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First published online 5 January 2006
doi: 10.1242/dev.02224
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1 Wallenberg Neuroscience Center, Department of Experimental Medical Science,
and Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund
University, BMC A11, SE-221 84 Lund, Sweden.
2 National Institute for Medical Research, Division of Molecular Neurobiology,
The Ridgeway, Mill Hill, London NW7 1AA, UK.
Author for correspondence (e-mail:
anders.bjorklund{at}med.lu.se)
Accepted 25 November 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Parkinson's disease, Proneural genes, Differentiation, Midbrain, Tyrosine hydroxylase, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ye et al.,
1998). In addition, loss-of-function studies have shown that genes
such as Nurr1 (Nr4a2 – Mouse Genome Informatics), Pitx3, Lmx1b, and
Engrailed (En) 1 and 2 are important for the later differentiation of the
mesDA neurons (Simon et al.,
2001; Smidt et al.,
2000; van den Munckhof et al.,
2003; Zetterstrom et al.,
1997). However, much is still unknown regarding the genes and
factors involved in generation of mesDA neurons in the developing VM.
The proneural gene neurogenin 2 (Ngn2; Neurog2 –
Mouse Genome Informatics) is member of a family of bHLH transcription factors
(Gradwohl et al., 1996;
Sommer et al., 1996), and is
important not only for neuronal differentiation
(Fode et al., 1998), but also
for neuronal subtype-specification in various regions of the nervous system
(Ma et al., 1999;
Scardigli et al., 2001).
Within the VM, Ngn2 expression is restricted to the ventricular zone (VZ), and
its expression correlates both spatially and temporally with the generation of
mesDA neurons, suggesting that Ngn2 is involved in DA neuron development
(Thompson et al., 2006). To
investigate the importance of Ngn2 in mesDA neuron development, we studied the
VM phenotype of a Ngn2-knock-out mutant mouse. Our analysis shows an
initial loss of DA neurons in the developing VM followed by a partial recovery
later during embryogenesis, such that the number of DA neurons in the
Ngn2 mutant amounts to less than half of the number in the wild type
at postnatal stages. Other populations of neurons in the VM are not affected,
suggesting that, in the VM, Ngn2 is involved in differentiation of the mesDA
neurons specifically.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For
genotyping of the mutant allele the following primers were used: Ngn2KI5
(5'-GGA CAT TCC CGG ACA CAC AC-3') and Ngn2KImutant3 (5'-GCA
TCA CCT TCA CCC TCT CC-3'; giving a 440 bp fragment). PCR conditions for
the primers were: 30 cycles of 94°C for 1 minute, 58°C for 1 minute
and 72°C for 1 minute. For staging of embryos, the morning of the vaginal
plug was considered as embryonic day (E) 0.5.
BrdU injections
Timed pregnant females were injected intraperitoneally (i.p.) with 50 µg
BrdU/g body weight, once at E13.5 (45-minute incorporation), or twice with a
three-hour interval at E15.5 and E16.5.
Immunohistochemistry
For histological analysis, heads or dissected brains (E11.5-P0) were
immersion fixed overnight with 4% paraformaldehyde (PFA) in PBS at 8°C.
P18 mice received lethal doses of pentobarbitone and were transcardially
perfused with 0.9% saline followed by 4% PFA. The brains were post-fixed for 2
hours. Following fixation, the tissue was cryoprotected overnight in 30%
(E11.5-E13.5) or 25% (E15.5-P18) sucrose. Sections were blocked in 2% normal
serum/0.25% Triton X-100/PBS prior to incubation with primary and secondary
antibodies. Primary antibodies used following this procedure were: rabbit
anti-AADC (1:1000; Chemicon), rat anti-BrdU (1:100; Oxford Biotechnology; DNA
was denaturated in 1 M HCl at 65°C for 30 minutes prior to preincubation,
incubation with antibody at 8°C), rabbit anti-En1/2 (1:1000; gift from A.
L. Joyner, New York, NY), rabbit anti-Isl1 (1:100; Abcam), rabbit anti-Isl1/2
(1:500; gift from H. Edlund and T. Edlund, Umeå, Sweden), chicken
anti-GFP (1:5000; Chemicon), mouse anti-nestin (1:1000; BD), goat anti-Neurod1
(1:400; Santa Cruz), rabbit anti-Nurr1 (1:1000; Santa Cruz), mouse
anti-PSANCAM (1:200; Chemicon), mouse anti-Th (1:4000; Chemicon), and rabbit
anti-Th (1:1000; Pelfreeze). The first blocking step was replaced by 20
minutes in boiling 10 mM citrate buffer (pH 6) for primary antibodies against:
rabbit anti-calbindin D28k (1:1000; Chemicon), rabbit anti-Girk2 (1:80;
Alomone Labs), mouse anti-Isl1 (1:100; Hybridoma Bank), rat anti-Ki67 (1:50;
DAKO), mouse anti-Ngn2 (1:20; gift from D. J. Anderson, Pasadena, CA), rabbit
anti-Pitx3 (1:400; gift from J. P. Burbach, Utrecht, The Netherlands), and
rabbit anti-VMAT (1:1000; Chemicon). The staining protocol for mouse
anti-Brn3a (1:50; Santa Cruz) included boiling in citrate buffer in addition
to a blocking step prior to both primary and secondary antibodies (blocking
solution: 1% milk/10% normal serum/1 mg/ml BSA/PBS). Cell nuclei were
visualized by staining with DAPI (1:1000; Sigma). Apoptotic cells were
detected by TUNEL staining using the ApopTag Red Kit (Chemicon).
Measurement of DA and DOPAC in forebrain tissue
Tissue samples were homogenized in 0.4 M perchloric acid, diluted in milliQ
water and filtered. The samples were injected into an ESA Coulochem III with
electrochemical detection by a cooled autosampler (Midas). The potential of
the second electrode was set at +320 mV. The mobile phase (sodium acetate 5
g/l, Na2-EDTA 30 mg/l, octane-sulfonic acid 100 mg/l, methanol 12%,
pH 4.2) was delivered at a flow rate of 500 µl/minute to a reverse-phase
C18 column (4.6 mm diameter, 150 mm length, CHROMPACK). The peaks were
processed by Azur chromatographic software.
In vitro differentiation
VM and dorsal midbrain (DM) were dissected from E11.5 wild-type embryos and
mechanically dissociated into single cell suspensions. Cells were plated on
Matrigel (BD Biosciences) at a density of 100,000 cells/0.8 cm2 in
DMEM/F12 medium supplemented with B27, 20 ng/ml EGF, 10 ng/ml bFGF, and 40 U
heparin. The cultures were transduced with VSV-G-pseudotyped retroviruses
containing a Ngn2-IRES-GFP vector
(Falk et al., 2002) or a
GFP control vector, 15 hours after plating. The medium was replaced
with differentiation medium (DMEM/F12 with 1% FCS, 10 ng/ml GDNF, and 100
µM ascorbic acid) after an additional 10 hours of incubation. The cultures
were fixed in 4% ice-cold PFA for 15 minutes after 5 days under
differentiation conditions.
Cell counts
Tyrosine hydroxylase (Th)-positive cells were quantified in P0 and P18
Ngn2 mutants and littermates. Every tenth section (16 µm) was
collected from P0 pups and every fifth section (30 µm) was collected from
P18 pups. Three sections were used for quantification using the procedure of
Sauer et al. (Sauer et al.,
1995), one containing the interpeduncular nucleus at the level of
oculomotor nerve exit, the one previous and the one following (indicated with
a star in Fig. 2J,K). The
average cell number per section was calculated bilaterally for each animal. An
estimation of the reduction in total Th-positive cell number in the P18 pups
was obtained by counting Th-positive cells in all sections containing SN and
VTA in the series (every fifth section stained). The average total numbers of
Th-positive cells counted were calculated. All Isl+ and
Brn3a+ cells within the oculomotor and red nuclei, respectively,
were counted bilaterally, and the mean total number was compared between
wild-type and Ngn2 mutant mice. At E13.5, all DAPI-stained nuclei
(within the MZ) and BrdU-positive cells within the mesDA neuron domain were
counted in a series of every tenth section. The number of GFP/GLAST
double-positive cells within a distance of 2 µm from the ventricular
surface at one confocal plan per animal was counted. All numbers were
corrected for split cell counts using the Abercrombie formula
(Abercrombie, 1946).
Statistical analysis
Cell counts were compared using an unpaired Student's t-test. The
statistical significance level was set at P<0.05. Data is
expressed as the group mean ±1 s.e.m.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In wild-type embryos at E11.5, some scattered Th-positive cells are seen at the ventral rim of the VM, and, at E13.5, Th-positive cells fill the mantle zone (MZ) forming the beginnings of the mesDA nuclei (Fig. 1D,F). In E11.5 Ngn2 mutants, only single Th-positive cells were seen (Fig. 1E), and some of the embryos analyzed (3/7) did not display any Th-positive cells at all. At E13.5, the number of Th-positive neurons in Ngn2 mutants was approximately one-tenth of that seen in wild-type mice. The Th-positive cells present in the mutants were primarily located in thin stripes at the lateral edges of the expected DA neuron domain (Fig. 1G). Later, at E15.5, the number of Th-positive cells in Ngn2 mutants was still dramatically reduced compared with in wild-type mice and the pattern of the existing Th-positive neurons was similar to that seen at E13.5 (Fig. 1H,I). At E17.5, however, more Th-positive neurons had been generated in the Ngn2 mutants and the discrepancy between wild-type and Ngn2 mutant mice, with respect to both number and distribution of the Th-positive cells, was less pronounced (Fig. 1N,O). Ngn2-GFP heterozygotes did not differ from wild-type mice at any stage of development. In further analyses, we therefore used the heterozygous animals interchangeably with wild-type mice.
The expression of Aadc (Ddc – Mouse Genome Informatics), which is expressed in both DA neuron precursors and mature DA neurons, and Vmat2 (Slc18a2 – Mouse Genome Informatics), a marker for more mature DA neurons, showed the same pattern of expression as Th did in the Ngn2 mutant (Fig. 1J-M). Therefore, the reduction of Th-positive neurons in the Ngn2 mutant does not appear to be caused by a specific downregulation of the Th gene but represents a reduction in the number of mesDA neurons.
Compromised mesDA neuron system in postnatal Ngn2 mutants
The Ngn2 mutants were born with the expected frequency (15/80),
but most of them died within the first days of birth. Because the
Ngn2 mutants were among the smaller pups it was possible to reduce
the size of the litter, which was beneficial for the survival of the mutants.
Reducing the litters to 
5 pups allowed for one mutant for every two
litters to survive (8 mutants in 15 litters). The mutants had a limited weight
gain when compared with wild-type and heterozygous pups, and weighed on
average one-third of what their littermates did at P18
(Fig. 2C). They had an
emaciated appearance but showed no obvious abnormal behavior. At approximately
3 weeks of age their condition began to deteriorate, and no mutants survived
beyond P25 (see also Fode et al.,
1998). To assess the fate of the mesDA system postnatally, the
expression of Th was analyzed in neonatal (P0) and P18 Ngn2 mutants
and littermates. At P0 there was no obvious difference in brain size between
mutant and wild-type pups. However, at P18 the size of Ngn2 mutant
brain was considerably reduced and weighed approximately 80% of that of
wild-type littermates. Notably, the number of sections retrieved from the
midbrain through the mesDA domain at P18 was roughly 20% fewer in the mutants,
as shown in Fig. 2J,K. In all
mutants analyzed at this stage (n=5), the ventricular system,
including the midbrain aqueduct, was enlarged. At P0, the distribution of
Th-positive cells was similar in wild-type and mutant mice; however the number
of Th-positive cells was markedly reduced both in VTA, SN
(Fig. 2A,B) and RRA (not
shown). This reduction was also apparent in P18 mutants when comparing
sections of similar rostrocaudal levels
(Fig. 2D,E,J,K). For
quantification, Th-positive cells were counted bilaterally in two to three
sections per animal and the average number of cells/section for the VTA and SN
was compared in mutants and littermates at P0 and P18. At P0, the number of
Th-positive cells in the Ngn2 mutants was reduced by about 50%, and
at P18 by about 60%, in both VTA and SN
(Fig. 2N,O; P<0.01
for all comparisons). Heterozygous littermates were also analyzed at P18.
There was a highly significant difference in Th-positive cell number between
homozygotes and heterozygotes (P<0.001 for both SN and VTA), but
no difference between heterozygotes and wild-type mice (P=0.47;
Fig. 2O). Because the SN-VTA
region was reduced in size in the Ngn2 mutants at P18 it is likely
that the above figure underestimates the reduction of the mesDA cell number.
In order to estimate the magnitude of the total cell loss at P18, we counted
Th-positive cells in all sections that included SN or VTA (7 sections in the
wild type and heterozygotes, and 5 sections in the mutants). The average
number of counted cells in wild type, heterozygotes and mutants were 815, 964
and 262 in SN, and 816, 954 and 254 in VTA, respectively, representing an
average mesDA neuron loss of about 70% in the Ngn2 mutant at P18.
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In Th immunostained sections from the forebrain, the distribution of Th-positive fibers in the striatum and the adjacent limbic and cortical forebrain areas in the Ngn2 mutants did not differ from that seen in wild-type littermates. No single forebrain area was deprived of Th-positive innervation, suggesting that the DA neurons remaining in the mutant SN and VTA were projecting to their appropriate target areas (Fig. 2L,M).
Neurochemical analysis at P18 showed reduced total levels of both DA (38% of wild type) and DOPAC (48% of wild type) in forebrain of Ngn2 mutants (Table 1). Even when correcting for the reduced weight of the brain, the tissue concentration of both DA and DOPAC in the mutant forebrain was less than 65% of that measured in wild-type mice. The turnover of DA, as assessed by the DOPAC/DA ratio, did not differ between wild-type and Ngn2 mutant mice.
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; Schein et al.,
1998). We compared the distribution of Girk2 and calbindin in
postnatal Ngn2 mutant and wild-type mice in order to investigate
whether the reduced number of mesDA neurons resulted from a selective loss of
one of the two DA neuron subtypes. In both wild type and Ngn2
mutants, Th-positive neurons in the VTA co-expressed calbindin, whereas the SN
DA neurons were negative for this marker (P0,
Fig. 3A-F). The typical
expression profile of Girk2 is first seen at later postnatal stages, we thus
studied the expression of Girk2 at P18. At this age, in both wild type and
Ngn2 mutants, Girk2 was preferentially expressed in the SN, and to a
lesser extent in DA neurons located in the zone between VTA and SN
(Fig. 3G-L).
In the ventral spinal cord, Ngn2 is required for the correct expression of
a number of homeodomain (HD) transcription factors involved in neuronal
subtype differentiation (Scardigli et al.,
2001). To study whether Ngn2 in a similar fashion is important for
the correct expression of HD transcription factors within the VM, we analysed
the expression of Pitx3, a HD transcription factor specifically expressed in
mesDA neurons (Smidt et al.,
1997). As previously reported
(Smidt et al., 2004;
Zhao et al., 2004), Pitx3 was
expressed in virtually all Th-positive neurons in the SN and VTA in wild-type
mice. Similarly, all remaining Th-positive DA neurons in the SN and VTA in the
postnatal Ngn2 mutant expressed Pitx3. Two other HD transcription
factors, En1 and En2, are also expressed in developing and mature mesDA
neurons, but are not as specific to this cell population as Pitx3
(Davis and Joyner, 1988;
Simon et al., 2001). We found
that En1/2 was co-expressed with Th in Ngn2 mutants to the same
extent as in wild-type mice (data not shown).
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Loss of medially located mesDA neuron precursors in the Ngn2 mutant
The analysis of the development of mesDA neurons in the Ngn2
mutants showed a substantial early loss of Th-positive neurons. To clarify if
the mesDA neuron precursors are generated normally in the mutant, but fail to
mature to more differentiated neurons, we studied the expression of the orphan
nuclear receptor Nurr1. MesDA neuron precursors express Nurr1 prior to Th, at
the time of migration through the postmitotic intermediate zone (IZ, defined
here as the zone between the proliferative VZ and the MZ)
(Zetterstrom et al., 1997). In
the Ngn2-GFP heterozygotes, at E11.5, the entire IZ is filled with
Nurr1-positive cells (Fig. 4A),
and at E13.5 the further differentiated mesDA neurons in the MZ also express
Nurr1 (Fig. 4G). In the
Ngn2 mutants, the region around the midline was completely void of
Nurr1-positive mesDA neuron precursors, at both E11.5 and E13.5
(Fig. 4D,J). Two streams of
Nurr1-positive cells were located at the lateral edges of the presumptive
mesDA precursor domain, similar to the Th expression pattern previously seen
at these stages (Fig. 4D,J). More Nurr1-positive cells were detected at E15.5, but their spatial
distribution remained the same (Fig.
4P). At E13.5, Pitx3 expression, which has an onset that is
slightly earlier than Th (Zhao et al.,
2004), showed the same loss of medially located mesDA neuron
precursors in the Ngn2 mutant mice as did Nurr1 (data not shown).
The Ngn2-GFP mouse used in this study, expresses GFP from the Ngn2 locus. In these mice, the GFP protein is detectable not only in the Ngn2-expressing cells in the VZ, but also in their immediate progeny in the IZ (Fig. 4H). This may be explained either by the longer half-life of the GFP protein, or by differences in the posttranscriptional regulation of Ngn2 and GFP. Because the progeny of Ngn2-expressing precursors continue to stain for GFP for a limited time, short-term lineage tracing of these cells is possible. Analysis of GFP expression in the Ngn2 mutants showed the presence of GFP-positive cells in the medial sector, as well as in more lateral positions (Fig. 4E,K,Q). This demonstrates that the VM cells lacking Ngn2 survive, at least initially. However, although the cells at the lateral edges of the mesDA neuron domain express Nurr1 as they migrate ventrally (Fig. 4L,R), the GFP-positive cells within the medial sector do not show any Nurr1 or Pitx3 expression. These results show that the absence of Th-positive neurons in the Ngn2 mutant mice is matched by a lack of Nurr1- and Pitx3-expressing mesDA neuron precursors.
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). The results showed a threefold increase in the percentage
of GFP/GLAST-positive cells within a 2 µm distance from the ventricular
surface in the Ngn2 mutants compared with in heterozygotes
(heterozygotes, 8.8±2.7%; mutants, 27.3±6.9%; n=3,
P<0.05), and a clear increase in the area of the ventricular
surface occupied by GFP/GLAST-positive endfeets (arrows in
Fig. 6C,K). Thus, the absence
of Ngn2 leads to an increase in the number of (GFP/GLAST-positive) neural
progenitor cells.
Next, we wanted to investigate the phenotype of the remaining cell
accumulation in the Ngn2 mutants in more detail. The proliferative
marker Ki67 (data not shown) and BrdU labeling at E13.5 showed that
proliferation was limited to a three to five cell-diameter-thick zone along
the ventricular surface, in a pattern indistinguishable from the distribution
in heterozygous mice, and that there was no increase in cell division within
the cell accumulation compared with heterozyotes (BrdU: heterozygotes,
299±87; mutants, 263±40; Fig.
6D,E). Thus, the border between the VZ and the IZ persists in the
Ngn2 mutants, and the absence of Ki67-positive and BrdU-labeled cells
in the mutant IZ indicates that the Nurr1-negative cells that accumulate in
this zone are postmitotic. The neuronal determination factor Neurod1
(Lee et al., 1995) was
expressed in the IZ of heterozygous mice at E13.5; however, similar to the
expression pattern of Nurr1, Neurod1-positive cells were only seen at lateral
positions in the mutant IZ (Fig.
6L,M,O,P). The lack of Neurod1 expression in the Ngn2
mutants was specific for the mesDA neuron domain, as Neurod1 expression was
unaffected in other parts of the midbrain (data not shown). At the same time,
none of the cells in the mutant IZ expressed the neuronal markers PSA-NCAM
(data not shown) and ß-III-tubulin
(Fig. 5B,G), which does not
support the presence of immature or mature neurons among the arrested cells
within the IZ. Finally, no expression of GFAP could be detected in this region
of the Ngn2 mutants (data not shown).
To determine the fate of the arrested cells in the mutant IZ, we performed a TUNEL assay. Single labeled cells were detected at E13.5 in the VM of wild-type mice, and up to six labeled cells in one section in Ngn2 mutant mice (n=6; data not shown). This suggests that some cells will go through apoptosis when lacking the differentiation cues provided by Ngn2; however most of them will remain. It seems likely, therefore, that cells appearing in the MZ from E15.5 (Fig. 6F,N) and onwards are derived from accumulated cells that are released from the differentiation arrest. The release of cells to the MZ also corresponds well with the catch-up of the mesDA neurons seen between E15.5 and E17.5. Alternatively, proliferating VZ cells could generate the late-appearing mesDA neurons. To investigate this possibility, we exposed E15.5 and E16.5 embryos to BrdU, i.e. at time-points when mesDA neurogenesis is complete in wild-type mice. Incorporation of BrdU in mesDA neurons was examined at P0. No BrdU/Th double-labeled mesDA neurons were observed in either wild-type or Ngn2 mutant mice, despite a high number of BrdU-labeled cells in this area (data not shown). Therefore, the increase in mesDA neurons between E15.5 and E17.5 is unlikely to be explained by a prolonged neurogenesis.
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;
Wallen et al., 1999). At
E11.5, the number of Isl1-positive neurons in Ngn2 mutants was
indistinguishable from that of wild-type mice
(Fig. 7A,D). When quantified at
P0, the number of Isl1-positive oculomotor neurons
(Fig. 7H,K) was
259.7±23.3 in the Ngn2 mutants compared with 255.9±14.2
in wild-type mice (n=3, P=0.90). Loss of Ngn2 thus did not
cause either a decrease or an increase of oculomotor neurons. Because the
neurogenesis of oculomotor neurons is ongoing at E11.5, these results also
indicate that genesis of these neurons is not delayed in the Ngn2
mutants, as is the case for mesDA neurons. Interestingly, Isl1-positive cells
show weak GFP expression at E11.5 (Fig.
7C,F). Hence, Ngn2 is likely to be expressed in oculomotor neuron
precursors without being fundamental for their development. Similarly, no
difference was detected in the number of Brn3a-positive neurons at E13.5
(Fig. 7G,J), and quantitative
analysis of Brn3a-positive neurons in the red nucleus at P0 did not reveal any
difference between wild-type and Ngn2 mutant mice (total cell number:
wild type, 1194±39; mutant, 1113±37; n=4,
P=0.19; Fig. 7I,L). Hence, within the VM, loss of Ngn2 function specifically affects the
differentiation of DA neurons, whereas the development of other neuronal
subtypes is unaltered.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the
VM, Ngn2 is expressed in the proliferative VZ in a spatiotemporal pattern that
correlates with mesDA neurogenesis, suggesting that Ngn2 is expressed in the
precursors of mesDA neurons (Fig.
1A) (Thompson et al.,
2006). In this study, we show that the generation of the mesDA
neuron system is greatly impaired in Ngn2 knockout mice, indicating
that Ngn2 function is important for the normal development of the mesDA neuron
system; other neuronal populations within the VM, notably the Isl1-expressing
oculomotor neurons and the Brn3a-expressing neurons of the red nucleus, are
unaffected. In the Ngn2 mutant, the number of Th-positive neurons in
the VM was initially reduced by about 90%, as seen at E11.5-E13.5. At later
stages of development, i.e. between E15.5 and E17.5, there was a partial
recovery to about 30-50% of normal levels, as seen postnatally. The remaining
mesDA neurons, most of which were formed with several days delay, were
correctly specified with respect to the expression of appropriate HD proteins
and other transcription factors, and differentiation into VTA and SN neuron
subtypes, as well as their axonal projections in the forebrain.
Specific loss of mesDA neurons in the absence of Ngn2
Ngn2 is known to be important for promoting neuronal differentiation in
many areas of the CNS. Our data show that the VM phenotype of Ngn2
mutants includes loss of mesDA neurons, a smaller midbrain and an enlarged
aqueduct, indicating that some cell populations are indeed lost or never
formed in the mutant VM. Within the VM, the reduction of neuron number appears
to be specific for the mesDA neurons, as the number of Isl1- and
Brn3a-positive neurons, i.e. the neurons forming the oculomotor and red
nuclei, respectively, is not affected. At early stages of mesDA neurogenesis
(E11.5), Ngn2 is expressed more broadly within the VZ of the VM, in a band
that extends lateral to the presumptive DA progenitor domain. In the
heterozygous Ngn2-GFP mice, where GFP expression can be used as a
short-term lineage tracer, Isl1-positive cells show weak GFP expression at
this stage, suggesting that they are derived from this lateral Ngn2-expressing
progenitor population. In the developing spinal cord, proneural genes, such as
Ngn2, interact with HD proteins to couple neurogenesis and subtype
specification (Lee and Pfaff,
2003), and in the Ngn2 mutant embryos, the expression of
the HD proteins Lim1/2, En1 and Chx10, which are markers for subtypes of
ventral spinal cord interneurons, is almost completely missing. In addition,
the number of neurons expressing the HD protein Isl1, a marker of the
cholinergic motoneurons, is reduced by about 50%
(Scardigli et al., 2001). Our
data show, that in contrast to the motoneurons in the ventral spinal cord, the
expression of Isl1 in oculomotor neurons in VM does not depend on Ngn2
expression. Interestingly, the mesDA neurons that are formed in the
Ngn2 mutants show the expected expression of Pitx3 and En1/2,
indicating that the expression of these characteristic HD proteins is
maintained also in the absence of Ngn2.
Although Ngn2 function is necessary for proper development of the mesDA
neuron system, our gain-of-function experiment showed that Ngn2 is not by
itself sufficient to ectopically promote mesDA neuron differentiation in
primary DM cultures. This is consistent with the view that Ngn2 has primarily
a permissive, rather than an instructive, role in neuronal subtype
specification, and that its function therefore, to a large extent, is
dependent on the cellular context (Bertrand
et al., 2002; Parras et al.,
2002).
Partial rescue of DA neurons in Ngn2 mutant mice
The initial loss of Th-positive neurons in the VM, seen at E11.5-E13.5, was
compensated for during later stages of development. A similar phenotype has
been reported for the large diameter sensory neurons of the dorsal root
ganglion (DRG) in Ngn2 mutants
(Ma et al., 1999). However, in
contrast to the DRG where the normal number of neurons is seen within a few
days after cessation of neurogenesis, the recovery of the mesDA neuron system
was only partial. It seems possible that the incomplete recovery could be
explained by an increase in apoptosis, as seen at E13.5 in the Ngn2
mutants. Alternatively, some of the accumulated precursors may have been
re-specified into other types of neurons. From our analysis, it is clear that
the numbers of neurons in the oculomotor and red nuclei are unchanged in the
absence of Ngn2. However, other types of neurons, such as GABAergic neurons
are present in the VM. In the absence of data relevant to the development of
these neuronal populations, we cannot exclude that they may be generated in
excess numbers in the mutant mice.
The mesDA neurons that were formed in the Ngn2 mutants at the
expected time of mesDA neurogenesis (E11.5-E13.5) were located in a distinct
lateral population, which suggested that they could represent a specific
subtype of mesDA neuron. In the PNS, Ngn2 is expressed in progenitors that
later form subclasses of sensory neurons, and in the Ngn2 mutants
only these neurons are affected (Fode et
al., 1998; Ma et al.,
1999). There are no markers to identify distinct subtypes of mesDA
neurons during neurogenesis; however, the number of neurons in the SN and VTA,
i.e. the two major subtypes that are discernible in the adult, were equally
affected. In addition, the remaining SN and VTA neurons were correctly
specified with respect to the expression of the distinguishing markers Girk2
and calbindin, and the distribution of Th-positive fibers in the striatum and
the adjacent limbic and cortical areas indicate that the SN and VTA
populations projected to their expected targets in the forebrain.
Delayed differentiation of mesDA neuron precursors in the absence of Ngn2
In the Ngn2 mutants, we noticed a retention of cells in the medial
part of the mesDA neuron domain, which was most pronounced at E12-E13.5. This
was observed as an accumulation of cells in the mutant VZ/IZ, matched by a
reduction of cells in the MZ, as evidenced by DAPI staining. Closer
examination of the mutant VZ/IZ indicated that Ngn2 is important at two steps
of the differentiation process. First, lack of Ngn2 function caused an
increase in GFP-positive radial glia cells, suggesting that a fraction of
cells remain as early neural progenitors in the absence of Ngn2. Importantly,
BrdU labeling showed that Ngn2 is not essential for cell cycle exit, as the VZ
was not increased in size in the Ngn2 mutants. Second, the
Nurr1-negative cells within the mutant IZ also lacked Neurod1 expression.
Neurod1 is a downstream target of Ngn2 and a known neuronal determination
factor (Lee et al., 1995;
Ma et al., 1996;
Mattar et al., 2004). Lack of
Neurod1 in the mutant IZ therefore implies that Ngn2 function is important for
the progenitor cells within the IZ to acquire a neuronal fate, which can
explain the lack of mesDA neuronal markers. It is worth noticing that this
requirement of Ngn2 for Neurod1 expression was specific for the mesDA neuron
domain, as Neurod1 expression was unaffected in other parts of the midbrain,
and that, despite an almost complete lack of mesDA neurons, a substantial
number of ß-III-tubulin-positive neurons were present at E13.5 in the
part of the MZ where the mesDA neurons are normally located.
It is likely that, due to the lack of specification, cells within the
mutant VZ/IZ cannot respond to the migratory cues present in the surrounding
environment and thus stay close to the ventricle. This is reminiscent of the
phenotype seen in distal cranial ganglia of Ngn2 mutants, where
delamination and migration of sensory neuron precursors from the epibranchial
placodes is blocked, and the expression of neuronal markers is missing
(Fode et al., 1998). In the
cranial sensory neurons, the block in neurogenesis is later compensated for
and the geniculate ganglia are present at birth in Ngn2 mutants,
although diminished in size. Although the recovery of geniculate ganglion
neurons is most likely due to neural crest-derived precursors populating the
structure, we investigated alternative explanations for the compensation in
the mesDA system. BrdU labeling at E15.5 and E16.5 showed that the increase of
Th-positive neurons between E15.5 and E17.5 is not due to a prolonged
neurogenesis of mesDA neurons in the Ngn2 mutants within this period.
Because the accumulation of cells in the VZ/IZ decreased as development
progressed, there is a possibility that some of these cells later
differentiate into mesDA neurons. In support of this idea, Kele et al. show
that another proneural gene, Mash1, is expressed within the VZ of the
mesDA neuron domain, and that the recovery of Th-positive neurons, seen at
E17.5 in the Ngn2 mutant, is abolished in mice lacking both
Ngn2 and Mash1 genes
(Kele et al., 2006). Moreover,
Kele et al. have observed that Mash1 when knocked into the
Ngn2 locus can partially restore the number of Th-positive neurons
that develop at this stage (Kele et al.,
2006).
Ngn2 functions upstream of Pitx3, Nurr1 and En1/2 in mesDA neuron differentiation
A number of developmental genes, such as En1/2, Lmx1b, Pitx3 and Nurr1, are
expressed in mesDA neuron precursors in the VM, and have been shown to be of
major importance for the development of the mesDA neuron system
(Simon et al., 2001;
Smidt et al., 2000;
van den Munckhof et al., 2003;
Zetterstrom et al., 1997). For
the most part, however, these genes are expressed at postmitotic stages, and
are important for the terminal differentiation, maintenance or survival of the
mesDA neurons. The only mutant where defects in the early differentiation into
mesDA neurons have been suggested is the Lmx1b mutant. In the absence
of Lmx1b, Pitx3 is never expressed, and Nurr1 and Th expression is lost after
E16.5. However, the defects are not limited to the mesDA neuron system, as the
VM is reported to show other major malformations
(Smidt et al., 2000). Hence,
Ngn2 is the first example of a gene, expressed in the VM VZ cells,
that is essential for proper mesDA neuron differentiation, and where loss of
function causes impaired mesDA neurogenesis without other major abnormalities
in the VM.
The proneural genes are known to be expressed in proliferating neural
progenitor cells in diverse regions of the nervous system, where they promote
neuronal differentiation and suppress glial phenotypes
(Bylund et al., 2003;
Sun et al., 2001). The
spatiotemporal pattern of Ngn2 expression in the developing VM suggests that
Ngn2 plays a role in the differentiation of VZ progenitors within the mesDA
neuron domain, and that it acts upstream of Nurr1, Pitx3 and En1/2 in mesDA
neuron differentiation. Ngn2 is thus the first gene shown to be
involved in the regulation of mesDA neuron differentiation at the level of the
early progenitor cell.
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ACKNOWLEDGMENTS |
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Footnotes |
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