Summary

Meso-diencephalic dopaminergic (mdDA) neurons control voluntary movement,
cognition and the reward response, and their degeneration is associated with
Parkinson's disease (PD). Prospective cell transplantation therapies for PD
require full knowledge of the developmental pathways that control mdDA
neurogenesis. We have previously shown that Otx2 is required for the
establishment of the mesencephalic field and molecular code of the entire
ventral mesencephalon (VM). Here, we investigate whether Otx2 is a specific
determinant of mesencephalic dopaminergic (mesDA) neurogenesis by studying
mouse mutants that conditionally overexpress or lack Otx2. Our data
show that Otx2 overexpression in the VM causes a dose-dependent and selective
increase in both mesDA progenitors and neurons, which correlates with a
remarkable and specific enhancement in the proliferating activity of mesDA
progenitors. Consistently, lack of Otx2 in the VM specifically affects the
proliferation of Sox2+ mesDA progenitors and causes their premature
post-mitotic transition. Analysis of the developmental pathway that controls
the differentiation of mesDA neurons shows that, in the absence of Otx2, the
expression of Lmx1a and Msx1, and the proneural genes
Ngn2 and Mash1 is not activated in Sox2+ mesDA
progenitors, which largely fail to differentiate into Nurr1+ mesDA
precursors. Furthermore, proliferation and differentiation abnormalities
exhibit increasing severity along the anterior-posterior (AP) axis of the VM.
These findings demonstrate that Otx2, through an AP graded effect, is
intrinsically required to control proliferation and differentiation of mesDA
progenitors. Thus, our data provide new insights into the mechanism of mesDA
neuron specification and suggest Otx2 as a potential target for cell
replacement-based therapeutic approaches in PD.

Generation of the tOtx2bov mutant and Otx2
overexpression in ES cells and mutant embryos. (A) The genomic
position at the chromosome 7 D2 region (upper line) where the
tOtx2bov cassette (second line) is inserted allows the
genotyping of mutant embryos by using two pairs of primers (open arrows in the
upper line for the wild type and open arrows in the second line for the
mutant). Cre-mediated removal of the Neo-triple polyA stop cassette generates
the tOtx2ov allele (third line) and this event is
monitored by the primers shown in the second and third line (black arrows).
(B) The R26CreER/+; tOtx2bov/+ ES clone
is treated with tamoxifen for 24 hours prior to monitoring the removal of the
Neo-triple polyA cassette (upper panel), the Otx2 protein level (second panel)
and the GFP activation (third panel). (C-K) Immunohistochemistry and in
situ hybridization performed to detect the expression of Otx2 (C-E),
En1-driven Cre recombinase (F-H) and GFP transcripts (I-K)
in adjacent sagittal sections of E10.5 En1Cre/+,
En1Cre/+; tOtx2ov/+ and En1Cre/+;
tOtx2ov/ov embryos show that Otx2 is ectopically activated in
the anterior hindbrain and cerebellum anlage (arrows in D,E) and, as revealed
by GFP expression, overexpressed in the whole mesencephalon (J,K).
Abbreviations: Mes, mesencephalon; Hb, hindbrain; MHB, midbrain-hindbrain
border.

MATERIALS AND METHODS

Mouse mutants and ES cells

For the inducible overexpression of Otx2, we designed a molecule where the
expression of the full-coding cDNA (49bp of 5′UTR-coding-204bp of
3′UTR), driven by the CMV enhancer-chicken β actin promoter, was
blocked by a removable loxP-neo-triple polyA-loxP `stop' cassette. In
addition, an ires-GFP sequence was introduced downstream of the Otx2 cDNA to
follow the transgene expression in vivo. The linearized construct was
electroporated into E14Tg4a2 ES cells. DNAs extracted from 30 randomly
integrated G418 resistant clones were digested with EcoRI and
BamHI restriction enzymes, and hybridized with two probes
(Neo and GFP) localized across the two restriction sites
(Fig. 1A). Only clones
detecting single EcoRI and BamHI bands longer than the
minimum expected length were chosen for further analysis. Then, in order to
detect potential disruption of any gene functions by the random integration of
the construct, and for genotyping, we identified the chromosomal site and
sequences flanking the transgene insertion in several ES clones by using the
Vectorette System (Sigma). We chose, as the best candidate to generate a mouse
line, a clone in which the transgenic and blocked Otx2 overexpressing
(tOtx2bov) construct was integrated in an intergenic
region of chromosome 7 D2, 51 kb downstream of the Isg20 gene and 111
kb upstream of the Agc1 gene (Fig.
1A). To test the functionality and efficacy of the transgene
expression, we re-transfected this clone to target the CreER-recombinase in
the Rosa26 (R26) locus.

The tOtx2bov/+; R26CreER/+ ES clone was
tested for excision of the loxP-Neo-triple polyA-loxP cassette by using
specific pairs of primers (black arrows in
Fig. 1A and upper panel in
Fig. 1B). The PCR products were
260 bp and 163 bp long for the excised and non-excised allele, respectively
(Fig. 1B).

The tOtx2bov/+; R26CreER/+ ES clone was also
tested to quantify the transgenic Otx2 and GFP gene products by western blot
of total protein extracts of ES cells treated 24 hours with 100 nM of
4-hydroxytamoxifen (Sigma) (Fig.
1B). Based on these tests, the original
tOtx2bov/+ ES cell clone was injected into C57 blastocysts
and chimeras were mated for germline transmission.

Litters were genotyped by using specific primers (open arrows in
Fig. 1A). Primers sequences
used in this study are available upon request. The En1Cre/+;
Otx2flox/flox mouse model to study Otx2 inactivation has been
previously described (Puelles et al.,
2004).

Dopaminergic cell counting

Cell counting of TH+ neurons was performed on eight E18.5
dissected brains for each genotype (wild type, En1Cre/+,
tOtx2bov/+, En1Cre/+; tOtx2ov/+ and
En1Cre/+; tOtx2ov/ov). TH cell counting was
also confirmed in three brains for each genotype by immunostaining with Pitx3
(data not shown). The procedure adopted for selection of sections and
TH+ cell-counting can be provided on request.

Cell proliferation experiments

To determine the labeling index (LI), pregnant females at E10.5 or E11.5
were intraperitoneally injected with 5′-bromo 2′-deoxyuridine
(BrdU) at a concentration of 50 mg/kg of body weight and embryos collected 30
minutes after injection. To determine the Quit fraction (Qf), BrdU was
administered as for LI experiments, but embryos were collected about 24 hours
later. Further information on these experiments can be provided on
request.

RESULTS

Generation of mouse mutants overexpressing Otx2

To assess the role of Otx2 in mesDA neurogenesis, we studied first the
effect of Otx2 overexpression in the VM. To achieve this, we generated a
transgenic mouse model that conditionally activates Otx2 by
En1-driven Cre activity (see Materials and methods). The construct
that conditionally overexpresses Otx2 (Fig.
1A) was transfected into ES cells to select the
tOtx2bov/+ clone that fulfils the following two
requirements: containing a single copy of the construct inserted in a bona
fide neutral genomic region; and overexpressing a moderate level of Otx2. The
selected tOtx2bov/+ ES cell clone contained a single
insertion of the construct in an intergenic area of chromosome 7 at the chr7D2
position, 51 kb downstream of the Isg20 gene and 111 kb upstream of
the Agc1 gene (Fig.
1A), and showed a moderate increase of about twofold of the Otx2
protein expressed in ES cells (Fig.
1B; see Materials and methods). In this assay, however, the level
of GFP was lower than that expected on the basis of its mRNA
(Fig. 1B; data not shown). This
low level of GFP protein was also confirmed in vivo and not further
investigated (data not shown). Based on these tests, the original
tOtx2bov/+ ES cell clone was used to generate the
tOtx2bov/+ strain. tOtx2bov/bov mice
were healthy and fertile, and did not exhibit abnormalities. Next, we analyzed
whether, at E10.5 in En1Cre/+; tOtx2ov/+ and
En1Cre/+; tOtx2ov/ov embryos, the
tOtx2bov transgene was properly activated by the
En1-driven Cre recombinase. As revealed by Otx2 immunostaining and
GFP transcription, the transgenic Otx2 protein was detected in the
mesencephalon and the rostral hindbrain
(Fig. 1C-K). Indeed, the
GFP mRNA and, consequently, the transgenic Otx2 were activated also
in those territories such as the posterior ventral pretectum and anterior
mesencephalon, where En1-driven Cre recombinase and the endogenous
En1 gene were not expressed at E10.5
(Fig. 1C-K; see Fig.
S1A-F″ in the supplementary material). This indicated that the
activation of the transgenic allele occurred earlier, when En1
expression was broader than at E10.5. Indeed, at E9.2, as revealed by
GFP transcripts, the Otx2 overexpressing allele was already activated
in early progenitors expressing En1 (see Fig. S1G-I″ in the
supplementary material).

Next, we analyzed whether the Otx2 overexpression affected the integrity of
the midbrain-hindbrain boundary (MHB) region by monitoring the expression of
Otx1, Fgf8 and Gbx2. Compared with E10.5 and E12.5
tOtx2bov/+ embryos, the expression of these three genes
was essentially unaffected in En1Cre/+;
tOtx2ov/+ embryos (see Fig. S2A-J in the supplementary
material), whereas in En1Cre/+; tOtx2ov/ov, the
posterior border of the Otx1 and Fgf8 expression domains was
moderately expanded in the anterior hindbrain at E10.5 (see Fig. S2K-M in the
supplementary material). However, at E12.5 in En1Cre/+;
tOtx2ov/ov mutants, these MHB abnormalities appeared much less
severe (see Fig. S2N,O in the supplementary material). Thus these data
indicate that the tOtx2bov allele was properly activated
by En1-driven Cre activity and that, probably owing to the moderate
level of Otx2 activation, the integrity of the MHB was not remarkably
affected. A more severe phenotype was instead observed in the cerebellum at
E18.5 (data not shown).

Otx2 overexpression induces AP graded increase of mdDA neurons.
(A-L″) Immunohistochemistry performed at six sequential
anatomical levels corresponding to the posterior pretectum and anterior
mesencephalon (A-B″,G-H″), the intermediate mesencephalon
(C-D″,I-J″) and the posterior mesencephalon
(E-F″,K-L″) of E12.5 wild-type, En1Cre/+;
tOtx2ov/+ and En1Cre/+;
tOtx2ov/ov embryos with Pitx3 and Nkx6.1 (A-F″) and Otx2
and Nkx2.2 (G-L″) shows that in Otx2-overexpressing embryos the number
of Pitx3+ neurons gradually increases, moving from the anterior
towards the posterior mesencephalon. This increase correlates with the copy
number of the Otx2-overexpressing allele. Conversely, the Otx2 overexpression
does not affect the extent of progenitor domains located dorsal to the mdDA
domain; indeed, while the Nkx6.1--Nkx2.2- mdDA domain
(ventral to the arrow) gradually expands, the
Nkx6.1+-Nkx2.2- (between arrow and arrowhead) and the
Nkx6.1+-Nkx2.2+ (dorsal to the arrowhead) domains retain
a similar distribution among the three genotypes at the different anatomical
levels. Abbreviation: Ant, anterior.

Analysis of mesDA neurons was performed at four AP anatomical levels in
E18.5 wild-type, tOtx2bov/+, En1Cre/+ and Otx2
overexpressing mice (n=8 per genotype). No difference in mesDA
neurons was detected among wild-type, tOtx2bov/+ and
En1Cre/+ embryos (data not shown)
(Sonnier et al., 2007),
whereas in En1Cre/+; tOtx2ov/+ embryos, the
number of TH+ neurons showed a graded AP increase
(Fig. 2E-H,M-P). In particular,
compared with control embryos, in En1Cre/+;
tOtx2ov/+ mutants, increases of about 20%, 35% and 50% of
TH+ neurons were detected, respectively, in the VTA of the
anterior, intermediate and posterior mesencephalon. We then studied the
phenotype of En1Cre/+; tOtx2ov/ov mutants
(n=8) to investigate whether, compared with En1Cre/+;
tOtx2ov/+ mutants, a more robust overexpression of Otx2
correlated with a higher number of TH+ neurons. We found that,
compared with control brains (Fig.
2A-D), En1Cre/+; tOtx2ov/ov mutants
generated up to about 80 and 100% more TH+ neurons in the
intermediate and posterior mesencephalon, respectively; and up to 25% and 40%
more TH+ neurons in the SNpc and anterior VTA, respectively
(Fig. 2I-P). These findings
were also confirmed by counting Pitx3+ cells (data not shown).

Selective expansion of the mesDA domain in Otx2-overexpressing
embryos. (A-O) Immunohistochemistry and in situ hybridization
performed on adjacent sections through the intermediate mesencephalon of E10.5
tOtx2bov/+, En1Cre/+; tOtx2ov/+ and
En1Cre/+; tOtx2ov/ov embryos with Otx2 and
Lmx1b (A,F,K), Nkx6.1 and Shh (B,G,L), Nkx2.2 and Shh (C,H,M), Nkx2.2 and
Foxa2 (D,I,N) antibodies, and Wnt1 probe (E,J,O). The arrow indicates
the dorsal border of Lmx1b expression, which is adjacent to the ventral border
of Nkx6.1 and the arrowhead indicates the dorsal border of Shh, which is
adjacent to or slightly overlapping the ventral border of Nkx2.2.
Abbreviations: rp, roof plate.

Next, we analyzed whether this AP differential response to Otx2
overexpression was detected in meso-diencephalic DA (mdDA) progenitors and in
early post-mitotic mdDA neurons by analyzing in wild type and Otx2
overexpressing embryos the expression of Foxa2, Nkx6.1, Otx2 and Nkx2.2 at
E10.75 and that of Pitx3, Nkx6.1, Otx2 and Nkx2.2 at E12.5. At E10.75, we
analyzed whether the relative extent of the mdDA domain
(Foxa2+-Nkx6.1-) was gradually expanded along the AP
axis and whether the dorsal
Nkx6.1+-Foxa2+-Nkx2.2- and
Foxa2+-Nkx6.1+-Nkx2.2+ domains were affected
by the Otx2 overexpression (Fig.
3; see Fig. S3 in the supplementary material). Compared with
wild-type embryos, a selective and AP graded expansion of the
Foxa2+-Nkx6.1- mdDA domain was detected in
Otx2-overexpressing mutants (Fig. S3A-F′; data not shown). In
particular, this expansion was much less evident in En1Cre/+;
tOtx2ov/+, where this expansion was detected in the posterior
region of the intermediate mesencephalon and in the posterior mesencephalon
(data not shown; Fig. 4).
Instead, in En1Cre/+; tOtx2ov/ov embryos, a
mild expansion of the Foxa2+-Nkx6.1--Nkx2.2-
domain was detected in the pretectum and anterior mesencephalon (see Fig.
S3A-B′ in the supplementary material); this expansion gradually
increased, moving towards the posterior mesencephalon (see Fig. S3C-F′
in the supplementary material). Interestingly, the relative extent of the
Nkx6.1+-Foxa2+-Nkx2.2- and
Nkx6.1+-Foxa2+-Nkx2.2+ progenitor domains
appeared not affected by the Otx2 overexpression along the AP axis (see Fig.
S3 in the supplementary material).

A similar experiment performed to detect early post-mitotic mdDA
Pitx3+ neurons showed an evident AP graded increase in the number
of the Pitx3+ neurons and in the extent of the Nkx6.1-
mdDA domain (Fig. 3A-F″).
Notably, the generation of Pitx3+ neurons was apparently unaffected
in the pretectum and anterior mesencephalon of En1Cre/+;
tOtx2ov/+ mutants and, similar to E10.75 embryos, the relative
extents of the Nkx6.1+-Nkx2.2- and
Nkx6.1+-Nkx2.2+ domains were very similar in control and
Otx2-overexpressing embryos (Fig.
3). Thus, these findings collectively suggest that mdDA
progenitors exhibit an AP differential and dose-dependent sensitivity in their
response to Otx2 overexpression through increased generation of mature mdDA
neurons.

Based on previous findings, we analyzed in detail at E10.5 and E12.5
whether the identity and/or relative extent of VM progenitor domains was
altered. At the anatomical level corresponding to the intermediate
mesencephalon of E10.5 wild-type (data not shown) or
tOtx2bov/+ control embryos, Lmx1b was restricted to the
mesDA domain (Fig. 4A); Shh
expression included the Lmx1b+ domain and the ventral half of the
Nkx6.1+ domain (Fig.
4B), and was adjacent to Nkx2.2
(Fig. 4C); the
Nkx2.2+ domain, in turn, partially overlapped with the dorsal
region of both the Foxa2+ and Nkx6.1+ domains
(Fig. 4D and compare
Fig. 4B to C); and
Wnt1 was co-expressed in the mesDA domain with Lmx1b with the
exception of the medialmost floor-plate region
(Fig. 4E). In
En1Cre/+; tOtx2ov/+ and
En1Cre/+; tOtx2ov/ov embryos, no obvious
abnormalities were identified in the boundary relationships between the
Nkx6.1+, Nkx2.2+,
Shh+, Lmx1b+,
Foxa2+ and Wnt1+ domains or in the
extent of the Nkx6.1+ and Nkx2.2+
domains (Fig. 4F-O), whereas a
selective expansion of the Lmx1b+ and
Wnt1+ domains was detected
(Fig. 4F,K,J,O). In particular,
this expansion was mild in En1Cre/+; tOtx2ov/+
embryos and more pronounced in En1Cre/+;
tOtx2ov/ov mutants. In addition to the analysis performed with
Pitx3, Nkx6.1 and Nkx2.2 (Fig.
3; see Fig. S3 in the supplementary material), the phenotype
described at E10.5 (Fig. 4) was
confirmed at E12.5 by assessing also the combined expression of Lmx1b,
Nkx6.1, Nkx2.2, Foxa2 and Shh (Fig. S4A-C″).

Next, we studied whether Otx2 overexpression in the VM had a specific
effect on the generation of mesDA neurons or also affected other neuronal
populations. To achieve this, we first compared the expression of Isl1 and
Pou4f1, two post-mitotic markers of the OM and RN neurons, with that of AADC,
Lmx1b and Pitx3; and then the expression of Nurr1 with that of Lim1/2, which
were, respectively, expressed in early post-mitotic mesDA and RN neurons. We
found that, in contrast to the increase in the mesDA neurons, the number of
Isl1+ and Pou4f1+ neurons, the size of the OM and RN and
the identity of RN early post-mitotic precursors were similar in
Otx2-overexpressing and control embryos (compare Fig.
5A-H with
5I-X). Collectively, these
findings indicate that Otx2 overexpression induces a selective expansion of
both mesDA progenitors and neurons, without affecting identity and size of
adjacent progenitor domains or their post-mitotic progeny.

Otx2 is required to control proliferating activity of mesDA
progenitors

Next, we studied whether Otx2 may control the extent of the mesDA domain by
regulating selectively the proliferation of mesDA progenitors and/or their
post-mitotic transition. To achieve this, we first determined the LI in the
mesDA and Nkx6.1+ domains by providing a short pulse of BrdU (30
minutes) and measuring the percentage of BrdU+ cells over the total
number of cells along the AP axis of the VM at E10.5 and E11.5 in control and
mutant embryos. We found that the fraction of progenitors in S phase was
remarkably increased in the mesDA domain of mutants at both stages analyzed,
and that this increase was dose dependent and gradually more pronounced in the
intermediate and posterior mesencephalon
(Fig. 6A-H). Remarkably, almost
no effect was detected in the proliferating activity of progenitors belonging
to the Nkx6.1+ domain (Fig.
6A-F,I,J).

We then investigated whether the increase of mesDA neurons may also be
contributed by a decreased number of early mesDA progenitors exiting the cell
cycle. The percentage of cycling progenitors quitting the cell cycle within 24
hours of BrdU administration at E10.5 was calculated by measuring the fraction
of BrdU+ cells that were Ki67- (Qf). Our data showed
that the Qfs measured in the mesDA domain, but not those in the
Nkx6.1+ domain, were significantly reduced and, also in this case,
correlated with the level of Otx2 overexpression (see Fig. S5 in the
supplementary material). These findings thus suggest that the expansion of the
mesDA domain may be caused by a selective enhancement in the proliferating
activity of mesDA progenitors coupled to a decrease in the percentage of mesDA
progenitors quitting the cell cycle. Based on these results, we studied the
proliferation in the VM of En1Cre/+;
Otx2flox/flox embryos
(Puelles et al., 2004).
Previously, we reported that, in this mutant, the lack of Otx2 generates
ventral de-repression of Nkx2.2, loss of Nkx6.1 expression in progenitors,
dorsal expansion of Shh and lack of Wnt1 expression. All these events
resulted in the generation of 5-HT-containing neurons from RN and dorsalmost
mesDA progenitors, and heavy reduction of mesDA neurons
(Puelles et al., 2004;
Prakash et al., 2006).
Interestingly, these abnormalities mildly affected the pretectum and anterior
mesencephalon (see Fig. S4D-I′ in the supplementary material), with
increasing severity in the intermediate and posterior mesencephalon (about 80%
less TH+ neurons) (see Fig. S4J-L′ in the supplementary
material) (Puelles et al.,
2004).

As previous studies have not addressed which process(es) was affected in
mesDA neurogenesis of En1Cre/+; Otx2flox/flox
mutants, we investigated whether the heavy reduction in mesDA neurons may be
caused by abnormality in proliferation and/or premature post-mitotic
transition and/or differentiation of their progenitors. First, we studied
proliferation in the mesDA (Nkx2.2-) and Nkx2.2+
domains. Proliferating activity determined at E10.5 and E11.5 showed that the
LI was heavily reduced in the mesDA domain of mutant embryos
(Fig. 7A-J). By contrast, the
LI detected in the Nkx6.1+ domain of control embryos and in the
Nkx2.2+ domain of En1Cre/+;
Otx2flox/flox mutants did not differ significantly
(Fig. 7A-H,K,L). Next, we
analyzed whether in these mutants mesDA progenitors quit the cell cycle
prematurely. Compared with wild-type embryos, the Qfs for the intermediate and
posterior (but not for the anterior) mesencephalon were dramatically increased
in mutants (Fig. 7M-Q) and,
remarkably, the expression of Ki67 was switched off in a relevant fraction of
mesDA progenitors of the intermediate and posterior VM
(Fig. 7M,N). As for the LI, the
Qfs determined in the Nkx2.2+ domain also showed only a mild
reduction when compared with those of the Nkx6.1+ domain of control
embryos (Fig. 7M-P,R). Next, as
Ki67 expression was heavily affected, we determined whether mesDA progenitors
lacking Otx2 retained the expression of Sox2, which is normally transcribed by
most of cycling CNS progenitors (Ki67+) and downregulated when they
exit the cell cycle and differentiate
(Graham et al., 2003;
Kele et al., 2006). We found
that, despite the severe lack of both Ki67+ and BrdU+
cells (Figs 7,
8), the expression of Sox2 was
retained (Fig. 8G,H),
indicating that the Otx2-dependent impairment in proliferation did not affect
Sox2 expression. Because this and previous studies showed that Wnt1
expression was lost in the intermediate and posterior mesencephalon of
En1Cre/+; Otx2flox/flox mutants
(Prakash et al., 2006)
(Fig. 8D,E) and expanded in
Otx2-overexpressing embryos (Fig.
8F), we studied whether in the absence of Wnt1,
Sox2+-Otx2- progenitors expressed Cyclin D1 (CycD1), a
direct target of the Wnt canonical pathway
(Shtutman et al., 1999;
Tetsu and McCormick, 1999). A
remarkable loss of CycD1 expression (Fig.
8K) was detected in the Sox2+-Nkx2.2-
(Fig. 8B,H) domain of
En1Cre/+; Otx2flox/flox embryos. Conversely,
p27kip1, which is normally expressed at high level in quiescent
post-mitotic neuronal cells (Lee et al.,
1996), was strongly activated in the
Sox2+-Nkx2.2- cells
(Fig. 8H). Compared with
En1Cre/+; Otx2flox/flox embryos, in mutants
overexpressing Otx2 an essentially opposite phenotype was detected. Indeed,
Wnt1 was expanded (Fig.
8F), BrdU+ (Fig.
8C,L) and CycD1+
(Fig. 8L) cells remarkably
increased in number, and a high level of p27kip1 was detected only
in Sox2- post-mitotic neurons
(Fig. 8I).

The proliferating activity of mesDA progenitors is enhanced in Otx2
overexpressing mutants. (A-F) Representative adjacent sections
through the intermediate mesencephalon of tOtx2bov/+
(A,B), En1Cre/+; tOtx2ov/+ (C,D) and
En1Cre/+; tOtx2ov/ov (E,F) embryos pulsed with
BrdU for 30 minutes at E10.5 or E11.5, are immunostained with BrdU and Nkx6.1,
and stained with Hoechst to determine the LI of progenitors in the mesDA and
Nkx6.1 domains. The arrow and the broken line indicate approximately the
Nkx6.1+ domain analyzed. (G-J) Graphic representation of the
LI detected along the mesDA (G,H) and the Nkx6.1+ (I,J) domains
shows a selective dose-dependent increase in the proliferating activity of
mesDA progenitors developing in the intermediate and posterior mesencephalon.
Abbreviations: Ant., anterior; Int., intermediate; Post., posterior.

We then investigated whether abnormalities in Wnt1 expression
correlated with the mesencephalic territory primarily affected in cell
proliferation by Otx2 (overexpression or inactivation). We found that,
although in the caudal mesencephalic area the expression of Wnt1 was
expanded in Otx2 overexpressing mutants and lost in embryos lacking Otx2 (see
Fig. S6C-D″ in the supplementary material), in the rostral mesencephalon
of both En1Cre/+; tOtx2ov/ov and
En1Cre/+; Otx2flox/flox mutants, the expression
of Wnt1 was slightly affected (see Fig. S6A-A″ in the
supplementary material), thus suggesting a close correlation between the
territory requiring Otx2 for proliferation of mesDA progenitors and
abnormalities in Wnt1 expression. Collectively, these findings
indicate that Otx2 selectively controls the proliferating activity of
intermediate and posterior mesDA progenitors and prevents their premature
post-mitotic transition, possibly, through activation/maintenance of the Wnt
canonical pathway.

Relevant studies have shown that Shh-dependent induction of Lmx1a is
required for Msx1 expression, and that Msx1 is necessary for Ngn2 activation,
which, in turn, promotes the differentiation of Sox2+ mesDA
progenitors into Nurr1+ post-mitotic DA precursors
(Kele et al., 2006;
Andersson et al., 2006a;
Andersson et al., 2006b;
Ono et al., 2007). On this
basis, we first analyzed the expression of Otx2 in relation to several markers
at E11.5 and E12.5 in wild-type embryos. This analysis showed that in the
Nkx6.1-Lmx1b+ mesDA domain (see Fig. S7A,A′ in the
supplementary material), Otx2 was co-expressed in progenitors with Sox2,
Mash1, Ngn2, Lmx1a, Msx1 and Lmx1b (see Fig. S7B-D′ in the
supplementary material; Fig.
9C,D,E,F,O,P), in early post-mitotic precursors with Ngn2, Nurr1
and Pitx3 (see Fig. S7D-F′ in the supplementary material), and in more
mature mesDA neurons with a large subset of Pitx3+ cells (see Fig.
S7F,F′ in the supplementary material). We then investigated whether the
expression of these genes and, consequently, the differentiation of mesDA
progenitor subsets was altered in response to increased level or lack of Otx2.
These experiments showed that, compared with wild-type embryos, in Otx2
overexpressing mutants Lmx1a and Msx1 expression was
expanded and the number of Ngn2+-Sox2+ and
Mash1+-Sox2+ progenitors was increased, whereas, in
En1Cre/+; Otx2flox/flox embryos, Lmx1a
and Msx1 transcription was silenced and the expression of Ngn2 and
Mash1 was confined to sporadic mesDA Sox2+ progenitors
(Fig. 9C-J″). In contrast
to these genes, the expression of Lmx1b was retained in
En1Cre/+; Otx2flox/flox mutants, where it was
also expanded dorsally within the Nkx2.2+ domain from which
5-HT+ neurons were generated
(Fig. 9O-P″)
(Puelles et al., 2004).
Finally, we analyzed the generation of Ngn2+-Nurr1+ and
Nurr1+-Pitx3+ subpopulations of post-mitotic mesDA
neurons, which, as expected, were remarkably expanded in
En1Cre/+; tOtx2ov/ov mutants and virtually
absent at E11.5 or heavily reduced at E12.5 in En1Cre/+;
Otx2flox/flox embryos (Fig.
9K-N″). These findings show that overexpression of Otx2
induces increased generation of mesDA neurons that correlates with a
corresponding expansion of the subpopulations of differentiating progenitors,
while lack of Otx2 results in the general failure of the mesDA differentiation
program.

Otx2 controls proliferation and post-mitotic transition of mesDA
progenitors. (A-H) Representative adjacent sections through the
intermediate mesencephalon of E10.5 (A-D) and E11.5 (E-H) wild-type and
En1Cre/+; Otx2flox/flox embryos immunostained
with Nkx6.1 and BrdU (A,B,E,F), Nkx2.2 and BrdU (C,D,G,H), and then stained
with Hoechst show that the number of BrdU+ cells is dramatically
and selectively reduced in the mesDA domain of mutants. (I-L) Graphic
representation showing the AP graded reduction of the LI in the mesDA domain
of mutants (I,J), whereas the LI measured in the Nkx2.2+ domain of
mutant embryos is similar to that detected in the Nkx6.1+ domain of
control embryos (K,L). (M-P) Representative adjacent sections through
the intermediate mesencephalon of E11.5 wild-type and En1Cre/+;
Otx2flox/flox mutants immunostained with Ki67 and BrdU (M,N),
and Nkx2.2 and Nkx6.1 (O,P) show that, in mutant embryos, Ki67 is switched off
in the majority of mesDA progenitors and that most of the mesDA
BrdU+ cells (labeled at E10.5) become post-mitotic
(Ki67-) at E11.5. Conversely, in the Nkx2.2+ domain,
most of the BrdU+ progenitors retain Ki67 expression as in the
Nkx6.1+ domain of control embryos. (Q,R) Graphic
representation of the Qfs in the mesDA (Q), Nkx6.1+ (in wild type)
and Nkx2.2+ (in En1Cre/+;
Otx2flox/flox embryos) domains (R) shows a selective increase
in the number of mesDA progenitors quitting the cell-cycle in mutant embryos,
whereas a mild reduction is detected in the Nkx2.2+ domain. The
arrow and the broken line in (A-H,M-P) indicate the Nkx6.1+ or the
Nkx2.2+ territories analyzed. Abbreviations: Ant., anterior; Int.,
intermediate; Post., posterior.

Otx2 controls selectively the generation of mesDA neurons through a
dose-dependent AP graded effect on the proliferation of mesDA progenitors

This study shows that Otx2 overexpression causes increased generation of
mesDA neurons. This increase correlated with the level of overexpressed Otx2
protein and was more dramatic in the posterior and intermediate mesencephalon
than in the anterior mesencephalon and pretectum. Consistently, embryos that
lack Otx2 exhibit heavy reduction of mesDA neurons in the intermediate and
posterior mesencephalon, whereas in the anterior mesencephalon and pretectum
their generation is less affected (Puelles
et al., 2004) (see Fig. S4 in the supplementary material). These
data, therefore, indicate that, depending on the position occupied along the
AP axis of VM, mesDA progenitors exhibit a differential response to Otx2. A
second finding of this study is that although Otx2 is overexpressed even in
the VM progenitor domains adjacent to the mesDA compartment, in these domains,
neurons generation is not increased. Indeed, boundary relationships among
Nkx6.1, Nkx2.2, Shh and Foxa2, as well as the number and identity of OM and RN
neurons are unaffected by Otx2 overexpression. This suggests that Otx2 may
exert a selective control on the generation of mesDA neurons by modulating
their number along the AP axis.

The selective effect on the generation of mesDA neurons has been
investigated by analyzing whether Otx2 is required to regulate the
proliferating activity and post-mitotic transition of mesDA progenitors. This
analysis has shown that Otx2 plays a major role in controlling proliferation
of mesDA progenitors. Indeed, in Otx2 overexpressing embryos, the LI is
significantly increased and the Qf is reduced. Conversely, in mutants that
lack Otx2, the LI exhibits a drastic reduction and the Qf a dramatic increase.
Furthermore, in En1Cre/+; Otx2flox/flox
embryos, a large fraction of Sox2+ progenitors in the intermediate
and posterior mesencephalon switch off Ki67 and induce high level of
p27kip1, suggesting that they represent a type of `frozen'
progenitor that prematurely exits the cell cycle. Remarkably, these
abnormalities are restricted to mesDA progenitors, as, in mutants that
overexpress or lacking Otx2, the adjacent Nkx6.1+ or
Nkx2.2+ domains are apparently unaffected or exhibit mild
impairments in LI, Qf and in expression of Ki67 and p27kip1. Thus,
our data provide the first evidence that (1) Otx2 may regulate selectively the
generation of mesDA neurons by controlling the proliferating activity of their
progenitors; and (2) this control exhibits an AP graded effect. This and
previous studies have shown that Wnt1 expression is lost in the VM of
En1Cre/+; Otx2flox/flox mice and that Wnt
molecules are differentially required, being indeed involved in promoting
proliferation (Wnt1) or mesDA differentiation (Wnt5a)
(Panhuysen et al., 2004;
Castelo-Branco et al., 2003;
Castelo-Branco et al., 2004).
Our data suggest that Otx2 may control mesDA proliferation through the
maintenance and/or activation of the Wnt/β-catenin pathway. In this
context, active Wnt pathway through the TCF/LEF/β-catenin nuclear complex
may modulate the transcription of a broad range of target genes, including
cyclins and, in particular, Cyclin D1, a major regulator of the cell cycle
progression (Fodde and Brabletz,
2007; Shtutman et al.,
1999; Tetsu and McCormick,
1999; Lin et al.,
2000; Arber et al.,
1997). We show that CycD1 expression is suppressed specifically in
the mesDA domain of mutants that lack Wnt1 expression, whereas in
embryos overexpressing Otx2, the expanded expression of Wnt1
correlates with a higher number of CycD1+ cells. Therefore, these
findings strongly support the possibility that Otx2 may control the
proliferation of mesDA progenitors through the maintenance/activation of the
Wnt canonical pathway by regulating the expression of Wnt1. In view
of the selective effect on mesDA progenitors, our data indicate propagation of
the Wnt1 proliferative signal over a very short range.

Differentiation of mesDA neurons requires Otx2. (A-P″)
Adjacent sections through the intermediate mesencephalon of E11.5 and E12.5
wild type, En1Cre/+; tOtx2ov/ov and
En1Cre/+; Otx2flox/flox embryos immunostained
with Nkx6.1 and Nkx2.2 (A-B″), Mash1 and Sox2 (G-H″), Ngn2 and
Sox2 (I-J″), Nurr1 and Ngn2 (K-L″), Nurr1 and Pitx3 (M-N″),
Lmx1b and Otx2 (O,O″), Lmx1b and 5-HT (P,P″) or hybridized with
Lmx1a (C-D″) or Msx1 (E-F″) probes. In
En1Cre/+; Otx2flox/flox mutants, Lmx1b is
dorsally expanded within the Nkx2.2+ domain that generates
5-HT+ neurons, and is abundant at E12.5 in the Sox2+
mesDA progenitors (O″,P″). The arrow indicates the ventral border
of Nkx6.1+ domain in wild-type and En1Cre/+;
tOtx2ov/ov embryos and the ventral border of the
Nkx2.2+ domain in En1Cre/+;
Otx2flox/flox mutants.

Otx2 is required for mesDA differentiation

Recent studies have indicated that the differentiation of mesDA progenitors
and their transition to post-mitotic mesDA neurons require an intricate
pathway of transcription factors (reviewed by
Smidt and Burbach, 2007).
Indeed, experiments performed in chick and analysis of mouse mutants
collectively suggest that early in mesDA differentiation Shh-dependent
expression of Lmx1a activates Msx1, which induces Ngn2; Ngn2, in turn, is
required for the differentiation of Sox2+ progenitors into
Nurr1+ post-mitotic young mesDA neurons
(Andersson et al., 2006a;
Andersson et al., 2006b;
Kele et al., 2006;
Ono et al., 2007). In this
context, however, although in chick embryos the silencing of Lmx1a abolishes
Nurr1 expression, mouse Lmx1a-null mutants exhibit only 30% fewer TH
neurons and, similarly, Msx1-null embryos lack 40% of
Ngn2+ progenitors and Nurr1+ mesDA neurons
(Ono et al., 2007;
Andersson et al., 2006b). As
reported, this might suggest the existence of compensatory functions and/or,
in addition to their sequential requirement, an Lmx1a-Msx1 synergistic action
(Andersson et al., 2006b). Our
data show that, compared with control embryos, in Otx2-overexpressing embryos,
the expression of Lmx1a, Lmx1b, Msx1, Ngn2 and Mash1 is
expanded in a higher number of mesDA progenitors, which generate more
Ngn2+-Nurr1+ immature precursors and Pitx3+
mature mesDA neurons; conversely, in embryos lacking Otx2, the expression of
Lmx1a, Msx1, Ngn2 and Mash1 is lost or severely affected in
Sox2+ mesDA progenitors, which are greatly impaired in their
ability to generate Nurr1+ post-mitotic mesDA neurons.

Thus, these findings indicate that Otx2 is required to activate the genetic
pathway leading to mesDA neuron generation. In this context, Otx2 might
directly control the activation of Lmx1a and, consequently, the
subsequent steps of mesDA differentiation. Supporting this possibility is the
finding that ectopic expression of Otx2 in the ventral hindbrain is sufficient
to induce Lmx1a, Msx1 and proneural gene expression, and the generation of
TH+-Pitx3+ DA neurons
(Ono et al., 2007). A second
possibility is that Otx2 is indirectly required for Lmx1a activation
by providing early mesDA progenitors with competence to respond to
Shh-mediated induction of Lmx1a. In molecular terms, Otx2 might be
necessary to repress a Shh-independent repressor of Lmx1a. A third
possibility is that Shh might activate in mesDA progenitors an Otx2
co-activator required for Otx2-mediated induction of Lmx1a. The
analysis of the mesDA genetic cascade in conditional mutants lacking Otx2 or
Shh or both functions might shed light on this important aspect in the future.
However, although our data suggest that Otx2-dependent expression of
Wnt1 is more likely to be required to control proliferation of mesDA
progenitors and prevent their premature exit from cell cycle, they do not
exclude a priori that Wnt1 signaling may be necessary for one or more steps of
mesDA differentiation, including competence of mesDA progenitors to
Shh-inducing activity. A final finding of this study is that in the mesDA
domain of En1Cre/+; Otx2flox/flox mutants most
of the Sox2+ progenitors are Ki67-p27kip1+
and that these `post-mitotic' progenitors do not activate any of the genes
required for post-mitotic maturation of mesDA neurons (e.g. Nurr1). This
suggests that, at least in the En1Cre/+;
Otx2flox/flox mouse model, Sox2 expression does not require
cell proliferation activity and that post-mitotic maturation of mesDA
progenitors is not activated only by a block in proliferating activity and/or
cell cycle exit, but, rather, depends on the correct progression of the
differentiation process culminating with the transition of
Sox2+-Ngn2+ progenitors into
Sox2-Ngn2+-Nurr1+ post-mitotic immature mesDA
neurons. Interestingly, it has been recently shown that Otx2 and Sox2
physically interact to activate Rax1 expression in the retina
(Danno et al., 2008). Whether
Otx2 and Sox2 may interact also in mesDA progenitors and regulate the
expression of target gene(s) remains to be investigated.

Concluding remarks

This and previous studies show that Otx2 is required for multiple steps of
mesDA neuron development. Indeed Otx2 controls early specification of VM by
both positioning Shh and Fgf8 signals, and maintaining the identity of
progenitors domains (Puelles et al.,
2003; Puelles et al.,
2004; Prakash et al.,
2006). Here, we have provided evidence that Otx2 exerts a crucial
influence over mesDA neurogenesis by regulating the proliferating activity and
differentiation of mesDA progenitors. Collectively, these and previous
findings suggest that Otx2 represents a potentially relevant genetic
determinant in future ES cell- or mesDA progenitor-based studies that are
focused on improving the generation of authentic mesDA neurons and providing
potential tools for the treatment of Parkinson's disease.

Supplementary material

Acknowledgments

We thank D. Grieco, C. Missero and T. Russo for helpful discussions and
criticisms on the manuscript, and the staff of the CEINGE animal house for
excellent animal care. We are also indebted to G. Corte for the Sox2 and Otx2
antibodies, and to D. J. Anderson for the Ngn2 antibody. This work was
supported by the Italian Association for Cancer Research (AIRC), the FP6 for
the EuTRACC Integrate Project (LSHG-CT-2007-037445) and the `Fondazione Cassa
di Risparmio' of Rome to A.S., and by the Deutsche Forschungsgemeinschaft, the
Bundesministerium für Bildung und Forschung and the European Communion to
W.W.

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