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First published online November 7, 2008
doi: 10.1242/10.1242/dev.025080
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1 Max-Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01307 Dresden, Germany.
2 Cold Spring Harbor Laboratory, Watson School of Biological Sciences and Howard
Hughes Medical Institute, Cold Spring Harbor, NY 11724, USA.
Authors for correspondence (e-mails:
davide.depietri{at}iit.it;
huttner{at}mpi-cbg.de)
Accepted 6 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Dicer (Dicer 1) knockout, MicroRNAs, Neurogenesis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Caviness et al., 1995;
Molnar et al., 2006;
Rakic, 1995;
Rakic, 2007). The concomitant
increase in neuron number is, in essence, due to an increase in neural
progenitors that undergo neurogenic divisions
(Götz and Huttner, 2005;
Kriegstein et al., 2006).
There are two principal classes of neural progenitors that generate the
neurons of the mammalian cerebral cortex: (1) the progenitors dividing at the
ventricular (apical) surface of the ventricular zone (VZ) (neuroepithelial
cells, radial glia and short neural precursors, collectively referred to as
apical progenitors); and (2) the progenitors that divide in the basal region
of the VZ and in the subventricular zone (SVZ) (referred to as basal
progenitors, also called intermediate, non-surface or SVZ progenitors)
(Götz and Huttner, 2005;
Kriegstein et al., 2006).
These distinct neural progenitors can divide to generate either progenitors,
neurons, or both. The molecular machinery that regulates the balance between
apical and basal progenitors, and between their neurogenic and non-neurogenic
divisions, is largely unknown.
MicroRNAs (miRNAs) are a class of small RNAs that bind to specific mRNA
targets, directing their degradation and/or repressing their translation
(Hannon et al., 2006;
Stefani and Slack, 2008).
Approximately 70% of known miRNAs are expressed in the mammalian brain
(Cao et al., 2006), and the
level of many miRNAs changes dramatically during brain development
(Krichevsky et al., 2003;
Miska et al., 2004;
Sempere et al., 2004). Indeed,
based on observations obtained with cell culture models in vitro, miRNAs have
been implicated in the control of neuronal differentiation
(Conaco et al., 2006;
Krichevsky et al., 2006;
Makeyev et al., 2007;
Smirnova et al., 2005;
Wu and Belasco, 2005). Many of
these investigations have focused on the in vitro role of miR-124,
one of the most abundant miRNAs in the brain, which is highly enriched in
neurons (De Pietri Tonelli et al.,
2006; Hohjoh and Fukushima,
2007; Lagos-Quintana et al.,
2002). These studies have revealed an important role of miRNAs in
the differentiation of postmitotic neurons in vitro.
To explore a possible role of miRNAs in neuronal differentiation during the
development of the mammalian nervous system in vivo, recent studies have
investigated the consequences of the genetic ablation of Dicer (Dicer1 - Mouse
Genome Informatics), one of the essential enzymes for the production of
endogenous small interfering RNAs (siRNAs)
(Watanabe et al., 2008) and
for miRNA maturation (Bernstein et al.,
2001; Hutvagner et al.,
2001). Dicer ablation in various specific subpopulations of
neurons has been found to impair neuronal differentiation and cause
neurodegeneration and neuronal cell death
(Cuellar et al., 2008;
Davis et al., 2008;
Kim et al., 2007;
Schaefer et al., 2007).
Although the most recent of these reports
[Cuellar et al., 2008;
Davis et al., 2008 (which
appeared while the present study was being prepared for publication)] includes
the analysis of neurons in the postnatal cerebral cortex, it has remained an
open issue to what extent miRNAs are essential for the early steps of neuronal
differentiation that occur during embryonic development of the neocortex.
Moreover, although Dicer has been ablated in neural progenitors
(Choi et al., 2008;
Makeyev et al., 2007), the
role of miRNAs in the progenitors that generate the neurons of the neocortex
is largely unexplored, and an analysis of miRNA-dependent functions in apical
versus basal progenitors and their neurogenic versus non-neurogenic divisions
is lacking. Here, we have ablated Dicer in the primary neural progenitors of
the neocortex, i.e. in the neuroepithelial cells of the dorsal telencephalon,
and have dissected the consequences of the resulting miRNA depletion for
apical and basal progenitor proliferation and differentiation, for
neurogenesis, and for neuronal differentiation and survival.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Emx1Cre/wt knock-in mice
(Iwasato et al., 2000) were
crossed with Z/EG mice (Novak et al.,
2000) to obtain Z/EG+ Emx1Cre/wt
for the analysis of Cre recombinase expression.
Dicerflox/wt mice
(Murchison et al., 2005) were
crossed with Emx1Cre/wt knock-in mice to obtain
Dicerflox/wt Emx1Cre/wt mice, and the
conditional ablation of Dicer was performed by crossing the latter with
Dicerflox/flox mice. The resulting
Dicerflox/wt Emx1Cre/wt (control) and
Dicerflox/flox Emx1Cre/wt (conditional
Dicer knockout) littermate mice were analyzed. For Tis21-GFP
analysis, Dicerflox/flox mice were first crossed with
Tis21-GFP knock-in mice
(Haubensak et al., 2004) to
obtain Dicerflox/flox Tis21-GFP+/+, which were
then crossed with Dicerflox/wt Emx1Cre/wt.
Genotyping of mice carrying the various Z/EG, Emx1Cre,
Dicerflox and Tis21-GFP alleles was performed by PCR
following published protocols (Andl et al.,
2006; Haubensak et al.,
2004; Iwasato et al.,
2000; Novak et al.,
2000).
BrdU labeling
Cumulative BrdU labeling was carried out by repeated intraperitoneal
injections, performed at 3-hour intervals, of pregnant females 12.5 days
post-coitum (dpc) (average mouse weight, 22-24 g), using either 1 mg (4-hour
time point), 800 µg (8-hour time point) or 600 µg (20-hour time point)
of BrdU (Sigma-Aldrich) in PBS per injection. Mice were sacrificed 1 hour
(4-hour time point) or 2 hours (8- and 20-hour time points) after the last
BrdU injection, and embryos processed for analysis of BrdU-labeled nuclei in
the dorsal telencephalon as described below.
For birthdating experiments, pregnant females were injected twice with 1 mg BrdU in PBS, either at 12.5 and 13.5 dpc, or at 17.5 and 18.5 dpc, and pups collected at P1 and processed for analysis of BrdU-labeled nuclei in the cortex as described below.
Immunohistochemistry and fluorescence microscopy
Immunohistochemistry and fluorescence microscopy on cryosections (10 µm)
and Vibratome sections (50-70 µm) of paraformaldehyde-fixed E10.5-16.5 and
P0-21 mouse brains were performed according to standard methods
(Kosodo et al., 2004).
Sections were incubated with primary antibodies (see below) in blocking
solution for 2 hours at room temperature (cryosections) or for 24 hours at
4°C (Vibratome sections), incubated with appropriate Cy2-, Cy3- or
Cy5-conjugated secondary antibodies (Jackson) in blocking solution containing
1 µg/ml DAPI (Molecular Probes) at room temperature for 1 hour
(cryosections) or 2 hours (Vibratome sections), and mounted in Mowiol 4-88
(Calbiochem).
The following primary antibodies were used. Chemicon: rabbit polyclonal anti-Tbr1 and anti-BLBP, 1:500; guinea pig polyclonal anti-GLAST, 1:500. Abcam: rat monoclonal clone HTA28 anti-phosphorylated histone H3, 1:200; rabbit polyclonal anti-Foxp2, 1:500; rabbit polyclonal anti-Tbr2, 1:1000. Covance: rabbit polyclonal anti-Pax6, 1:500; mouse monoclonal anti-βIII-tubulin, 1:1000. Sigma: rabbit polyclonal anti-activated caspase 3, 1:500; mouse monoclonal anti-βIII-tubulin, 1:300. Santa Cruz: goat polyclonal anti-Brn1, 1:200. Developmental Studies Hybridoma Bank, University of Iowa, USA: mouse monoclonal clone 2H3 anti-neurofilament, 165 kDa form, 1:500. Swant: rabbit polyclonal anti-calretinin, 1:500.
For the detection of Pax6, Tbr2, Tbr1 and Brn1, cryosections prior to
permeabilization were heated in 0.01 M sodium citrate at pH 6.0 in a standard
microwave oven for 1 minute at 800 W, then for 10 minutes at 80 W, and allowed
to cool to room temperature. BrdU detection was performed as described
previously (Calegari et al.,
2005). TUNEL staining was performed following the manufacturer's
instructions (Roche). Nissl staining of P21 cerebral cortex Vibratome sections
was performed with Cresyl-Violet acetate (Sigma) following standard protocols
(Banny and Clark, 1950;
Brinks et al., 2004).
Sections were analyzed by conventional (Olympus BX61) or confocal (Zeiss Axiovert 200M LSM 510) fluorescence microscopy. EGFP (Z/EG mice) and Tis21-GFP were detected via their intrinsic fluorescence. Images were recorded digitally and processed using Adobe Photoshop software.
Detection of miRNAs by in situ hybridization
In situ hybridization on cryosections was performed using
3'-digoxigenin-labeled LNA antisense probes (Exiqon) to mouse
miR-124 and miR-9, as previously described
(De Pietri Tonelli et al.,
2006).
Quantification and statistical analyses
Quantifications concerning the embryonic dorsal telencephalon were carried
out in its lateral region, as indicated by the dashed white box in
Fig. 1A, part a.
Quantifications concerning the postnatal cerebral cortex were carried out in
the region described in the Fig.
1 legend. Comparisons between control and Dicer-ablated animals
involved littermate embryos and pups; considering the size differences between
control and Dicer-ablated embryonic dorsal telencephalon and postnatal
cerebral cortex that emerged during development, care was taken that
corresponding regions were analyzed and that the fields compared covered the
same amount of ventricular surface. Morphological measurements were performed
with ImageJ version 1.33u (Wayne Rasband, National Institutes of Health,
USA).
Analysis of embryonic dorsal telencephalon
For the determination of progenitor and neuronal layer thickness, the area
of the neuronal layer as revealed by Tbr1 immunostaining, and that of the
entire cortical wall as revealed by Tbr1 immunostaining and DAPI staining,
were determined. The progenitor layer (VZ and SVZ) area was then calculated by
subtracting the neuronal layer area from the entire cortical wall area. The
surface of the lateral ventricle was determined in DAPI-stained sections by
measuring the distance from the pallial-subpallial boundary (where the
ganglionic eminence starts) to the dorsal-most point of the telencephalon (see
arrowheads in Fig. 2F). Tbr2-
and Tis21-GFP-positive nuclei in the VZ and SVZ were counted in
fields, obtained with a 40x objective, the right-hand edge of which was
near the pallial-subpallial boundary, and their numbers expressed as a
proportion of total nuclei as revealed by DAPI staining. In similar fields,
phosphohistone H3-positive cells at the ventricular surface (mitotic apical
progenitors) and in the basal region of the VZ and in the SVZ (mitotic basal
progenitors) were counted and their numbers expressed per field (i.e. per
equal length of ventricular surface). TUNEL-positive cells were counted across
the entire cortical wall and their numbers expressed per field.
Analysis of postnatal cerebral cortex
DAPI-, BrdU-, Tbr1- and Brn1-stained nuclei were counted across the entire
cortical wall, except for the VZ. Numbers of Tbr1- and Brn1-positive nuclei
were expressed per DAPI-stained nuclei. Numbers of Tbr1-BrdU and Brn1-BrdU
double-positive nuclei, as well as nuclei stained only for BrdU, were
expressed per field.
Cell cycle parameters were calculated from cumulative BrdU labeling data as
previously described (Calegari et al.,
2005). Statistical analysis was performed with Excel (Microsoft)
using Student's t-test.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), in
which Cre recombinase under the control of the Emx1 promoter is
specifically expressed in the dorsal telencephalon starting at embryonic day
9.5 (E9.5) (Simeone et al.,
1992), with a mouse line carrying a conditional allele for
Dicer (Dicerflox), in which the essential exons
22 and 23, encoding the majority of the two ribonuclease III (RNase III)
domains, are flanked by loxP sites
(Murchison et al., 2005). To
confirm the spatial specificity of Emx1-Cre-mediated recombination,
we crossed the Emx1-Cre mice with the Z/EG reporter line
(Novak et al., 2000) to reveal
loxP recombination via EGFP expression. Indeed, in the developing brain at
E13.5, EGFP was specifically expressed in the dorsal telencephalon
(Fig. 1Aa, compare EGFP with
DAPI staining) in both the primary target cells of Emx1-Cre-mediated
recombination, the neuroepithelial cells of the VZ, and in the neurons derived
from them (Fig. 1Ab). This
indicated that Cre expression and, consequently, recombination, had
selectively occurred in this region of the brain.
|
). As
controls in these and all subsequent experiments, we used conditional
heterozygous mice bearing a single Dicer copy
(Emx1Cre/wt Dicerflox/wt) in the dorsal
telencephalon rather than two (Emx1wt/wt
Dicerflox/wt or Emx1wt/wt
Dicerflox/flox), as we did not observe any obvious difference
between single- and double-copy Dicer mice with regard to the initial
parameters examined (viability, fertility, body weight, brain size), nor any
obvious phenotype in the single-copy Dicer mice with regard to
cortical development in any of the parameters analyzed.
Analysis of the E10.5-14.5 control telencephalon revealed the presence of
mature miR-9 throughout the cortical wall
(Fig. 1B,C; data not shown),
with the highest level in the VZ (Fig.
1C), and the presence of mature miR-124 almost
exclusively in the preplate and cortical plate
(Fig. 1D,E), as previously
reported (De Pietri Tonelli et al.,
2006). Consistent with the Emx1-driven Cre-mediated
recombination occurring specifically in the dorsal telencephalon (see
Fig. 1A), we did observe normal
levels of mature miR-9 and miR-124 in other brain regions,
such as the mesencephalon, in both control and Dicer knockout
littermate embryos at all stages analyzed (see
Fig. 1C,E for representative
examples). By contrast, upon Dicer ablation in the dorsal telencephalon, these
two miRNAs were barely, if at all, detectable from E10.5 onwards
(Fig. 1B-E). As miR-9
and miR-124 are amongst the most abundant miRNAs in the mouse brain
(Hohjoh and Fukushima, 2007;
Lagos-Quintana et al., 2002),
our observations indicate that the conditional Dicer ablation in the dorsal
telencephalon resulted in the depletion of mature miRNAs from E10.5 onwards
(although persistence of low levels of some miRNAs cannot be completely
excluded). Importantly, the Emx1-driven Cre-mediated ablation in
neuroepithelial cells (Simeone et al.,
1992) resulted in the depletion of mature miRNAs in both the
neural progenitors themselves (as evidenced by the depletion of mature
miR-9 in the VZ) and in the neurons derived from these progenitors
(as evidenced by the depletion of mature miR-124 in the preplate and
cortical plate).
To investigate the consequences of Dicer ablation during neocortical
development, we first examined the postnatal cortex. At postnatal day 0 (P0),
the size of the Dicer-ablated hemispheres and olfactory bulbs was clearly
reduced compared with control littermate brains
(Fig. 1F, top). DAPI staining
of cryosections revealed, consistent with previous observations
(Makeyev et al., 2007), a
major reduction in the radial thickness and lateral expansion of the cortex,
with the former being more evident rostrally
(Fig. 1F') than caudally
(Fig. 1F'') [which perhaps
reflects the gradient of Emx1 expression
(Simeone et al., 1992)]. In
addition, Dicer ablation resulted in a smaller, massively disorganized
hippocampus (Fig. 1F'').
Consistent with the specific pattern of Emx1-driven Cre-mediated
recombination (see Fig. 1A),
the size of the midbrain was unaffected
(Fig. 1F'').
At P21, the Dicer-ablated cortex was dramatically hypotrophic (see Fig. S1
in the supplementary material). Mice with Dicer-ablated cortex were viable
until weaning (P21) (although their postnatal growth was markedly reduced; see
Fig. S2 in the supplementary material), but died shortly thereafter
(
P24-25), presumably owing to starvation and dehydration.
miRNAs are required for the proper formation of neuronal layers but not for the early development of progenitor layers
Following these observations with the postnatal cortex, we investigated the
dorsal telencephalon of conditional Dicer knockout mice during
embryonic development, distinguishing between effects on progenitors and
neurons. In order to detect neurons, we performed immunofluorescence for
βIII-tubulin (Tuj1; Tubb3) at E12.5, E13.5 and E14.5. At E12.5, we did
not observe any obvious difference between control and conditional
Dicer knockout embryos with respect to the thickness of the neuronal
(Fig. 2A, arrowheads) or
progenitor (Fig. 2A) layers as
revealed by DAPI staining. By contrast, at E13.5, the Dicer-ablated cortex
showed a reduced thickness of the neuronal layers
(Fig. 2B, arrowheads). This
phenotype was even more pronounced at E14.5, when neither a cortical plate, a
subplate, nor an intermediate zone was distinguishable
(Fig. 2C) (because of this
disturbed cortical architecture, we use the term `neuronal layers' for all
layers basal to the SVZ, i.e. layers containing migrating as well as resident
neurons). Remarkably, the massive reduction in the thickness of the neuronal
layers at E13.5 and E14.5 upon Dicer ablation was not matched by a
corresponding decrease in the progenitor layers, i.e. the VZ and SVZ
(Fig. 2B,C).
To quantify these observations, we immunostained the E13.5 dorsal
telencephalon for the neuron-specific transcription factor Tbr1
(Hevner et al., 2001) in order
to distinguish neuronal and progenitor layers from each other and to judge the
effects on neuron number versus neuronal cell volume
(Fig. 2D). The thickness of the
neuronal layers was found to be reduced by one-third upon Dicer ablation
(Fig. 2E, white column
segments), without any obvious change in nuclear density
(Fig. 2D), indicating that
neuron number rather than neuronal cell volume was decreased by the depletion
of mature miRNAs. Consistent with the results of DAPI staining and
βIII-tubulin immunofluorescence (Fig.
2B), there was no significant reduction in the thickness of the
progenitor layers (Fig. 2E,
black column segments).
We next compared the lateral extension of the E13.5 control and Dicer-ablated cortex. The length of the ventricular surface of the dorsolateral telencephalon was determined in consecutive DAPI-stained coronal Vibratome sections (Fig. 2F, arrowheads) and summed for an equal number of sections (12) along the rostrocaudal axis, which covered virtually all of the dorsal telencephalon for either condition. This showed that the ventricular extension of the Dicer-ablated cortex was the same as in the control.
The reduction, upon Dicer ablation, in neuron number and, consequently, in the radial thickness of the neuronal layers observed at E13.5 and E14.5 (Fig. 2) could be due to either (1) a decrease in the number of the neurons generated, (2) a loss of neurons by cell death, or (3) both. We therefore concentrated next on the process of neuron generation from progenitors.
The reduced thickness of the neuronal layer in miRNA-depleted E13.5 cortex is not due to a decrease in progenitor numbers or to alterations in progenitor lineage
Given the lack of effect on progenitor layer thickness and overall
progenitor number as revealed by DAPI staining
(Fig. 2), we first investigated
whether Dicer ablation affected the proportion of neurogenic progenitors
relative to the entire progenitor population. There are two principal classes
of neural progenitors in the telencephalon: (1) the progenitors that divide at
the ventricular (apical) surface of the VZ (neuroepithelial cells, radial glia
and short neural precursors, collectively referred to as apical progenitors);
and (2) the progenitors that divide in the basal region of the VZ and in the
SVZ (referred to as basal progenitors, also called intermediate progenitors),
which originate from apical progenitors
(Götz and Huttner, 2005;
Kriegstein et al., 2006). At
E13.5, the proportion of apical progenitors that are neurogenic is relatively
small (15%), whereas the overwhelming majority of basal progenitors are
neurogenic (Haubensak et al.,
2004).
Apical progenitors (including the neurogenic subpopulation) can be
identified by the expression of the transcription factor Pax6
(Götz and Barde, 2005),
and basal progenitors by the transcription factor Tbr2 (Eomes - Mouse Genome
Informatics) (Englund et al.,
2005). Pax6 immunostaining of the E13.5 dorsal telencephalon
revealed that the radial thickness of the VZ and its nuclear density were
unaffected by Dicer ablation (Fig.
3A-D). Similarly, the distribution of basal progenitors between VZ
and SVZ, as revealed by Tbr2 immunostaining
(Fig. 3E,F), and their
proportion relative to the total progenitor population
(Fig. 3I), were unaltered.
To specifically study the neurogenic subpopulations of apical and basal
progenitors, we made use of the previously described Tis21-GFP
(Tis21 is also known as Btg2 - Mouse Genome Informatics)
knock-in mouse line in which these subpopulations can be identified by their
nuclear GFP fluorescence (Haubensak et
al., 2004). Comparison of control and Dicer-ablated mouse embryos
carrying a Tis21-GFP knock-in allele revealed that the abundance of
Tis21-GFP-positive (i.e. neurogenic) progenitors in the VZ and SVZ of
the E13.5 dorsal telencephalon was unaltered
(Fig. 3G,H,J). Taken together,
these observations imply that the reduction in neuronal layer thickness at
E13.5 that results from Dicer ablation in cortical progenitors was not due to
a decrease in progenitor numbers, or to their switch to the neurogenic
lineage.
|
To directly investigate cell cycle progression of VZ progenitors, we
carried out cumulative BrdU labeling of control and Dicer-ablated E12.5
embryos in utero and quantitated the proportion of BrdU-positive nuclei in the
VZ of the dorsal telencephalon after 4, 8 and 20 hours
(Fig. 4J-L). For the control
(Fig. 4L, black triangles),
this confirmed previously reported
(Calegari et al., 2005) cell
cycle parameters, such as the proportion of cycling cells in the VZ (growth
fraction), their cell cycle length, and the proportion of cells in S phase. In
VZ progenitors of Dicer-ablated dorsal telencephalon, these cell cycle
parameters were found to be very similar
(Fig. 4L, white diamonds). The
slightly greater proportion of BrdU-positive cells in the Dicer-ablated VZ at
the three time points analyzed might not necessarily indicate a real
difference to the control, but could reflect an overestimation owing, for
example, to an increased presence in the VZ of neurons that were born from
BrdU-positive progenitors (but did not yet express Tbr1, see
Fig. 4J,K) and migrated more
slowly from the VZ than in the control. Taken together, the results shown in
Fig. 4A-F,J-L indicate that the
cell cycle progression and division of apical and basal progenitors were
unaffected by Dicer ablation until E13.5.
At E14.5 miRNA depletion decreases the abundance of mitotic apical and basal progenitors
In contrast to E12.5 and E13.5 (Fig.
4A-F), the E14.5 dorsal telencephalon showed the first clear-cut
signs of Dicer ablation at the level of progenitors. Specifically, as revealed
by phosphohistone H3 immunostaining, the abundance of mitotic apical
(Fig. 4G-I, white arrows) and
basal (Fig. 4G-I, arrowheads)
progenitors was significantly reduced by miRNA depletion. Concomitant with
this reduction, we noticed an increased appearance of pycnotic nuclei in the
VZ by DAPI staining (Fig. 4H,
yellow arrows). This suggested that the reduction in mitotic progenitors was
due to cell death. Interestingly, mitotic basal progenitors
(Fig. 4, right-hand columns)
were reduced to a greater extent than mitotic apical progenitors
(Fig. 4, left columns),
indicating that the former were more sensitive to the depletion of miRNAs.
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The massive apoptosis in the Dicer-ablated VZ and SVZ at E14.5 (Fig. 5H,J) suggested that at this stage not only newborn neurons, but also progenitors, underwent apoptosis, consistent with the observed reduction in mitotic apical and basal progenitors (see Fig. 4H,I). In support of this, in the E16.5 Dicer-ablated dorsal telencephalon, we observed a dramatic reduction in the radial thickness of the progenitor layers, in BrdU incorporation, and in the expression of the radial glia markers BLBP and GLAST (Fabp7 and Slc1a3, respectively - Mouse Genome Informatics) (data not shown). Thus, concomitant with the progression of neurogenesis, neural progenitors become increasingly sensitive to miRNA depletion.
Decreased generation of upper layer neurons and lack of cortical layering in the miRNA-depleted P1 cortex
Given the loss of progenitors from E14.5 onwards, we investigated the
effects of Dicer ablation on the generation of early-born neurons versus
late-born neurons. For this purpose, we analyzed the P1 cortex, specifically
its caudal region, in which the reduction in overall radial thickness due to
Dicer ablation is less pronounced than rostrally (see
Fig. 1, F' versus
F''). During mouse cortical development, early-born neurons
(E12.5-13.5) form the deep cortical layers, notably layer VI, which contains
strongly Tbr1-positive neurons (Hevner et
al., 2001). Later-born neurons (generated between E14.5 and E18.5)
accumulate in an inside-out manner above the deep layers, forming the upper
layers, notably layers III and II, which contain neurons expressing the
transcription factor Brn1 (Pou3f3 - Mouse Genome Informatics)
(He et al., 1989). To relate
the expression of the Tbr1 and Brn1 markers to the birthdate of neurons, we
labeled control and Dicer-ablated embryos in utero with BrdU either at E12.5
and E13.5, when predominantly Tbr1-positive neurons and few Brn1-positive
neurons are being generated (Molyneaux et
al., 2007) (Fig.
6A), or at E17.5 and E18.5, when the last upper layer
Brn1-positive neurons but hardly any deep layer Tbr1-positive neurons are
being generated (Molyneaux et al.,
2007) (Fig. 6D). We
then analyzed the abundance and localization of the total population, as well
the BrdU-labeled subpopulation, of Tbr1-positive and Brn1-positive neurons in
the caudal region of the P1 cortex. This analysis yielded four major
differences between the control and Dicer-ablated cortex.
|
4-fold) reduced and,
correspondingly, that of BrdU-labeled nuclei containing neither Tbr1 nor Brn1
massively increased (Fig. 6I).
Fourth, when BrdU labeling had been carried out at E17.5 and E18.5
(Fig. 6D-F), the total number
of BrdU-labeled nuclei in the Dicer-ablated P1 cortex was markedly reduced (by
60%), which was largely due to a decrease in Brn1-positive BrdU-labeled nuclei
(Fig. 6J).
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miRNAs are required for proper neuronal differentiation in vivo
The deficient or altered neuronal subtype specification at the early stage
of cortical neurogenesis (Fig.
6I), and the defective cortical layering as indicated by the
intermixing of the few remaining Brn1-positive neurons with the Tbr1-positive
neurons observed at P1 (Fig.
6C,F), raised the possibility that miRNA depletion not only
decreased upper layer neuron production quantitatively, but also affected
certain aspects of neuronal differentiation. The transcription factor Foxp2,
which like Tbr1 is expressed by layer VI neurons, has been implicated in
postmigratory neuronal differentiation
(Ferland et al., 2003). We
therefore examined Foxp2 expression in the control and Dicer-ablated P0 and P7
cortex. In contrast to Tbr1, which was abundantly expressed in the P1 cortex
(Fig. 6), Dicer ablation almost
completely abolished Foxp2 expression in the postnatal cortex
(Fig. 7). This implies that
miRNAs are required for proper neuronal differentiation.
Lack of interneurons and defective cortical connections in the miRNA-depleted P7 cortex
The increase in BrdU-labeled nuclei containing neither Tbr1 nor Brn1
(Fig. 6I), besides suggesting
deficient or altered neuronal subtype specification, could also reflect an
increased entry into the cortex of interneurons that were generated in
germinal zones not affected by Dicer ablation (i.e. outside of the
Emx1 domain) (Wonders and
Anderson, 2006). We investigated this issue by immunostaining the
control and Dicer-ablated P7 cortex for calretinin (calbindin 2), a marker for
a subset of cortical interneurons (Wonders
and Anderson, 2006). This revealed a dramatic decrease, rather
than an increase, in these interneurons in the Dicer-ablated cortex (see Fig.
S3 in the supplementary material). Moreover, the few calretinin-positive
interneurons observed were scattered throughout the cortical wall, and their
neurites appeared to be poorly developed.
To obtain more-representative information about the formation of cortical
connections, control and Dicer-ablated P7 cortex was immunostained for
neurofilament protein. The miRNA-deficient cortex showed a massive reduction
in, and a disorganized architecture of, cortical connections (see Fig. S3 in
the supplementary material). In line with the lack of Foxp2 expression
(Fig. 7), this provided further
evidence that key aspects of neuronal differentiation were greatly perturbed
in the miRNA-depleted cortex (Molyneaux et
al., 2007; Price et al.,
2006).
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|
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), and
hence dilution of pre-existing miRNAs should have occurred earlier in the
former than in the latter. Why are neurons and neurogenic progenitors more sensitive to miRNA depletion than the stem-cell-like neural progenitors? An intriguing possibility is that when cells alter their state (e.g. from uncommitted to committed progenitor, or from cycling neurogenic progenitor to postmitotic neuron) and therefore carry out major changes in their gene expression profile, they are more dependent on miRNA-mediated regulation than when the progenitors and their progeny are very similar.
This concept is not only consistent with the general idea that `miRNAs
confer precision and robustness to developmental processes'
(Stark et al., 2005),
specifically with regard to brain development, but it is also in line with the
prevailing notion (Choi et al.,
2008; Conaco et al.,
2006; Krichevsky et al.,
2003; Krichevsky et al.,
2006; Makeyev et al.,
2007; Miska et al.,
2004; Sempere et al.,
2004; Smirnova et al.,
2005; Wu and Belasco,
2005) that miRNAs are required for neuronal differentiation, for
which the present study provides two further lines of evidence. One is the
deficient or altered neuronal subtype specification that is suggested by the
increase, upon Dicer ablation, in BrdU-labeled nuclei containing neither Tbr1
nor Brn1. The other is the lack of Foxp2 expression in layer VI neurons of the
miRNA-deficient cortex. Both phenotypes are indicative of a perturbation of
the gene expression changes that are normally concomitant with neuronal
differentiation.
The idea that the proliferation of neural progenitors is less sensitive to
miRNA depletion than their differentiation, together with our observation that
the cell cycle progression and division of Pax6-positive apical progenitors
were unaffected until E13.5, i.e. for 8 cell cycles after the onset of
Dicer ablation (Calegari et al.,
2005; Simeone et al.,
1992), raise interesting perspectives with regard to the expansion
of neural progenitor cells. This expansion is typically curtailed as a
consequence of the progeny derived from the initial progenitor population
becoming increasingly differentiated. Our observations imply that the
physiological expansion of Pax6-positive apical progenitors proceeds normally
upon depletion of miRNAs. Perhaps, interfering with the miRNA-mediated
regulation of the changes in gene expression that accompany differentiation
will increase our repertoire of approaches to achieve expansion of neural
progenitors, and possibly of mammalian somatic stem cells in general.
Consistent with this proposal, a recent study has shown that germline stem
cell maintenance in the Drosophila ovary is not impaired by the
absence of miRNAs (Shcherbata et al.,
2007).
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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20x objective). (B-E) In situ
hybridization on 10-µm cryosections through the heads of E10.5-12.5 control
(Emx1Cre/wt Dicerflox/wt) and conditional
Dicer knockout (Dicer KO, Emx1Cre/wt
Dicerflox/flox) littermate embryos, using LNA antisense probes
for miR-9 (B,C) and miR-124 (D,E). (B,D) Low-magnification
overviews. Brackets indicate the region of the dorsal telencephalon used for
subsequent analyses; dotted lines and arrows indicate the ventral
telencephalon that is unaffected by Emx1-Cre-mediated Dicer ablation.
(C,E) Higher magnification of the E10.5 (E) and E11.5 (C) mesencephalon and of
the E10.5-12.5 dorsal telencephalon. Note the lack of accumulation of mature
miR-9 (B,C) and miR-124 (D,E) specifically in the dorsal
telencephalon upon Dicer ablation. VZ, ventricular zone; BL,
basal lamina; brackets in C,E indicate the pre-plate (E11.5) and cortical
plate (E12.5). (F) Comparison of P0 brains of control
(Emx1Cre/wt Dicerflox/wt, left) and conditional
Dicer knockout (Dicer KO, Emx1Cre/wt
Dicerflox/flox, right) littermate mice. (Top) Dissected
brains. Note the reduced size of the cerebral cortex (Cx) and
olfactory bulbs (OB) in the Dicer KO brains. Dashed lines indicate
the location of the coronal cryosections shown in F' and F''.
(F',F'') DAPI staining of 10 µm coronal
cryosections. Note the reduced size of the cortex (Cx) and
hippocampus (Hp) (which lacks its typical structure), but not
midbrain (Mb), in the Dicer KO brains. Scale bars: 500 µm in
F',F''; 250 µm in Aa,B,D; 100 µm in Ab; 50 µm in C,E.
