|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online September 26, 2008
doi: 10.1242/10.1242/dev.027003
1 CEINGE Biotecnologie Avanzate, via Comunale Margherita 482, 80145 Naples,
Italy and SEMM European School of Molecular Medicine - Naples site,
Italy.
2 Institute of Genetics and Biophysics `A. Buzzati-Traverso', CNR, Via P.
Castellino 111, 80131 Naples, Italy.
3 Max-Planck-Insitute of Psychiatry, Molecular, Neurogenetics, Kraepelinstr.
2-16, 80804 Munich, Germany and Helmholtz Zentrum München, German
Research Center for Environmental Health, Institute of Developmental Genetics,
Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
Authors for correspondence (e-mails:
simeone{at}ceinge.unina.it;
wurst{at}helmholtz-muenchen.de)
Accepted 2 September 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Dopaminergic neuron, Cell proliferation, Cell differentiation, Otx2, Wnt1, Sox2, Lmx1a, Msx1, Ngn2, Parkinson's disease
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Björklund and Lindvall,
1984; Smidt and Burbach,
2007; Smits et al.,
2006; Marín et al.,
2005). These neurons play a crucial role in the control of motor,
sensorimotor and motivated behaviors
(Björklund and Lindvall,
1984; Kelley and Berridge,
2002; Isacson,
1993). Degeneration of the DA neurons in the SN leads to the
characteristic symptoms of Parkinson's disease (PD), whereas abnormal
functioning of those in the VTA is involved in psychiatric and affective
dysfunction (Jellinger, 2001;
Egan and Weinberger, 1997;
von Bohlen und Halbach et al.,
2004). These disorders highlight the relevance of this population
of neurons and the enormous effort to understand the molecular basis that
controls their neurogenesis.
During development, neural cell-type specification is controlled by
inducing signals and depends on the responsiveness of target progenitor cells
(Lumsden and Krumlauf, 1996;
Wurst and Bally-Cuif, 2001;
Jessell, 2000;
Briscoe and Ericson, 2001).
Mesencephalic DA (mesDA) progenitors require the inducing properties of Fgf8
and Shh signals that are emitted at the isthmic organizer and floor-plate
region, respectively, whereas competence in interpreting these inducing
signals is provided by the molecular code that is defined by the expression of
different transcription factors (Briscoe
and Ericson, 2001; Ye et al.,
1998; Simeone,
2005; Smidt and Burbach,
2007; Smits et al.,
2006; Prakash and Wurst,
2006; Liu and Joyner,
2001). Several genes, including the transcription factors Pitx3,
Lmx1a, Lmx1b, En, Msx1, Foxa2, Ngn2, Otx2, the orphan nuclear receptor Nurr1,
the Wnt1 and Wnt5a members of the Wnt family, and retinoic acid play a
relevant role in differentiation and survival of mesDA neurons
(Saucedo-Cardenas et al.,
1998; Semina et al.,
1998; Smidt et al.,
1997; Smidt et al.,
2000; Smidt et al.,
2004; Kele et al.,
2006; Ferri et al.,
2007; Andersson et al.,
2006a; Andersson et al.,
2006b; Ono et al.,
2007; McCaffery and
Dräger, 1994; van den
Munckhof et al., 2003; Nunes
et al., 2003; Simon et al.,
2001; Zetterstrom et al.,
1997; Castelo-Branco et al.,
2003; Puelles et al.,
2003; Puelles et al.,
2004; Prakash et al.,
2006). Among these, our attention has been focused on Otx2, a
transcription factor containing a bicoid-like homeodomain that plays a crucial
role in specification, regionalization and differentiation of forebrain and
midbrain (Puelles et al.,
2006; Acampora and Simeone,
1999; Simeone et al.,
2002; Simeone,
2005). Accordingly, previous studies have shown that: (1) Otx2
together with Otx1 is required to control the positioning of Shh and
Fgf8 expression, and that failure in this control generates profound
alteration in the identity code of progenitor domains in the ventral
mesencephalon (VM), which exhibits a dorsal expansion of the mesDA compartment
at the expense of the red nucleus (RN) domain
(Puelles et al., 2003); (2)
Otx2 is required in the VM to suppress the transcription factor Nkx2.2 and
prevents the generation of 5-HT-containing neurons in place of RN and
dorsalmost mesDA neurons (Puelles et al.,
2004); and (3) Wnt1 and Otx2 may be engaged in a positive-feedback
loop that, in turn, is required for proper development of mesDA neurons
(Prakash et al., 2006).
However, although these studies have indicated that Otx2 plays a relevant role
in the establishment of the mesencephalic field and molecular code of the
entire VM, they have not provided direct evidence on whether Otx2 is
intrinsically required for mesDA neurogenesis. Here, we have investigated this
crucial aspect by analyzing mutant mice that conditionally overexpress or lack
Otx2 using En1-driven Cre recombinase. Our findings indicate that
Otx2 controls selectively the proliferating activity of mesDA progenitors
through a dose-dependent anterior-posterior (AP) graded effect and regulates
the differentiation of mesDA neurons by inducing the expression of crucial
determinants required to promote the transition of Sox2+ mesDA
progenitors into Nurr1+ immature mesDA neurons. In general, these
findings provide new insights into the regulatory mechanism that controls
mesDA neurogenesis.
|
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
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).
In situ hybridization and immunohistochemistry
In situ hybridization on sections and whole embryos was performed as
described (Simeone, 1999;
Puelles et al., 2006) with
GFP, Msx1, Lmx1a probes, each corresponding to a PCR fragment of
variable length between 300 bp and 500 bp or with Wnt1, En1, Otx1,
Gbx2 and Fgf8 probes
(Puelles et al., 2004).
Immunohistochemistry was performed as previously reported
(Puelles et al., 2004;
Prakash et al., 2006). Details
of primary antibodies can be provided on request.
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.
|
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
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).
|
),
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).
|
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.
Otx2 overexpression induces a selective expansion of the mesDA progenitor domain
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'').
|
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).
|
MesDA differentiation in En1Cre/+; tOtx2ov/ov and En1Cre/+; Otx2flox/flox mutants
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.
|
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Smits et
al., 2006; Prakash and Wurst,
2006). In this process, Otx2 is early required to control the
positioning of Fgf8 and Shh expression, to regulate
Wnt1 expression and to maintain the integrity of progenitor domains
in the whole VM (Puelles et al.,
2003; Puelles et al.,
2004; Simeone et al.,
2002; Prakash et al.,
2006; Liu and Joyner,
2001; Smidt and Burbach,
2007). In this study, we have investigated for the first time the
potential role of Otx2 as an intrinsic determinant of the developmental
program of mesDA neurogenesis, and have discovered that Otx2 controls
selectively the proliferating activity of mesDA progenitors and is required to
promote their differentiation by directly or indirectly activating the
Lmx1a-Msx1-Ngn2 genetic cascade (Kele et
al., 2006; Andersson et al.,
2006a; Andersson et al.,
2006b; Ono et al.,
2007).
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.
|
).
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
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/20/3459/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Acampora, D. and Simeone, A. (1999). The
TINS lecture. Understanding the roles of Otx1 and Otx2 in the control
of brain morphogenesis. Trends Neurosci
22,116
-122.[CrossRef][Medline]
Andersson, E., Jensen, J. B., Parmar, M., Guillemot, F. and
Björklund, A. (2006a). Development of the mesencephalic
dopaminergic neuron system is compromised in the absence of neurogenin 2.
Development 133,507
-516.
Andersson, E., Tryggvason, U., Deng, Q., Friling, S.,
Alekseenko, Z., Robert, B., Perlmann, T. and Ericson, J.
(2006b). Identification of intrinsic determinants of midbrain
dopamine neurons. Cell
124,393
-405.[CrossRef][Medline]
Arber, N., Doki, Y., Han, E. K., Sgambato, A., Zhou, P., Kim, N.
H., Delohery, T., Klein, M. G., Holt, P. R. and Weinstein, I. B.
(1997). Antisense to cyclin D1 inhibits the growth and
tumorigenicity of human colon cancer cells. Cancer
Res. 57,1569
-1574.
Björklund, A. and Lindvall, O. (1984).
Dopamine-containing systems in the CNS. In Handbook of Chemical
Neuroanatomy: Classical Transmitters in the CNS (ed. A.
Biörklund and T. Hökfelt), Vol. 2, pp.55
-121. Amsterdam, The Netherlands:
Elsevier.
Briscoe, J. and Ericson, J. (2001).
Specification of neuronal fates in the ventral neural tube. Curr.
Opin Neurobiol 11,43
-49.[CrossRef][Medline]
Castelo-Branco, G., Wagner, J., Rodriguez, F. J., Kele, J.,
Sousa, K., Rawal, N., Pasolli, H. A., Fuchs, E., Kitajewski, J. and Arenas,
E. (2003). Differential regulation of midbrain dopaminergic
neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc. Natl. Acad.
Sci. USA 100,12747
-12752.
Castelo-Branco, G., Rawal, N. and Arenas, E.
(2004). GSK-3beta inhibition/beta-catenin stabilization in
ventral midbrain precursors increases differentiation into dopamine neurons.
J. Cell Sci. 117,5731
-5737.
Danno, H., Michiue, T., Hitachi, K., Yukita, A., Ishiura, S. and
Asashima, M. (2008). Molecular links among the causative
genes for ocular malformation: Otx2 and Sox2 coregulate Rax expression.
Proc. Natl. Acad. Sci. USA
105,5408
-5413.
Egan, M. F. and Weinberger, D. R. (1997).
Neurobiology of schizophrenia. Curr. Opin Neurobiol
7, 701-707.[CrossRef][Medline]
Ferri, A. L., Lin, W., Mavromatakis, Y. E., Wang, J. C., Sasaki,
H., Whitsett, J. A. and Ang, S. L. (2007). Foxa1 and Foxa2
regulate multiple phases of midbrain dopaminergic neuron development in a
dosage-dependent manner. Development
134,2761
-2769.
Fodde, R. and Brabletz, T. (2007).
Wnt/beta-catenin signaling in cancer stemness and malignant behavior.
Curr. Opin Cell Biol.
19,150
-158.[CrossRef][Medline]
Graham, V., Khudyakov, J., Ellis, P. and Pevny, L.
(2003). SOX2 functions to maintain neural progenitor identity.
Neuron 39,749
-765.[CrossRef][Medline]
Hökfelt, T., Matensson, A., Björklund, S., Kleinau, S.
and Goldstein, M. (1984). Distributional maps of tyrosine
hydroxylase-immunoreactive neurons in the rat brain. In Handbook of
Chemical Neuroanatomy: Classical Transmitters in the CNS (ed. A.
Biörklund and T. Hökfelt), Vol. 2, pp.227
-379. Amsterdam, The Netherlands:
Elsevier.
Isacson, O. (1993). On neuronal health.
Trends Neurosci. 16,306
-308.[CrossRef][Medline]
Jellinger, K. A. (2001). The pathology of
Parkinson's disease. Adv. Neurol.
86, 55-72.[Medline]
Jessell, T. M. (2000). Neuronal specification
in the spinal cord: inductive signals and transcriptional codes.
Nat. Rev. Genet. 1,20
-29.[CrossRef][Medline]
Kele, J., Simplicio, N., Ferri, A. L., Mira, H., Guillemot, F.,
Arenas, E. and Ang, S. L. (2006). Neurogenin 2 is required
for the development of ventral midbrain dopaminergic neurons.
Development 133,495
-505.
Kelley, A. E. and Berridge, K. C. (2002). The
neuroscience of natural rewards: relevance to addictive drugs. J.
Neurosci. 22,3306
-3311.
Lee, M. H., Nikolic, M., Baptista, C. A., Lai, E., Tsai, L. H.
and Massagué, J. (1996). The brain-specific activator
p35 allows Cdk5 to escape inhibition by p27Kip1 in neurons.
Proc. Natl. Acad. Sci. USA
93,3259
-3263.
Lin, S. Y., Xia, W., Wang, J. C., Kwong, K. Y., Spohn, B., Wen,
Y., Pestell, R. G. and Hung, M. C. (2000). Beta-catenin, a
novel prognostic marker for breast cancer: its roles in cyclin D1 expression
and cancer progression. Proc. Natl. Acad. Sci. USA
97,4262
-4266.
Liu, A. and Joyner, A. (2001). Early
anterior/posterior patterning of the midbrain and cerebellum. Annu.
Rev. Neurosci. 24,869
-896.[CrossRef][Medline]
Lumsden, A. and Krumlauf, R. (1996). Patterning
the vertebrate neuraxis. Science
274,1109
-1115.
Marín, F., Herrero, M. T., Vyas, S. and Puelles, L.
(2005). Ontogeny of tyrosine hydroxylase mRNA expression in mid-
and forebrain: neuromeric pattern and novel positive regions. Dev.
Dyn. 234,709
-717.[CrossRef][Medline]
McCaffery, P. and Dräger, U. C. (1994).
High levels of a retinoic acid-generating dehydrogenase in the
meso-telencephalic dopamine system. Proc. Natl Acad. Sci.
USA 91,7772
-7776.
Nunes, I., Tovmasian, L. T., Silva, R. M., Burke, R. E. and
Goff, S. P. (2003). Pitx3 is required for development of
substantia nigra dopaminergic neurons. Proc. Natl. Acad. Sci.
USA 100,4245
-4250.
Ono, Y., Nakatani, T., Sakamoto, Y., Mizuhara, E., Minaki, Y.,
Kumai, M., Hamaguchi, A., Nishimura, M., Inoue, Y., Hayashi, H. et al.
(2007). Differences in neurogenic potential in floor plate cells
along an anteroposterior location: midbrain dopaminergic neurons originate
from mesencephalic floor plate cells. Development
134,3213
-3225.
Panhuysen, M., Vogt Weisenhorn, D. M., Blanquet, V., Brodski,
C., Heinzmann, U., Beisker, W. and Wurst, W. (2004). Effects
of Wnt1 signaling on proliferation in the developing mid-/hindbrain region.
Mol. Cell. Neurosci. 26,101
-111.[CrossRef][Medline]
Prakash, N. and Wurst, W. (2006). Development
of dopaminergic neurons in the mammalian brain. Cell Mol. Life
Sci. 63,187
-206.[CrossRef][Medline]
Prakash, N., Brodski, C., Naserke, T., Puelles, E., Gogoi, R.,
Hall, A., Panhuysen, M., Echevarria, D., Sussel, L., Weisenhorn, D. M. et
al. (2006). A Wnt1-regulated genetic network controls the
identity and fate of midbrain-dopaminergic progenitors in vivo.
Development 133,89
-98.
Puelles, E., Acampora, D., Lacroix, E., Signore, M., Annino, A.,
Tuorto, F., Filosa, S., Corte, G., Wurst, W., Ang, S. L. et al.
(2003). Otx dose-dependent integrated control of antero-posterior
and dorso-ventral patterning of midbrain. Nat.
Neurosci 6,453
-460.[Medline]
Puelles, E., Annino, A., Tuorto, F., Usiello, A., Acampora, D.,
Czerny, T., Brodski, C., Ang, S. L., Wurst, W. and Simeone, A.
(2004). Otx2 regulates the extent, identity and fate of neuronal
progenitor domains in the ventral midbrain.
Development 131,2037
-2048.
Puelles, E., Acampora, D., Gogoi, R., Tuorto, F., Papalia, A.,
Guillemot, F., Ang, S. L. and Simeone, A. (2006). Otx2
controls identity and fate of glutamatergic progenitors of the thalamus by
repressing GABAergic differentiation. J. Neurosci.
26,5955
-5964.
Saucedo-Cardenas, O., Quintana-Hau, J. D., Le, W. D., Smidt, M.
P., Cox, J. J., De Mayo, F., Burbach, J. P. and Conneely, O. M.
(1998). Nurr1 is essential for the induction of the dopaminergic
phenotype and the survival of ventral mesencephalic late dopaminergic
precursor neurons. Proc. Natl. Acad. Sci. USA
95,4013
-4018.
Semina, E. V., Ferrell, R. E., Mintz-Hittner, H. A., Bitoun, P.,
Alward, W. L., Reiter, R. S., Funkhauser, C., Daack-Hirsch, S. and Murray, J.
C. (1998). A novel homeobox gene PITX3 is mutated in families
with autosomal-dominant cataracts and ASMD. Nat.
Genet. 19,167
-170.[CrossRef][Medline]
Shtutman, M., Zhurinsky, J., Simcha, I., Albanese, C., D'Amico,
M., Pestell, R. and Ben-Ze'ev, A. (1999). The cyclin D1 gene
is a target of the beta-catenin/LEF-1 pathway. Proc. Natl. Acad.
Sci. USA 96,5522
-5527.
Simeone, A. (1999). Detection of mRNA in tissue
sections with radiolabelled riboprobes. In In Situ Hybridization. A
Practical Approach, 2nd edn (ed. D. G. Wilkinson), pp.69
-86. Oxford: IRL, Oxford University
Press.
Simeone, A. (2005). Genetic control of
dopaminergic neuron differentiation. Trends Neurosci.
28, 62-66.[CrossRef][Medline]
Simeone, A., Puelles, E. and Acampora, D.
(2002). The Otx family. Curr. Opin. Genet.
Dev. 12,409
-415.[CrossRef][Medline]
Simon, H. H., Saueressig, H., Wurst, W., Goulding, M. D. and
O'Leary, D. D. (2001). Fate of midbrain dopaminergic neurons
controlled by the engrailed genes. J. Neurosci.
21,3126
-3134.
Smidt, M. P. and Burbach, J. P. (2007). How to
make a mesodiencephalic dopaminergic neuron. Nat. Rev
Neurosci. 8,21
-32.[CrossRef][Medline]
Smidt, M. P., van Schaick, H. S., Lanctôt, C., Tremblay,
J. J., Cox, J. J., van der Kleij, A. A., Wolterink, G., Drouin, J. and
Burbach, J. P. (1997). A homeodomain gene Ptx3 has highly
restricted brain expression in mesencephalic dopaminergic neurons.
Proc. Natl. Acad. Sci. USA
94,13305
-13310.
Smidt, M. P., Asbreuk, C. H., Cox, J. J., Chen, H., Johnson, R.
L. and Burbach, J. P. (2000). A second independent pathway
for development of mesencephalic dopaminergic neurons requires Lmx1b.
Nat. Neurosci 3,337
-341.[CrossRef][Medline]
Smidt, M. P., Smits, S. M., Bouwmeester, H., Hamers, F. P., van
der Linden, A. J., Hellemons, A. J., Graw, J. and Burbach, J. P.
(2004). Early developmental failure of substantia nigra dopamine
neurons in mice lacking the homeodomain gene Pitx3.
Development 131,1145
-1155.
Smits, S. M., Burbach, J. P. and Smidt, M. P.
(2006). Developmental origin and fate of meso-diencephalic
dopamine neurons. Prog. Neurobiol.
78, 1-16.[CrossRef][Medline]
Sonnier, L., Le Pen, G., Hartmann, A., Bizot, J. C., Trovero,
F., Krebs, M. O. and Prochiantz, A. (2007). Progressive loss
of dopaminergic neurons in the ventral midbrain of adult mice heterozygote for
Engrailed1. J. Neurosci.
27,1063
-1071.
Tetsu, O. and McCormick, F. (1999).
Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells.
Nature 398,422
-426.[CrossRef][Medline]
van den Munckhof, P., Luk, K. C., Ste-Marie, L., Montgomery, J.,
Blanchet, P. J., Sadikot, A. F. and Drouin, J. (2003). Pitx3
is required for motor activity and for survival of a subset of midbrain
dopaminergic neurons. Development
130,2535
-2542.
von Bohlen und Halbach, O., Schober, A. and Krieglstein, K.
(2004). Genes, proteins, and neurotoxins involved in Parkinson's
disease. Prog. Neurobiol.
73,151
-177.[CrossRef][Medline]
Wurst, W. and Bally-Cuif, L. (2001). Neural
plate patterning: upstream and downstream of the isthmic organizer.
Nat. Rev Neurosci 2,99
-108.[CrossRef][Medline]
Ye, W., Shimamura, K., Rubenstein, J. L., Hynes, M. A. and
Rosenthal, A. (1998). FGF and Shh signals control
dopaminergic and serotonergic cell fate in the anterior neural plate.
Cell 93,755
-766.[CrossRef][Medline]
Zetterström, R. H., Solomin, L., Jansson, L., Hoffer, B.
J., Olson, L. and Perlmann, T. (1997). Dopamine neuron
agenesis in Nurr1-deficient mice. Science
276,248
-250.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  N. Prakash, E. Puelles, K. Freude, D. Trumbach, D. Omodei, M. Di Salvio, L. Sussel, J. Ericson, M. Sander, A. Simeone, et al. Nkx6-1 controls the identity and fate of red nucleus and oculomotor neurons in the mouse midbrain Development, August 1, 2009; 136(15): 2545 - 2555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tang, Y. Miyamoto, and E. J. Huang Multiple roles of {beta}-catenin in controlling the neurogenic niche for midbrain dopamine neurons Development, June 15, 2009; 136(12): 2027 - 2038. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.S. Layman, D.P. McEwen, L.A. Beyer, S.R. Lalani, S.D. Fernbach, E. Oh, A. Swaroop, C.C. Hegg, Y. Raphael, J.R. Martens, et al. Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome Hum. Mol. Genet., June 1, 2009; 18(11): 1909 - 1923. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Omodei, D. Acampora, P. Mancuso, N. Prakash, L. G. Di Giovannantonio, W. Wurst, and A. Simeone Anterior-posterior graded response to Otx2 controls proliferation and differentiation of dopaminergic progenitors in the ventral mesencephalon J. Cell Sci., October 15, 2008; 121(20): e1 - e1. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
