1 Department of Psychiatry, Weill Medical College of Cornell University, New
York, NY 10021, USA
2 Graduate Program in Neuroscience, Weill Medical College of Cornell University,
New York, NY 10021, USA
Author for correspondence (e-mail:
SAA2007{at}med.cornell.edu)
Accepted 14 September 2005
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SUMMARY |
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Key words: Nkx2.1, Parvalbumin, Somatostatin, Conditional knockout, Smoothened
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Introduction |
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Recently, we demonstrated the existence of regional specificity for the
genesis of cortical interneuron subgroups
(Xu et al., 2004). Parvalbumin
(Pv; Pvalb Mouse Genome Informatics)- and somatostatin (Som; Sst
Mouse Genome Informatics)-expressing interneuron subgroups require the
transcription factor Nkx2.1 for their specification, and originate primarily
in the MGE. Calretinin-expressing subgroups are not Nkx2.1 dependent and
appear to originate primarily in the dorsal, non-Nkx2.1-expressing region of
the caudal ganglionic eminence (CGE).
Despite recent advances in our understanding of the origins and regulation
of cortical interneuron migrations (Marin
et al., 2001; Marin et al.,
2003
; Alifragis et al.,
2004
), apart from the dependence on Nkx2.1, little is known about
the specification of interneuron fate. One factor that appears to function
upstream of Nkx2.1 is sonic hedgehog (Shh). In the telencephalon, Shh is
initially secreted from the prechordal mesoderm; it then induces its own
expression in the overlying neural tube, in the proliferative zone of the
preoptic area and in the mantle region of the MGE
(Ericson et al., 1995
;
Shimamura et al., 1995
). Shh
permits the initial induction of transcription factors with ventrally
restricted patterns of expression, including Dlx1, Dlx2, Mash1 and
Nkx2.1 (Kohtz et al.,
1998
; Gunhaga et al.,
2000
), and it is normally required for the formation of the
ventral telencephalon (Chiang et al.,
1996
; Rallu et al.,
2002
).
Recently, an analysis of the role of Shh signaling on the initial
patterning of the telencephalon was made using a
FoxG1-Cre:SmoothenedFl/Fl conditional knockout, which loses Shh
signaling by about embryonic day (E) 9
(Fuccillo et al., 2004). In
these embryos, initial patterning of the ventral telencephalon fails, such
that ventrally derived cell types, including cortical interneurons, are
absent. However, the functions of later telencephalic Shh expression during
the age range of neurogenesis are less clear, although, throughout the
neuroaxis, Shh support of progenitor proliferation maintains multipotent
progenitor `niches' (Britto et al.,
2002
; Machold et al.,
2003
). In vitro studies suggest that Shh signaling can direct
telencephalic progenitors towards a GABAergic phenotype
(Yung et al., 2002
;
Gulacsi and Lillien, 2003
;
Watanabe et al., 2005
),
although the downstream effectors of this process remain unclear.
In this paper, we provide evidence for a new function of Shh during the genesis of cortical interneurons, the maintenance of progenitor identity. Analyses of nestin-Cre (NsCre):ShhFl/Fl mouse embryos at E12.5 reveal that there is a dramatic reduction in the proportion of S-phase progenitors that co-express Nkx2.1. Consequently, two Nkx2.1-dependent interneuron subgroups, those expressing Pv or Som, are greatly reduced in layers II to IV of the cortex at postnatal day 12. Several lines of evidence suggest that the postnatal loss of these interneuron subgroups primarily results from a loss of Shh signaling in the MGE, rather than from a later influence of Shh on interneuron migration, differentiation or survival. These findings suggest that Shh signaling during neurogenesis maintains cortical interneuron progenitor identity through its regulation of Nkx2.1 expression, and thus plays a crucial role in determining the relative composition of excitatory and inhibitory neurons of the cerebral cortex.
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Materials and methods |
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Telencephalic slice cultures
Coronal telencephalic slices were generated and cultured as described
(Xu et al., 2004). For
cyclopamine experiments, 5 µM of cyclopamine dissolved in ethanol or an
equivalent amount of ethanol (0.05% volume) was added to the medium. For the
exogenous Shh experiments, recombinant modified N-terminal Shh protein (Shh-N;
Curis) was diluted in Neurobasal/B27 (Nb/B27; Gibco) and used at a final
concentration of 10 nM. Slices were maintained in vitro for 24-36 hours with
no change of medium.
BrdU labeling
For in vivo labeling of S-phase cells, one injection of BrdU (100 mg/kg,
intraperitoneally) was made one hour prior to sacrificing the dam (unless
indicated otherwise). For in vitro studies, BrdU (100 ng/ml) was added to the
culture medium six hours prior to fixation or transplantation.
Slice electroporation
Mouse patched 1 with a loop2 deletion
[mPtc1loop2, a gift from Dr
Gary Struhl (Briscoe et al.,
2001
)] was subcloned into
pCAGGS-mPtc
loop2IRES-GFP. The
same vector without mPtc1
loop2
was used as control. DNA was purified with the Endofree Plasmid Maxi Kit
(Qiagen) and electroporated into the MGE region of E12.5 slices as described
(Stuhmer et al., 2002a
).
Co-cultures of MGE-derived cells on cortical feeder cells
Primary cortical feeder cultures (100,000 cells per 36 mm2 well
of 16-well chamber slides, Lab-Tek) were prepared from the dissociated
cortices of neonatal pups as described (Xu
et al., 2004). For the cyclopamine treatment experiments,
telencephalic slice cultures were made from pan-GFP expressing reporter mice
(Hadjantonakis et al., 1998
).
Following one day in vitro (DIV), the periventricular proliferative zone of
the MGE was dissected free, gently triturated and resuspended in Nb/B27
medium. Two thousand cells per well were added to cortical feeder cultures
prepared 1 day earlier, and the cultures were maintained as described
(Xu et al., 2004
).
In situ hybridization and immunohistochemistry
Postnatal mice were perfused with 4% paraformaldehyde (PFA) and processed
for cryosectioning (Xu et al.,
2004). Cryostat sections were obtained at 12 µm thickness for
immunohistochemistry, and at 12 or 20 µm for in situ hybridization using
digoxigenin-labeled riboprobes (Wilkinson
and Nieto, 1993
;
Schaeren-Wiemers and Gerfin-Moser
1993
). cDNA probes used were Shh (BC063087 from
Open Biosystems), Gli1 (Kinzler
et al., 1988
), Nkx2.1
(Kimura et al., 1996
),
Nkx6.2 (gift from Dr Gord Fishell) and Oct6.
The primary antibodies for immunofluorescence labeling included anti-BrdU
(mouse, Chemicon, 1:400; rat, Serotec, 1:200), calretinin (rabbit, Chemicon,
1:2000; mouse, Swant, 1:5000), Calbindin (rabbit, Swant, 1:5000), Dlx2 [rabbit
(Porteus et al., 1994)], GABA
(rabbit, Sigma, 1:5000), GFP (rabbit or chick, Molecular Probes, 1:2000), Gsh2
(rabbit, Kenneth Campbell, 1:5000), islet 1 (mouse, DSHB, 1:1000), NeuN
(mouse, Chemicon, 1:1000), Nkx2.1 (rabbit, Biopat, 1:1000; mouse, Neomarkers,
1:100), Npy (rabbit, Immunostar, 1:2000), Olig2 (rabbit, 1:2000), somatostatin
(rat, Chemicon, 1:400), parvalbumin (mouse, Chemicon, 1:5000), phospho-histone
H3 (rabbit, Upstate Biotech 1:200), and Tbr1 [rabbit
(Englund et al., 2005
),
1:1000]. Fluorescent secondary antibodies were Alexa line (Molecular Probes,
1:500). Triple labeling of in vitro cell cultures was performed using a
Cy5-conjugated secondary (Jackson ImmunoReseach)
(Xu et al., 2004
). The nuclear
marker DAPI (300 nM) was applied with the secondary antibodies. Signal was
detected by epifluorescence microscopy (Nikon E800), and images acquired
(Coolsnap HQ, Roper; Metamorph software, Universal Imaging).
Data collection and statistical analysis
Co-labeling of Nkx2.1 and BrdU was counted using a 60x oil immersion
lens (n.a. 1.4) on a Nikon E600 microscope fitted with a motorized stage and
Stereo Investigator software (MicroBrightField). Cell counts in a specified
region were obtained using the Fractionator probe and systematic random
sampling. For each embryo, data was pooled from both hemispheres of four
non-consecutive sections.
For postnatal analyses, coronal cryosections were evaluated from the genu of the corpus callosum to the hippocampal commissure. The medial boundary of primary somatosensory cortex was identified in cresyl violet-stained sections, and the counting region extended about 2 mm lateral to that point, to the lateral boundary of the primary and secondary somatosensory cortex. The boundary between layers four and five was identified by Cresyl violet and DAPI staining, and the cortex was divided into two bins containing layers two to four, and layers five and six. For all counts, both hemispheres of at least four non-consecutive coronal sections for were examined. First, the relative areal densities (counts/mm2) of cell profiles labeled for DAPI (all cells) and Tbr1 (most or all projection neurons), and of NeuN-labeled cells (most or all neurons), were estimated using the Fractionator probe and systematic random sampling (Microbrightfield Stereoinvestigator Software). Other markers were quantified as total profile counts in the defined region (profiles/mm2), and are presented as the ratio of these counts with all cells (DAPI), or with all NeuN+ profiles per unit area. Thus, these data provide relative counts for the comparison of cortical composition between mutant and control brains. Pilot studies revealed no consistent differences in profile size between any marker in mutant and wild-type sections. Statistical analysis (unpaired t-test) was performed with Excel software on data obtained from at least three mutants and three controls.
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Results |
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As noted previously, these NsCre:ShhFl/Fl mutants have a marked
reduction of Nkx2.1 expression, even though the density of S-phase cells is
not grossly reduced and there is no increase in the numbers of apoptotic cells
(Machold et al., 2003),
leading to the possibility that loss of Shh signaling reduces the expansion of
Nkx2.1-expressing progenitors. To examine this possibility, Nkx2.1 expression
was examined in E12.5 embryos that had received the S-phase marker BrdU 1 hour
prior to sacrifice. The BrdU-labeling index has subtle abnormalities in the
mutant MGE at this age, with a trend (not significant after correction for
multiple comparisons) towards reduced BrdU-labeling index in the ventricular
zone (VZ) of both the dorsal and ventral halves of MGE
(Fig. 1G). In the SVZ region of
the MGE there is a significant increase of the BrdU-labeling index in the
dorsal MGE (n=3, P<0.01). These results are difficult to
interpret in absence of data on the total cell-cycle time, but could suggest
that the loss of Shh results in a decrease in symmetrical,
progenitor-expanding divisions within the VZ and an increase in asymmetrical
VZ divisions that then undergo terminal, `transient-amplifying' divisions
within the SVZ (Haubensak et al.,
2004
; Noctor et al.,
2004
).
Regardless of subtle changes in BrdU-labeling index, the percentage of S-phase cells that co-expressed Nkx2.1 in the MGE drops from over 90% in NsCre():ShhFl/Wt controls to less than 50% in the NsCre:ShhFl/Fl embryos (Fig. 1A-F). Phospho-histone H3 (PH3) staining that primarily labels cells in M-phase appears to be grossly normal, suggesting that cell-cycle progression is occurring in the mutant telencephalon (see Fig. S2 in the supplementary material). These findings suggest that progenitor cells in the VZ and SVZ of the MGE respond to a reduction in Shh signaling by dramatically reducing their expression of Nkx2.1.
Loss of Shh signaling in the nestin-Cre:ShhFl/Fl telencephalon results in a subtle disruption of MGE patterning
Because a reduction in Shh signaling leads to a decrease in Nkx2.1
expression, we examined whether this reduction is indicative of a gross
re-patterning of the MGE or is an effect specific to certain Shh-responsive
genes. First the expression of Gli1, a marker of Shh signaling
(Lee et al., 1997), was
investigated (Fig. 2A). At
E12.5, two of the three mutants showed no detectable Gli1 expression,
while the other showed a marked reduction of expression in cells located in
the dorsal MGE when compared with wild types. By E14.5, all
NsCre:ShhFl/Fl mice lack Gli1 expression (not shown),
consistent with a loss of Shh signaling. We also examined the expression of
Nkx6.2, a target of Shh signaling in the spinal cord, which is also
expressed in the dorsal-most MGE
(Vallstedt et al., 2001
;
Stenman et al., 2003b
). At
E12.5, Nkx6.2 expression was not detected in any
NsCre:ShhFl/Fl mutant (Fig.
2B; n=3). By contrast, more general markers of pallidal
telencephalic patterning appear to be normal in the VZ, SVZ and mantle, as the
expression of Gsh2 (Fig. 2D),
Dlx2 (Fig. 2E) and islet 1
protein (see Fig. S3 in the supplementary material) is unchanged in the
mutants (see also Machold et al.,
2003
).
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Because cyclopamine treatment of slice cultures replicates the observed
decrease in Nkx2.1 expression in the NsCre:ShhFl/Fl mice, we
examined whether inhibition of Shh signaling alters the fate of MGE
progenitors and whether this alteration differentially affects cells that
entered S-phase after the longest exposure to cyclopamine. To address this
issue, we used a co-culture system in which the fates of progenitors from the
embryonic MGE are assessed 2-4 weeks after culturing on a feeder layer made
from neonatal cortex. In this system, MGE progenitors from wild-type embryos,
but not from Nkx2.1/ embryos, give rise to Pv and to
Som-expressing interneurons (Xu et al.,
2004). Slice cultures from E12.5 GFP-expressing donor mice were
treated with cyclopamine and then BrdU as above, then the MGEs of these slices
were dissociated and cells were plated over cortical feeder cells
(Xu et al., 2004
). After 2-4
weeks in vitro, the fates of all MGE cells (GFP+), and of those that were in
S-phase within 6 hours of transplantation (BrdU+), were examined by
co-labeling with neurochemical markers of differentiated interneurons
(Fig. 3). Cyclopamine treatment
of the slices has no effect on the total number of GFP-expressing cells with
neuronal morphology that survive 14 or 28 days in vitro (DIV;
Fig. 3J). By contrast,
cyclopamine treatment results in a significant reduction in the percentage of
these cells that express Som (at 14 DIV, 36.6±1.2% versus
27.8±1.7%; n=5; P<0.005) or Pv (at 28 DIV,
32.9±0.8% versus 21.2±2.3%, n=3;
P<0.05).
To examine whether there is a differential effect of cyclopamine on the fate of cells that were in S-phase during the final 6 hours of the treatment period, triple immunolabeling of GFP, Som and BrdU was performed on cultures after 14 DIV. Cyclopamine pre-treatment has no influence on the numbers of BrdU-labeled cells after 5 DIV, and a modest but statistically significant effect after 14 DIV (13.4±0.9% versus 11.5±1.2%, n=3, P<0.05). However, cyclopamine pre-treatment results in a dramatic reduction in the percentage of BrdU-labeled, MGE progenitors with neuronal morphology that expressed Som (Fig. 3J, 36.4±3.9% versus 6.8±1.9%, n=3, P<0.005).
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Cell-autonomous blockade of Shh signaling reduces Nkx2.1 expression in vitro
The results presented above suggest that Shh signaling maintains Nkx2.1
expression in the MGE and thereby promotes Pv/Som interneuron fate
determination. To confirm that Shh signaling blockade results in a
cell-autonomous downregulation of Nkx2.1, telencephalic slices were
transfected with a dominant-negative version of the Shh receptor Patched,
mPTC1loop2 (a gift from Gary Struhl), that is insensitive to
Shh, causing a blockade of Shh signal transduction
(Briscoe et al., 2001
).
Electroporation of pCAGGS-mPTC1
loop2-ires-GFP
(mPTC1
loop2) into the MGE of telencephalic slices (E12.5 + 1
DIV) produces a large decrease in the percentage of GFP+ cells that co-label
with Nkx2.1 relative to pCAGGS-ires-GFP controls
(Fig. 4). In summary, although
we cannot rule out the possibility that Shh blockade results in the relative
expansion of a non-Nkx2.1 expressing lineage, these results strongly support
the idea that MGE progenitors downregulate Nkx2.1 but continue to cycle in
response to a cell-autonomous reduction of Shh signaling.
Exogenous Shh rescues the loss of Nkx2.1 expression and interneuron fate effects in slices from NsCre:ShhFl/Fl mice
Because cyclopamine treatment reduces the number of Pv- or Som-expressing
interneurons generated from cultured MGE progenitors, we next investigated
whether a similar loss occurs in MGE transplants from
NsCre:ShhFl/Fl mutant mice. Telencephalic slices from
NsCre:ShhFl/Fl litters were cultured for 24 hours, with BrdU added
for the last 6 hours. The proliferative region of the MGE was transplanted
onto cortical feeders as described above. After 10 DIV, these cultures were
fixed and processed for the immunodetection of BrdU and Som, the interneuron
subgroup marker that is most affected in NsCre:ShhFl/Fl mutants in
vivo (Fig. 6). Whereas
wild-type transplants showed an expected co-localization of BrdU with Som, the
transplants made from NsCre:ShhFl/Fl brains showed little
co-labeling (Fig. 5E;
17.5±4.5% versus 2.4±0.4%, P<0.03).
If Shh signaling maintains Nkx2.1 expression within MGE progenitors, the addition of exogenous Shh to slices from NsCre:ShhFl/Fl mutants should restore Nkx2.1 expression in S-phase cells. Thus, slices from E13.5 NsCre:ShhFl/Fl embryos were cultured for 24 hours in the presence or absence of exogenous Shh (10 nM). BrdU was again added six hours prior to fixation. The mutant slices were then re-sectioned at 12 µm, and immunolabeled for BrdU and Nkx2.1. As shown in Fig. 5C, Shh-treatment of NsCre:ShhFl/Fl slices does not alter the density of MGE cells in S-phase (BrdU/DAPI). By contrast, this treatment results in a nearly threefold increase in the percentage of BrdU+ cells expressing detectable levels of Nkx2.1 (Fig. 5A-C; 67.0% versus 24.6%; P<0.008).
Because Shh-treatment of NsCre:ShhFl/Fl slices rescues Nkx2.1 expression, this treatment might also rescue the reduction of interneurons generated from NsCre:ShhFl/Fl MGE transplants. Slices were treated as above and then cultured on cortical feeder cells for 10 days. BrdU+ cells from mutant slices treated with Shh showed a 4-fold increase in the percentage of double labeling for Som (Fig. 5; 2.4±0.4% versus 9.6±1.3%, P<0.03, n=3).
|
To examine the differential effects on excitatory versus inhibitory
neurons, the density of markers for projection neurons [Tbr1
(Hevner et al., 2001;
Englund et al., 2005
)] or
interneurons (GABA) were determined in both superficial (layers 2-4) and deep
(layers 5 and 6) cortical layers and expressed as a percentage of NeuN
(Neuna60 Mouse Genome Informatics)-expressing profiles. In both
wild-type and NsCre:ShhFl/Fl mice, about 70% of all cells are
neurons at P12. As shown in Fig.
6, NsCre:ShhFl/Fl mice show a significant increase in
the percentage of Tbr1+ profiles in the superficial cortex when
compared with wild type (n=3; P<0.009). This increase is
accompanied by a decrease in the percentage of GABA+ profiles
(n=3; P<0.003), although, overall, a lower than expected
number of profiles labeled for GABA in both wild-type and mutant brains,
probably due to the absence of glutaraldehyde in the fixative. In the deep
layers of the cortex, a similar decrease in the percentage of GABA+
profiles is observed (n=3; P<0.009), although the small
increase in Tbr1 expression in the deep cortical layers is not statistically
significant.
The observed decrease in GABA-expressing profiles in the postnatal cortex
of NsCre:ShhFl/Fl mice raises the question of whether specific
interneuron subgroups are preferentially affected by reduced Shh signaling.
The MGE has previously been shown to generate at least two neurochemically
distinct subgroups of cortical interneurons those expressing Som and
those expressing Pv (Wichterle et al.,
2001; Anderson et al.,
2002
; Valcanis and Tan,
2003
). Nkx2.1 expression within the MGE is required for the
specification of these subgroups (Xu et
al., 2004
). To determine whether these subgroups are reduced in
the cortex of NsCre:ShhFl/Fl mice, we counted profiles of Pv- and
Som-expressing cells in somatosensory cortex at P12. Indeed, there is a
dramatic reduction of Som-expressing profiles, particularly in the superficial
cortex (Fig. 6E,E',I).
Pv-expressing profiles are also greatly reduced
(Fig. 6D,D',I), although
detection of this effect in superficial cortex is probably compromised by the
protracted differentiation of this subgroup into the third postnatal week
(Alcantara et al., 1996
). Npy,
also expressed in a Nkx2.1-dependent subgroup of cortical interneurons
(Anderson et al., 2002
;
Xu et al., 2004
), is also
reduced in the superficial cortical layers
(Fig. 6F,F',I). Similar
interneuron deficits are present in the somatosensory cortex of
NsCre:SmoothenedFl/Fl mutants (see Fig. S4 in the supplementary
material). Consistent with evidence that GABAergic interneurons of the cortex
and striatum share common progenitors
(Marin et al., 2000
;
Reid and Walsh, 2002
), Som-
and Npy-expressing interneurons are also reduced in the striatum of
NsCre:ShhFl/Fl mice (see Fig. S5 in the supplementary
material).
|
|
To determine whether Shh signaling has a cell-autonomous effect on
postmitotic interneuron development, Dlx5/6Cre:SmoFl/Fl mice were
generated. In marked contrast to the NsCre:ShhFl/Fl mutants, and to
the NsCre:SmoFl/ mutants
(Machold et al., 2003) (see
also Fig. S4 in the supplementary material), Dlx5/6Cre:SmoFl/Fl
mice are viable with no gross phenotype. Cortical interneuron profiles labeled
with GABA, Som, Pv or Npy showed no significant change when compared with
controls in layers 2-4 (Fig.
7G), although there was a trend towards a decrease of GABA and Pv
(n=3, P<0.14 for either marker, not corrected for
multiple comparisons). Fewer differences between mutants and controls in the
percentage of profiles expressing GABA or Pv were found in the deeper cortical
layers (not shown; n=3, P<0.18 for either marker, not
corrected for multiple comparisons). These results suggest that
cell-autonomous Shh signaling is not crucial for the migration,
differentiation or survival of most MGE-derived cortical interneurons.
Interestingly, grossly normal expression of Nkx2.1 in the striatum of
Dlx5/6Cre:SmoFl/Fl mutants at E16.5 and P25 suggest that factors
other than Shh mediate Nkx2.1 expression in postmitotic interneurons (see Fig.
S7 in the supplementary material).
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Discussion |
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Shh signaling maintains Nkx2.1 expression in interneuron progenitors of the MGE
Loss of Shh signaling in the telencephalon of NsCre:ShhFl/Fl
mutants occurs by E12.5 (Fig.
2) (Machold et al.,
2003), after initial patterning of the telencephalon has been
established (Fuccillo et al.,
2004
). Indeed, in these mutants, general aspects of ventral
telencephalon identity are preserved (Fig.
2) (see also Machold et al.,
2003
), including the LGE-MGE boundary (based on the expression of
Olig2 in the VZ, and Oct6 in the SVZ and mantle zone;
Fig. 2). Conversely, the
NsCre:ShhFl/Fl embryos also show reduced Nkx2.1 expression that is
most pronounced in the dorsal MGE, where there is also a loss of
Nkx6.2 expression (Figs
1,
2).
|
Also, similar to findings in the spinal cord
(Ericson et al., 1996), our
results suggest that Shh signaling during the final cell cycle determines both
the expression of Nkx2.1 and the interneuron fate of MGE progenitors. There
was a striking loss of Som-expression in cultured cells that entered S-phase
during the last 6 hours of treatment with cyclopamine
(Fig. 3). However, the fate of
MGE progenitors that no longer express Nkx2.1 remains to be determined.
Consistent with the continued expression of ventral markers in these mutants
(Fig. 2)
(Machold et al., 2003
),
cyclopamine-treated, BrdU-labeled cells continue to express GABA
(Fig. 3). In the light of
evidence that the LGE-MGE boundary is intact
(Fig. 2), these cells may be
partially-specified interneurons that fail to express the neurochemical
subgroup marker but that retain other characteristics of these cells.
Three actions of Shh on embryonic telencephalon development
The above results suggest the presence of three distinct actions of Shh
signaling on embryonic development of the telencephalon, with the bulk of
evidence suggesting that the main role is played by Shh
(Fig. 8). First, between E9 and
E12.5, Shh, acting mainly by inhibiting the formation of the Gli3 repressor
(Rallu et al., 2002),
contributes to the establishment of dorsoventral patterning
(Chiang et al., 1996
;
Fuccillo et al., 2004
).
Second, Shh signaling also supports the expansion of progenitors of both the
dorsal and ventral telencephalon (Dahmane
et al., 2001
; Britto et al.,
2002
; Machold et al.,
2003
). Third, Shh signaling maintains the expression of Nkx2.1 and
possibly also Nkx6.2. Based on the rescue of Nkx2.1/BrdU co-labeled
cells in slices from NsCre:ShhFl/Fl mutants cultured from E13.5 + 1
DIV (Fig. 5), this period
extends at least until E14, well into the period of neuronogenesis.
Alterations in Shh-responsive gene expression correlate with alterations in neuronal fate in vivo
We have previously shown that expression of Nkx2.1 in the MGE is required
for the specification of several neurochemically defined subgroups of cortical
interneurons that originate there (Xu et
al., 2004). Thus, the reduction in Nkx2.1-expressing progenitors
in the MGE would be expected to result in reductions of Pv, Som and
Npy-expressing interneurons in the postnatal cortex of the
NsCre:ShhFl/Fl mutants. Indeed, losses in each of these subgroups
have been found, with the strongest effect on the superficial cortical layers
(Fig. 6). Interneurons destined
for the superficial cortical layers are later born, and thus are most likely
to be affected in these mutants (Miller,
1985
; Fairén et al.,
1986
; Cavanagh and Parnavelas,
1988
). The correlation between the reduction in the proportion of
all neurons (NeuN expressing) that express GABA, and the increased proportion
of neurons that co-label for the projection neuron marker Tbr1, further
supports the idea that NsCre:ShhFl/Fl mutants have an actual
decrease in cortical interneurons, as predicted by the loss of
Nkx2.1-expressing progenitors in the MGE, rather than a reduction in the
expression of the interneuron markers.
To what extent is this decrease attributable to the loss of Nkx2.1 expression in the embryonic MGE? As shown in Fig. 7, Shh expression in the cortex at P0 is also lost in the NsCre:ShhFl/Fl mutants, raising the possibility that in addition to the effects of Shh loss on interneuron specification in the MGE, the NsCre:ShhFl/Fl cortical interneuron phenotype may reflect later effects on interneuron migration, survival and differentiation. However, several lines of evidence do not support this interpretation. First, by E16.5, calbindin immunolabeling already reveals a profound reduction of migrating interneurons in the SVZ/lower intermediate zone stream of NsCre:ShhFl/Fl mutants (Fig. 7), suggesting that the interneuron phenotype in these mutants is established during the age range of neurogenesis (E12.5-E16.5). Second, postnatal analysis of mice in which the Shh receptor smoothened has been inactivated in postmitotic interneurons by Dlx5/6Cre reveals normal densities of Som- and Npy-expressing interneuron profiles, suggesting that cell autonomous Shh signaling is not required for the migration, differentiation or survival of these subgroups.
In summary, during embryonic neuronogenesis, Shh signaling appears to
maintain Nkx2.1 expression, and thereby interneuron fate determination, in MGE
progenitors. These findings suggest that prenatal alterations in Shh signaling
could disrupt the generation of cortical interneurons without causing a more
gross disruption of telencephalic patterning. Such disruptions could result in
the subtle alterations in interneurons that are implicated in a variety of
cortical illnesses. Indeed, although loss-of-function mutations in SHH in
humans can result in severe holoprosencephalies, more subtle mutations or
variable penetrance can result in a spectrum of disorders that includes
abnormalities of learning and attention, and seizures
(Muenke and Beachy, 2000;
Heussler et al., 2002
;
Lazaro et al., 2004
;
Nieuwenhuis and Hui,
2005
).
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/22/4987/DC1
* These authors contributed equally to this work
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