Department of Neurology, Northwestern University Feinberg School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611, USA
* Authors for correspondence (e-mail: j-otero{at}northwestern.edu and jakessler{at}northwestern.edu)
Accepted 13 April 2004
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SUMMARY |
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Key words: Embryonic stem cells, ß-Catenin, Neurogenesis, Retinoic acid, Tyrosine hydroxylase, Cell density
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Introduction |
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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/ß-catenin pathway that has been shown to be a cell-cell (and/or
cell-matrix) contact-regulated inducer of neurogenesis
(Patapoutian and Reichardt,
2000). ß-catenin exists in three cellular pools: membrane
bound, cytoplasmic and nuclear. Membrane-bound ß-catenin is associated
with E-cadherin/adherens junctions and functions to bridge E-cadherin to the
cytoskeleton (Aberle et al.,
1996
; Gumbiner and McCrea,
1993
). In the cytoplasm, ß-catenin turnover is regulated by a
stepwise phosphorylation on its N terminus. Initially, ß-catenin is
phosphorylated on Ser45 by a `priming' kinase
(Liu et al., 2002
).
Phosphorylation at Ser45 primes ß-catenin for phosphorylation at
Ser33/37;Thr41 by Gsk3ß (Kang et al.,
2002
). Phospho-Ser33/37;Thr41-ß-catenin is then targeted for
ubiquitin-directed proteolysis (Salic et
al., 2000a
), and increased phosphorylation by Gsk3ß decreases
the nuclear pool of ß-catenin (Lucas
et al., 2001
). During Wnt signaling, Gsk3ß is inhibited,
leading to the accumulation of unphosphorylated ß-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
ß-catenin with TCF/LEF transcription factors causes both inhibition of
repression and activation of transcription. However, both Wnt and
ß-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 ß-catenin signaling in neural lineage commitment by ES cells. We find that induction of differentiation at high density inhibits both ß-catenin signaling and neural differentiation. However, overexpression of ß-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.
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Materials and methods |
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BrdU incorporation assay
To test for differences in proliferation in undifferentiated ES cells
stably transfected with either empty vector or ß-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 ß-catenin
N during neural differentiation, cells were induced at 104
cells/cm2 without 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 ß-catenin on lineage commitment by ES cells were
examined by transfecting the cells with various constructs of the
ß-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
ß-catenin and increase ß-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: ß-catenin
total, full-length protein; ß-catenin
N, first 128 amino acids
truncated; ß-catenin
C, last 113 amino acids truncated; and
ß-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.
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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. Conditioned media was used instead of
induction media for the 4-/6+ protocol, with ES cells seeded at high density
(106 cells/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-ß-catenin (Santa
Cruz), anti-N-terminal-phospho ß-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 antibodies used were as follows: anti-ß-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, ß-tubulin 3, BrdU; numerator=stain, denominator=
Hoechst-positive nuclei) were counted.
Confocal microscopy
Day 2 EBs were fixed, permeabilized, and stained with anti-ß-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 106
cells/cm2 on 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 ß-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/LEFTOPFLASH and treated with control conditioned, Wnt3a-conditioned media
for 3 days, or co-transfected with H2kd-E-cadherin with TCF/LEF-luc. For
ß-catenin experiments, HEK293 cells were cotransfected with 5 ng
TK-renilla luciferase, 0.3 µg TCF/LEF-Luciferase with or without different
ß-catenin expression constructs (0.1 µg transfected for low dosage and
0.3 µg transfected for high dosage)
(Fig. 4). ß-catenin
constructs used were pcDNA3.1 (CMV-ß-cat) or the retroviral pCLE
(ß-cat, ß-cat
N, ß-cat
C, ß-cat
NC and
ß-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 y axis 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 builtin
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.
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Results |
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Culture at high density reduces TCF/LEF dependent transcription
To determine whether the differences in phosphorylation and localization of
ß-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/ß-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 ß-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 ß-tubulin 3 (red) and 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 105 cells/ml
(Fig. 2A). As the seeding
density was increased, the percentage of ß-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 (ß-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 (105 cells/ml) with media preconditioned by
high-density cultures (106 cells/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).
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As both treatment with Wnt3a-conditioned media or overexpression of H2Kd-E-cadherin could activate several different signaling pathways, we directly tested whether increasing ß-catenin signaling alone can overcome the inhibitory effects of increased cell density by stably transfecting ES cells with ß-catenin. Control cells were transfected with a vector containing only the antibiotic resistance gene. Lif was then withdrawn and the ß-catenin overproducing cells cultured at high density in the 4-/4+ EB protocol (Fig. 3G). Overexpression of ß-catenin resulted in differentiation into ß-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 ß-catenin pathways can prevent the density-dependent inhibition of neural and neuronal differentiation in RA-treated cultures.
ß-catenin induced neuronal differentiation of ES cells requires the armadillo domain and does not require RA treatment
To determine whether ß-catenin signaling can by itself induce
neurogenesis in ES cells in the absence of RA, and to define the domains of
the ß-catenin molecule that might be involved, we constructed various
full-length and truncated constructs of ß-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 ß-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 ß-catenin, as well as
with an N-terminal truncation of ß-catenin (ß-catenin
N),
increased transcription of the reporter gene. The activity of ß-catenin
N was several orders of magnitude higher than the activity of the
full-length ß-catenin construct as expected. By contrast, transfection of
constructs with a truncation of the C-terminal portion of the molecule
(ß-catenin
C), or with a truncation of the armadillo domains
(ß-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
ß-catenin, as luciferase activity increased when the amount of
transfected ß-catenin pCLE plasmid (insert controlled by a retroviral LTR
promoter) was increased or when ß-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
Gsk3ß-targeted degradation or by artificially increasing levels of total
ß-catenin.
To directly test the effects of ß-catenin on ES cell differentiation
and to determine which domains of ß-catenin were necessary for its
function, we then stably transfected ES cells with these various constructs of
ß-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 ß-catenin
N or ß-catenin
C differentiated into
ß-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 ß-catenin-transfected cultures potentiated the neurogenic
effects of ß-catenin (Fig.
6A). By contrast, ES cells transfected with empty vector or with
the ß-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). ß-tubulin
3-immunoreactive cells induced by overexpression of ß-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 ß-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
ß-tubulin 3, NeuN, neurofilament, Map2 and synaptophysin, and their
expression of voltage-gated channels, substantiate the conclusion that these
cells are neurons.
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Increased neuronal differentiation by ß-catenin is not mediated by increased proliferation
ß-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 ß-catenin
N or with the empty vector were
therefore tested for BrdU incorporation in order to study the effects of
ß-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 ß-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 ß-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 ß-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 ß-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 ß-tubulin 3-immunoreactive cells, suggesting that
activation of ß-catenin signaling resulted in increased exit from the
cell cycle and differentiation.
Phenotypic analysis of neurons generated byß-catenin overexpression
To determine whether the phenotypes of neurons induced by ß-catenin
and RA are the same, profiles of gene expression were compared in neurons
derived from ES cells that overexpress ß-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 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 ß-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
ß-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 ß-catenin
(Fig. 8I-L).
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Discussion |
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Similar to observations with keratinocytes
(Dietrich et al., 2002),
culture of ES cells at high density promotes membrane localization of
ß-catenin with a consequent decrease in signaling. Furthermore, although
the levels of total ß-catenin were not influenced by cell density,
high-density cultures had a higher proportion of N-terminally phosphorylated
ß-catenin (Ser33/37;Thr41), which targets the molecule for degradation
(Salic et al., 2000b
). The
sequestration of ß-catenin by membrane binding and the targeting for
degradation reduces the nuclear pool of ß-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 ß-catenin signaling is diminished in these cultures.
Although there are extensive data indicating that Wnt/ß-catenin
signaling enhances neuronal differentiation in the developing embryo
(Baker et al., 1999;
Dorsky et al., 1998
;
McGrew et al., 1999
;
Megason and McMahon, 2002
) the
effects of ß-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
ß-catenin in maintaining the proliferative state of neural stem cells.
Overexpression of constitutively active ß-catenin in neural stem cells
increased neurogenesis primarily by decreasing cell cycle exit of neural
progenitors (Chenn and Walsh,
2002
), and ß-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
ß-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 ß-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 ß-catenin in the pluripotent P19 cell line
induces neuronal differentiation (Israsena
et al., 2004), whereas pharmacological inhibition of Gsk3ß
facilitated neuronal differentiation in P19 cells
(Ding et al., 2003
). Other
investigators have also shown that ß-catenin signaling can result in
increased differentiation without affecting proliferation
(Jin et al., 2001
). It is
possible that ß-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, ß-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
ß-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 ß-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 underlie all of its actions. Nestin immunoreactive neural progenitor cells did not develop in high-density cultures in the absence of ß-catenin signaling, but they did develop in response to increased ß-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, ß-catenin signaling promotes commitment of these progenitors to the neuronal lineage, indicating an effect on the neural stem cells as well.
The domains of ß-catenin mediating its effects on neurogenesis in ES
cells and the signaling pathways involved are unclear. In the canonical Wnt
pathway, ß-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 ß-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 ß-catenin and the
C-terminal transactivation domain. By contrast, although the armadillo domain
of ß-catenin was required to induce neural lineage commitment by ES
cells, the C-terminal transactivation domain was not. Yet, ß-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
ß-catenin on neurogenesis might not be mediated by the classical TCF/LEF
pathway, consistent with prior observations that ß-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 ß-catenin
C may lead to displacement of
endogenous ß-catenin from adherins junctions to the nucleus. However,
this seems unlikely as the replacement of endogenous membrane bound
ß-catenin with ß-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
ß-catenin antibody or an N-terminal ß-catenin antibody was used.
RA signaling was incapable of inducing neural differentiation in
high-density cultures in the absence of ß-catenin signaling. There is a
precedent for such dependence on ß-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 ß-catenin
signaling (Lui et al., 2002),
and inhibition of axin, an auxiliary factor to Gsk3ß that promotes
ß-catenin degradation, inhibits RA-mediated differentiation of P19 cells
(Lyu et al., 2003
).
Nevertheless RA treatment enhanced neural differentiation in ES cells
overexpressing ß-catenin, suggesting that there is a synergistic
interaction between the two signaling pathways. There are numerous types of
crosstalk between RA and ß-catenin signaling (for a review, see
Katoh, 2002
). For example, RA
increases ß-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
ß-catenin with the retinoic acid receptor and an increase in
ß-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 ß-catenin and RA signaling
(Lickert and Kemler, 2002
).
Interestingly, neurons generated by either ß-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 ß-catenin transfected cells with or without RA treatment. Nevertheless
RA treatment inhibited the generation of TH-positive neurons by
ß-catenin, suggesting that RA exerts at least some effects independent of
ß-catenin signaling. Although ß-catenin signaling has been
demonstrated in the caudal neural tube it has not been shown previously that
ß-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
ß-catenin signaling can lead to the development of caudal neurons in a
RA-independent fashion.
In addition to inhibiting ß-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 ß-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 ß-catenin signaling enhances neural lineage commitment by ES cells. Furthermore, ß-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 ß-catenin at the cell membrane and by increasing phosphorylation of ß-catenin. However, enhanced ß-catenin signaling can overcome the inhibitory effects of increased cell density. These observations illustrate the importance of ß-catenin signaling in neural lineage commitment by ES cells, and the synergy between RA and ß-catenin signaling indicates a method for obtaining large numbers of neural species for possible use in therapeutic strategies involving ES cell transplantation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Aberle, H., Schwartz, H. and Kemler, R. (1996). Cadherin-catenin complex, protein interactions and their implications for cadherin function. J. Cell Biochem. 61,514 -523.[CrossRef][Medline]
Aubert, J., Dunstan, H., Chambers, I. and Smith, A. (2002). Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat. Biotech. 20,1240 -1245.[CrossRef][Medline]
Bain, G., Kitchens, D., Yao, M., Huettner, J. E. and Gottlieb, D. I. (1995). Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168,342 -357.[CrossRef][Medline]
Baker, J. C., Beddington, R. S. and Harland, R. M.
(1999). Wnt signaling in Xenopus embryos inhibits bmp4 expression
and activates neural development. Genes Dev.
13,3149
-3159.
Bartlett, P. F., Brooker, G. J., Faux, C. H., Dutton, R., Murphy, M., Turnley, A. and Kilpatrick, T. J. (1998). Regulation of neural stem cell differentiation in the forebrain. Immunol. Cell Biol. 76,414 -418.[CrossRef][Medline]
Brantjes, H., van Barker, N. E. J. and Clevers, H. (2002). TCF, Lady Justice casting the final verdict on the outcome of Wnt signalling. Biol. Chem. 383,255 -261.[Medline]
Brewer, G. J., Torricelli, J. R., Evege, E. K. and Price, P. J. (1993). Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35,567 -576.[Medline]
Brustle, O., Jones, K. N., Learish, R. D., Karram, K.,
Choudhary, K., Wiestler, O. D., Duncan, I. D. and McKay, R. D.
(1999). Embryonic stem cell-derived glial precursors, a source of
myelinating transplants. Science
285,754
-756.
Byers, S., Pishvaian, M., Crockett, C., Peer, C., Tozeren, A., Sporn, M., Anzano, M. and Lechleider, R. (1996). Retinoids increase cell-cell adhesion strength, beta-catenin protein stability, and localization to the cell membrane in a breast cancer cell line, a role for serine kinase activity. Endocrinology 137,3265 -3273.[Abstract]
Chazaud, C., Chambon, P. and Dolle, P. (1999).
Retinoic acid is required in the mouse embryo for left-right asymmetry
determination and heart morphogenesis. Development
126,2589
-2596.
Chen, U. and Mok, H. (1995). Development of mouse embryonic stem (ES) cells, IV. Differentiation to mature T and B lymphocytes after implantation of embryoid bodies into nude mice. Dev. Immunol. 4,79 -84.[Medline]
Chenn, A. and Walsh, C. A. (2002). Regulation
of cerebral cortical size by control of cell cycle exit in neural precursors.
Science 297,365
-369.
Cho, E. A. and Dressler, G. R. (1998). TCF-4 binds beta-catenin and is expressed in distinct regions of the embryonic brain and limbs. Mech. Dev. 77, 9-18.[CrossRef][Medline]
Chung, S., Sonntag, K. C., Andersson, T., Bjorklund, L. M., Park, J. J., Kim, D. W., Kang, U. J., Isacson, O. and Kim, K. S. (2002). Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons. Eur. J. Neurosci. 16,1829 -1838.[CrossRef][Medline]
Collins, M. D. and Mao, G. E. (1999). Teratology of retinoids. Annu. Rev. Pharmacol. Toxicol. 39,399 -430.[CrossRef][Medline]
Dietrich, C., Scherwat, J., Faust, D. and Oesch, F. (2002). Subcellular localization of beta-catenin is regulated by cell density. Biochem. Biophys. Res. Commun. 292,195 -199.[CrossRef][Medline]
Ding, S., Wu, T. Y., Brinker, A., Peters, E. C., Hur, W., Gray,
N. S. and Schultz, P. G. (2003). Synthetic small
molecules that control stem cell fate. Proc. Natl. Acad. Sci.
USA 100,7632
-7637.
Dinsmore, J., Ratliff, J., Deacon, T., Pakzaban, P., Jacoby, D., Galpern, W. and Isacson, O. (1996). Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation. Cell Transplant. 5,131 -143.[CrossRef][Medline]
Dorsky, R. I., Moon, R. T. and Raible, D. W. (1998). Control of neural crest cell fate by the Wnt signalling pathway. Nature 396,370 -373.[CrossRef][Medline]
Easwaran, V., Pishvaian, M., Salimuddin, X. and Byers, S. (1999). Cross-regulation of beta-catenin-LEF/TCF and retinoid signaling pathways. Curr. Biol. 9,1415 -1418.[CrossRef][Medline]
Gratsch, T. E. and O'Shea, K. S. (2002). Noggin and chordin have distinct activities in promoting lineage commitment of mouse embryonic stem (ES) cells. Dev. Biol. 245, 83-94.[CrossRef][Medline]
Gumbiner, B. M. and McCrea, P. D. (1993). Catenins as mediators of the cytoplasmic functions of cadherins. J. Cell Sci. Suppl. 17,155 -158.[Medline]
Haegele, L., Ingold, B., Naumann, H., Tabatabai, G., Ledermann, B. and Brandner, S. (2003). Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression. Mol. Cell. Neurosci. 24,696 -708.[CrossRef][Medline]
Hsu, S. C., Galceran, J. and Grosschedl, R.
(1998). Modulation of transcriptional regulation by LEF-1 in
response to Wnt-1 signaling and association with beta-catenin. Mol.
Cell. Biol. 18,4807
-4818.
Ioffe, E., Liu, Y., Bhaumik, M., Poirier, F., Factor, S. M. and Stanley, P. (1995). WW6, an embryonic stem cell line with an inert genetic marker that can be traced in chimeras. Proc. Natl. Acad. Sci. USA 92,7357 -7361.[Abstract]
Israsena, N., Hu, M., Fu, W., Kan, L. and Kessler, J. A. (2004). The presence of FGF2 signaling determines whether beta-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells. Dev. Biol. 268,220 -231.[CrossRef][Medline]
Jamora, C., DasGupta, R., Kocieniewski, P. and Fuchs, E. (2003). Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 422,317 -322.[CrossRef][Medline]
Jin, E. J., Erickson, C. A., Takada, S. and Burrus, L. W. (2001). Wnt and BMP signaling govern lineage segregation of melanocytes in the avian embryo. Dev. Biol. 233, 22-37.[CrossRef][Medline]
Kang, D. E., Soriano, S., Xia, X., Eberhart, C. G., De Strooper, B., Zheng, H. and Koo, E. H. (2002). Presenilin couples the paired phosphorylation of beta-catenin independent of axin, implications for beta-catenin activation in tumorigenesis. Cell 110,751 -762.[Medline]
Katoh, M. (2002). Regulation of WNT signaling molecules by retinoic acid during neuronal differentiation in NT2 cells, threshold model of WNT action. Int. J. Mol. Med. 10,683 -687.[Medline]
Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa, S. I. and Sasai, Y. (2000). Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31-40.[Medline]
Kielman, M. F., Rindapaa, M., Gaspar, C., van Poppel, N., Breukel, C., van Leeuwen, S., Taketo, M. M., Roberts, S., Smits, R. and Fodde, R. (2002). Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling. Nat. Genet. 32,594 -605.[CrossRef][Medline]
Kikuchi, A. (1999). Roles of Axin in the Wnt signalling pathway. Cell Signal 11,777 -788.[CrossRef][Medline]
Kioussi, C., Briata, P., Baek, S. H., Rose, D. W., Hamblet, N.
S., Herman, T., Ohgi, K. A., Lin, C., Gleiberman, A., Wang, J. et
al. (2002). Identification of a
Wnt/Dvl/beta-cateninPitx2 pathway mediating cell-type-specific
proliferation during development. Cell
111,673
-685.[Medline]
Korswagen, H. C. (2002). Canonical and non-canonical Wnt signaling pathways in Caenorhabditis elegans, variations on a common signaling theme. BioEssays 24,801 -810.[CrossRef][Medline]
Kudoh, T., Wilson, S. W. and Dawid, I. B. (2002). Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129,4335 -4346.[Medline]
Kuhl, M. (2002). Non-canonical Wnt signaling in Xenopus, regulation of axis formation and gastrulation. Semin. Cell Dev. Biol. 13,243 -249.[CrossRef][Medline]
Lickert, H. and Kemler, R. (2002). Functional analysis of cis-regulatory elements controlling initiation and maintenance of early Cdx1 gene expression in the mouse. Dev. Dyn. 225,216 -220.[CrossRef][Medline]
Liu, C., Li, Y., Semenov, M., Han, C., Baeg, G. H., Tan, Y., Zhang, Z., Lin, X. and He, X. (2002). Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108,837 -847.[Medline]
Liu, S., Qu, Y., Stewart, T. J., Howard, M. J., Chakrabortty,
S., Holekamp, T. F. and McDonald, J. W. (2000).
Embryonic stem cells differentiate into oligodendrocytes and myelinate in
culture and after spinal cord transplantation. Proc. Natl. Acad.
Sci. USA 97,6126
-6131.
Lucas, J. J., Hernandez, F., Gomez-Ramos, P., Moran, M. A., Hen,
R. and Avila, J. (2001). Decreased nuclear
beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta
conditional transgenic mice. EMBO J.
20, 27-39.
Lui, T., Lee, Y. N., Malbon, C. C. and Wang, H. Y.
(2002). Activation of the beta-catenin/LEF-TCF pathway is
obligate for formation of primitive endoderm by mouse F9 totipotent
teratocarcinoma cells in response to retinoic acid. J. Biol.
Chem. 277,30887
-30891.
Lyu, J., Costantini, F., Jho, E. H. and Joo, C. K.
(2003). Ectopic expression of axin blocks neuronal
differentiation of embryonic carcinoma p19 cells. J. Biol.
Chem. 278,13487
-13495.
Martin, D. M., Skidmore, J. M., Fox, S. E., Gage, P. J. and Camper, S. A. (2002). Pitx2 distinguishes subtypes of terminally differentiated neurons in the developing mouse neuroepithelium. Dev. Biol. 252,84 -99.[CrossRef][Medline]
McGrew, L. L., Takemaru, K., Bates, R. and Moon, R. T. (1999). Direct regulation of the Xenopus engrailed-2 promoter by the Wnt signaling pathway, and a molecular screen for Wnt-responsive genes, confirm a role for Wnt signaling during neural patterning in Xenopus. Mech. Dev. 87,21 -32.[CrossRef][Medline]
Megason, S. G. and McMahon, A. P. (2002). A
mitogen gradient of dorsal midline Wnts organizes growth in the CNS.
Development 129,2087
-2098.
Novak, A. and Dedhar, S. (1999). Signaling through beta-catenin and Lef/Tcf. Cell. Mol. Life Sci. 56,523 -537.[CrossRef][Medline]
Pandur, P., Lasche, M., Eisenberg, L. M. and Kuhl, M. (2002a). Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Nature 418,636 -641.[CrossRef][Medline]
Pandur, P., Maurus, D. and Kuhl, M. (2002b). Increasingly complex, New players enter the Wnt signaling network. BioEssays 24,881 -884.[CrossRef][Medline]
Patapoutian, A. and Reichardt, L. F. (2000). Roles of Wnt proteins in neural development and maintenance. Curr. Opin. Neurobiol. 10,392 -399.[CrossRef][Medline]
Peifer, M., Rauskolb, C., Williams, M., Riggleman, B. and Wieschaus, E. (1991). The segment polarity gene armadillo interacts with the wingless signaling pathway in both embryonic and adult pattern formation. Development 111,1029 -1043.[Abstract]
Salic, A., Lee, E., Mayer, L. and Kirschner, M. W. (2000a). Control of beta-catenin stability, reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts. Mol. Cell 5,523 -532.[Medline]
Salic, A., Lee, E., Mayer, L. and Kirschner, M. W. (2000b). Control of beta-catenin stability, reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts. Mol. Cell 5,523 -532.[Medline]
Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. and Brivanlou, A. H. (2004). Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nature Med. 10, 55-63.[CrossRef][Medline]
Tada, M., Concha, M. L. and Heisenberg, C. P. (2002). Non-canonical Wnt signalling and regulation of gastrulation movements. Semin. Cell Dev. Biol. 13,251 -260.[CrossRef][Medline]
Tropepe, V., Hitoshi, S., Sirard, C., Mak, T. W., Rossant, J. and van der Kooy, D. (2001). Direct neural fate specification from embryonic stem cells, a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 30,65 -78.[CrossRef][Medline]
Vaccarino, F. M., Schwartz, M. L., Raballo, R., Nilsen, J., Rhee, J., Zhou, M., Doetschman, T., Coffin, J. D., Wyland, J. J. and Hung, Y. T. (1999). Changes in cerebral cortex size are governed by fibroblast growth factor during embryogenesis. Nat. Neurosci. 2,246 -253.[CrossRef][Medline]
van de Wetering, M., Cavallo, R., Dooijes, D., van Beest, M., van Es, J., Loureiro, J., Ypma, A., Hursh, D., Jones, T. and Bejsovec, A. (1997). Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88,789 -799.[Medline]
Vizirianakis, I. S., Chen, Y. Q., Kantak, S. S., Tsiftsoglou, A. S. and Kramer, R. H. (2002). Dominant-negative E-cadherin alters adhesion and reverses contact inhibition of growth in breast carcinoma cells. Int. J. Oncol. 21,135 -144.[Medline]
Vleminckx, K., Kemler, R. and Hecht, A. (1999). The C-terminal transactivation domain of beta-catenin is necessary and sufficient for signaling by the LEF-1/beta-catenin complex in Xenopus laevis. Mech. Dev. 81,65 -74.[CrossRef][Medline]
Westmoreland, J. J., McEwen, J., Moore, B. A., Jin, Y. and
Condie, B. G. (2001). Conserved function of Caenorhabditis
elegans UNC-30 and mouse Pitx2 in controlling GABAergic neuron
differentiation. J. Neurosci.
21,6810
-6819.
Yamanaka, H., Moriguchi, T., Masuyama, N., Kusakabe, M.,
Hanafusa, H., Takada, R., Takada, S. and Nishida, E.
(2002). JNK functions in the non-canonical Wnt pathway to
regulate convergent extension movements in vertebrates. EMBO
Rep. 3,69
-75.
Ying, Q. L., Stavridis, M., Griffiths, D., Li, M. and Smith, A. (2003). Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21,183 -186.[CrossRef][Medline]
Zechner, D., Fujita, Y., Hulsken, J., Muller, T., Walther, I., Taketo, M. M., Crenshaw, E. B., 3rd, Birchmeier, W. and Birchmeier, C. (2003). beta-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system. Dev. Biol. 258,406 -418.[CrossRef][Medline]
Zhu, A. J. and Watt, F. M. (1996). Expression
of a dominant negative cadherin mutant inhibits proliferation and stimulates
terminal differentiation of human epidermal keratinocytes. J. Cell
Sci. 109,3013
-3023.
Zhu, G., Mehler, M. F., Zhao, J., Yu Yung, S. and Kessler, J. A. (1999). Sonic hedgehog and BMP2 exert opposing actions on proliferation and differentiation of embryonic neural progenitor cells. Dev. Biol. 215,118 -129.[CrossRef][Medline]