1 MRC Centre for Developmental Neurobiology, New Hunt's House, 4th Floor, King's
College London, Guy's Campus, London Bridge, London SE1 1UL, UK
2 Institute of Genetics and Biophysics `A. Buzzati-Traverso', CNR, Via G.
Marconi 12, 80125 Naples, Italy
3 Department of Neuroscience, Retzius väg 8, Karolinska Institutet, S-17177
Stockholm, Sweden
4 Institute of Animal Breeding and Genetics, University of Veterinary Medicine,
Veterinärplatz 1, A-1210 Wien, Austria
5 Division of Developmental Neurobiology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London, UK
6 GSF-Research Center, Institute of Developmental Genetics, 85764
Munich/Neuherberg, Max-Planck-Institute of Psychiatry, Molecular
Neurogenetics, Kraepelinstrasse 2-16, 80804 Munich, Germany
* Authors for correspondence (e-mail: antonio.simeone{at}kcl.ac.uk and wurst{at}gsf.de)
Accepted 31 January 2004
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SUMMARY |
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To gain insights into this process, we investigated the role of Otx2 in the specification of identity and fate of neuronal progenitors in the ventral midbrain. To achieve this, Otx2 was inactivated by Cre recombinase under the transcriptional control of En1. Lack of Otx2 in the ventrolateral and posterior midbrain results in a dorsal expansion of Shh expression and in a dorsal and anterior rotation of the midbrain-hindbrain boundary and Fgf8 expression. Indeed, in this mutant correct positioning of the ventral site of midbrain-hindbrain boundary and Fgf8 expression are efficiently controlled by Otx1 function, thus allowing the study of the identity and fate of neuronal progenitors of the ventral midbrain in the absence of Otx2. Our results suggest that Otx2 acts in two ways: by repressing Nkx2.2 in the ventral midbrain and maintaining the Nkx6.1-expressing domain through dorsal antagonism on Shh. Failure of this control affects the identity code and fate of midbrain progenitors, which exhibit features in common with neuronal precursors of the rostral hindbrain even though the midbrain retains its regional identity and these neuronal precursors are rostral to Fgf8 expression. Dopaminergic neurons are greatly reduced in number, red nucleus precursors disappear from the ventral midbrain where a relevant number of serotonergic neurons are generated. These results indicate that Otx2 is an essential regulator of the identity, extent and fate of neuronal progenitor domains in the ventral midbrain and provide novel insights into the mechanisms by which neuronal diversity is generated in the central nervous system.
Key words: Otx2, Midbrain, Neuronal precursors, Dopaminergic neurons, Serotonergic neurons, Mouse
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Introduction |
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The homeoproteins encoded by Otx1 and Otx2 play crucial
multiple roles in brain development, and during regionalisation they are
required to control AP and DV patterning of the midbrain through a
dose-dependent antagonism exerted on Fgf8 and Shh
expression, respectively (Acampora et al.,
1997; Li and Joyner,
2001
; Martinez-Barbera et al.,
2001
; Puelles et al.,
2003
; Simeone et al.,
2002
). However, very little is known about the role of Otx genes
in further decisions involving the allocation of midbrain neuronal fates under
the influence of Fgf8 and Shh induction. To address this issue, we generated
conditional mutants inactivating Otx2 by Cre recombinase expressed
under the transcriptional control of the En1 gene
(En1cre) (Kimmel et
al., 2000
). Otx2 inactivation in the ventral and
posterior midbrain resulted in a dorsal expansion of Shh expression
and in a dorsal and anterior rotation of the midbrain-hindbrain boundary
(MHB). In En1cre/+; Otx2flox/flox
embryos, the ventral territory rostral to the domain of Fgf8
expression retained a midbrain identity but exhibited dramatic abnormalities
in the identity and fate of neuronal precursors. DA neurons were greatly
reduced in number and the precursor domain that normally generates red nucleus
(RN) neurons gave rise to Ser neurons. This abnormality correlated with
altered expression of Shh, Nkx2.2 and Nkx6.1, which define an expression code
in the ventral midbrain similar to that normally observed in the rostral
hindbrain.
These findings support an essential role for Otx2 in controlling the extent, identity and fate of neuronal progenitor domains of the ventral midbrain, thus providing novel insights into the molecular mechanisms that regulate neuronal diversity in the CNS.
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Materials and methods |
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The amplification products are 301, 150, 197, 290, 235, 302 bp long,
respectively. For the Otx1- and Otx2-null alleles, primers
and conditions were previously reported
(Acampora et al., 1995;
Acampora et al., 1996
)
In situ hybridisation, immunohistochemistry and apoptosis
In situ hybridisation and immunohistochemistry were performed as previously
described (Acampora et al.,
1998; Simeone,
1999
). Probes for Shh, Fgf8, Gbx2, Otx1, Grg4, Foxa2, Pet1,
Pou4f1, Th, Isl1 have been already reported
(Puelles et al., 2003
) and
correspond to PCR fragments ranging in length between 0.2 and 1 kb. The
Otx2
probe corresponds to the Otx2 exon 2 and has
been previously described (Puelles et al.,
2003
); the Otx2-5' probe is a PCR fragment including the
last 150 bp of the exon containing the methionine; the probe for the
Otx1 null allele corresponds to a 700 bp lacZ DNA fragment
(Acampora et al., 1996
).
Immunohistochemistry was performed as described
(Puelles et al., 2003
) with
antibodies directed against Otx2 (1:2000), Nkx2.2 (1:100), Pou4f1 (
Brn3a) (1:100), Shh (1:200), Isl1 (1:100), Th (1:300), 5-HT (1:100) and Nkx6.1
(1:100) proteins. The
Nkx2.2,
2H3 and
Isl1 are from
Hybridoma Bank; the
Shh and
Pou4f1 from Santa Cruz
Biotechnology; the
Th and
5-HT from Chemicon and the
Nkx6.1 is a rabbit polyclonal serum kindly provided by J. Ericson. The
2H3 is a monoclonal antibody recognising the 165 kDa neurofilament
subunit.
Apoptosis was detected according to the TUNEL method
(Puelles et al., 2003).
Dopaminergic cell counting
The general procedure was essentially as previously reported
(Acampora et al., 1999).
Frontal sections through the midbrain of En1cre/+;
Otx2flox/flox (n=4) and
En1cre/+ (n=3) adult mice were immunostained with
Th antibody. For each brain a total of four sections (one every four
consecutive sections) were selected along a comparable area and photographed
at high magnification. Th-positive cell bodies were counted and the mean value
for each genotype was calculated. The mean value of mutant brains was compared
with that of control animals and the cell number reduction reported as a
percentage.
Transfections, RNAse protection experiments and coimmunoprecipitation assays
A series of Otx1 and Otx2 cDNA molecules carrying
nonoverlapping deletions along the entire Otx1- or
Otx2-coding region were generated by PCR. Wild-type and mutant
versions of Otx1 and Otx2 were cloned in the pCT expression
vector downstream of a CMV enhancer-promoter
(Simeone et al., 1993;
Thali et al., 1988
). The
Grg4 expression plasmid has been previously reported
(Eberhard et al., 2000
). HeLa
cells were electroporated with mouse Otx1 and Otx2
constructs, alone or in combination with the Grg4 expressing plasmid.
The amount of expression plasmids was equalised by addition of an empty CMV
topping plasmid. Transactivation of a cotransfected multimerised bts
or np target site was monitored by RNAse protection using as probe a
fragment of the rabbit ß-globin reporter gene
(Simeone et al., 1993
;
Thali et al., 1988
).
Transactivations were normalised by monitoring the amount of the mRNA
transcribed by the Otx and/or Grg4 expression vectors (data not
shown).
For co-immunoprecipitation assays, the Grg4-coding sequence was
cloned in the pKW2T vector and its C terminus was fused to the Flag epitope by
PCR. Transiently transfected HeLa cells were processed as described
(Eberhard et al., 2000).
Lysates were incubated for 30 minutes on ice, cleared from cellular debris and
mixed with 10 µl of anti-Flag M2 affinity beads (Sigma) for 2 hours at
4°C under constant rotation. After extensive washing of the beads in
buffer B (20 mM Tris-HCl pH 7.9, 200 mM NaCl, 5 mM EDTA, 0.5% NP-40, 0.1%
SDS), the precipitated proteins were analysed by SDS-PAGE and western blotting
using polyclonal Flag, Otx2 and Grg4 antibodies. The input contains between 2%
and 5% of the transfection.
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Results |
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At E8.75 (around 10 somites), En1cre/+;
Otx2flox/flox embryos showed a normal expression pattern
of these markers according to a very mild inactivation of Otx2 at
this early stage (data not shown). At E9.5, the dorsal expression of
Fgf8 and Gbx2 at the MHB was rostrally shifted and appeared
expanded (Fig. 3B,C); the
dorsolateral part of the ring of Wnt1 expression was also shifted
rostrally, while its expression in the roof plate and at ventral site of the
MHB was unaltered (Fig. 3D);
Otx1 (Fig. 3E) was
transcribed in the midbrain also in the area adjacent to the ventral site of
the MHB and where Otx2 was inactivated (compare
Fig. 3E with 3A); the
expression domain of En1 (Fig.
3F) was also anteriorly shifted with an anterior border in close
proximity with the posterior one of the functional Otx2
(Fig. 3A); and Pax6
was expressed in the pretectal domain (Fig.
3H). At E10.5 (around 35 somites), the Fgf8 and
Gbx2 expression at the MHB (Fig.
3J,K,R,S) was sharpened ventrolaterally while along the dorsal
edge of the neural tube remained rostrally expanded up to the caudal border of
the functional Otx2 domain (Fig.
3I). A corresponding rostral shift similar to that described at
E9.5 was observed for Wnt1 (Fig.
3L,T); Otx1 transcription persisted at E10.5 in the
Otx2-depleted region with a caudal and ventral border in close proximity of
the Fgf8 and Gbx2 expression domains
(Fig. 3M,U); the expression
domain of En1 included the ventral domain of Fgf8 (arrow in
Fig. 3N-V) and was adjacent to
that of the functional Otx2; and Pax6 demarcated the
pretectal area (Fig. 3P), thus
suggesting that the Otx2-positive territory should also
include the dorsolateral anterior midbrain. Therefore, a fairly normal
positioning of both MHB and its molecular code were retained only ventrally in
proximity of the strongest domain of Otx1 expression while, dorsally,
the MHB was rostrally shifted and the caudal and dorsal midbrain was
repatterned into cerebellum (Fig.
3X). This dorsal and rostral shift of the MHB can be visualised as
a rotation of its DV axis describing an arc of about 45°. In this rotation
the ventral site of the MHB represents the fixed point
(Fig. 3X). To test the
possibility that in the absence of Otx2, Otx1 may efficiently control the
positioning of ventral MHB, we generated triple mutants in which Otx2
inactivation by En1cre was achieved in an
Otx1-null background (Acampora et
al., 1996
). Compared with conditional mutants,
En1cre/+; Otx2flox/flox;
Otx1-/- embryos revealed at E10.5 an anterior shift of the
ventral domain of Fgf8 expression
(Fig. 4B) up to the border with
the domain expressing functional Otx2 transcripts in the ventral
pretectum (Fig. 4A).
Surprisingly, the ventral domain of Gbx2 expression at the MHB did
not move rostrally into the ventral midbrain and rather it was lost
(Fig. 4C), raising the issue on
the time-competence in responding to the lack of Otx proteins. Importantly,
transcription of the Otx1 null allele (lacZ)
(Acampora et al., 1996
) was
retained along the presumptive ventral midbrain
(Fig. 4D). The dorsal
expression of Fgf8 and Gbx2 was very similar in triple and
conditional mutants. Besides supporting a role for Otx1 in controlling at
least the ventral positioning of Fgf8 expression, these findings
suggest that the rostral shift of the ventral domain of Gbx2
expression at the MHB is sensitive to the lack of Otx gene products prior to
E9-9.5. Next, to assess whether in conditional and triple mutants
Otx2 was transcribed in the Otx2-depleted territory, we analysed its
expression with an Otx2 probe (Otx2-5') unaffected by
Cre activity. In both mutants, robust transcription of Otx2 was
detected with this probe in the territory where Otx2 was inactivated
(Fig. 3G,O,W;
Fig. 4E). Together these data
indicate that in conditional mutants the Otx2-depleted territory rostral to
Fgf8 expression exhibits a midbrain regional identity and similarly,
in triple mutants the Otx-depleted territory, although caudal to Fgf8
expression, still retains relevant midbrain molecular features (transcription
of Otx1 and Otx2 null alleles and absence of Gbx2
expression).
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Discussion |
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A second relevant feature of En1cre/+;
Otx2flox/flox mutants is represented by the dorsal
expansion of Shh expression in response to the Otx2
inactivation in lateral and ventral midbrain. A similar expansion of the Shh
domain has been reported recently in embryos with reduced level of Otx1 and
Otx2 in the lateral midbrain (Puelles et
al., 2003). Apart from confirming these previous findings, the
data reported here indicate that Otx2 is not required to regulate Shh
expression in the floor-plate region ventral to the ABB. Moreover, we provide
evidence that the Grg4-co-repressor may interact with Otx proteins to
attenuate their transactivating ability. Together with expression data, these
findings strengthen the possibility that dorsal antagonism on
Shh/Foxa2 expression at the ABB may require direct or indirect
interaction with the Otx-Grg4 repressing complex. Grg4 is able to interact
with different classes of transcription factors including Pax and Nkx
homeodomain proteins (Muhr et al.,
2001
; Ye et al.,
2001
). Thus, our findings provide further support for the general
idea that a combinatorial series of interactions between co-repressor
molecules and transcription factors belonging to different gene families
define a sophisticated regulatory network controlling the transcription of
signaling molecules and cellular determinants.
The role of Otx2 in formation and maintenance of ventral progenitor domains
Studies on Shh and Fgf8 have provided crucial information for understanding
molecular events controlling the sequential steps of neuronal development
(Agarwala et al., 2001;
Briscoe and Ericson, 2001
;
Hynes and Rosenthal, 1999
;
Jessell, 2000
).
In the spinal cord and hindbrain, graded distribution of Shh activity is
interpreted by class II Nkx factors, which, in turn, are crucial
intermediaries in the assignment of the identity and fate of neuronal
progenitor domains (Briscoe et al.,
1999; Briscoe et al.,
2000
; Sander et al.,
2000
). Interestingly, in the spinal cord, rostral hindbrain and
midbrain the molecular code defined by Shh and Nkx expression patterns is not
uniform but exhibits a characteristic, regionally restricted profile. In
particular, in the rostral hindbrain Nkx2.2 is co-expressed with Shh and is
ventral to the Nkx6.1 domain, while in the ventral midbrain the Nkx6.1 domain
is located between that expressing Shh and that positive for Nkx2.2
(Fig. 7
). Adjacent to
the MHB, Shh and Fgf8 signaling activities induce and position the Ser and DA
neuronal populations in the rostral hindbrain and in the midbrain,
respectively (Hynes and Rosenthal,
1999
; Ye et al.,
1998
). Ser neurons originate from the progenitor domain expressing
Shh and Nkx2.2, while most if not all DA neurons arise from the ventralmost
neuroepithelium positive only for Shh. This suggests that the different
expression code of these two progenitor domains may be relevant in the
establishment of the Ser and DA neuronal phenotype. Indeed, it has been shown
that Nkx2.2 is essential for the coordinated generation of hindbrain Ser
neurons (Briscoe et al., 1999
;
Pattyn et al., 2003
).
Therefore, a crucial issue was to elucidate the regulatory mechanism(s) and
factor(s) controlling the identity code of midbrain and hindbrain progenitor
domains. Our study provides in vivo evidence that Otx2 is a major genetic
determinant of this process in the ventral midbrain. Indeed, lack of Otx2 from
E9.5 produces relevant abnormalities in the expression pattern of Shh, Nkx6.1
and Nkx2.2. This event generates a major change in the identity and fate of DA
and RN progenitors, which, in turn, exhibit a molecular code similar to that
observed in the rostral hindbrain (Fig.
7). This strongly suggests that Otx2 is required to provide
midbrain neuronal precursors with a specific differentiation code suppressing
that of the anterior hindbrain. To perform this role, Otx2 exerts a dual
control; that is, repression of Nkx2.2 in the ventral midbrain and maintenance
of the Nkx6.1 expression domain through dorsal antagonism on Shh expression.
Failure of this dual control in En1cre/+;
Otx2flox/flox embryos affects identity and fate of dorsal
DA and RN neuronal precursors which, as in the hindbrain, co-express Shh and
Nkx2.2 and generate Ser neurons (Fig.
7
). However, in Otx1cre/+;
Otx2flox/ embryos, failed antagonism on
Shh expression and consequent lack of Nkx6.1 generates a remarkable increase
of DA neurons (Puelles et al.,
2003
) because in this case presumptive RN precursors (normally
positive for Nkx6.1 and negative for Shh and Nkx2.2) acquire the identity and
fate of presumptive DA progenitors (positive for Shh and negative for Nkx6.1
and Nkx2.2). Therefore, in En1cre/+;
Otx2flox/flox embryos where Otx2 is inactivated in ventral
and lateral midbrain, progenitor domains undergo an anterior into posterior
change of identity and fate, while in Otx1cre/+;
Otx2flox/- mutants
(Puelles et al., 2003
), where
Otx2 is inactivated only in the lateral midbrain, they undergo a dorsal into
ventral transformation.
In En1cre/+; Otx2flox/flox embryos,
OM neurons are not severely affected and the DA cell type is never completely
abolished. For OM neurons, a likely explanation is based on the fact that, as
revealed by BrdU experiments and Isl1 immunodetection (data not shown), this
neuronal cell type is generated quite early (between E9.5 and E10) and,
therefore should not be severely affected by the Otx2 inactivation. For
midbrain DA, our data suggest that the ventralmost fraction of DA precursors
is excluded from the Nkx2.2 ventralisation, and thereby retains its proper
identity and fate. Why these neuronal precursors are not permissive to express
Nkx2.2 remains to be determined. Complete suppression of the DA phenotype is
observed only in Otx mutants exhibiting full transformation of midbrain into
rostral hindbrain and coordinated anterior shift of both MHB and expression of
Fgf8 and Gbx2 at early somite stage
(Acampora et al., 1997;
Brodski et al., 2003
).
Finally, in conditional and triple mutants, the presumptive ventral midbrain
showed a similar and abnormal differentiation program, regardless of the site
of Fgf8 expression. Together, these data indicate that Fgf8- and
Shh-inducing signals require Otx2 in the ventral midbrain to be properly
interpreted. This suggests that Otx2 should play a crucial role in the
establishment of the cellular competence to respond to these inducing signals.
This supports the idea that midbrain-polarised activity of Shh and Fgf8
depends on the molecular identity of the responding tissue and might not
represent an intrinsic property of these inducing molecules.
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ACKNOWLEDGMENTS |
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