, we examined the histomorphology of the Pax2-positive cell aggregates induced by stabilized ß-catenin. However, even with extended incubation in vitro for up to 7 days (a time period after which tubulogenesis is robustly observed with all known epithelial inducers), none of these aggregates displayed evidence of tubulogenesis. In contrast to Gsk3ß inhibitors, which induced either unsegmented (10 mM lithium chloride) or segmented (4 µM BIO) renal epithelia (Fig. 7D,E), Ad-CTNNBS37A induced the formation of virtually unorganized cellular clusters (n=5, Fig. 7D) and Cdh1, a marker of polarized epithelial cells in the kidney, remained absent in these aggregates (n=10, Fig. 7E). In addition, by real-time RT-PCR, several markers of mature renal epithelial cells, including Cdh1, Lama1 and Cdh16, failed to be upregulated, even after 7 days in culture (Fig. 7F). To exclude the possibility that this response was related to an excess level of ß-catenin signaling in the presence of the mutant stabilized ß-catenin construct, we also tested the effect of Ad-CTNNBWT, a virus expressing wild-type non-stabilized ß-catenin. Although this treatment was sufficient to induce survival of Pax2-positive cell aggregates, they were substantially smaller in size than those induced by stabilized ß-catenin, but also did not initiate tubulogenesis or express the above markers of differentiated epithelial cells (data not shown). These results indicated that ß-catenin signaling alone, as opposed to Gsk3ß inhibition, was insufficient to induce the complete program of epithelial differentiation in the metanephric mesenchyme.
Metanephric progenitors with compromised TCF/Lef signaling are progressively depleted from the epithelial lineage
Although the above results indicated sufficiency of ß-catenin/TCF/Lef
signaling to maintain viability and control cellularity of the metanephric
mesenchymal progenitor pool in the absence of exogenous growth factors, it was
unclear whether this pathway was also required for these processes in the
experimental setting of epithelial differentiation. To test this, we examined
the effect of compromised TCF/Lef signaling in epithelial progenitors in the
organ culture system. We infected freshly isolated metanephric mesenchymes
with either Ad-DN-TCF or Ad-GFP and cultured them in the presence of
Tgf, Fgf2 and Lif to induce epithelial conversion. Viruses were removed
after 14 hours and culture was continued in virus-free inductive media to
prevent continuous infection of differentiating cells during the course of the
experiment. The GFP tags on both constructs enabled us to follow the fate of
infected progenitor cells during the process of epithelial differentiation.
Initial infection rates of Pax2-positive epithelial progenitors by Ad-GFP and
Ad-DN-TCF (determined 14 hours after infection) were similar (n=5,
Fig. 8A,B). However, during the
course of the experiment, the fraction of Pax2-positive progenitors expressing
DN-TCF was progressively reduced, whereas the fraction of GFP-expressing
Pax2-positive progenitors remained similar
(Fig. 8A,B). Following
epithelial differentiation after 6 days of culture, DN-TCF-expressing cells
were depleted from both the Pax2-positive and Cdh1-negative epithelial
progenitor pool and from Pax2-positive Cdh1-positive epithelial progeny
(n=22, Fig. 8A,B).
Co-staining for activated caspase 3 after 48 hours of culture revealed
frequent apoptosis in cells expressing dominant-negative TCF, but not in
control cells expressing GFP, suggesting that compromised TCF/Lef signaling in
these cells promoted apoptosis (n=3,
Fig. 8A). Cells expressing
dominant-negative TCF were occasionally observed in Cdh1-positive epithelia
(Fig. 8B), suggesting that they
were still competent to undergo epithelial conversion, but we cannot exclude
the possibility that they were infected subsequent to epithelial conversion by
small amounts of residual virus in the culture media. In summary, these data
unequivocally demonstrated that TCF/Lef signaling is crucially involved in the
regulation of cellularity in the epithelial lineage and suggested a
requirement of this pathway for cell survival and stage progression during
epithelial differentiation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Herzlinger et al., 1994;
Kispert et al., 1998;
Stark et al., 1994), these
data provide the first evidence that TCF/Lef signaling constitutes a central
downstream signal.
Although part of our data rely on experiments on cultured explants of rat
metanephric mesenchyme, several facts point to the relevance of our
observations in vivo and in other species, e.g. mouse. First, the
transcriptional program induced in the course of epithelial differentiation in
our organ culture system is associated with an activation of a transcriptional
signature that closely recapitulates gene expression in the developing kidney
in vivo. Second, the ß-catenin/TCF/Lef target genes identified in the in
vitro system are activated in close temporal and spatial correlation in vivo
in pretubular aggregates and in the emerging early epithelia and their sites
correlate with that of a TCF/Lef reporter in vivo. Third, TCF/Lef target gene
expression in vivo is identical in rat and mouse kidneys pointing towards a
conserved process. Fourth, the promoter region of ß-catenin/TCF/Lef
target genes identified in vitro is occupied by ß-catenin-containing
transcriptional complexes in vivo, implying that ß-catenin and its
downstream targets might be directly involved in their regulation.
Furthermore, Wnt1, which triggers ß-catenin-dependent signaling in many
cell types (although this remains to be confirmed in the case of the
metanephric mesenchyme), when misexpressed from the ureteric bud of
Wnt9b-deficient mice, rescues a transcriptional program downstream of Wnt9b
(Carroll et al., 2005). This
program includes Pax8, a gene that we identify here to be a
TCF/Lef-dependent target of ß-catenin signaling in the metanephric
mesenchyme. Together, these observations suggest that ß-catenin signaling
controls a differentiation-associated transcriptional subprogram in
differentiating cells of the renal epithelial lineage.
Previous studies have suggested a role for ß-catenin signaling during
differentiation of the developing renal epithelial lineage in vitro. First, in
the isolated metanephric mesenchyme from rats or mice, co-cultivation of
fibroblasts expressing Wnt1 triggers survival and differentiation of tubular
and glomerular-like epithelia (Herzlinger
et al., 1994; Kispert et al.,
1998). Secondly, lithium chloride and BIO, which increase cellular
ß-catenin levels by inhibiting Gsk3ß
(Klein and Melton, 1996;
Sato et al., 2004), induce
epithelial structures in isolated metanephric mesenchymes from rats or mice
(Davies and Garrod, 1995;
Kuure et al., 2007). Third, in
the model of Lif-induced differentiation of the rat metanephric mesenchyme,
epithelial differentiation is accompanied by an occupation of TCF/Lef sites in
mobility shift assays and is blocked by exogenous application of Sfrp1, a
secreted WNT antagonist (Plisov et al.,
2001). Now, our study provides the first functional link between
ß-catenin/TCF/Lef signaling and anti-apoptotic and proliferative effects
in the epithelial lineage and defines a set of target genes in epithelial
progenitors. However, ß-catenin signaling in our model phenocopies only
part of the effects of Gsk3ß inhibitors or WNT-expressing cell lines in
that we detect neither epithelial cells nor polarized tubules, hallmarks
induced by Gsk3ß inhibitors and co-expressed WNTs. In a recent study,
lotus lectin- and peanut agglutinin-positive structures were detected
following homozygous deletion of exon 3 of ß-catenin in metanephric
mesenchyme suggesting that stabilization of ß-catenin might be sufficient
to trigger expression of surface markers of proximal tubules and glomeruli
(Kuure et al., 2007). Their
data and ours strongly support the concept that ß-catenin signaling
induces stage progression in the epithelial lineage. However, additional
pathways downstream of WNT signaling and Gsk3ß inhibitors appear to
cooperate with ß-catenin/TCF/Lef signaling to induce polarized and
segmented renal epithelia.
An important remaining question is the nature of the extracellular signal
that triggers ß-catenin/TCF/Lef signaling in the organ culture system and
in vivo. Wnt4 seems an obvious candidate based on an upregulation we observe
in differentiating metanephric explants and co-expression of Wnt4 and TCF/Lef
target genes in vivo. Furthermore, in the present study, inhibition of
ß-catenin/TCF/Lef signaling in epithelial progenitors decreases but
incompletely abolishes their participation in epithelial differentiation,
which is reminiscent of the reduction, but not complete absence, of epithelial
progeny in Wnt4-deficient kidneys
(Kobayashi et al., 2005).
Wnt9b and Wnt7b, both of which are formed by the ureteric bud, may participate
in controlling ß-catenin signaling, but the fact that we observe
activation of the TCF/Lef-responsive transcriptional program in the absence of
the ureteric bud and surrogate WNT molecules argues for a mesenchymal-derived
autocrine signal that activates ß-catenin/TCF/Lef signaling. In this
regard, it is also notable that Frzb, a class B gene expressed in
pretubular aggregates, has been shown to act as an antagonist of
WNT/ß-catenin signaling (Lin et al.,
1997). This secreted molecule might act to sharpen the gradient of
TCF/Lef transcriptional activity in the vicinity of the pretubular
aggregate.
In summary, our data suggest that ß-catenin/TCF/Lef drives a transcriptional program in differentiating renal epithelia that participates in maintenance, proliferation and stage progression of the renal epithelial lineage. Additional transcriptional programs downstream or in parallel to this pathway might participate in epithelial differentiation, and the global transcriptional profile of epithelial conversion identified herein will provide a valuable tool to elucidate their molecular nature.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/17/3177/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work 
Present address: Bioinformatics Centre and Department of Psychiatry,
University of British Columbia, Vancouver, BC, Canada
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
