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3545
Introduction
Embryonic stem (ES) cells are pluripotent cells derived from
the inner cell mass of the blastocyst stage embryo that can
differentiate in vivo and in vitro into all cell lineages of the
adult animal. The ability of these cells to adopt multiple fates
makes ES cells prime candidates for use in stem cell therapies.
However, unmodified ES cells cannot be used in regenerative
therapies because they form teratomas after transplantation.
In successful transplantation studies, ES cells are
predifferentiated into the desired cell types, which may then
integrate into the transplanted tissue (Brustle et al., 1999; Chen
and Mok, 1995; Dinsmore et al., 1996; Liu et al., 2000). Hence,
an understanding of how ES cells differentiate in vitro
into neural cells is essential for developing and refining
transplantation strategies for the nervous system, as well as for
understanding the molecular mechanisms underlying
neurogenesis.
A commonly used technique for differentiating ES cells into
the neural lineage involves treatment of the cells with all-
trans retinoic acid (RA) (Bain et al., 1995). However, the
mechanisms by which ES cells are committed to the neural
lineage using this technique are poorly understood. RA-
mediated neural differentiation requires growth of ES cells as
embryoid bodies (EBs) for 4 days prior to the RA treatment
(Bain et al., 1995), indicating that certain as yet undefined
events must occur within the EB to make the cells responsive
to the neurogenic effects of RA. Other techniques for
promoting neural differentiation of ES cells include inhibition
of BMP signaling (Gratsch and O’Shea, 2002), growth at low
density (Tropepe et al., 2001; Ying et al., 2003) and co-culture
with PA6 cells (Kawasaki et al., 2000). However, these
protocols all require culture at very low densities and cell-
cell/matrix interactions for neuronal differentiation, and the
mechanisms underlying neuronal lineage commitment induced
by these techniques remain unclear. Furthermore, the need
to culture the cells at low density to achieve neuronal
differentiation limits the number of cells that could potentially
be obtained for transplantation strategies.
One candidate pathway for neural lineage commitment by
ES cells is the Wnt/!-catenin pathway that has been shown to
be a cell-cell (and/or cell-matrix) contact-regulated inducer of
neurogenesis (Patapoutian and Reichardt, 2000). !-catenin
exists in three cellular pools: membrane bound, cytoplasmic
and nuclear. Membrane-bound !-catenin is associated with
E-cadherin/adherens junctions and functions to bridge E-
cadherin to the cytoskeleton (Aberle et al., 1996; Gumbiner
Culture of embryonic stem (ES) cells at high density
inhibits both !-catenin signaling and neural differentiation.
ES cell density does not influence !-catenin expression,
but a greater proportion of !-catenin is targeted for
degradation in high-density cultures. Moreover, in high-
density cultures, !-catenin is preferentially localized to the
membrane further reducing !-catenin signaling. Increasing
!-catenin signaling by treatment with Wnt3a-conditioned
medium, by overexpression of !-catenin, or by
overexpression of a dominant-negative form of E-cadherin
promotes neurogenesis. Furthermore, !-catenin signaling
is sufficient to induce neurogenesis in high-density cultures
even in the absence of retinoic acid (RA), although RA
potentiates the effects of !-catenin. By contrast, RA does
not induce neurogenesis in high-density cultures in the
absence of !-catenin signaling. Truncation of the armadillo
domain of !-catenin, but not the C terminus or the N
terminus, eliminates its proneural effects. The proneural
effects of !-catenin reflect enhanced lineage commitment
rather than proliferation of neural progenitor cells.
Neurons induced by !-catenin overexpression either alone
or in association with RA express the caudal neuronal
marker Hoxc4. However, RA treatment inhibits the !-
catenin-mediated generation of tyrosine hydroxylase-
positive neurons, suggesting that not all of the effects of
RA are dependent upon !-catenin signaling. These
observations suggest that !-catenin signaling promotes
neural lineage commitment by ES cells, and that !-catenin
signaling may be a necessary co-factor for RA-mediated
neuronal differentiation. Further, enhancement of !-
catenin signaling with RA treatment significantly increases
the numbers of neurons generated from ES cells, thus
suggesting a method for obtaining large numbers of neural
species for possible use in for ES cell transplantation.
Key words: Embryonic stem cells, !-Catenin, Neurogenesis,
Retinoic acid, Tyrosine hydroxylase, Cell density
Summary
!-Catenin signaling is required for neural differentiation of
embryonic stem cells
José Javier Otero*, Weimin Fu, Lixin Kan, Adolfo E. Cuadra and John A. Kessler*
Department of Neurology, Northwestern University Feinberg School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611,
USA
*Authors for correspondence (e-mail: j-otero@northwestern.edu and jakessler@northwestern.edu)
Accepted 13 April 2004
Development 131, 3545-3557
Published by The Company of Biologists 2004
doi:10.1242/dev.01218
Research article
3546
and McCrea, 1993). In the cytoplasm, !-catenin turnover is
regulated by a stepwise phosphorylation on its N terminus.
Initially, !-catenin is phosphorylated on Ser45 by a ‘priming’
kinase (Liu et al., 2002). Phosphorylation at Ser45 primes !-
catenin for phosphorylation at Ser33/37;Thr41 by Gsk3!
(Kang et al., 2002). Phospho-Ser33/37;Thr41-!-catenin is then
targeted for ubiquitin-directed proteolysis (Salic et al., 2000a),
and increased phosphorylation by Gsk3!decreases the nuclear
pool of !-catenin (Lucas et al., 2001). During Wnt signaling,
Gsk3!is inhibited, leading to the accumulation of
unphosphorylated !-catenin in the cytoplasm and its
translocation to the nucleus, where it interacts with members
of the TCF/LEF family of transcription factors. The interaction
of !-catenin with TCF/LEF transcription factors causes both
inhibition of repression and activation of transcription.
However, both Wnt and !-catenin can signal through other
independent pathways (Korswagen, 2002; Kuhl, 2002; Pandur
et al., 2002a; Pandur et al., 2002b; Tada et al., 2002; Yamanaka
et al., 2002).
This study addresses the role of !-catenin signaling in neural
lineage commitment by ES cells. We find that induction of
differentiation at high density inhibits both !-catenin signaling
and neural differentiation. However, overexpression of !-
catenin is sufficient to induce neuronal lineage commitment
even in the absence of RA or EB formation, and, unlike RA,
can induce neuronal differentiation in high-density cultures.
Materials and methods
Cell culture
The WW6 ES cell (Ioffe et al., 1995) line was obtained from Dr
Winfred Edelman (Albert Einstein College of Medicine). ES cells
were cultured without feeder cells on gelatin-coated tissue culture
flasks, in media described previously (Bain et al., 1995) with Lif
(ESGRO, Chemicon). Cells were routinely observed for SSEA-1
(Fut4 – Mouse Genome Informatics) and Oct4 staining by
immunofluorescence, and were never kept in continued cell culture
for more than 15 passages. For EB inductions, dissociated ES cells
were counted by Trypan Blue exclusion and seeded at indicated
densities into 10 cm petri dishes (Falcon) in induction media (4–/4+
protocol) without feeder cells (Bain et al., 1995). Induction media was
identical to the cell culture media except that Lif and !-
mercaptoethanol was not added. On day 4, 5"10–7 M all-trans RA
(Sigma) was added. On the eighth day of the induction, EBs were
dissociated by incubation with trypsin for 5-10 minutes and plated
onto PDL/laminin-coated glass coverslips, in Neurobasal Media
supplemented with B27 and L-glutamine (Life Technologies). As
serum and B27 supplement contain retinoids that could be converted
to active RA by the cells (Brewer et al., 1993), Knock Out Serum
Replacement induction media was used [serum in induction media
was replaced with 15% Knock Out Serum Replacement (Life
Technologies)] for retinoid starvation studies. Knock Out Serum
Relpacement (KOSR) is a serum supplement designed to culture
undifferentiated ES cells in the presence of Lif. For the retinoid
starvation studies, cells were plated on PDL/laminin-coated coverslips
in media composed of 50% Knock Out DMEM/50% neurobasal
media, supplemented with N2 supplement and 7.5% KOSR.
Cells were fixed and stained 3-4 days after plating on the PDL/
laminin-coated coverslips. In addition to EB-mediated neuronal
differentiation, neuronal induction protocols were performed as
described previously (Gratsch and O’Shea, 2002; Ying et al., 2003).
In the EB-independent protocol, cells were seeded onto gelatinized
coverslips at 0.5-1.5"104cells/cm2in DMEM/F12 with N2 and B27
supplements. In order to test density-dependent regulation of this EB-
independent induction protocol, ES cells were plated at 1"106
cells/cm2in DMEM/F12 with N2 and B27 supplements, and fixed for
immunostaining 8-10 days after plating.
BrdU incorporation assay
To test for differences in proliferation in undifferentiated ES cells
stably transfected with either empty vector or !-catenin #N, cells were
grown on gelatin-coated coverslips in ES cell culture media plus Lif
and pulsed for 3 hours with BrdU. To test for differences in
proliferation between ES cells stably transfected with either empty
vector or !-catenin #N during neural differentiation, cells were
induced at 104cells/cm2without Lif, as described previously (Ying et
al., 2003). Cells were pulsed for 3 hours with BrdU at day 6 post-Lif
withdrawal. BrdU-labeled cells were detected by staining with anti-
BrdU antibodies (Chemicon).
Generation of stable cell lines
The effects of !-catenin on lineage commitment by ES cells were
examined by transfecting the cells with various constructs of the !-
catenin molecule and H2kd-E-cadherin [dominant-negative E-
cadherin, a kind gift of Dr Fiona Watt (Zhu and Watt, 1996)]. H2kd-
E-cadherin is a chimeric protein composed of the extracellular domain
of the murine MHC class I antigen H2kd fused to the cytoplasmic and
transmembrane domains of E-cadherin (16 amino acids of the
extracellular domain of E-cadherin are also present). H2kd-E-
cadherin has been shown to decrease endogenous levels of E-cadherin,
increase protein levels of !-catenin and increase !-catenin signaling
(Vizirianakis et al., 2002; Zhu and Watt, 1996). As ES cells undergo
several rounds of mitosis during the process of differentiation in vitro,
stably transfected cell lines were established to avoid possible effects
due to dilution of transiently transfected cells and/or preferential
proliferation of nontransfected cells. The genes of interest were placed
in the pcDNA3.1 expression plasmid (Invitrogen) under the control of
a CMV promoter. Constitutively active promoters are widely used to
study in vitro differentiation of ES cells (Chung et al., 2002; Gratsch
and O’Shea, 2002). The truncation mutations were as follows: !-
catenin total, full-length protein; !-catenin #N, first 128 amino acids
truncated; !-catenin #C, last 113 amino acids truncated; and !-catenin
#Armadillo, armadillo repeats deleted (Fig. 4). Cells were transfected
by electroporation. Selection media containing 250 µg/ml geneticin
(Life Technologies) was added 2-3 days post-electroporation.
Colonies of geneticin-resistant ES cells were picked and expanded.
Genomic DNA was extracted by proteinase K digestion and analyzed
for insertion of plasmid DNA by PCR. PCR-positive cells were
expanded in selection media, whereas differentiation experiments
were performed in media without geneticin. Stable cell lines were
routinely tested by PCR of genomic DNA to ensure continued
integration of the construct (data not shown). None of the cell lines
were observed to alter expression of SSEA-1 and Oct4, or to alter the
undifferentiated morphology (data not shown). All experiments were
repeated in independently derived clones to control for possible
positional effects.
Noggin treatment
Noggin-Fc chimera (R&D Systems, ED50=0.1-1 µg/ml) was used to
inhibit BMP function at concentrations from 50 ng/ml to 2 µg/ml in
the induction media. The higher concentration is sufficient to block
the effects of 100 ng/ml of BMP added exogenously to cultures of
neural stem cells (data not shown). The ES cells were seeded at high
density (106cells/ml) in induction media and treated with noggin-Fc
from day 0 to day 8. EBs were plated onto PDL/laminin-coated
coverslips in Neurobasal plus B27 supplement.
Wnt treatment
Fibroblasts secreting Wnt3a (ATCC CRL-2647) and control,
untransfected fibroblasts (ATCC CRL-2648) were used to create
conditioned media following the protocol recommended by ATCC.
Development 131 (15) Research article
3547!-catenin induces neuronal lineage commitment by ES cells
Conditioned media was used instead of induction media for the 4–/6+
protocol, with ES cells seeded at high density (106cells/ml). EBs
were dissociated and plated on PDL/laminin-coated coverslips in
Neurobasal plus B27 supplement.
Western blotting
Cell lysates were isolated by using Cell Lysis Buffer (Roche
Molecular Biochemicals) and protein concentration was quantified by
using the BCA Protein Assay Kit (Pierce). Antibodies used were anti-
!-catenin (Santa Cruz), anti-N-terminal-phospho !-catenin (phospho-
Ser33/37/Thr41 and phospho-Ser45/Thr41 sites were blotted, Cell
Signaling), and anti-actin (Santa Cruz). Secondary antibodies
conjugated to horse radish peroxidase were purchased from Santa
Cruz. Detection of signal was performed by the Luminol
Chemiluminescence Kit (Roche Molecular Biochemicals).
Immunocytochemistry
Cells plated on coverslips (Carolina Science and Math) were fixed in
freshly prepared 4% paraformaldehyde and permeabilized with 0.2%
Triton X-100. Primary antibodie s used were as foll ows: anti-!-tubulin
3 (Sigma), anti-GABA (Sigma), anti-GFAP (Sigma), anti-Hoxc4
(BABCo), anti-neurofilament 200 (Sigma), anti-nestin (Chemicon),
anti-NeuN (Chemicon), anti-Oct4 (Chemicon), anti-tyrosine
hydroxylase (Sigma) and anti-SSEA-1 (Developmental Studies
Hybridoma Bank). Appropriate secondary antibodies were purchased
from Southern Biotechnology Associates. Coverslips were mounted
onto glass slides (Fisher) with Antifade Kit (Molecular Probes).
Quantification of percentage of cells immunoreactive for specific
antigens was determined by capturing images from random fields.
Hoechst-staining nuclei and cells positive for the markers indicated
(e.g. nestin, !-tubulin 3, BrdU; numerator=stain, denominator=
Hoechst-positive nuclei) were counted.
Confocal microscopy
Day 2 EBs were fixed, permeabilized, and stained with anti-!-catenin
(Santa Cruz) antibody in aggregate form in 15 ml conical centrifuge
tubes. Cells were then resuspended in Antifade reagent (Molecular
Probes) and placed on glass slides. For dissociated cultures, cells were
plated at 106cells/cm2on gelatin-coated coverslips. Analysis was
performed on a Zeiss LSM 510 Laser Scanning Confocal Microscope.
Fluorescence intensity over cross-sections of the cells was analyzed
by Metamorph software (v6.0).
Pitx2
RT-PCR
Total RNA was extracted using trizol (Invitrogen) and first strand
cDNA synthesis was performed using the Thermoscropt rt-PCR kit
(Invitrogen). Pitx2 forward primer, ACG GAT CCA TGA ACT GCA
TGA AAG GCC CGC TG; Pitx2 reverse primer, TTT CTA GAT CAC
ACC GGC CGG TCG ACT GC; actin forward primer, GTG AAA
AGA TGA CCC AGA TC; actin reverse primer, TCA TGG ATG CCA
CAG GAT TC. PCR was performed for 25 cycles.
TOPFLASH assay
In order to assay !-catenin signaling, the TCF/LEF-TOPflash
construct [a kind gift of H. Clevers (van de Wetering et al., 1997),
from hereon referred to as TCF/LEF-luciferase] was used. This
promoter has multiple TCF/LEF consensus sites driving luciferase
transcription. Total luminescence from the lysates was normalized by
the activity of TK-renilla luciferase to control for the difference in
transfection efficiencies between the experimental samples. Cell
lysates were then used to measure luciferase activity. Luciferase
assays were performed in EBs by transiently transfecting
undifferentiated ES cells with TCF/LEF luciferase and TK-renilla
luciferase (Fugene 6, Roche Molecular Biochemicals). After transient
transfection, the cells were induced by EB formation at either high
or low densities and incubated overnight. For the Wnt-conditioned
media experiment, HEK293 cells were transfected with TCF/LEF-
TOPFLASH and treated with control conditioned, Wnt3a-conditioned
media for 3 days, or co-transfected with H2kd-E-cadherin with
TCF/LEF-luc. For !-catenin experiments, HEK293 cells were co-
transfected with 5 ng TK-renilla luciferase, 0.3 µg TCF/LEF-
Luciferase with or without different !-catenin expression constructs
(0.1 µg transfected for low dosage and 0.3 µg transfected for high
dosage) (Fig. 4). !-catenin constructs used were pcDNA3.1 (CMV-!-
cat) or the retroviral pCLE (!-cat, !-cat#N, !-cat#C, !-cat#NC and
!-cat#Arm). The promoter in the pCLE plasmid is known to be
weaker than the CMV promoter in pcDNA3.1. Activity was detected
by using a Berthold Luminomitor. The yaxis in all TOPFLASH assays
is defined as Relative Luciferase units, which we define as total
luciferase activity from the tested promoter divided by TK-renilla
luciferase activity so as to normalize for variability in transfection
efficiency.
Electrophysiology
Cells were examined for voltage gated-channel function using the
whole-cell patch clamp technique. All recordings were performed at
room temperature using a Leica DM-IRB inverted microscope.
Electrodes were fashioned from borosillicate glass, G150-F4 (Warner
Instruments, Hampden, CT), pulled from Sutter instruments P97
horizontal pipette puller (Novato, CA) and fire polished with
Narishigue MF-83 microforge (Setayaga-Ku, Japan). Pipettes had a
resistance of 4-8 m$when filled with pipette solution containing 105
mM KCl, 5 mM K-EGTA, 0.5 mM MgCl2, 5 mM Mg-ATP, 5 mM
Na-phosphocreatinine, 5 mM K-phophoenopyruvate, with osmolarity
set to 255 milliosmoles and pH adjusted pH 7.3 with KOH. Cells were
perfused in bath solution containing 115 mM NaCl, 5 mM HEPES, 5
mM KCl, 2 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, with
osmolarity set to 260 milliosmoles and pH adjusted to 7.4 with NaOH.
Recordings were obtained with Axon, Axopatch 200B amplifier onto
a PC using Axon pClamp 9.0 Acquisition Software (Union City, CA).
Recordings were sampled at 5 kHz using an eight pole lowpass bessel
filter set at 2 kHz. Capacitative currents were compensated using built-
in analog circuits with series resistance error corrected to a minimum
of 70%. For voltage gated-channel experiments, cells were maintained
at –80 mV holding potential and depolarized stepwise for 100 ms,
from –60 to +50 mV in 10 mV increments applied at 0.1 Hz.
Results
Culture at high density inhibits !-catenin signaling
The Wnt/!-catenin signaling cascade is a crucial contact-
regulated process that is involved in neurogenesis (Baker et al.,
1999; Cho and Dressler, 1998; Dorsky et al., 1998). We found
that when ES cells were induced to differentiate by EB
formation at high density (106cells/ml), a greater proportion
of !-catenin was phosphorylated on its N terminus, although
the total amount of !-catenin remained unchanged (Fig. 1A).
Phosphorylation of !-catenin at residues Ser33/37;Thr41
targets it for ubiquitin-directed proteolysis (Brantjes et al.,
2002) and inhibits nuclear !-catenin (Lucas et al., 2001),
therefore suggesting that !-catenin signaling is more active
when ES cells are differentiated at low density.
As the subcellular localization of !-catenin is regulated by
cell density in some cell types (Dietrich et al., 2002), the
localization of !-catenin was also compared between low- and
high-density cultures. Day 2 EBs seeded from either low (105
cells/ml) or high (106 cells/ml) densities were fixed and stained
with anti-!-catenin antibodies and analyzed by confocal
microscopy (Fig. 1B,C). In the high-density cultures, !-catenin
was localized primarily on the cell membrane (Fig. 1C) with
lower levels in the cytoplasm, whereas in low-density cultures
3548
it was diffusely localized throughout the cell (Fig. 1B). To
analyze the distribution of !-catenin objectively and
quantitatively, optical sections were analyzed for fluorescence
intensity (see Materials and methods). Graphing the
fluorescence intensity over a cross-section of high-density EBs
revealed many peaks in fluorescence intensity at the cell
membrane, and valleys in other cellular compartments (Fig.
1C,C%). By contrast, low-density EBs showed a very
diffuse/nonlocalized pattern of staining, indicating that !-
catenin was not localized to any specific subcellular
compartment (Fig. 1B,B%). Differences between the peaks and
the valleys of fluorescence intensity in the low-density and
high-density seeded cultures were plotted graphically (Fig. 1D)
and analyzed. The peak-valley differences in high-density
seeded EBs were significantly larger than in the low-density
seeded EBs, substantiating the visual observation of increased
membrane localization of !-catenin in high-density cultures
(Fig. 1D).
Culture at high density reduces TCF/LEF dependent
transcription
To determine whether the differences in phosphorylation and
localization of !-catenin between high and low density cultures
resulted in differences in TCF/LEF-dependent transcription,
undifferentiated ES cells were transiently transfected with a
TCF/LEF-luciferase plasmid (see Materials and methods) and
then seeded for EB formation at either high or low densities.
A dual luciferase reporting system with sufficient sensitivity
and reproducibility to detect possible decreases in baseline
TCF/LEF transcription was used. In fact, seeding EBs at high
density resulted in a 2-fold repression of baseline luciferase
activity when compared with luciferase activity in low-density
cultures (Fig. 1E). In order to confirm this result, we examined
the expression of Pitx2, a homeobox transcription factor
involved in gabaergic neuron differentiation that is downstream
of Wnt/!-catenin (Chazaud et al., 1999; Kioussi et al., 2002;
Martin et al., 2002; Westmoreland et al., 2001). RT-PCR
analysis revealed that levels of Pitx2 mRNA were significantly
higher in the low-density cultures compared with the high-
density cultures (Fig. 1F), supporting the conclusion that !-
catenin signaling is more active in low-density cultures.
Neural differentiation of ES cells is inhibited at high
density
To define the effects of cell density on neuronal lineage
commitment, ES cells dissociated from monolayer cultures
were induced by the 4–/4+ protocol (see Materials and
methods) at various densities. Cells were then plated onto
PDL/laminin-coated coverslips. Four days after plating the
cells were stained with antibodies to !-tubulin 3 (red) and
Development 131 (15) Research article
Fig. 1. Phosphorylation of !-catenin is inhibited
in low-density cultures. (A) Western blot analyses
of total cell lysates extracted on days 2 (d2) and 4
(d4) after ES cells were seeded for EB formation
at low (LD, 105cells/ml) and high densities (HD,
106cells/ml). Density did not alter the levels of
total !-catenin protein. However, the levels of
phospho-!-catenin were significantly reduced in
the low-density cultures when compared with the
same time point in the high-density cultures using
both antibodies (lower two blots). ES cells were
seeded for EB formation at either low densities
(B) or high densities (C) and stained with anti-!-
catenin antibody. Fluorescence intensity over a
random cross-section of an EB demonstrated
diffuse staining in low-density EBs (B%), whereas
high-density EBs resulted in peaks and valleys in
fluorescent intensities (C%). (D) The difference in
fluorescence intensity (*P<0.05, **P<0.01)
between the peaks and valleys was quantified and
plotted graphically. HD EBs had a higher average
difference between the peaks and the valleys,
demonstrating that !-catenin staining is more
localized (B%,C%,D, yaxis shows fluorescence
intensity). (E) Undifferentiated ES cells were
transiently transfected with an artificial TCF/LEF
promoter and then seeded at high and low
densities. Total luciferase activity was then
assayed at day 1 post-seeding. High-density EBs
repressed basal levels of TCF/LEF activity 2-fold
(yaxis shows relative luciferase units). (F) ES
cells were differentiated by EB formation at low
(105cells/ml) and high (106cells/ml) densities.
On day 4, RT-PCR for Pitx2 was performed in
cells treated with RA for 2 hours. Pitx2 is
upregulated at low density by RA treatment but
not at high density.
3549!-catenin induces neuronal lineage commitment by ES cells
nestin (green), and counterstained with Hoechst dye (blue)
(Fig. 2A-D). Plating at higher densities resulted in formation
of EBs that were larger than those formed at low plating
densities, and contact between EBs was increased in the high-
density cultures. The maximal percentage of neurons was
obtained when the cells were seeded at 105cells/ml (Fig. 2A).
As the seeding density was increased, the percentage of !-
tubulin 3-positive cells in the cultures decreased in a linear
fashion (Fig. 2A-D). Furthermore, as density increased, the
number of nestin-positive cells similarly decreased, indicating
that elevated density inhibited both neural and neuronal
differentiation (Fig. 2D). At low density without RA treatment,
there is a significant amount of neural differentiation, as
demonstrated by nestin immunoreactivity. However, RA
potentiates neural differentiation at low density by increasing
the percentage of cells that are committed to the neural lineage
(!-tubulin 3 + nestin-positive cells were 36% without RA and
73% with RA). If the RA-treated cultures are maintained for
more than three days post-plating, virtually all of the nestin-
positive cells observed go on to differentiate into GFAP-
positive cells. As BMP signaling has been reported to inhibit
neuronal differentiation of ES cells (Gratsch and O’Shea, 2002;
Kawasaki et al., 2000; Tropepe et al., 2001) high-density RA-
treated cultures were also treated with a BMP antagonist,
Noggin-Fc, to determine whether BMP signaling mediated the
inhibitory effects of high density. Treatment of the high-density
cultures with 50 ng/ml Noggin-Fc did not result in any
detectable neuronal differentiation, and treatment with a
saturating dose (2 µg/ml) resulted in minimal (<0.1% of cells)
neuronal differentiation. Furthermore, treatment of low-density
cultures (105cells/ml) with media preconditioned by high-
density cultures (106cells/ml) did not significantly inhibit
neuronal differentiation (data not shown), suggesting that the
inhibitory effect was not mediated solely by soluble factors.
Finally, using two different EB-independent protocols (Gratsch
and O’Shea, 2002; Ying et al., 2003), neuronal differentiation
was also found to be inhibited by culture at high density
(Fig. 7).
Stimulation of endogenous !-catenin overcomes
density-dependent inhibition of neural
differentiation in ES cells
Stimulation of endogenous !-catenin signaling was performed
by treatment with Wnt3a-conditioned media or by generation
of stably transfected ES cells expressing H2kd-E-cadherin, a
dominant-negative form of E-cadherin that has been shown
previously to increase cellular levels of !-catenin (Vizirianakis
et al., 2002; Zhu and Watt, 1996). The activity of these reagents
in the stimulation of !-catenin signaling was assayed both by
co-transfecting HEK293 cells with the TCF/LEF-luciferase
plasmid with H2kd-E-cadherin and by treating TCF/LEF-
luciferase-transfected HEK-293 cells with Wnt3a-conditioned
media (Fig. 3A). H2kd-E-cadherin increased TCF/LEF-
luciferase activity 2-fold, whereas treatment with Wnt3a-
conditioned media resulted in more than 3-fold activation. To
test whether Wnt3a-conditioned media could activate !-catenin
in ES cells, we seeded ES cells for EB formation at high
density in either control-conditioned media or Wnt3a-
conditioned media and total cell lysates were analyzed by
western blotting (Fig. 3B). Culture with Wnt3a-conditioned
media resulted in a decrease in the levels of P-Ser33/37;Thr41
!-catenin (Fig. 3B, lower blot). In addition, H2kd-E-cadherin-
expressing ES cells had higher levels of !-catenin protein
relative to controls, as described previously (Vizirianakis et al.,
2002; Zhu and Watt, 1996).
To determine whether increasing endogenous !-catenin
Fig. 2. Neural differentiation of ES cells is
regulated by cell density. ES cells were
induced to differentiate by the 4–/4+
protocol at increasing seeding densities.
After the induction, cells were plated onto
PDL/laminin-coated coverslips and fixed 3-4
days post-plating. Coverslips were stained
with anti-!-tubulin 3 (red) and anti-nestin
(green), and counterstained with Hoechst
dye (blue) to visualize nuclei. (A) 105
cells/ml seeding density; (B) 106 cells/ml
seeding density; (C) quantification of
neuronal differentiation at different seeding
densities (ND, none detected; error bars are
s.d.). (D) Quantification of nestin-positive
cells at low (105cells/ml) and high (106
cells/ml) densities with or without RA
treatment. Seeding at low density allows
significant neural differentiation. Higher
seeding density inhibits both neural and
neuronal differentiation (LD, low density;
HD, high density).
3550
signaling can overcome the inhibitory effects on differentiation
of culture at high density, ES cells treated with Wnt3a-
conditioned media or those expressing H2kd-E-cadherin were
induced at high density by the 4–/4+ protocol (Fig. 4C-G).
Conditioned media from untransfected cells was used as
acontrol. Cultures treated with Wnt3a-conditioned media
contained many !-tubulin 3-positive (Fig. 3D) and nestin-
positive cells. By contrast, virtually no cells immunoreactive
for !-tubulin 3 (Fig. 3E), GFAP or nestin were detected in
cultures treated with control media. Similarly, overexpression
of H2Kd-E-cadherin in ES cells also promoted neuronal
differentiation in high-density cultures (Fig. 3F), whereas cells
transfected with an empty vector did not (Fig. 3E). Taken
together, these observations suggest that increased !-catenin
signaling in RA-treated ES cells can largely prevent the
density-dependent inhibition of neural differentiation.
As both treatment with Wnt3a-conditioned media or
overexpression of H2Kd-E-cadherin could activate several
different signaling pathways, we directly tested whether
increasing !-catenin signaling alone can overcome the
inhibitory effects of increased cell density by stably
Development 131 (15) Research article
Fig. 3. Stimulation of endogenous !-catenin signaling overcomes the inhibitory effects of high density on RA-treated cultures. Stimulation of
endogenous signaling was mediated by treatment with Wnt3a-conditioned media and by overexpression of H2kd-E-cadherin. (A) HEK293 cells
were transfected with TCF/LEF-luciferase and tested for luciferase activity after treatment with Wnt3a-conditioned media or after co-
transfection of H2kd-E-cadherin with TCF/LEF-luc. H2kd-E-cadherin was able to increase luciferase activity, although Wnt3a treatment had a
greater effect. (B) Changes in !-catenin degradation were analyzed in ES cells after Wnt3a treatment. ES cells were induced by EB formation
in high density and were either treated with control-conditioned media or with Wnt3a-conditioned media. Total cell lysates were extracted on
day 2 of the differentiation (2 days Wnt3a treatment) and analyzed by western blotting (B). Treatment with Wnt3a-conditioned media reduced
the amount of phospho-!-catenin while not significantly altering the levels of total !-catenin. Wnt3a treatment resulted in a decrease in the
levels of phopshorylated !-catenin signaling. ES cells were induced by EB formation at high density in either control-conditioned media (C) or
Wnt3a-conditioned media (D), and differentiated by the 4–/4+ protocol. Wnt3a treatment resulted in many !-tubulin 3-positive cells (D),
whereas control media did not (C). Cells were stably transfected with an empty vector (E), H2kd-E-cadherin (F) or full-length !-catenin (G),
and induced by EB formation at high density using the 4–/4+ protocol. Overexpression of H2kd-E-cadherin and !-catenin resulted in neuronal
differentiation in high-density cultures, but transfection with empty vector did not. (C-G) All cells were fixed 3-4 days post-plating, and stained
with anti-!-tubulin 3 antibodies (green) and counterstained with Hoechst (blue).
Fig. 4. Modulation of !-catenin signaling regulates TCF/LEF
signaling. ES cells were stably transfected with the constructs
indicated (A). To test the activity of these constructs, we transiently
transfected HEK293 cells with the constructs indicated and a
TOPFLASH plasmid containing the TCF/LEF promoter driving a
luciferase reporter gene (B). The full-length and N-terminal
truncations were able to stimulate the TCF/LEF promoter but the C-
terminal truncation, the armadillo truncation, and the N- plus C-
terminal truncation constructs did not stimulate the promoter.
TCF/LEF driven transcription was also increased by increasing the
amount of !-catenin transfected into the cells.
3551!-catenin induces neuronal lineage commitment by ES cells
transfecting ES cells with !-catenin. Control cells were
transfected with a vector containing only the antibiotic
resistance gene. Lif was then withdrawn and the !-catenin
overproducing cells cultured at high density in the 4–/4+ EB
protocol (Fig. 3G). Overexpression of !-catenin resulted in
differentiation into !-tubulin 3-positive (Fig. 3G) and nestin-
positive (data not shown) cells whereas virtually no such cells
were present in the control cultures (Fig. 3E). This directly
demonstrates that stimulation of !-catenin pathways can
prevent the density-dependent inhibition of neural and
neuronal differentiation in RA-treated cultures.
!-catenin induced neuronal differentiation of ES
cells requires the armadillo domain and does not
require RA treatment
To determine whether !-catenin signaling can by itself induce
neurogenesis in ES cells in the absence of RA, and to define
the domains of the !-catenin molecule that might be involved,
we constructed various full-length and truncated constructs
of !-catenin (Fig. 4A). We first tested the ability of these
constructs to stimulate an artificial TCF/LEF-TOPFLASH
promoter (Fig. 4B). HEK293 cells were co-transfected with the
different !-catenin constructs and with a TCF/LEF promoter
driving a luciferase reporter gene. In agreement with other
studies (Peifer et al., 1991; van de Wetering et al., 1997;
Vleminckx et al., 1999), transfection with full-length !-
catenin, as well as with an N-terminal truncation of !-catenin
(!-catenin #N), increased transcription of the reporter gene.
The activity of !-catenin #N was several orders of magnitude
higher than the activity of the full-length !-catenin construct
as expected. By contrast, transfection of constructs with a
truncation of the C-terminal portion of the molecule (!-catenin
#C), or with a truncation of the armadillo domains (!-catenin
#Armadillo), did not transactivate the reporter gene in this
assay, suggesting that both the armadillo domain and the C
terminus are necessary for the transactivation of this promoter
in HEK293 cells. In addition, stimulation of the promoter was
dependent on the dosage of available !-catenin, as luciferase
activity increased when the amount of transfected !-catenin
pCLE plasmid (insert controlled by a retroviral LTR promoter)
was increased or when !-catenin-pcDNA3.1 (insert
transcription controlled by a stronger CMV promoter) was
used. These data demonstrate that TCF/LEF driven
transcription can be increased either by inhibiting Gsk3!-
targeted degradation or by artificially increasing levels of total
!-catenin.
To d ir ec tl y t est t he e ff ec ts of !-catenin on ES cell
differentiation and to determine which domains of !-catenin
were necessary for its function, we then stably transfected ES
cells with these various constructs of !-catenin. As serum and
B27 supplement contain retinoids that could be converted to
active RA by the cells (Brewer et al., 1993), the induction
protocol was slightly altered so that ES cells cultured in the
absence of RA were induced in a medium that lacks serum and
any retinoids (see Materials and methods). As mammals cannot
synthesize retinoids de novo, retinoid starvation has been
widely used as a simple way to study retinoid dependent
processes (Collins and Mao, 1999). After withdrawal of Lif and
the formation of EBs, the cells that overexpressed either !-
catenin #N or !-catenin #C differentiated into !-tubulin 3-
immunoreactive cells in the high-density cultures with or
without RA at all seeding densities tested (Fig. 5I-P). However,
RA treatment of !-catenin-transfected cultures potentiated the
neurogenic effects of !-catenin (Fig. 6A). By contrast, ES cells
transfected with empty vector or with the !-catenin #Armadillo
failed to differentiate into neurons in the high-density cultures,
although they were still able to differentiate into neurons in low-
density cultures treated with RA (Fig. 5A-H). !-tubulin 3-
Fig. 5. The armadillo domain mediates
neurogenesis promoted by !-catenin and RA
acts synergistically to enhance !-catenin-
mediated neurogenesis. Cells were seeded at
high (106cells/ml, HD) or low densities
(105cells/ml, LD) and treated with or without
RA (cells in panels C,G,K,O were grown in
KOSR media, see Materials and methods).
Cells were fixed and stained 3-4 days post-
plating with !-tubulin 3 (green) and
counterstained with Hoechst dye (blue).
(A-D) Cells transfected with empty vector.
(E-H) Cells transfected with !-catenin
#Armadillo. (I-L) Cells transfected with
!-catenin #C. (M-P) Cells transfected with
!-catenin #N. The armadillo domain of
!-catenin, but not the C terminus, was required
to overcome density-dependent inhibition of
neurogenesis.
3552
immunoreactive cells induced by overexpression of !-catenin
with or without RA treatment were also found to be
immunoreactive for NeuN (Neuna60 – Mouse Genome
Informatics; Fig. 8E-H), neurofilament 200 (data not shown),
Map2 (Fig. 8M-P), and synaptophysin (Fig. 8Q-T). Cells from
high-density cultures transfected with !-catenin were patch
clamped and were found to possess voltage-gated ion channels,
whereas cells from control cultures did not (Fig. 6B and data
not shown). The neuronal morphology of these cells, their
expression of !-tubulin 3, NeuN, neurofilament, Map2 and
synaptophysin, and their expression of voltage-gated channels,
substantiate the conclusion that these cells are neurons.
!-catenin induced neural and neuronal lineage
commitment does not require EB formation
Recently it has been demonstrated that ES cells can differentiate
into neuron-like cells without having to go through an EB step
(Gratsch and O’Shea, 2002; Ying et al., 2003). However, both
of these protocols require culture at very low densities to induce
differentiation. When ES cells were plated at 106cells/cm2[a
concentration 100-fold greater than that used by Ying and
colleagues (Ying et al., 2003)] in DMEM/F12 with N2 and B27
supplements, cells immunoreactive to nestin and !-tubulin 3
were not detectable in either untransfected cells (data not
shown) or cells transfected with an empty vector (Fig. 7A).
However, overexpression of !-catenin #N (Fig. 7B) overcame
the density-dependent inhibition of neural differentiation (for
quantification, see Fig. 7C). Interestingly, GFAP- and CNPase-
immunoreactive cells were not detected in !-catenin #N
transfected cells seeded at high density. These cultures were
stained with anti-!-catenin antibodies and the localization of !-
catenin staining was analyzed by optical sectioning in a
confocal microscope (Fig. 7E,F). In control cells most staining
was localized to the cell membrane, whereas in cells stably
transfected with !-catenin #N there was a more diffuse pattern
of staining. These studies demonstrate that the stimulatory
effects of !-catenin on neuronal lineage commitment by ES
cells do not require formation of EBs.
Increased neuronal differentiation by !-catenin is
not mediated by increased proliferation
!-catenin is a known mitogen (Kikuchi, 1999; Megason and
McMahon, 2002), and it is therefore possible that the increased
neuronal differentiation was due to increased proliferation of
neural progenitor cells and/or increased re-entry of neural
progenitors into the cell cycle. ES cells stably transfected with
!-catenin #N or with the empty vector were therefore tested
for BrdU incorporation in order to study the effects of !-
catenin #N expression on the proliferation of ES cells.
Undifferentiated ES cells grown with Lif (see Materials and
methods) were pulsed for 3 hours with BrdU and stained with
anti-BrdU antibodies (Fig. 7D, left graph). BrdU incorporation
by cells stably transfected with !-catenin #N did not differ
significantly from cells transfected with the empty vector,
although there was a slight trend in that direction (P=0.11, by
t-test). This is not surprising as undifferentiated ES cells
cultured with Lif already have a very high rate of proliferation.
More importantly, we investigated whether ES cells stably
transfected with !-catenin #N increased proliferation during
neural differentiation by plating the cells at low density (104
cells/cm2) on gelatin-coated tissue culture dishes in
DMEM/F12 with B27 and N2 supplements without Lif. This
has been shown previously by Ying and colleagues (Ying et
al., 2003) to result in neural differentiation, with nestin-
immunoreactive cells appearing by day 5 post-Lif withdrawal.
Cells stably transfected with !-catenin #N or with empty
vector were differentiated in vitro with this protocol and pulsed
for 3 hours with BrdU (Fig. 7D, right graph) on day 6 post-Lif
withdrawal. Interestingly, the cells transfected with !-catenin
#N were found to have a statistically significant decrease in
BrdU incorporation (P<0.01, by t-test) and had a decrease in
the number of cells but an increase in the percentage of !-
tubulin 3-immunoreactive cells, suggesting that activation of !-
catenin signaling resulted in increased exit from the cell cycle
and differentiation.
Phenotypic analysis of neurons generated by
!-catenin overexpression
To determine whether the phenotypes of neurons induced by
!-catenin and RA are the same, profiles of gene expression
were compared in neurons derived from ES cells that
overexpress !-catenin and in neurons induced by the 4–/4+
protocol with untransfected cells cultured at low density. We
first used Hox gene expression profiles to characterize the
Development 131 (15) Research article
Fig. 6. RA acts synergistically with !-catenin to
induce neuronal differentiation in ES cells.
(A) The percentage of !-tubulin 3-
immunoreactive cells was quantified from high
density inductions with or without RA
treatment (see Materials and methods). !-
catenin #N induced a greater proportion of
neurons than !-catenin #C did. Treatment with
RA, along with either !-catenin #C or #N,
resulted in an increase in the number of
neurons, suggesting that there is synergy
between RA and !-catenin signaling (ND, none
detected; error bars show s.d.). (B) Cells with
neuronal morphology from these cultures were
patch clamped to determine whether they
expressed voltage-gated ion channels (see
Materials and methods). All cultures showed voltage-gated ion channel activity. A representative tracing from neurons induced from !-catenin
#C transfected ES cells at high density without RA is shown. Cells from control conditions did not exhibit this activity (data not shown). Cells
were held at –90 mV holding potential and activated at –30 mV.
3553!-catenin induces neuronal lineage commitment by ES cells
phenotype of the cells (Fig. 8). Virtually all neurons expressed
Hoxc4 after induction of neuronal lineage commitment by RA
treatment (Fig. 8V), by overexpression of !-catenin (Fig.
8A,C), or by both (Fig. 8B,D). This suggests that the neurons
generated in all conditions are caudal in character.
Furthermore, GABAergic neurons were detected in the culture
in all conditions (Fig. 8E-H). Some neurons (<5%) induced by
overexpression of !-catenin were immunoreactive for tyrosine
hydroxylase (TH), the rate-limiting enzyme in dopamine
biosynthesis. However, treatment with RA suppressed the
generation of TH-positive cells, suggesting that not all of the
effects of RA are dependent on !-catenin (Fig. 8I-L).
Discussion
We have found that !-catenin signaling and neural
differentiation of ES cells are inhibited by culture at high
density. This observation is consistent with prior studies of
neural/neuronal differentiation of ES cells that have all used
relatively low densities irrespective of whether EB or
dissociated cell culture techniques were used (Gratsch
and O’Shea, 2002; Ying et al., 2003). The need to culture
the cells at low density to achieve neuronal
differentiation limits the number of cells that could
potentially be obtained for transplantation strategies and
raises questions about the mechanisms mediating
neuronal differentiation of the cells. Our studies suggest
that !-catenin signaling promotes both neural and neuronal
differentiation of ES cells, and that the effects of increased cell
density are mediated at least in part by inhibition of !-catenin
signaling.
Similar to observations with keratinocytes (Dietrich et al.,
2002), culture of ES cells at high density promotes membrane
localization of !-catenin with a consequent decrease in
signaling. Furthermore, although the levels of total !-catenin
were not influenced by cell density, high-density cultures had
a higher proportion of N-terminally phosphorylated !-catenin
(Ser33/37;Thr41), which targets the molecule for degradation
(Salic et al., 2000b). The sequestration of !-catenin by
membrane binding and the targeting for degradation reduces
the nuclear pool of !-catenin thereby reducing signaling
(Novak and Dedhar, 1999). The suppression of baseline
TCF/LEF activity and decreased Pitx2 transcription in our
high-density cultures supports the conclusion that !-catenin
signaling is diminished in these cultures.
Although there are extensive data indicating that Wnt/!-
catenin signaling enhances neuronal differentiation in the
Fig. 7. Overexpression of !-catenin overcomes density-
dependent inhibition of neural differentiation in an EB-
independent protocol. ES cells transfected with the constructs
indicated were induced to differentiate into the neural lineage
in an EB-independent protocol (see Materials and methods) at
a density of 106cells/cm2. Cells were fixed and stained 8-10
days post-plating, with Hoechst dye (blue), and anti-nestin
(green) and anti-!-tubulin 3 (red) antibodies. (A) Empty
vector transfected cells; (B) !-catenin #N transfected cells.
Control cultures were devoid of nestin- and !-tubulin 3-
positive cells, whereas overexpression of !-catenin #N
resulted in many nestin- and !-tubulin 3-positive cells,
suggesting that !-catenin exerts both proneural and
proneuronal effects. (C) Quantification of cells
immunoreactive to antibodies specific to the antigens
indicated in the key. (D) BrdU incorporation of ES cells. Left
panel (plus Lif): undifferentiated ES cells stably transfected
with either empty vector (black) or !-catenin #N (gray) were
pulsed for 3 hours with BrdU and stained with anti-BrdU
antibodies. No statistical difference was found. Right panel:
ES cells stably transfected with either empty vector (black) or
!-catenin #N (gray) were differentiated in vitro and pulsed
with BrdU on day 6 post-Lif withdrawal. !-catenin #N
expression resulted in decreased BrdU incorporation
(*P<0.05; **P<0.01, by t-test; ND, none detected). Cells
transfected with empty vector (E) or !-catenin #N (F) seeded
at high density were analyzed for !-catenin localization by
optical sectioning. High-density cultures had a highly
membrane localized stain for !-catenin, whereas in the !-
catenin #N overexpressor, the staining was more diffuse. To
substantiate the visual observation, fluorescent intensities over
a random cross section of the cells were plotted. In the control
cells, there are clean peaks and valleys (E%), whereas in the !-
catenin #N-transfected cells, the fluorescence is noisier (F%).
3554
developing embryo (Baker et al., 1999; Dorsky et al., 1998;
McGrew et al., 1999; Megason and McMahon, 2002) the
effects of !-catenin on ES cells could also reflect enhanced
proliferation and/or reduced exit of progenitor cells from the
cell cycle, or survival of neural species, as well as an instructive
effect on neural lineage commitment. In fact, recent studies
have demonstrated a role for !-catenin in maintaining the
proliferative state of neural stem cells. Overexpression of
constitutively active !-catenin in neural stem cells increased
neurogenesis primarily by decreasing cell cycle exit of neural
progenitors (Chenn and Walsh, 2002), and !-catenin
expression in the developing spinal cord maintained neural
progenitor cells in a proliferative state with decreased neuronal
differentiation (Zechner et al., 2003). Furthermore, it has been
suggested that stabilization of !-catenin results in maintenance
of pluripotency in human embryonic stem cells (Sato et al.,
2004) and inhibition of differentiation of murine ES cells
(Aubert et al., 2002; Haegele et al., 2003; Kielman et al., 2002).
By contrast, our study shows that !-catenin facilitates neural
and neuronal differentiation of ES cells while not
increasing proliferation of neural progenitors, and
that it is associated with enhanced exit from
cell cycle. Overexpression of !-catenin in the
pluripotent P19 cell line induces neuronal
differentiation (Israsena et al., 2004), whereas
pharmacological inhibition of Gsk3!facilitated
neuronal differentiation in P19 cells (Ding et al.,
2003). Other investigators have also shown that
!-catenin signaling can result in increased
differentiation without affecting proliferation (Jin
et al., 2001). It is possible that !-catenin exerts an
effect either on proliferation, on differentiation, or on both,
depending on the context of other signaling cascades. For
example, many other molecules such as Shh, Lif and FGF have
been shown to be potent mitogens or potent differentiation
signals (Bartlett et al., 1998; Zhu et al., 1999) depending upon
the cellular context. In the Chenn and Walsh study, and in the
Zechner study, !-catenin was overexpressed in a setting where
they were also exposed to significant levels of Fgf2 present in
the ventricular zone (Vaccarino et al., 1999). By contrast, in
our studies serum was removed from the cultures after EB
dissociation. In fact, in other studies we find that the effects of
!-catenin signaling in cultured neural progenitor cells are
modified from pro-differentiation to maintenance of the
proliferative state by the presence of Fgf2 (Israsena et al.,
2004).
Finally, it should also be emphasized that we found that the
stimulatory effects of !-catenin on neurogenesis reflect effects
on pre-neural cells, as well as on neural progenitor cells, so
that mitogenic effects on neural progenitors could not possibly
Development 131 (15) Research article
Fig. 8. !-catenin overexpression results in a less
restricted population of neurons compared with RA-
derived neurons. The phenotype of neurons generated
by !-catenin #N and #C overexpression in high-density
EB cultures was compared with neurons generated by
RA treatment of low-density EB cultures.
(A-D) Hoxc4, red; !-tubulin 3, green; (E-H) NeuN,
green; GABA, red; (I-L) tyrosine hydroxylase, red; !-
tubulin 3, green; Hoechst stain blue (white arrows
indicate TH-positive neurons); (M-P) Map2, green; !-
tubulin 3, red; (Q-T) synaptophysin, green; !-tubulin 3,
red. (A-D) All neurons generated by either !-catenin
overexpression or RA treatment were positive for
Hoxc4, a homeobox gene specific for caudal neurons.
(E-H) Many neurons were positive for the
neurotransmitter GABA and all neurons were positive
for NeuN. (I-L) Some neurons induced by
overexpression of !-catenin were immunoreactive for
tyrosine hydroxylase (TH), the rate-limiting enzyme in
catecholamine biosynthesis. By contrast,
TH immunoreactivity was not observed in RA-treated
conditions. (M-T) All !-tubulin 3-immunoreactive
cells also expressed Map2 and synaptophysin.
(U) Quantification of the percentage of gabaergic
neurons found in the cultures. There was no statistically
significant difference between the RA-treated and
untreated cultures (by ANOVA). (V) Neurons generated
from untransfected ES cells differentiated at low
density also express Hoxc4, suggesting that these are
caudal neurons (green, Hoxc4; red, !-tubulin 3).
3555!-catenin induces neuronal lineage commitment by ES cells
underlie all of its actions. Nestin immunoreactive neural
progenitor cells did not develop in high-density cultures in the
absence of !-catenin signaling, but they did develop in
response to increased !-catenin signaling, indicating an effect
on pre-neural cells. However, in low-density cultures, which
develop nestin-immunoreactive cells in the absence of
exogenous RA treatment, !-catenin signaling promotes
commitment of these progenitors to the neuronal lineage,
indicating an effect on the neural stem cells as well.
The domains of !-catenin mediating its effects on
neurogenesis in ES cells and the signaling pathways involved
are unclear. In the canonical Wnt pathway, !-catenin interacts
with members of the TCF/LEF family of transcription factors
leading to both relief of repression and activation of
transcription. Numerous genetic and biochemical studies
suggest that the C-terminal domain of !-catenin is the primary
transactivation domain (Peifer et al., 1991; van de Wetering et
al., 1997), although it has been reported that a second
transactivation domain may be present in the N terminus (Hsu
et al., 1998). We found that in HEK293 cells the transactivation
of TCF/LEF genes requires both the armadillo domain of !-
catenin and the C-terminal transactivation domain. By contrast,
although the armadillo domain of !-catenin was required to
induce neural lineage commitment by ES cells, the C-terminal
transactivation domain was not. Yet, !-catenin #C was unable
to enhance TCF/LEF driven transcription in an artificial
promoter system. This raises the possibility that some of the
effects of !-catenin on neurogenesis might not be mediated
by the classical TCF/LEF pathway, consistent with prior
observations that !-catenin can signal through other
transcription factors (Easwaran et al., 1999; Kioussi et al.,
2002). However, there are several possible alternative
explanations for the results of the truncation experiments. First,
in some cell types the armadillo domain partly activates
TCF/LEF pathways (N. Israsena and J.A.K., unpublished),
raising the possibility that this is the case with ES cells.
Alternatively, overexpression of !-catenin #C may lead to
displacement of endogenous !-catenin from adherins junctions
to the nucleus. However, this seems unlikely as the replacement
of endogenous membrane bound !-catenin with !-catenin #C
would be expected to reduce C-terminal immunostaining on
the cell membrane, and immunohistochemical studies did not
show any change in the relative staining when a C-terminal !-
catenin antibody or an N-terminal !-catenin antibody was
used.
RA signaling was incapable of inducing neural
differentiation in high-density cultures in the absence of !-
catenin signaling. There is a precedent for such dependence
on !-catenin signaling of RA-mediated differentiation. In
addition, the effects of RA in inducing endoderm in the F9
teratocarcinoma cell line are absolutely dependent upon !-
catenin signaling (Lui et al., 2002), and inhibition of axin, an
auxiliary factor to Gsk3!that promotes !-catenin degradation,
inhibits RA-mediated differentiation of P19 cells (Lyu et
al., 2003). Nevertheless RA treatment enhanced neural
differentiation in ES cells overexpressing !-catenin,
suggesting that there is a synergistic interaction between the
two signaling pathways. There are numerous types of
crosstalk between RA and !-catenin signaling (for a review,
see Katoh, 2002). For example, RA increases !-catenin
protein stability and affinity for adherins junctions in a breast
cancer cell line (Byers et al., 1996), and RA treatment results
in co-immunoprecipitation of !-catenin with the retinoic
acid receptor and an increase in !-catenin-RAR driven
transcription (Easwaran et al., 1999). Interestingly, RA has
been shown to upregulate the Wnt receptor frizzled (Katoh,
2002), although we did not find a change in the expression of
frizzled by western blotting in EBs after RA treatment (data
not shown). Moreover, some caudal homeobox genes contain
response elements for both !-catenin and RA signaling
(Lickert and Kemler, 2002). Interestingly, neurons generated
by either !-catenin overexpression or RA treatment were
caudal in nature as evidenced by expression of Hoxc4 (Fig.
8). In addition, we found no statistically significant difference
in the proportion of gabaergic neurons in !-catenin transfected
cells with or without RA treatment. Nevertheless RA
treatment inhibited the generation of TH-positive neurons by
!-catenin, suggesting that RA exerts at least some effects
independent of !-catenin signaling. Although !-catenin
signaling has been demonstrated in the caudal neural tube it
has not been shown previously that !-catenin can induce
neurogenesis in an RA-independent manner. Wnt and FGF
signaling inhibited the expression of cyp26, a cytochrome
P450 oxidase that degrades RA and which is in part
responsible for the spatially restricted signaling of RA in the
caudal neural tube (Kudoh et al., 2002). Furthermore, the
expression of caudal neural genes by Wnt signaling was
mediated through RA signaling. By contrast, our studies
demonstrate that !-catenin signaling can lead to the
development of caudal neurons in a RA-independent fashion.
In addition to inhibiting !-catenin signaling, it is possible
that high-density culture increases BMP signaling, a known
inhibitor of neural differentiation in ES cells (Kawasaki et al.,
2000; Tropepe et al., 2001). However treatment with noggin-
Fc or low-density inductions using media conditioned by high
density inductions did not inhibit neural differentiation.
Interestingly, it has been shown that inhibition of BMP
signaling in epithelial bud development results in the
upregulation of Lef1 and an increase in !-catenin signaling
(Jamora et al., 2003). We, however, were unable to find any
difference in Lef1 protein levels between high and low density
cultures (data not shown).
In summary, our observations indicate that !-catenin
signaling enhances neural lineage commitment by ES cells.
Furthermore, !-catenin signaling may be a necessary co-factor
for RA-induced neural differentiation. Culture of ES cells at
increased density inhibits neurogenesis mediated by all of the
previously described protocols for inducing neurogenesis (RA,
antagonism of BMP signaling, or treatment with stromal cell
membranes), apparently by both sequestering !-catenin at the
cell membrane and by increasing phosphorylation of !-catenin.
However, enhanced !-catenin signaling can overcome the
inhibitory effects of increased cell density. These observations
illustrate the importance of !-catenin signaling in neural
lineage commitment by ES cells, and the synergy between RA
and !-catenin signaling indicates a method for obtaining large
numbers of neural species for possible use in therapeutic
strategies involving ES cell transplantation.
This work was supported by NIH grants NS 34758 and NS 20778.
We would also like to thank Dr Richard Miller and Dr Anjen Chenn
for fruitful discussions.
3556
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