(vMB) dopamine neurons. However, the interactions between these two mechanisms and how such interactions can be targeted to
promote a maximal production of dopamine neurons are not fully understood. Here we show that conditional mouse mutants with
region-specific activation of ?-catenin signaling in vMB using the Shh–Cre mice show a marked expansion of Sox2-, Ngn2-, and Otx2-
positive progenitors but perturbs their cell cycle exit and reduces the generation of dopamine neurons. Furthermore, activation of
?-catenin in vMB also results in a progressive loss of Shh expression and Shh target genes. Such antagonistic effects between the
neurons. Persistent activation of ?-catenin in early progenitors perturbs their cell cycle progression and antagonizes Shh expression,
neurogenic niche that is enriched with progenitor cells for dopa-
mine (DA) neurons (Bjorklund and Lindvall, 1984). Within this
migration, and differentiation to become mature DA neurons
(Ang, 2006; Prakash and Wurst, 2006; Smidt and Burbach, 2007;
Arenas, 2008). Several lines of evidence indicate that two distinct
genetic networks critically regulate the development of DA neu-
rons. Sonic hedgehog (Shh) induces the expression of forkhead
transcription factor Foxa2 in vMB through specific Gli (glioma-
associated oncogene homolog) transcription factor binding ele-
ments in the enhancer sequence of Foxa2 (Sasaki et al., 1997).
Interestingly, the enhancer elements in Shh contain highly con-
in vMB (Jeong and Epstein, 2003), supporting the notion that
Shh and Foxa2 constitute a feedback transcriptional mechanism
for mutual expression. Consistent with this notion, mouse mu-
loss of Shh (Lin et al., 2009). In addition to the Shh–Foxa2 regu-
latory loop, the canonical Wnt/?-catenin signaling mechanism
controls a distinct set of transcription factors critical for the de-
velopment of DA neurons. Specifically, genetic studies in several
mouse mutants indicate that Wnt1 and Otx2 (Orthodenticle ho-
meobox 2) form a feedback mechanism to regulate the expres-
sion for each gene (Puelles et al., 2004; Vernay et al., 2005;
Prakash et al., 2006). Furthermore, in mouse embryonic stem
cells (mESCs), Wnt1 and Lmx1a (LIM homeobox transcription
that in Shh–Foxa2 (Chung et al., 2009).
Several Wnts regulate the development of DA neurons in
vMB. For instance, Wnt1 regulates proliferation, specification,
neurogenesis in vMB DA progenitors, as well as the survival of
This work was supported by National Institutes of Health Grants NS48393 and RR24858 and Department of
Veterans Affairs Merit Review (E.J.H.), as well as grants from the European Commission (Neurostemcell) and the
Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121. E-mail: email@example.com,
9280 • TheJournalofNeuroscience,July7,2010 • 30(27):9280–9291
DA neurons (McMahon and Bradley, 1990; Thomas and Capec-
chi, 1990; Danielian and McMahon, 1996; Prakash et al., 2006).
Other components of the Wnt signaling pathway, including
Lrp6, have been found to regulate the development of DA neu-
rons (Castelo-Branco et al., 2010; Sousa et al., 2010; Stuebner et
al., 2010). Similarly, ?-catenin, a critical Wnt signaling compo-
nent, is expressed in vMB DA progenitors and is required for the
maintenance of adherent junctions, the integrity of radial glia pro-
cesses, and cell cycle progression of DA progenitors (Joksimovic et
To further investigate the role of canonical Wnt signaling in
DA neurogenesis, we generated conditional mouse mutants in
sites in ?-catenin (?-CtnEx3) was removed from the neurogenic
niche in vMB. Our results indicate that the activation of
?-catenin in vMB promoted a marked expansion of DA progen-
the antagonistic interaction between the Wnt and Shh pathways
of DA progenitors and mESCs. Conversely, cell-type-specific acti-
esis. These results provide strong evidence that Wnt/?-catenin and
Shh signaling pathways control a delicate balance of target gene ex-
Animals. To generate conditional activation of ?-catenin in mice,
?-cateninExon3mice (?-CtnEx3) were crossed with Shh–Cre (stock
#005622; The Jackson Laboratory) or tyrosine hydroxylase-internal ribo-
somal entry site-Cre (Th-IRES-Cre) (Harada et al., 1999; Harfe et al.,
by the Institutional of Animal Care and Use Committee and followed
National Institutes of Health guidelines.
Histology and immunohistochemistry. The protocols for histology and
10.5 (E10.5) to E12.5, were fixed with 1% paraformaldehyde (PFA) in
PBS (4% PFA for Nkx6.1 antibody). Mice at E18.5, postnatal day 0 (P0),
sucrose solution, and sectioned in the coronal plane using a Leica cryo-
ary antibodies for 1 h, followed by incubation in DAB solution to detect
signals. The primary antibodies in this study included the following:
anti-bromodeoxyuridine (BrdU) antibody (1:500; MAB3222; Millipore
Bioscience Research Reagents), anti-Foxa2 [1:20; 4C7; Developmental
Hybridoma Study Bank (DHSB)], anti-Ki67 (1:200; RM9106-S0;
Thermo Fisher Scientific), anti-Lmx1a (1:1000; gift from Dr. Mike Ger-
man, University of California, San Francisco, San Francisco, CA), anti-
Ngn2 (Neurogenin 2) (1:10; gift from Dr. David Anderson, California
3) (1:300; gift from Dr. Marten Smidt, University Medical Center Utre-
cht, Utrecht, The Netherlands), anti-Nkx2.2 (NK2 transcription factor
related, locus 2) (1:50; 74.5A5; DHSB), anti-Nkx6.1 (NK6 homeobox 1)
(1:1000; gift from Dr. Mike German), anti-Nurr1 (Nuclear receptor re-
lated 1 protein) (1:500; sc-990; Santa Cruz Biotechnology), anti-Otx2
(1:200; ab21990; Abcam), anti-phospho-histone H3 (PH3) (1:200; 06-
ing Technology), anti-TuJ1 class III ?-tubulin (1:2000; MMS435P;
Covance), anti-tyrosine hydroxylase (1:1000; AB157; Millipore Bio-
cam), and anti-?-catenin (1:200; catalog #9587; Cell Signaling
Technology). For stereology counting, sections were incubated for 1 h
Images were captured using a Nikon Eclipse E800 fluorescent micro-
scope connected to a SPOT RT camera (Diagnostic Instruments) or a
Images were captured using Spot Advance or Olympus DP Controller
schemes. In the first scheme, the pregnant mice were injected with BrdU
(50 ?g/g) (BD Biosciences) at E10.5 and E12.5, respectively, and killed
2 h later. In the second scheme, the pregnant mice were injected with
BrdU at E10.5 and E11.5, respectively, and killed 24 h later (Zhang et al.,
2007; Tang et al., 2009).
In situ hybridization. In situ hybridization were the same as described
previously (Zhang and Huang, 2006). Briefly, RNA probes for in situ
hybridization were prepared using plasmid cDNA clones for Shh, cyclin
D1, and Lmx1b transcribed with T7 or T3 polymerase using digoxigenin
(DIG)-labeling reagents and a DIG RNA labeling kit (Roche). Embryos
were fixed overnight at room temperature in 4% PFA in DEPC-treated
PBS, cryoprotected in 15 and 30% sucrose in DEPC PBS, and embedded
in OCT. Sections were processed at 14 ?m. During hybridization, sec-
tions were first postfixed with 4% PFA and then washed with acetylation
solution and 1% Triton X-100. Then sections were prehybridized with
hybridization buffer (Amresco) for 2–4 h before applying hybridization
buffer containing DIG-labeled riboprobes (200–400 ng/ml) at 55°C
with 2? SSC, 1? SSC, and 0.5? SSC at room temperature. For visualiz-
kit (Roche). Finally, the slides were dried under room temperature and
mounted with Crystal Mount (Biomeda).
Stereology. The total number of TH-positive (TH?) neurons in sub-
stantia nigra pars compacta (SNpc) and ventral tegmental area (VTA)
was determined using the optical fractionator, an unbiased cell counting
size of the counted elements (i.e., neurons) (Zhang et al., 2007; Tang et
al., 2009). Neuronal counts were performed using a computer-assisted
image analysis system consisting of an Olympus BX-51 microscope
equipped with a x–y–z computer-controlled motorized stage and the
StereoInvestigator software (MicroBrightField). TH?neurons were
counted in SNpc or VTA of every third section throughout the entire
a random start, the numbers of TH-stained cells were counted at high
power (60? oil; numerical aperture 1.4) using a 50 ? 50 ?m counting
Ventral midbrain DA progenitor cultures. Primary cultures for dopa-
mine neurons were prepared from vMB using microisland methods ac-
embryos were collected from time-pregnant CD-1 (for E10.5 and E13.5
wild-type cultures) or Shh–Cre (for E12.5 Shh–Cre;?-CtnEx3/?cultures)
females. The ventral midbrain was dissected, dissociated after treatment
with trypsin, and cultured on coverslips coated with poly-D-ornithine
(Sigma) and laminin (Sigma) at the density of 1.2 ? 106/ml. The disso-
ciated cells were maintained in the DMEM/F-12 (1:1) medium contain-
ing 10% FBS overnight. Then, the differentiated neurons were changed
20 ng/ml FGF2 (Millipore Corporation), 100 ng/ml FGF8 (Peprotech),
and designated factors, including Shh (Peprotech), Wnt1 (Peprotech),
chem) before they were fixed with 4% PFA. The number of mature DA
neurons in culture were determined by counting the total number of
TH?neurons per 20? field (Parish et al., 2008).
Mouse embryonic stem cell cultures. Differentiation of R1 mESCs into
al., 2003). Briefly, R1 mESCs were seeded at a density of 50 cells/cm2
on mitomycin-treated stromal cell PA6 and cultured in ES-Serum Re-
placement Media, composed by KnockOut-DMEM (Invitrogen), 15%
KnockOut serum replacement (Invitrogen), 0.1 mM ?-mercaptoethanol
(Biochrom AG), and 2000 U/ml penicillin/streptomycin (Invitrogen).
Tangetal.•Wnt/?-CateninandShhinDANeurogenesisJ.Neurosci.,July7,2010 • 30(27):9280–9291 • 9281
After 5 d, medium was changed and supplemented with 25 ng/ml FGF8
(R & D Systems) and different concentrations of Shh (R & D Systems)
and the GSK3? inhibitor CT99021. From day 8 to day 11, cells were
cultured in N2 medium consisting of F-12 and MEM mixture at 1:1
(Invitrogen), glucose, N2 supplement (Invitrogen), 15 mM HEPES (In-
vitrogen), 200 mM L-glutamine, and 3 mg/ml AlbuMax I (Invitrogen)
supplemented with 50 ng/ml FGF8 and 10 ng/ml FGF2 (R & D Systems)
and the same concentration of Shh and CT99021 as in days 5–8. From
derived neurotrophic factor (GDNF) (both from R & D Systems), and
200 ?M ascorbic acid (Sigma).
After in vitro differentiation, cells were fixed in 4% PFA (10?, RT),
serum blocked, and incubated in the appropriate primary and subse-
quently secondary antibodies as described previously (Parish et al.,
2005). Nuclear counterstaining was performed using Hoechst. The fol-
lowing antibodies were used: mouse monoclonal anti-III-tubulin (TuJ1;
Freez Biologicals), mouse monoclonal anti-tyrosine hydroxylase (1:500;
ImmunoStar), rabbit anti-Foxa2 (1:500; Cell Signaling Technology),
Santa Cruz Biotechnology), and Alexa Fluor 488 goat anti-mouse and
Alexa Fluor 555 donkey anti-rabbit (1:500; Invitrogen).
Statistical analyses. Data were analyzed by two-tailed Student’s t test.
Values were expressed as mean ? SEM. Changes were identified as sig-
nificant if the p value was ?0.05.
To determine whether activation of canonical Wnt/?-catenin
signal in vMB affects the development of DA neurons, we gener-
ated conditional mutant mice in which the floxed exon 3 of
?-catenin (?-CtnEX3) was removed using Shh–Cre (named Shh–
Cre;?-CtnEx3/?). Expression of one copy of ?-CtnEX3allele using
mental Fig. S1, available at www.jneurosci.org as supplemental
Cre (Tang et al., 2009), Shh–Cre;?-CtnEx3/?mutants showed a
much higher level of ?-catenin protein in vMB at E12.5, with a
significant accumulation of the mutant proteins in the nuclei of
the neural progenitors (Fig. 1A,B) (data not shown). Compared
with control (?-CtnEx3/?) embryos, the vMB of Shh–Cre;?-
CtnEx3/?embryos showed a marked expansion of Sox2-, Ngn2-,
and Otx2-positive progenitors in the ventricular zone (VZ) (Fig.
1C–G). In addition, DA progenitors expressing Lmx1a, Lmx1b,
and Nurr1 also showed significant increases in the intermediate
zone and marginal zone (Fig. 1H–N).
We next examined whether the constitutive activation of
Wnt/?-catenin in vMB could have altered cell cycle progression
performed a short-term (2 h) BrdU labeling to determine the
and E11.5 Shh–Cre;?-CtnEx3/?mutants showed no detectable
difference in the number of BrdU?progenitors in the vMB VZ
(Fig. 2A–D), a significant increase was detected at E12.5 (Fig.
2E,F,M). Furthermore, a longer BrdU labeling time interval
(24 h) showed an even more drastic increase in the number of
progenitors that incorporated BrdU (Fig. 2G,H,N). In contrast,
9282 • J.Neurosci.,July7,2010 • 30(27):9280–9291 Tangetal.•Wnt/?-CateninandShhinDANeurogenesis
much fewer BrdU and TH double-positive neurons were gener-
ated in the Shh–Cre;?-CtnEx3/?mutants within the same time
interval (Fig. 2G,H, insets, O). Many of the apical progenitors in
the VZ of Shh–Cre;?-CtnEx3/?mutants continued to show posi-
of cell cycle in the vMB of Shh–Cre;?-CtnEx3/?mutants at E12.5
suggested that the constitutive activation of Wnt/?-catenin sig-
address this, we performed birthdating of DA neurons by pulse
the number of progenitors that have exited cell cycle [BrdU-
positive; Ki67-negative (BrdU?; Ki67?)] within this time inter-
val. Consistent with our prediction, there were much fewer
exited the cell cycle during the 24 h time interval (Fig. 2K,L,Q).
Together, these results supported the notion that constitutive
activation of Wnt/?-catenin signal in vMB led to the expansion
DA progenitors by reducing their exit from the cell cycle.
In analyzing the phenotype of the constitutive activation of
Wnt/?-catenin signaling in DA progenitors, we noticed that the
number of newly born DA neurons, marked by TH-positive
at E12.5 (Fig. 2E–H). To provide a more quantitative analysis of
DA neurons in Shh–Cre;?-CtnEx3/?mutants, we used stereology
to E18.5. Our results showed that, compared with control litter-
mates, there were consistently fewer DA neurons in the vMB of
Shh–Cre;?-CtnEx3/?mutants (Fig. 3A–G). Interestingly, a small
ectopic cluster of DA neurons was identified the interpedun-
cular nucleus (Fig. 3D,F). At E18.5, the reduction in DA neu-
To characterize the reduced DA neuron phenotype in Shh–
Cre;?-CtnEx3/?mutants, we first determined whether there was
?-CtnEx3/?mutants (supplemental Fig. S2, available at www.
jneurosci.org as supplemental material). We next examined
whether the ability of DA progenitors to differentiate was im-
paired in Shh–Cre;?-CtnEx3/?mutants. To test this hypothesis,
we cultured vMB progenitors from E12.5 control and Shh–Cre;
?-CtnEx3/?embryos in conditions that have been shown previ-
ously to promote differentiation of DA neurons (Takeshima et
al., 1996; Ye et al., 1998; Schulte et al., 2005). Consistent with the
in vivo phenotype, progenitors from Shh–Cre;?-CtnEx3/?mu-
Tangetal.•Wnt/?-CateninandShhinDANeurogenesisJ.Neurosci.,July7,2010 • 30(27):9280–9291 • 9283
tants gave rise to fewer number of DA neurons under basal cul-
ture conditions (Fig. 3H,I). Interestingly, the addition of
increasing doses of Shh only promoted a very modest increase
in the number of DA neurons in progenitors from Shh–Cre;?-
CtnEx3/?mutants (Fig. 3J,K,N). However, when treated with
Wnt5a, progenitors from Shh–Cre;?-CtnEx3/?mutant em-
bryos showed an increase in DA neuron numbers in a manner
similar to those from control (Fig. 3L–N).
The results from Figure 3 supported the notion that treatments
with additional exogenous factors, such as Shh or Wnt5a, can
indeed promote the generation of DA neurons from the progen-
itors of Shh–Cre;?-CtnEx3/?mutants. However, the fewer num-
ber of DA neurons from Shh–Cre;?-CtnEx3/?mutants suggested
that the regional activation of canonical Wnt/?-catenin signal
may have altered the milieu in the neurogenic niche of DA neu-
rons or the intrinsic properties of DA progenitors in Shh–Cre;?-
CtnEx3/?mutants. To test these hypotheses, we examined Shh
expression, an important exogenous factor that regulates the
neurogenesis of DA neurons (Hynes et al., 1995). Our results
at E10.5 (Fig. 4A). By E12.5, Shh mRNA became more restricted
to the VZ of vMB, immediately adjacent to the neurogenic niche
of DA progenitors (Fig. 4C). Despite the restricted expression
vMB, extending from VZ to the pia surface, suggesting that Shh
proteins may be transported along the radial glia (Fig. 4E). This
was confirmed by confocal imaging, which showed an extensive
Ventral midbrain progenitors in Shh–Cre;?-CtnEx3/?mutants show a significant reduction in DA neurons in vivo and in vitro. A–F, Compared with control (?-CtnEx3/?), vMB of
9284 • J.Neurosci.,July7,2010 • 30(27):9280–9291Tangetal.•Wnt/?-CateninandShhinDANeurogenesis
colocalization of Shh proteins with radial glia markers, e.g., Nes-
tin, RC-2, and Glast (glutamate–aspartate transporter) (Fig.
4G,H and data not shown). Unlike the wild-type embryos, con-
stitutive activation of Wnt/?-catenin led to a modest decrease of
Shh mRNA at E10.5 (Fig. 4B) but a near complete loss of Shh
of Shh–Cre;?-CtnEx3/?mutants at E12.5 but not at E10.5 (Fig.
4I–N). In contrast, the expression of other regional vMB mark-
ers, such as Nkx2.2 and Nkx6.1, showed no detectable change
(Fig. 4O,P). These results supported the hypothesis that persis-
for DA neurons by antagonizing the expression of Shh and Shh
target genes in the progenitors.
To further characterize the interactions between canonical
Wnt/?-catenin and Shh in the generation of DA neurons, we
cultured progenitors from the vMB of wild-type E10.5 embryos
Activation of ?-catenin in vMB antagonizes Shh expression and reduces Shh target genes. A, B, Shh mRNA expression is slightly reduced at E10.5 in vMB of Shh–Cre;?-CtnEx3/?
Tangetal.•Wnt/?-CateninandShhinDANeurogenesis J.Neurosci.,July7,2010 • 30(27):9280–9291 • 9285
and treated these progenitors with single, combined, or sequen-
tial treatment of Shh, Wnt1, or the GSK3? inhibitor CT99021
with increasing amount of recombinant Wnt1 or Shh led to a
dose-dependent increase in DA neuron numbers, with the opti-
with these results, the selective GSK3? inhibitor CT99021 also
promoted the generation of DA neurons (Fig. 5J). Surprisingly,
combined treatments of Wnt1 and Shh did not show an additive
or synergistic effect on the generation of DA neurons. Rather,
higher doses of Wnt1 (1250 ng/ml) appeared to reduce DA neu-
Shh (250 ng/ml) (Fig. 5K). Similarly, the GSK3? inhibitor
CT99021 also showed inhibitory effects on the generation of DA
neurons in the optimal conditions for Shh (250 ng/ml) (Fig. 5L).
Such antagonistic effects between Wnt1 and Shh in the genera-
tion of DA neurons were also detected in cultures obtained from
the vMB of E13.5 embryos (supplemental Fig. S3, available at
www.jneurosci.org as supplemental material).
The lack of additive or synergistic effect between Wnt1 and
recapitulate the in vivo conditions of DA neurogenesis and max-
imize the yield of DA neuron generation in cultures. To test this
hypothesis, we cultured progenitors from E10.5 embryos and
first treated them with optimal concentration of the GSK3? in-
hibitor CT99021 or Shh (the “priming” stage), followed by
switching culture conditions to optimal concentration of Shh or
CT99021 (the “maintenance” stage) (Fig. 5A). Contrary to our
expectations, sequential treatments with CT99021 followed by
Shh, or Shh followed by CT99021, reduced the number of DA
neurons compared with cultures treated with CT99021 or Shh
alone (Fig. 5M,N).
The antagonistic interaction between Shh and Wnt1 in the
generation of DA neurons from stem/progenitor cells was also
examined in a previously established culture condition to gener-
ate DA neurons from mESCs (Barberi et al., 2003). This culture
protocol consisted of a four-step protocol of treating mESCs
cocultured with mitomycin-treated stromal cells PA6 in serum
replacement media (SRM) (days 0–5), SRM plus FGF8 (days
media plus ascorbic acid, BDNF, and GDNF (days 11–14) (Fig.
6A). Under this condition, the majority of the TH?neurons
expressed additional dopaminergic markers, including Foxa2,
Nurr1, and Pitx3a (supplemental Fig. S4, available at www.
jneurosci.org as supplemental material) (Rodríguez-Go ´mez et
al., 2007; Hedlund et al., 2008). These results supported the no-
tion that most TH?neurons derived from mESCs using this
protocol exhibited a phenotype consistent with that of vMB DA
neurons. Our results also showed that the addition of Shh (200
ng/ml) from days 5 to 11 further promoted the generation of
TH?neurons from mESCs (Fig. 6B). Unlike the primary cul-
effect on DA neurons (Fig. 6B). Here it is important to note that
the baseline generation of DA neurons (and possibly the level of
9286 • J.Neurosci.,July7,2010 • 30(27):9280–9291Tangetal.•Wnt/?-CateninandShhinDANeurogenesis
E10.5 embryos. Despite this difference, and similar to the obser-
vation in progenitor cultures from E10.5 embryos, combined
treatments of Shh and CT99021 did not show additive or syner-
gistic effects (Fig. 6C,D). Rather, higher doses of Shh suppressed
DA neurogenesis from mESCs (Fig. 6C), and high doses of
(Fig. 6D). Moreover, we also found that high doses of CT99021
by a reduction in the number of Tuj1?cells. Interestingly, Tuj1-
positive neurons were mainly detected outside the colonies (Fig.
DAneurogenesis in vivo
The results in Shh–Cre;?-CtnEx3/?mutants indicated that the
constitutive activation of the canonical Wnt/?-catenin signaling
in the vMB led to the expansion of DA progenitors but reduced
the neurogenesis of DA neurons (Figs. 1, 2). Based on these data,
we reasoned that cell-type-specific activation of the Wnt/?-
catenin signaling in midline progenitors may avoid the defect in
DA neurogenesis seen in Shh–Cre;?-Ct-
IRES–Cre mediates recombination in
essentially all postmitotic DA neurons
and a subpopulation of midline progeni-
tors at E10.5 (Tang et al., 2009). Unlike
the phenotype in Th–IRES–Cre;?-Ctnfl/fl
IRES–Cre;?-CtnEx3/?mutants showed a
significant increase at E11.5 and E12.5 (Fig.
7A–D,I). By P0 and P21, Th–IRES–Cre;?-
CtnEx3/?mutants showed an ?20% in-
crease in DA neuron numbers compared
increase in DA neurons, Th–IRES–Cre;?-
CtnEx3/?mutants also showed a persistent
increase in the number of committed pro-
genitors (Nurr1?;TH?cells) in vMB at
E11.5 and E12.5 (Fig. 7J–N). Further-
more, we performed 24 h neuronal birth-
progenitors with BrdU at E10.5 or E11.5
and allowed them to become TH?post-
mitotic DA neurons until E11.5 and
E12.5, respectively. Our results showed
that the number of newly born TH?
neurons was significantly increased
To further investigate the mechanisms
of the increased Nurr1?;TH?progeni-
we performed birthdating experiments in
this population by labeling the progeni-
tors with BrdU at E10.5 or E11.5 and al-
lowed them to develop for 24 h. Our
results showed an increase in the number
of newly born Nurr1?precursors within
and from E11.5 to E12.5 (Fig. 8A–E). To-
gether, these results indicated that the ac-
tivation of Wnt/?-catenin signaling in a subpopulation of
midline progenitors using the Th–IRES–Cre led to a significant
increase in neurogenesis and DA neurons.
The results from this study reveal an intricate, albeit primarily
DA neurogenesis in vMB progenitors as well as in mESCs (Fig.
8F). Activation of Wnt/?-catenin can promote the expansion of
DA progenitors and the generation of DA neurons. However,
these effects appear to be cell-context dependent such that con-
stitutive activation of Wnt/?-catenin in vMB using Shh–Cre ex-
pands early progenitors but perturbs cell cycle progression in
these progenitors and antagonizes the expression of Shh and
ber of DA neurons. In contrast, a cell-type-specific activation of
Wnt/?-catenin in the midline progenitors using Th–IRES–Cre
circumvents these adverse effects and leads to a significant in-
crease in DA neuron numbers.
day 5 to 11. B, Treatment with Shh (200 ng/ml) or CT99021 (2 ?M) alone increases the DA neuron numbers. C, Combined
treatments with Shh and CT99021 do not show additive or synergistic effects in generating more DA neurons from mESCs. In
Tangetal.•Wnt/?-CateninandShhinDANeurogenesis J.Neurosci.,July7,2010 • 30(27):9280–9291 • 9287
Several members of the Wnt family have been shown to regulate
instance, the canonical Wnt signaling mechanisms, mediated by
Wnt1, Wnt2, and Wnt3a, control the patterning of midbrain–
hindbrain junction and the initial generation of DA progenitors
in vMB, whereas Wnt5a regulates the differentiation of DA neu-
rons (Danielian and McMahon, 1996; Castelo-Branco et al.,
2003; Castelo-Branco and Arenas, 2006; Andersson et al., 2008;
Sousa et al., 2010). Consistent with these findings, analyses of
Wnt1?/?and En1Wnt1/?mutant mice reveal a genetic network
controlled by Wnt1 to regulate the establishment of DA progen-
itor domain and the full differentiation of DA neurons (Prakash
et al., 2006; Omodei et al., 2008). Moreover, targeted deletion of
?-catenin using either region-specific Shh–Cre in vMB or cell-
type-specific Th–IRES–Cre in midline progenitors further dem-
onstrate the essential role of Wnt/?-catenin signaling in the
control of gene expression and in cell cycle progression during
DA neurogenesis (Joksimovic et al., 2009; Tang et al., 2009). Re-
conserved in mESCs in which ?-catenin and Lmx1a cooperatively
controls the differentiation of DA neurons through an autoregula-
9288 • J.Neurosci.,July7,2010 • 30(27):9280–9291 Tangetal.•Wnt/?-CateninandShhinDANeurogenesis
Our current study provides additional in vivo evidence that
activation of Wnt/?-catenin signaling leads to a marked expan-
sion of early DA progenitors that express Sox2, Ngn2, and Otx2,
as well as an increase in the progenitors that express Lmx1a,
Lmx1b, and Nurr1 (Figs. 1, 8F). Despite the expansion of these
progenitors, however, activation of Wnt/?-catenin perturbs
cell cycle progression and reduces the production of TH?DA
neurons in vMB (Figs. 2, 3). Interestingly, when cultured in the
presence of Wnt5a, the progenitors from Shh–Cre;?-CtnEx3/?
mutants differentiate into DA neurons in a manner similar to
those from control (Fig. 3N). These results provide important in-
of Lmx1a in mESCs alone induces robust expression of Nurr1 and
Pitx3, but only a limited number of these cells show properties of
differentiated DA neurons (Chung et al., 2009). Furthermore, our
results provide additional support that, when given the optimal
growth conditions, such as excess Wnt5a, the progenitors ex-
panded by the Wnt/?-catenin signaling mechanisms have the
potential to differentiate into mature DA neurons.
Several explanations can account for the failure for constitutive
activation of Wnt/?-catenin signaling to promote the differenti-
ation of vMB progenitors into mature DA neurons in Shh–Cre;
?-CtnEx3/?mutants. First, as indicated above, analyses of the
proliferation and cell cycle progression in the DA progenitors in
Shh–Cre;?-CtnEx3/?mutants show much more progenitors in
itors show reduced cell cycle exit (Fig. 2). Although the underly-
ing cause(s) for the dysregulation of cell cycle progression in the
DA progenitors of Shh–Cre;?-CtnEx3/?mutants is not entirely
clear, it is possible that the reduced expression of cyclin D1 and
perhaps other cell cycle genes in the vMB of these mutants may
have contributed to this phenotype. Second, the expanded pro-
genitors may be exposed to a different environment that may
or postmitotic neurons. Consistent with this notion, progenitors
from Shh–Cre;?-CtnEx3/?mutants can
differentiate into DA neurons in the pres-
ence of Wnt5a just like those progenitors
from control embryos (Fig. 3N).
The third explanation for the reduced
production of DA neurons in Shh–Cre;
?-CtnEx3/?mutants is the significant
downregulation of Shh and forkhead
transcription factor Foxa2 expression in
the vMB (Fig. 4). The downregulation of
no detectable Shh is present in vMB in
these mutants. In contrast, no detectable
downregulation of Foxa2 is present until
E12.5. The downregulation of Foxa2 may
be attributable to the loss of Shh. Alterna-
tively, activation of Wnt/?-catenin may
directly or indirectly suppress the expres-
sion of Foxa2. Consistent with these re-
sults, expanded progenitors from Shh–
Cre;?-CtnEx3/?mutants show limited
even when cultured in the presence of ex-
cess Shh, probably because of the severe
reduction in Foxa2 expression (Figs. 3N,
4L). Similar antagonistic effects of Wnt/?-catenin activation on
the expression of Shh in the developing hindbrain have been
reported in a recent study (Joksimovic et al., 2009). Remarkably,
the antagonistic effects between Wnt/?-catenin and Shh can be
demonstrated in the differentiation of DA neurons using in vitro
the development of DA neurons (Fig. 8F) (Chung et al., 2009).
Constitutive activation of one signaling mechanism may perturb
a delicate balance between Wnt/?-catenin and Shh signaling
mechanisms in the process of DA neurogenesis (Fig. 8F). Curi-
tectable effects on the expression of Lmx1a, Lmx1b, Foxa1, or
Foxa2 (Ferri et al., 2007; Lin et al., 2009). These studies raise the
is possible that loss of Shh and Foxa2 in the Shh–Cre;?-CtnEx3/?
mutants cooperatively block the differentiation of DA neurons.
Alternatively, activation of Wnt/?-catenin in the vMB of Shh–
Cre;?-CtnEx3/?mutants may suppress additional target genes
that influence the generation of DA neurons.
The phenotype that Shh–Cre;?-CtnEx3/?mutants show a sig-
nificant reduction in Foxa2 expression in vMB is reminiscent of
those in Nestin–Cre;Foxa2flox/floxmutants, which show an expan-
TH?DA neurons from E12.5 to E18.5 (Ferri et al., 2007).
Although Foxa1 null mutants also show a similar phenotype at
E12.5, this deficit appears to be transient at E12.5 and is not
detected at later developmental stages. It is unclear whether the
possible that downstream targets activated by ?-catenin may
negatively regulate the expression of Foxa2. Regardless of the
Tangetal.•Wnt/?-CateninandShhinDANeurogenesisJ.Neurosci.,July7,2010 • 30(27):9280–9291 • 9289
naling may be a feasible target to promote the generation of DA
the GSK3? inhibitor CT99021 promote the production of DA
neurons in vMB progenitor cell and mESC cultures (Figs. 5, 6).
Despite these encouraging results, simultaneous treatment with
optimal doses of Wnt1 or CT99021 and Shh in vMB progenitors
or mESCs shows no additive or synergistic effects, whereas treat-
ments with higher doses of CT99021 and Shh actually suppress
the generation of DA neurons (Figs. 5, 6). Furthermore, sequen-
tial treatments of Shh and CT99021 also do not show additional
in different vMB cells may further aid in the development of
improved protocols for the generation of DA neurons in embry-
onic stem cell cultures. As a proof of principle, we report that
cell-type-specific activation of Wnt/?-catenin in midline DA
progenitors, using Th–IRES–Cre, leads to increases in Nurr1?
precursors as well as in mature DA neurons both in prenatal and
postnatal brains (Figs. 7, 8). Together, these encouraging results
support the notion that, although a broad activation of Wnt/?-
catenin remains an effective means to promoting the expansion
of DA progenitors, a restricted activation in midline progenitors
provides beneficial effects in promoting the generation of DA
neurons. We suggest that Wnt/?-catenin activation in specific
cell types may become a valuable strategy to improve the DA
differentiation of embryonic stem cells.
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