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First published online January 12, 2006
doi: 10.1242/10.1242/dev.02223
,
,
1 Laboratory of Molecular Neurobiology, MBB, Karolinska Institutet,
Scheelesväg 1, Retzius building A1, 17 177 Stockholm, Sweden.
2 Division of Molecular Neurobiology, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
3 Division of Developmental Neurobiology, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Authors for correspondence (e-mail:
Ernest.Arenas{at}mbb.ki.se
and
sang{at}nimr.mrc.ac.uk)
Accepted 25 November 2005
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Proneural genes, Cell fate specification, Differentiation, Sox2, Nurr1, Stem cells, Parkinson's disease
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The proneural genes of the bHLH class were first identified in
Drosophila as key regulators of neural lineage development
(Brunet and Ghysen, 1999
;
Guillemot, 1999). The three
most extensively studied genes in rodents are the mouse achaete-scute
homologue (Mash1) and the members of the atonal-related
family of genes, neurogenins (Ngn) 1 and 2 (Neurog1 and 2 –
Mouse Genome Informatics). Mash1 and Ngns are sufficient for the initiation of
a generic neurogenic program in a variety of progenitor cells, both in vitro
(Lo et al., 1998;
Farah et al., 2000;
Sun et al., 2001) and in vivo
(Ma et al., 1996;
Blader et al., 1997; Mizugushi
et al., 2001). At a mechanistic level, the proneural activity of Ngns involves
the promotion of neurogenesis and the concomitant repression of the
alternative glial fate (Tomita et al.,
2000; Nieto et al.,
2001; Sun et al.,
2001). Interestingly, proneural bHLH genes also contribute to the
specification of diverse neurotransmitter identities/neuronal subtypes
(Bertrand et al., 2002). In the
mammalian peripheral nervous system, for example, Ngns, but not
Mash1, promote a sensory neuron identity
(Perez et al., 1999;
Lo et al., 2002). In the
central nervous system, Mash1 influences neuronal fate decisions in
noradrenergic neurons (Hirsch et al.,
1998; Lo et al.,
1998), ventral and dorsal telencephalic GABAergic neurons
(Fode et al., 2000;
Casarosa et al., 1999), spinal
cord interneurons (Parras et al.,
2002; Helms et al.,
2005) and serotonergic neurons
(Pattyn et al., 2004), whereas
Ngns are involved in the differentiation of dorsal telencephalic
glutamatergic neurons (Fode et al.,
2000; Schuurmans et al.,
2004) and the specification of motoneurons in the ventral spinal
cord (Mizuguchi et al., 2001;
Novitch et al., 2001). These
studies indicate that proneural bHLH genes contribute to a unique
transcriptional code for generating neuronal diversity, and coordinate generic
and cell-type-specific neurogenesis in a region-specific manner.
In recent years, stem cells have raised important expectations and have
been considered as attractive candidates in cell replacement therapies for
neurodegenerative disorders (Lindvall et
al., 2004). The promise of stem cell therapies in diseases such as
Parkinson's disease has renewed the interest in gaining a deeper understanding
of the signals and mechanisms that regulate the differentiation of
stem/progenitor cells into specific neuronal populations, such as dopaminergic
(DA) neurons. It is known that DA neurons in the ventral midbrain (VM) require
for their development sonic hedgehog (Shh), as a ventralizing signal
(Hynes et al., 1995), and
signals derived from the isthmic organizer for anteroposterior specification
(Wurst and Bally-Cuif, 2001;
Rhinn and Brand, 2001). The
isthmic organizer is induced at the midbrain-hindbrain border and its position
is controlled by two homeodomain transcription factors, Otx2 in the midbrain
and Gbx2 in the hindbrain. Organizer-derived signals, such as Fgf8
(Ye et al., 1998) and Wnt1
(McMahon and Bradley,
1990; Thomas and Cappechi,
1990), maintain the expression pattern of a number of
genes, including engrailed 1 and 2
(Würst et al., 1994;
Hanks et al., 1995; Danielian
et al., 1996), and Pax2 and Pax5
(Urbanek et al., 1997), that
contribute to the development of most neuronal cell types in the mid- and
hindbrain region. DA neurons are first detected in the mantle zone of the
mouse VM midline at embryonic day (E) 10.5 and DA neurogenesis continues until
E13 (DiPorzio et al., 1990).
These cells are generated from proliferating precursor cells in the
ventricular zone (VZ) of the VM. Proliferating precursors express Aldh1 and
give rise to postmitotic DA precursors that express Nurr1, an orphan nuclear
receptor required for the differentiation of DA precursors into tyrosine
hydroxylase (Th+) DA neurons
(Zetterstrom et al., 1997;
Saucedo-Cárdenas et al.,
1998; Castillo et al.,
1998). Additional genes required for DA neuron development include
the LIM homeodomain Lmx1b (Smidt
et al., 2000) and the Pitx3 homeodomain genes, essential
for DA neuron survival in the substantia nigra pars compacta (SNc)
(Nunes et al., 2003;
Van den Munckhof et al., 2003;
Hwang et al., 2003;
Smidt et al., 2004).
Surprisingly, no study has yet investigated the role of proneural bHLH genes
in DA neurogenesis, in the specification of the VM DA neuronal identity or in
the differentiation of DA precursors into neurons. We hereby report that
Ngn2, but not Ngn1 or Mash1, is required for the
generation of DA neurons. Our findings indicate about 86% and 66% of
Th+ DA neurons fail to develop in Ngn2 mutants at the end
of the neurogenic period (E14.5) and at E17.5, respectively. The partial
rescue of Th+ DA neurons between E14.5 and E17.5 requires Mash1
activity, as this rescue does not occur in Ngn2;Mash1 double mutants.
Importantly, endogenous Mash1 can partially compensate, after a
delay, for the loss of Th+ neurons in Ngn2 mutants.
However, even ectopic expression of Mash1 from the Ngn2
promoter could not completely rescue the generation of DA neurons, indicating
that Ngn2 has a unique role in DA neurogenesis. Thus, our results
indicate that Ngn2 is required to for the development of midbrain DA
neurons.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
Ngn2KIGFP (Seibt et
al., 2003), Mash1;Ngn2KIGFP,
Mash1KINgn2 (Parras et
al., 2002), Ngn2KIMash1
(Parras et al., 2002) and
Ngn1;Ngn2 (Fode et al.,
2000) were genotyped and processed as previously described. For
analysis of BrdU incorporation, 6-hour pulses were performed with 100 µg
BrdU /gram of animal.
In situ hybridization and immunohistochemistry
For in situ hybridization (ISH), embryos were fixed (4% paraformaldehyde in
phosphate-buffered saline; PBS at 4°C) for 20 minutes (E10.5), 30 minutes
(E11.5), 90 minutes or overnight (E14.5) before being cryopreserved in 20%
sucrose, frozen in OCT and coronally sectioned (12-14 µm) onto slides
(SuperFrost®Plus). ISH was performed as described
(Conlon and Herrmann, 1993).
ISH was performed on fresh frozen or fixed tissue with digoxigenin-labelled
single-stranded RNA probes at 55°C or at 70°C, followed by incubation
with nitroblue tetrazolium (NBT) plus 5-bromo-Ychloro-3-indolyl phosphate
(BCIP) (purple) substrates. The following mouse antisense RNA probes were
used: Th (Perlmann and Jansson,
1995), Lmx1b (Chen et
al., 1998), Mash1
(Guillemot and Joyner, 1993),
Ngn1 (Fode et al.,
1998), Ngn2 (Fode et
al., 1998), Dll1
(Bettenhausen et al., 1995) and
Hes5 (Akazawa et al.,
1992).
For immunohistochemistry, coronal sections (12-14 µm thick) were pre-incubated for 1 hour in blocking solution [PBS, 0-1% bovine serum albumin (BSA), 0.1-0.3% Triton-X 100 and 5-10% normal serum] followed by incubation at 4°C overnight with one or more of the following primary antibodies diluted in blocking solution: mouse monoclonal anti-Mash1 (1:1, gift from D. J. Anderson); mouse anti-Ngn2 (1:20, gift from D. J. Anderson), rabbit anti-GFP (1:1000, Molecular Probes), rat anti-bromodeoxyuridine (BrdU; 1:20, Immunological Direct), rat anti-BrdU (1:150, Abcam), rabbit anti-Cleaved Caspase-3 (1:100, Cell Signaling), guinea pig anti-glutamate transporter GLAST (1:200-1:2000, Chemicon), mouse anti-MAP2 (1:750, Sigma), rat anti-Ki67 (1:80, Dako), rabbit anti-Nurr1 (1:100, gift from T. Perlmann, Karolinska Institute, Stockholm), rabbit anti-Pitx3 (1:200, gift from P. Burbach, Rudolf Magnus Institute of Neuroscience, Utrecht); mouse anti-RC2, (1:200, Developmental Studies Hybridoma Bank); rabbit anti-Sox2 (1:500, Chemicon; 1:25, R&D Systems; 1:3000, gift from T. Edlund, Umea University, Umea); rabbit anti-Th, (1:250, PelFreeze), sheep and rabbit anti-Th (1:1000, Chemicon), mouse anti ßIII-tubulin (1:1000, Sigma) followed by nuclear staining with Toto-3 iodide (1:1000, Molecular Probes). Pre-treatment with 2N HCl for 15 minutes prior to pre-incubation with primary antibody was needed for the detection of BrdU. After washing, slides were incubated for 1-2 hours at room temperature with the appropriate secondary antibodies: biotinylated (1:400, Jackson Laboratories), fluorophore conjugated (Cy2-, Cy3- and Cy5-, 1:300, Jackson Laboratories), or secondary antibodies conjugated with a fluorochrome (Molecular Probes). Hoechst nuclear stain (5 mg/ml, 1:5000, Sigma) was performed for visualization of all cells. Biotinylated secondary antibodies were visualized with the Vector Laboratories ABC immunoperoxidase kit, using 3-3' diaminobenzidine tetrahydrochloride (DAB 0.5 mg/ml)/nickel chloride (1.6 mg/ml) substrate. Where appropriate, endogenous peroxidase activity was quenched for 20 minutes with 5% H2O2 prior to pre-incubation with secondary antibody. Sections were washed and mounted using glycerol or Aquapolymount mounting media (Poly-Labo). Cresyl violet staining solution was 0.25%. Quantitative immunocytochemical data represent mean ±standard deviation for cell counts in consecutive sections through the entire substantia nigra, every 70 µm, in three to eight animals per condition. Photos were acquired with a Zeiss Axioplan 100M microscope and collected with a Hamamatsu camera C4742-95 (with the OpenlabTM 3.1.7 imaging software). Confocal pictures were taken with a LSM510 Zeiss microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). At E11.5
and E13.5, Ngn1, Ngn2 and Mash1 showed distinct profiles of
expression in the VM; however, Ngn1 expression was extinguished by
the latter stage (Fig.
1C-E,H-J). The combined expression patterns of these genes
revealed the existence of three distinct zones differing in their expression
patterns of proneural genes at E11.5, referred to in the rest of the
manuscript as zones 1, 2 and 3. Ngn2 and Mash1 were
expressed in zone 1 (Fig.
1D,E,R), which also expressed Lmx1b
(Fig. 1B). Ngn1, Ngn2
and Mash1 were all expressed in zone 2
(Fig. 1C-E,R), while
Mash1 was the only proneural gene expressed in zone 3
(Fig. 1E,R). The dorsal
boundary of zone 3 corresponds to the alar-basal boundary. As both
Ngn2 and Mash1 mRNAs were detected in the VZ of zone 1, we
examined whether both proteins were expressed in the same or distinct
precursor pools. Double immunohistochemistry at E11.5 showed that Mash1 and
Ngn2 proteins had a similar distribution to the corresponding transcripts. In
particular, Mash1 was expressed at high levels in zone 3, and both Mash1 and
Ngn2 proteins were expressed in zones 1 and 2, where they were co-expressed in
some VZ cells (Fig. 1K-M).
|
). Co-expression of Sox2 and Ki67, a marker of
proliferating cells, indicates that all Sox2+ VZ progenitors
correspond to proliferating progenitors in the VM at E11.5
(Fig. 4C). To determine whether
Mash1 and Ngn2 are expressed in proliferating progenitors, we therefore
colocalised the expression of these proteins with Sox2 in the VM in wild-type
embryos at E11.5 by double immunohistochemistry. All Mash1+ cells
expressed Sox2, indicating that Mash1 is only expressed in proliferating VZ
progenitors (Fig. 1N). By
contrast, Ngn2 was co-expressed with Sox2+ proliferating
progenitors, as well as in Sox2– postmitotic cells outside
the VZ (Fig. 1O). Consistent
with this result, Ngn2 was also co-expressed with some postmitotic DA
precursors in the intermediate zone (IZ) of the ventral midbrain
(Fig. 1P,Q) that express the
orphan nuclear receptor Nurr1 (Wallen et
al., 1999). The IZ is defined here as the zone between the
Sox2+ VZ and the marginal zone (MZ) containing Th+
neurons. By contrast, Ngn2 was not expressed in
Nurr1+Th+ cells in the marginal zone (data not shown).
Our data therefore indicate that Mash1 and Ngn2 are expressed in some
Sox2+ VZ progenitors, and Ngn2 is also expressed in postmitotic
Nurr1+ DA precursors, but not in differentiated Th+ DA
neurons (Fig. 1R), suggesting
possible functions for these genes in VM DA neurogenesis.
|
Analysis of Ngn2 null mutant mice revealed a near complete loss of
DA neurons at E11.5, as assessed by the expression of Th
(Fig. 2A-C) and Pitx3
(Fig. 2D,E)
(Van der Munckhof et al.,
2003). Examination of the expression of the pan-neuronal marker
ßIII tubulin, together with the nuclear marker Toto, revealed the absence
of Tuj1+/Toto+ cells normally positioned in the MZ
(Fig. 2F). This domain was
acellular and only Tuj1+/Toto-fibers also present in wild-type
embryos were found in mutant embryos. By contrast,
Tuj1+/Toto+ neurons were observed in the Ngn2
null embryos at E14.5, but were reduced in number compared with in wild-type
embryos (Fig. 2G). Similarly,
the number of microtubule-associated protein 2 (MAP2+)
(Mtap2+)/Hoechst+ cells decreased in the Ngn2
null embryos at E14.5 (Fig.
2H), indicating that only a few neurons are generated in the
absence of Ngn2. Accordingly, a very substantial loss of DA neurons
was detected, with only 17% of the Th+ neurons normally present in
the developing VM remaining (Fig.
2I-K). These residual Th+ cells were found in a lateral
position, suggesting that the loss of Ngn2 primarily affected
midline, but, to a lesser extent, also lateral DA neurons. Previous studies
have proposed that prospective substantia nigra (SN) DA neurons could be born
in a more lateral position than ventral tegmental area DA neurons
(Hanaway et al., 1971;
Smidt et al., 2004). We thus
examined whether the expression of Pitx3 was differentially affected. The
expression pattern of Pitx3 at E14.5 (Fig.
2L,M) revealed no difference from Th staining, suggesting that the
loss of DA neurons in the Ngn2 null mice move did not affect the
Pitx3-expressing DA neurons to a greater extent.
As 17% of VM DA neurons are still present at E14.5 in the absence of Ngn2, we next examined whether Ngn1 or Mash1, which are also expressed in the VZ of the VM, could be responsible for the birth of a subset of DA neurons in the absence of Ngn2. Analysis of Ngn1;Ngn2 double mutant mice showed no additional loss of Th+ DA neurons (10%) when compared with Ngn2 single mutants (11%, Fig. 3A-C) at E14.5. Consistently, Ngn1 single mutants showed no change in the number of Th+ neurons (data not shown) at E14.5. Similarly, analysis of Mash1;Ngn2 double mutant embryos showed a complete loss of DA neurons (Fig. 3D-F), identical to that observed in Ngn2 single mutants at E11.5. Moreover, analysis of Th expression in Mash1 mice revealed no abnormality in the staining pattern or the number of Th-positive cells during DA neurogenesis at E11.5 (Fig. 3G-I) or E14.5 (Fig. 3J-L), indicating that Mash1 is dispensable for VM DA neurogenesis. Thus, our results demonstrate that Ngn2, but not Ngn1 or Mash1, is required for DA neurogenesis.
Ngn2 promotes the generation of DA precursors and their differentiation into DA neurons
We next examined whether the loss of Th+ cells in the
Ngn2 null mice at E11.5 was due to a failure in precursor survival,
proliferation, fate specification or differentiation. We first assessed the
possibility that VM precursors would die in the absence of Ngn2. The
number of pyknotic nuclei and of active caspase 3 immunoreactive cells did not
differ between wild-type and Ngn2 mutant mice, when the loss of
Th+ cells was first detected at E11.5 (data not shown). We also
examined whether the reduction in the number of Th+ cells was due
to a defect in the proliferation of VM precursors. The number of proliferating
cells that incorporated BrdU during S phase in zones 1 and 2 of the VZ did not
differ between wild-type (1378±218, n=5) and Ngn2
null mice (1130.75±128, n=5) at E11.5
(Fig. 4A,B). In agreement with
this, Sox2 and Ki67 double labeling also showed a similar expression in the VZ
of the Ngn2 mutant when compared with wild-type embryos at this stage
(Fig. 4C,D). Thus cell death or
defects in cell proliferation are not responsible for the reduction in the
number of Th+ cells in Ngn2 mutants at E11.5.
|
|
) and GFP
therefore mimicked the expression domain of Ngn2 in the midbrain (data not
shown). We made use of the stability of the GFP protein to transiently follow
Ngn2+ cells in the VM of Ngn2KIGFP/+ and
Ngn2KIGFP/KIGFP embryos. Double immunohistochemistry with
GFP and Nurr1 revealed that VM progenitors give rise to
GFP+Nurr1+ cells that span the ventral midline in
wild-type embryos at E11.5 (Fig.
4H). By contrast, only lateral GFP+Nurr1+
cells are observed in Ngn2 mutants at E11.5, indicating a failure to
generate DA precursors and neurons in the medial region of the VM
(Fig. 4I). This result is in
agreement with the loss of TOTO+ cells underneath the ventral
midline in Ngn2 mutants (Fig.
2F). Taken together, these data indicate that more medially
located zone 1 VM progenitors fail to generate postmitotic Nurr1+
DA precursors and neurons at E11.5. By E13.5, Hes5 and Dll1 expression in zone 1 VM recovered, suggesting a partial rescue of neurogenesis (Fig. 5A-D). Accordingly, Toto+/Tuj1+ cells were seen in mutant embryos albeit significantly reduced when compared with wild-type embryos at E14.5 (Fig. 2G). Despite this delayed recovery, a ventral expansion of the medial VZ was visualized in cresyl violet-stained histological sections of the Ngn2 mutant compared with wild-type embryos at E14.5 (Fig. 5E,F). In order to further characterize the cells that were accumulating in the Ngn2 mutant embryos, we performed double immunohistochemistry for radial glial progenitor markers, GLAST (Slc1a3 – Mouse Genome Informatics) and RC2 (Ifaprc2 – Mouse Genome Informatics) (Fig. 5G,H), and also for Sox2 and GLAST (Fig. 5I,J). A dramatic increase in the number of GLAST+/RC2+ and Sox2+/GLAST+ cells in Ngn2 mutant compared with wild-type embryos at E14.5 indicated an expansion of radial glial VZ progenitors in the mutant zone 1. The partial rescue of neurogenesis also leads to an increase in the number of Nurr1+ cells (38%) in Ngn2 mutants at E14.5 (Fig. 5K-M).
Despite this overall increase in the fraction of Nurr1+ cells occurring in mutant mice between E11.5 and E14.5, the number of Th+ cells did not increase to the same extent, and there were twice as many Nurr1+ cells than Th+ cells in Ngn2 mutants at E14.5 (Fig. 5K and Fig. 2I, respectively). Thus, our results indicate that the loss of Ngn2 not only affects the early differentiation step of Sox2+ precursors into Nurr1+ postmitotic cells, but also the later differentiation of Nurr1+/Th– cells into Nurr1+/Th+ DA neurons. This difference in numbers of Nurr1+ cells and Th+ cells at E14.5 may reflect a delayed and partial rescue of Nurr1+ DA precursors that have not yet differentiated into Th+ DA neurons.
Mash1 partially compensates for the loss of Ngn2
We next examined whether the defects in the generation of DA neurons were
solely attributable to the loss of Ngn2 or also involved
misregulation of other proneural genes. We found expression of Ngn1
mRNA in zone 2 in both wild-type and Ngn2–/–
mice at E11.5 (Fig. 6A).
However, Mash1 mRNA was clearly reduced in zone 1 in Ngn2
mutant mice at E11.5, whereas it had partially recovered by E13.5
(Fig. 6A). Thus, our results
suggested that: (1) part of the loss of Nurr1+ and Th+
cells observed at E11.5 could be contributed by a reduction of Mash1
in VZ precursors; and (2) the partial rescue in Nurr1+ cells
observed at E14.5 could be mediated by Mash1. In order to examine
these possibilities, we enhanced or decreased Mash1 levels in
Ngn2 mutant mice by substituting Ngn2 expression with that
of Mash1 in knock-in mice (Ngn2KIMash1/KIMash1)
(Fode et al., 2000), or by
deleting both genes in double mutant mice
(Ngn2–/–;Mash1–/–).
We then analyzed the differentiation of Sox2 VZ precursors into
Nurr1+ postmitotic precursors and into Th+ DA neurons.
The number of Th+ cells in Ngn2KIMash1/KIMash1
mice at E11.5 was similar to that found in
Ngn2–/– mice (5% of wild-type numbers,
Fig. 6B), but the number of
Nurr1+ cells was 56% (data not shown) of that observed in wild-type
embryos, instead of 9% (data not shown) in
Ngn2–/– mice. By the end of the neurogenic
period (E14.5), the number of Nurr1+ cells had reached 38% in
Ngn2 mutant embryos (Fig.
6D), and this was slightly improved in
Ngn2KIMash1/KIMash1 mice (52% Nurr1+ cells
compared with wild-type littermates, Fig.
6D,J). However, the number of Th+ cells had increased
by then, from 11% in Ngn2 null mutants, to 43% and 63% in
Ngn2KIMash1/KIMash1 mutants
(Fig. 6C,E,G) at E14.5 and
E17.5, respectively (Fig.
7A-D).
|
Altogether, these findings demonstrate that Mash1 can partially
compensate for the loss of Ngn2 in the generation of
Nurr1+ precursors and Th+ DA neurons. Mash1
endogenous expression is dependent on Ngn2 function at E11.5, and the
loss of both Ngn2 and Mash1 results in an almost complete
loss of Nurr1+ and Th+ cells at this stage. When
endogenous Mash1 expression increases in a Ngn2-independent
manner, it results in a partial rescue of Nurr1+ cells at E14.5 and
of Th+ neurons at E17.5. Moreover, when Mash1 is
exogenously provided by expression from the Ngn2 locus as early as
E11.5, it results in a partial rescue of Nurr1+ cells at E11.5, and
a delayed and partial rescue of Th+ DA neurons at E14.5 and E17.5,
which leaves a permanent deficit in Th+ cells
(Andersson et al., 2006).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ngn2 is required for the differentiation of Sox2+ ventricular zone progenitors into Nurr1+ IZ precursors
Our immunohistochemical studies and data from the literature indicate that
zone 1 VM progenitors
(Ngn2+/Sox2+/GLAST+/Ki67+)
generate postmitotic DA precursors (Ngn2+, Nurr1+,
En1+) in the IZ, which subsequently differentiate further into
postmitotic DA neurons
(Nurr1+/En1+/Pitx3+/Th+/Tuj1+/MAP2+,
see Fig. 7E) in the MZ. Ngn2 is
required for the generation of postmitotic DA precursors, as zone 1
progenitors accumulate as
Sox2+/Ki67+/GLAST+/GFP+ cells in
the VZ, indicating that these progenitors fail to exit the cell cycle and
migrate into the IZ in Ngn2 mutants at E11.5. This requirement is
partially rescued by endogenous Mash1 or by Mash1 expressed
under the control of the Ngn2 promoter, indicating that part of
Ngn2 function in the DA neuron lineage is not unique to this gene and
can be partly compensated by Mash1. In agreement with this finding,
Mash1 also partially rescued the generation of postmitotic cells in
the IZ of Ngn2 mutant embryos at E14.5. In addition, the recovery of
Hes5 and Dll1 expression, which is likely to be due to the
recovery of Mash1 expression in zone 1 of Ngn2 mutant
embryos at E13.5, also support a role for Ngn2 in a general program
of neurogenesis underlying the production of DA precursors and DA neurons.
Altogether, these results demonstrate that Ngn2 is required by some
VZ progenitors to generate postmitotic precursors in the IZ. We therefore
examined whether Ngn2 is sufficient to ectopically induce DA
precursors or neurons. Preliminary studies indicate that when Ngn2 is
ectopically expressed in the dorsal midbrain of mouse embryos at E10.5 by
electroporation, enhanced neurogenesis measured by the number of ßIII
tubulin+ cells is observed two days later, but these cells do not
express DA neuron markers such as Pitx3 and Nurr1 (W. Lin
and S.-L.A., unpublished). Altogether, our findings indicate that
Ngn2 is required, but not sufficient, for the generation of the
majority of DA precursors.
|
).
This posibility is further suggested by the finding that ß-catenin binds
directly to the promoter and activates the expression of proneural bHLH genes
in neural progenitors (Israsena et al.,
2004). Mash1 and Ngn2 double mutants showed a similar reduction in the number of Th+ neurons, when compared with Ngn2 single mutants, indicating that Mash1 was not required for the differentiation of Nurr1+ precursors into Th+ cells. This finding is consistent with the lack of expression of Mash1 in postmitotic precursors. However, in Ngn2KIMash1/KIMash1 embryos, expression of Mash1 in postmitotic DA precursors partially accelerated their differentiation into Th+ neurons at E14.5 and E17.5, suggesting Mash1 is also able to substitute for this later role of Ngn2 when ectopically expressed in postmitotic precursors.
Is there a function of Mash1 in DA neuron development?
Mash1 substitution into the Ngn2 locus was previously
reported to misdirect cortical progenitors to become GABAergic instead of
glutamatergic (Fode et al.,
2000). Unlike Ngn2, Mash1 has been attributed an
instructive role in other systems, because it can re-specify neuronal lineages
when expressed in Ngn2–/– precursors in the
cortex and hindbrain (Parras et al.,
2002; Bertrand et al.,
2002). Mash1 has also been implicated in the
specification of GABAergic neurons in the dorsal midbrain
(Miyoshi et al., 2004). Our
findings, however, indicate that Mash1 is not sufficient to
re-specify VM progenitors in Ngn2KIMash1/KIMash1 embryos
into GABA or serotonin neurons, two phenotypes known to be specified by
Mash1 (Fode et al.,
2000; Pattyn et al.,
2004). Thus, our results indicate that the function of
Mash1 in normal VM development is more permissive than instructive,
unlike in other neural systems (Parras et
al., 2002; Bertrand et al.,
2002).
|
It should also be noted that DA neuronal generation was not completely
blocked by the deletion of Ngn2, as about 10-15% of Th+
cells were observed in the Ngn2 single, Ngn2;Ngn1 double and
Ngn2;Mash1 double mutants at E14.5. Thus additional factor(s) are
likely to be involved in the specification of DA neurons in the absence of
Ngn2. Alternative and complementary neurogenic pathways have recently
been described for cortical neuron specification
(Schuurmans et al., 2004).
First, a Ngn1/2-dependent process, and, second, a
Ngn-independent but Pax6- and Tlx-dependent
process, result in the generation of early-born and late-born glutamatergic
cortical neurons, respectively. Preliminary results indicate that
Pax6 is upregulated in zone 1 of the Ngn2 null mice (data
not shown). Thus, it remains to be determined whether Pax6 could
contribute to DA neurogenesis by a mechanism similar to that previously
described in the cerebral cortex.
Concluding remarks
Our findings show that Ngn2 is a key regulator of midbrain DA
neuron development and suggest that overexpression of Ngn2 in DA
progenitors or stem cells, together with other transcription factors, may
contribute to enhancing the DA differentiation of stem/precursor cells. Such
strategies could contribute to the future development of transplantation- or
endogenous neurogenesis-based cell replacement strategies for the treatment of
neurodegenerative diseases such as Parkinson's disease.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Cell Biology, Universitat de Valencia,
Spain
These authors contributed equally to this work
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