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First published online 21 November 2007
doi: 10.1242/dev.009951
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1 GSF-National Research Institute for Environment and Health, Institute for Stem
Cell Research, Ingolstädter Landstr. 1, 85764 Neuherberg/Munich,
Germany.
2 Physiological Genomics, University of Munich, Schillerstr. 46, 80639 Munich,
Germany.
* Author for correspondence (e-mail: Magdalena.goetz{at}gsf.de)
Accepted 23 September 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
overexpression promoted the generation of Pax6+ self-renewing progenitors in
vitro and in vivo and increased the clonal progeny of single progenitors in
vitro. Time-lapse video microscopy revealed that a change in the mode of cell
division, rather than an alteration of the cell cycle length, causes the
Par-complex-mediated increase in progenitors. Taken together, our data
demonstrate a key role for the apically located Par-complex proteins in
promoting self-renewing progenitor cell divisions at the expense of neurogenic
differentiation in the developing cerebral cortex.
Key words: Cortical progenitors, Cell lineage, Cell proliferation, Pax6, Tbr2 (Eomes)
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), tries to
explain the enlargement of the cerebral cortex observed during evolution as
well as the differences in size of distinct brain areas. It is predicted that
the number of symmetric proliferative divisions determines the progenitor pool
and thereby the size of the cerebral cortex, whereas the number of
differentiative divisions generating either one or two neurons determines the
number of neurons per surface area, i.e. the thickness of the cerebral cortex.
Indeed, this concept has been well supported through the identification of
sets of progenitors dividing mostly in a proliferative mode (neuroepithelial
cells) and others mostly in a differentiative mode (basal progenitors).
Prior to neurogenesis, neuroepithelial cells divide exclusively in the
symmetric proliferative mode, giving rise to two progenitor cells
(Chenn and McConnell, 1995). At
the onset of neurogenesis, two sets of progenitors arise, the apically located
radial glial cells forming the ventricular zone (VZ) and the basally located
subventricular zone (SVZ) cells. The latter largely divide in a symmetric
differentiative mode generating two postmitotic neurons
(Miyata et al., 2004;
Haubensak et al., 2004;
Noctor et al., 2004). By
contrast, radial glial cells located in the VZ either divide in a symmetric
proliferative mode or divide asymmetrically, generating a radial glial cell
and a postmitotic neuron or a basal progenitor cell
(Noctor et al., 2001;
Noctor et al., 2004;
Miyata et al., 2001;
Miyata et al., 2004;
Tamamaki et al., 2001) and
thereby contributing to the generation of neurons destined for different
cortical layers (Takahashi et al.,
1999; Desai and McConnell,
2000; Shen et al.,
2006). Thus, during cortical neurogenesis, distinct sets of
progenitors contribute differently to the differentiative and proliferative
cell divisions, suggesting that the generation of these specific types of
progenitors might actually regulate the size and thickness of the cerebral
cortex. Indeed, considerably enlarged upper cortical layers characterize the
primary visual area of the cortex in primates (for a review, see
Dehay and Kennedy, 2007). They
are formed by basal SVZ progenitors as this layer dominates the progenitor
pool during the formation of the upper cortical layers
(Smart et al., 2002;
Lukaszewicz et al., 2005).
Moreover, faster cell cycle kinetics regulate the enlargement of the primary
visual area as compared with other neighboring cortical areas in primates
(Lukaszewicz et al., 2005).
Thus, the speed of the cell cycle and the type of cortical progenitors appear
to be critical for the size and thickness of the cortex or of a specific
cortical area. However, although the dynamic progression in the cellular
composition of the developing cerebral cortex has been extensively examined,
the molecular mechanisms controlling the generation of specific progenitor
subtypes and their mode of cell division are not well understood.
One of the molecular characteristics distinguishing the apically located VZ
progenitors from the basally located SVZ progenitors is their apico-basal
polarity. The VZ progenitors are highly polarized (for a review, see
Götz and Huttner, 2005),
with an apical domain enriched in the Par-complex proteins Par3, Par6,
aPKC
(also known as Pard3, Pard6 and Prkci, respectively -
Mouse Genome Informatics) and Cdc42
(Manabe et al., 2002;
Kosodo et al., 2004;
Cappello et al., 2006;
Imai et al., 2006;
von Trotha et al., 2006;
Afonso and Henrique, 2006;
Kovac et al., 2007) that is
separated from the basolateral membrane domain by adherens junctions
(Mollgard et al., 1987;
Astrom and Webster, 1991) (for
a review, see Götz and Huttner,
2005). By contrast, the basal SVZ progenitors display none of
these features. Unlike in Drosophila, however, the role of
apico-basal polarity and adherens junctions in vertebrate VZ and SVZ
progenitor cell division has not yet been elucidated (reviewed by
Wodarz and Huttner, 2003).
Recently, it has been shown that conditional deletion of Cdc42 in early VZ
progenitors leads to an increased number of basal Tbr2-positive progenitors
(Tbr2 is also known as Eomes - Mouse Genome Informatics) and a premature
increase in neuron production (Cappello et
al., 2006). As Cdc42 is well known to activate the apically
localized Par complex, we proposed that the Par complex might contribute to
maintain apical progenitors, thereby promoting symmetric proliferative or
asymmetric cell divisions. However, the rate of neuron generation remained
unaffected when aPKC (a Par-complex protein) was inactivated at later
developmental stages (Imai et al.,
2006). Two explanations could account for these observations:
either Cdc42 does not act via the Par complex to increase basal progenitors,
or the Par complex plays a prominent role only at earlier developmental
stages. We were therefore prompted to examine the expression levels of Par
proteins during corticogenesis and elucidate their function by Par3 and Par6
gain- or loss-of-function experiments in vitro and in vivo.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmids
The full-length cDNA of Par3 (accession number AY_026057) and Par6 alpha
(accession number NM_019695) were cloned into the retroviral plasmid pMXIG
(Mizuguchi et al., 2001). The
short-hairpin RNA (shRNA) duplexes were designed against the full-length cDNA
of Par3. They contain the respective sequence in sense orientation, followed
by eight nucleotides CAAGCTTC as loop, followed by the respective sequence in
antisense orientation and six nucleotides T as termination. The synthesized
double-stranded DNA nucleotides were annealed and ligated into pbs-U6 plasmid
with same cloning site immediately downstream of the U6 promoter. The U6-shRNA
cassette was generated by PCR using the forward primer U6_E22
(5'-GCAGGAATTCCCTGCAGGCATGCAAGCG-3') and the reverse primer U6_C22
(5'-CGGTATCGATGCTCTAGAACTAGGATTGGC-3') containing EcoRI
and ClaI sites and subcloned into the lentiviral plasmid pLVTH
(Wiznerowicz and Trono, 2003)
after deleting the H1 promoter. The three oligonucleotides designed were Par3a
(5'-GGAGATCTTCGAAACAGAAGA-3'; 21 bp), Par3b
(5'-GCAGCAAACAAGGAGCAATAT-3'; 21 bp) and Par3c
(5'-GAAGAAAGTAGGCAAGAGGCT-3'; 21 bp).
Virus production
Replication-incompetent enhanced-GFP-expressing retrovirus was produced
from a stably transfected packaging cell line (GPG-293) transiently
transfected with the retroviral expression plasmid. Lentiviral vectors were
produced by transient cotransfection of 293T cells with the following
plasmids: lentiviral packaging plasmid pCMVdelta8.9, pseudotyping plasmid
pVSVG (both kindly provided by Pavel Osten, Northwestern University, Feinberg
Medical School, Chicago, IL) and lentiviral expression plasmid. Supernatant
was harvested 48 and 72 hours after transfection, centrifuged at low speed and
filtered through a 0.45 µm low-protein-binding PVDF filter (Millipore).
Viral particles were pelleted at 50,000 g at 4°C for 2
hours, resuspended in a large volume of TBS-5 (50 mM Tris-HCl pH 7.8, 130 mM
NaCl, 10 mM KCl, 5 mM MgCl2) and stored at -80°C.
Histology and immunohistochemistry
Timed pregnant mice were sacrificed by cervical dislocation. Embryonic
brains were removed and fixed in 4% paraformaldehyde (PFA) in PBS,
cryoprotected in 30% sucrose solution (in PBS), embedded in Tissue-tek and
cryosectioned (20-30 µm).
Sections or coverslips (see below) were incubated in primary antibody
overnight at 4°C in 0.5% Triton X-100 and 10% normal goat serum in PBS.
Primary antibodies were: anti-Par3 (rabbit, Upstate, 1:500), anti-aPKC
(mouse, BD Transduction, 1:200), anti-pan-cadherin (mouse, Sigma, 1:100),
anti-Ki67 (rat, Dako, 1:50), anti-Tbr2 (rabbit, Chemicon, 1:100), anti-Pax6
(rabbit, Chemicon, 1:750), anti-Tbr1 (rabbit, Chemicon, 1:100),
anti-βIII-tubulin (mouse IgG2b, Sigma, 1:100), anti-MAP2 (mouse IgG1,
Sigma, 1:100), anti-GFP (rabbit, Clontech, 1:500; chicken, Sigma, 1:500),
anti-nestin (mouse IgG1, Hybridoma Bank, 1:10). Nuclei were visualized by
incubating sections for 10 minutes with 0.1 µg/ml DAPI (4',6'
diamidino-2-phenylindole, Sigma) in PBS. Fluorescent secondary antibodies were
used according to the manufacturer's protocol (Jackson ImmunoResearch or
Southern Biotechnologies). Sections or coverslips were mounted in Aqua
Polymount (Polyscience) and analyzed using Zeiss Axioplan 2 and Olympus
confocal laser-scanning microscopes.
In-utero injection
E12- and E13-timed pregnant mice were anaesthetized with a mixture of
fentanyl (0.05 mg/kg), midazolam (5 mg/kg) and metedomidine (0.5 mg/kg). Only
when deep anesthesia was verified by the absence of pain reflexes were the
uterine horns exposed after caesarean incision. Under fiber optic lighting,
each embryo was manipulated through the uterine wall until the position of the
lateral ventricle was discernible. Viral vector suspension (0.5±1.0
µl) with Fast Green (2.5 mg/µl, Sigma) was injected into the cerebral
ventricles through a bevelled glass micropipette. After injection, the
peritoneal cavity was rinsed with warmed 0.9% NaCl, the uterine horns were
replaced and the wound was closed. Anesthesia was terminated by injection of
naloxon (1.2 mg/kg), flumacenil (0.5 mg/kg) and atipamezol (2.5 mg/kg).
Post-operative care involved application of painkillers and surveillance
several times a day. Animals recovered very well after the operation and no
signs of distress could be observed 1 day later.
Primary cell culture
Embryonic brains were isolated from E12-14 timed pregnant mice. The lateral
portion of the dorsal telencephalon was dissected and dissociated as described
previously (Heins et al.,
2001). After 2 hours, cultures were infected with a low titer
(<30 particles) of the viruses described above and medium changes and
fixation after 7 days in vitro were performed as previously described
(Heins et al., 2001).
By using a low number of viral particles to transduce the cells, we could
identify well-separated cell clusters at 20x magnification and the mean
number of clusters per coverslip in the experiments described was found to be
24.7±9.2 (mean±s.e.m.). This allowed us to infer that each
cluster was derived from a single progenitor cell and could therefore be
defined as an individual clone (Williams
et al., 1991; Haubst et al.,
2004). This was also confirmed by time-lapse video microscopy. The
mean number of neuronal and non-neuronal cells within different types of
clones, as well as the frequency of clones with a given size, were plotted and
are shown as mean±s.e.m. Data were derived from at least three
experimental batches with four to six coverslips analyzed per experiment.
Time-lapse video microscopy
Primary E12/13 cortical cell cultures were prepared as described above and
infected with a greater number of virus particles. Twenty hours after viral
infection and 2 hours after medium replacement, the tissue culture plate was
tightly sealed and time-lapse microscopy was performed with a cell observer
(Zeiss) at a constant 37°C. Phase-contrast images were acquired every 2
minutes and fluorescence images every 3 hours for 5 days with Axiovision Rel.
4.5 software (Zeiss) and an AxioCam HRm camera. Images were assembled into a
movie and analyzed using Axiovision Rel. 4.5 and MetaMorph Offline (Version
6.1r4, Molecular Devices) software. Transduced cells were recognized by GFP
fluorescence and tracked in phase-contrast images, enabling the identification
of single-cell lineages.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the embryonic cortex at different developmental stages.
Consistent with previous data (Manabe et
al., 2002; Kosodo et al.,
2004; Cappello et al.,
2006), we observed a strong enrichment of Par3, Par6 and
aPKC immunoreactivity at the apical surface of the VZ at E12
(Fig. 1A-C,G-I,M-O). The
expression level gradually weakened at subsequent developmental stages (E14,
data not shown) and was almost absent by E16
(Fig. 1D-F,J-L,P-R). This
decrease in Par3, Par6 and aPKC-immunoreactivity at the apical surface
correlates with the decrease in proliferative cell divisions towards
mid-neurogenesis (E14-16) in the developing cortex
(Takahashi et al., 1995;
Chenn and McConnell, 1995;
Haydar et al., 2003;
Noctor et al., 2004;
Haubensak et al., 2004).
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). A
reduction in Par3 immunostaining was observed upon Par3b shRNA transduction in
E12 cortical cells cultured for 3 days in vitro (see Fig. S1 in the
supplementary material). Next we transduced cells isolated from E12 cortices
in vitro with a low titer of each Par3 shRNA construct (shPar3a, b, c) or
control plasmids. This allowed us to detect the transduced cells and/or their
progeny by GFP fluorescence in individual clusters, i.e. the progeny derived
from a single cell - a clone. Both the number of cells per clone and the
proportion of neurons within clones were affected after transduction with Par3
shRNAs, in comparison to control, after 7 days in vitro
(Fig. 2A-F). Par3a proved to be
most efficient in reducing the mean clone size (to 24% of that of the
control), whereas Par3c, the shRNA previously shown to be effective
(Plusa et al., 2005), reduced
the mean clone size to about half (56%) the size observed upon control
transduction (Fig. 2F). It is
noteworthy that the mean clone size obtained with the Par3a shRNA was close to
the maximum achievable reduction, as virtually every clone (84.5±13.5%
of all clones) consisted of only a single cell, whereas this was the case for
just 43±15% of clones transduced with control virus. The relatively
high proportion of single-cell clones amongst control-infected cells was due
to the use of lentiviral vectors that transduce postmitotic neurons as well as
proliferating cells present at the beginning of the cultures. In addition, all
Par3 shRNA constructs significantly reduced the number of clones that
contained three or more cells (Fig.
2E). Examination of the cell fate after control transduction or
Par3 knockdown revealed a significant increase, as compared with controls, in
the proportion of transduced cells differentiating into neurons within the
culture period (Fig. 2G). These
results therefore suggest that knockdown of Par3 by three independent shRNAs
potently reduced clone size and drove many more cells towards a neuronal
fate.
Time-lapse video microscopy reveals increased cell cycle exit upon Par3 knockdown
Our observations following Par3 knockdown using shRNA constructs could
reflect increased exit from the cell cycle or cell death. To discriminate
between these possibilities, we performed live time-lapse video microscopy of
control and Par3b shRNA-transduced E12 cortical cells in vitro. We chose Par3b
shRNA for these experiments, because it significantly decreased the number of
cells per clone, but still allowed some cell divisions to occur
(Fig. 2E). Transduced cells
were identified by GFP fluorescence and their behavior was followed in
phase-contrast images taken every 2 minutes during the culture period (see
Materials and methods). The GFP fluorescence signal was first detected 2-3
days after lentiviral infection (see Movies 1, 2 in the supplementary
material). At this time, we observed about 23±6% of control-transduced
cells as single cells. These cells were present as single cells from the
beginning of the time-lapse imaging, and proved to be postmitotic neurons by
subsequent immunolabeling for the neuronal marker MAP2 (also known as Mtap2 -
Mouse Genome Informatics) (data not shown). Apart from these postmitotic
neurons, most cells in the control-infected cultures belonged to clones that
were shown to derive from single progenitors undergoing one or more rounds of
cell division (see Movie 1 in the supplementary material). By contrast, cells
transduced with Par3b shRNA were mostly found as single cells (77±20%).
None of these cells ever divided (46 cases analyzed). Thus, a significantly
higher proportion of Par3b-transduced cells leaves the cell cycle and
differentiates into neurons (see Movie 2 and Fig. S2 in the supplementary
material). Moreover, those clones generated by Par3 shRNA-transduced cells
that did contain a few more cells were usually small (2-6 cells) and the cell
divisions took place during the first 2 days in vitro, with virtually all
cells differentiating into neurons, as previously indicated in our clonal
analysis (Fig. 2). Taken
together, Par3 loss-of-function inhibits cell proliferation in cultured
cortical progenitors, leading to premature cell cycle exit and neuronal
differentiation.
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Par3 and Par6 overexpression in vitro
To examine whether Par3 gain-of-function might have the opposite effect to
the Par3 shRNA-mediated effects, we transduced E12 cortical progenitors with
the full-length Par3 (180 kDa form, see Materials and methods for details)
coexpressed with IRES GFP or a GFP-containing control retroviral vector. These
viral vectors require breakdown of the nuclear envelope for viral genome
integration and hence incorporate their genome only in proliferating cells
(Price and Thurlow, 1988). As
such, postmitotic neurons do not integrate these viral vectors, resulting in a
smaller proportion of clones containing 1-2 cells than with the control
lentivirus (compare controls in Figs
2,
4). Notably, transduction with
Par3-containing retrovirus resulted in a significant increase (150%) in the
mean size of clones in comparison to control-transduced clones after 7 days in
vitro (Fig. 4A-F). This
increase in clone size by Par3 transduction was mostly due to an increase in
the number of large clones, containing more than 20 cells
(Fig. 4E). Moreover,
Par3-transduced cells comprised fewer neurons
(Fig. 4G), indicating that a
higher proportion of Par3-transduced cells (about 60%,
Fig. 4G) maintained an
undifferentiated state in comparison to controls (35%,
Fig. 4G). Taken together,
Par3-transduced cells exhibit the opposite effect in clone size (an increase)
to those subject to Par3 knockdown (a decrease).
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), might have similar effects upon overexpression in
cortical progenitors. Indeed, Par6 transduction resulted in a significant
increase in the clone size 7 days after transduction of cells derived from
both E12 (Fig. 5A,B) and E14
cortices (Fig. 5D,E). As
observed for Par3-transduced cells, the increase in clone size upon Par6
transduction was mostly due to an increased proportion of clones containing
more than 10 cells (Fig. 5A,D),
and the proportion of neurons originating from E12 cortical progenitors was
also reduced (Fig. 5C).
Consistent with the hypothesized role of the Par complex in cell
proliferation/differentiation and the diminished levels of Par proteins at
mid-neurogenesis (Fig. 1), E14
progenitors produced smaller clones than E12 progenitors (compare the controls
in Fig. 5A,B and D,E), and the
proportion of neurons between E14 control and Par6 clones was not affected
(Fig. 5F). However, after Par6
overexpression, E14 cortical progenitors generated clones similar in size to
those derived from E12 progenitors (Fig.
5B,E), indicating that the activation of the Par complex at later
developmental stages is sufficient to promote proliferation.
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Par6 overexpression in vitro increases the proportion of Pax6-expressing progenitors
To further examine the molecular identity of the progenitor cells generated
upon Par6 overexpression, we analyzed pairs of cells 2 days after transduction
in vitro (Shen et al., 2002;
Kawaguchi et al., 2004;
Clayton et al., 2007). We used
low numbers of viral particles in order to obtain a small number of pairs that
could thereby be inferred as being derived from a single progenitor. First, we
examined progenitor identity by staining for Ki67. Consistent with the
time-lapse analysis, the number of pairs with both cells expressing Ki67 was
significantly increased, in comparison to the control, after Par6
overexpression (see Fig. S4 in the supplementary material). Thus, the pair
analysis provided a reliable read-out of cell divisions as observed by
time-lapse video microscopy. Next, we examined the molecular identity of the
progenitors by staining for Pax6, a transcription factor expressed in
virtually all self-renewing VZ cells
(Götz et al., 1998;
Englund et al., 2005;
Cappello et al., 2006), versus
Tbr2, a transcription factor expressed in the basal SVZ progenitor cells
(Englund et al., 2005;
Cappello et al., 2006).
Strikingly, whereas the frequency of Pax6-positive pairs significantly
increased after Par6 overexpression (Fig.
7A-J; P<0.01, unpaired t-test), the frequency
of Tbr2-positive pairs was not, and the proportion of pairs with both
daughters expressing Tbr2 was higher (by almost double) in the
control-transduced cells (Fig.
7K; P<0.01, unpaired t-test). These data
demonstrate for the first time that the Par complex is upstream of these fate
determinants, demonstrating that Par6 overexpression is sufficient to maintain
progenitors in a Pax6-expressing self-renewing state.
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), and/or to the maintenance of a progenitor state by Par6
overexpression. In support of the latter, double staining for GFP and Ki67
showed that the vast majority of GFP-positive cells in the VZ of Par6-injected
mice were colabeled (Fig. 8D),
supporting their progenitor-cell identity. Moreover, in virtually every case,
Par6-transduced VZ cells also double labeled with the VZ progenitor marker
Pax6 (Fig. 8E). At SVZ
positions, some cells also contained Tbr2
(Fig. 8F), but the proportion
of such cells was not increased upon Par6 overexpression
(Fig. 8C). Moreover, neither VZ
nor SVZ cells transduced with Par6 contained the neuron-specific antigen MAP2
(Fig. 8G), consistent with
their progenitor fate. Taken together, Par6 overexpression promotes the
maintenance of Pax6-positive VZ progenitors both in vitro and in vivo. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Role of the Par complex in proliferation and the mode of cell division
Much of our knowledge on the function of polarity genes is derived from
studies of Drosophila neuroblasts, which give rise to two daughter
cells of distinct size and fate. The smaller basal daughter cell (ganglion
mother cell, GMC) undergoes terminal division to generate two neurons or glia,
whereas the larger apical daughter maintains a neuroblast identity and
continues to divide, generating new neuroblasts and GMCs (reviewed by
Betschinger and Knoblich,
2004). The molecular mechanisms controlling the specification of
these two types of cells have been extensively studied and are reviewed
elsewhere (e.g. Betschinger and Knoblich,
2004; Yu et al.,
2006). Particularly relevant for our results, the Par-complex
proteins Bazooka (the fly homolog of Par3), Par6 and aPKC have been shown to
localize apically in the neuroblasts and to be inherited by the large apical
daughter cell, which continues to divide in a stem-cell-like fashion. Our
results indicate similarities and differences in the role of Par-complex
proteins in the mouse cerebral cortex. Par3 and Par6 also play an important
role in the control of proliferation and differentiation, but they differ with
regard to their effects on asymmetric cell division. Overexpression of Par6
did not significantly affect the number of asymmetric cell divisions. If
asymmetric distribution of Par6 was required for asymmetric cell division,
overexpression of Par6 should result in inheritance by both daughter cells and
hence should abolish asymmetric cell divisions. Indeed, it has been shown that
ectopic expression of Par3 is sufficient to mislocalize the entire Par complex
in transduced neuroepithelial cells in chicken embryos
(Afonso and Henrique, 2006). As
neither Par3 nor Par6 overexpression abolished asymmetric cell divisions in
cortical progenitors, it appears that the role of Par3 and Par6 in regulating
proliferation in the mouse cerebral cortex may be independent of asymmetric
cell division and/or the asymmetric inheritance of the Par protein itself.
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) (but see Horne-Badovinac
et al., 2001), whereas defects in Par-complex-mediated signaling
affect spindle orientation in Drosophila
(Kuchinke et el., 1998;
Wodarz et al., 2000;
Petronczki and Knoblich,
2001). Indeed, a major difference between vertebrate and
invertebrate neurogenesis appears to be the mode of cell division. Only a
minority of neural progenitors divide obliquely to the apico-basal axis in
vertebrate neuroepithelia (Lyons et al.,
2003; Götz and Huttner,
2005; Das et al.,
2003; Kosodo et al.,
2004; Stricker et al.,
2006), in contrast to the predominant asymmetric mode of cell
division of Drosophila neuroblasts (for a review, see
Yu et al., 2006). Asymmetry in
daughter cell fate also occurs in the vertebrate nervous system, but whether
or not this is achieved by the asymmetric inheritance of fate determinants
(Kosodo et al., 2004),
extrinsic signals or yet unresolved mechanisms, is still not clear.
Importantly, the characteristically low proportion of asymmetric cell
divisions in the mammalian cortex also persists in vitro - in slice cultures
(Miyata et al., 2004),
single-cell cultures (Qian et al.,
1998) and in our high-density dissociated cortical cultures. Live
imaging in cortical slice cultures demonstrated that in most cases (73%)
apical VZ progenitors divide symmetrically to generate two progenitors, and in
only 27% of cases asymmetrically (Miyata
et al., 2004). In addition to these VZ progenitors, about 30% of
all progenitors at this stage are basal SVZ progenitors that divide in a
terminally symmetric cell division (Haubst
et al., 2004; Capello et al., 2006;
Noctor et al., 2004;
Miyata et al., 2004;
Haubensak et al., 2004).
Therefore, amongst all progenitor cells, a third divides in a terminally
symmetric manner (all SVZ cells), half divide in a proliferative symmetric
manner (70% of all VZ cells), and only 20% (30% of VZ cells) divide in an
asymmetric manner. These proportions are strikingly similar to those observed
in our time-lapse analysis.
The similarity in the effect of Par protein manipulations in vivo and in
dissociated-cell cultures in our study implies that tissue polarity and
coordinated apico-basal fate determinants are not required for the Par complex
to influence cell proliferation. As cell polarity is not required for the Par
complex to exert its effect, and as asymmetric cell divisions were not
abolished by Par6 overexpression, we suggest that the overall level and
activity of the Par complex might determine cell proliferation in a threshold
manner. Interestingly, the apically located Par complex is mostly
symmetrically distributed in the zebrafish neuroepithelium
(von Trotha et al., 2006;
Tawk et al., 2007), where it
is required for the mirror-symmetric cell divisions that occur during neural
keel formation (Tawk et al.,
2007), as well as in the mouse VZ
(Kosodo et al., 2004). A
unifying model might therefore be one in which the amount of apically located
Par protein determines whether both cells continue to proliferate (high levels
of Par protein, as upon overexpression or early in development), or only one
cell continues to proliferate (stochastically, upon lower Par protein levels),
or no cells continue to proliferate (as observed after Par3 knockdown or at
the end of neurogenesis). This model not only fits very well with the results
obtained by our manipulation of Par protein levels, but also with our
observation that the Par proteins decrease considerably at the apical surface
during cortical development, thereby causing the reduction in the progenitor
pool. Moreover, the low level of Par proteins around mid-neurogenesis in
apical progenitors explains the mild phenotype upon deletion of aPKC
or Cdc42 at these later stages (Cappello et
al., 2006; Imai et al.,
2006), in contrast to the much stronger effects upon their
deletion at earlier stages (Cappello et
al., 2006). Taken together, these results suggest a role of the
Par complex in proliferation that appears to be rather independent on an
asymmetric distribution of Par3 and Par6, but which also employs Cdc42 and
aPKC (Rolls et al., 2003;
Castoria et al., 2004;
Imai et al., 2006;
Cappello et al., 2006).
Role of the Par complex in cell-fate specification
One of the most striking results obtained by the manipulation of Par
proteins in our study is their instruction of a specific type of progenitor,
namely the Pax6-positive VZ progenitors. Par3 and Par6 overexpression promote
progenitors that express Pax6 and not Tbr2. Whereas VZ progenitors that
contain different levels of Pax6 proliferate to different degrees, the basal
SVZ progenitors that are characterized by expression of a different set of
transcription factors [Tbr2, Svet1, Cux2 (Cutl2), or Prox1]
(Tarabykin et al., 2001;
Nieto et al., 2004;
Zimmer et al., 2004;
Englund et al., 2005;
Lavado and Oliver, 2007) most
often generate two postmitotic daughters. Thus, overexpression of Par6 may
exert its effect by instructing a VZ fate, an indicator of which is the
expression of Pax6. However, deletion of Pax6 results in a greater number of
progenitors (Haubst et al.,
2004), suggesting that Pax6 itself is not mediating the
proliferative function exerted by Par3 or Par6 overexpression. Indeed,
overexpression of Pax6 rather promotes exit from the cell cycle
(Quinn et al., 2007) and
differentiation into neurons (Haubst et
al., 2004; Hack et al.,
2004; Heins et al.,
2002). However, Pax6 is expressed at different levels in all
VZ/radial glial cells and recent experiments have shown differences between
Pax6 isoforms present in the self-renewing radial glia versus the radial glia
that directly generate postmitotic neurons (L. Pinto and M.G., unpublished).
Thus, the increased proportion of Pax6+ cells indicates an increase of
progenitors with a radial glia/VZ fate, at the expense of SVZ progenitors
(Tbr2+ cells). Interestingly, upper-layer neurons have been proposed to derive
largely via basal SVZ progenitor cells
(Tarabykin et al., 2001;
Nieto et al., 2004;
Zimmer et al., 2004;
Schuurmans et al., 2004;
Wu et al., 2005). This
suggests that the maintenance of progenitors in the VZ fate, as achieved upon
Par6 overexpression in vivo, might interfere with upper-layer neuron
generation. Conversely, however, the knockdown of Par proteins does not
increase the generation of upper-layer neurons, most likely because they exit
from the cell cycle too prematurely. Our observation that the overexpression
of Par-complex proteins promotes the fate of Pax6+ VZ progenitors is also
consistent with the results obtained after deletion of Cdc42, the activator of
the Par complex, upon which Pax6-positive self-renewing progenitors are lost
and Tbr2-positive progenitors dividing in a differentiative mode are increased
(Cappello et al., 2006).
Notably, the reduction in Pax6 levels is very mild upon deletion of Cdc42 at
later stages (Cappello et al.,
2006) (data not shown), consistent with the reduced amount of Par
proteins at this later stage. Interestingly, Par6 has recently been observed
in the nucleus (Cline and Nelson,
2007), implying the possibility that Par proteins might indeed
participate in the regulation of gene transcription. Thus, our data not only
demonstrate the key role of Par proteins in regulating self-renewing
progenitors, but also suggest a novel role for Par proteins in regulating the
expression of transcriptional cell-fate determinants.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/1/11/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Afonso, C. and Henrique, D. (2006). PAR3 acts
as a molecular organizer to define the apical domain of chick neuroepithelial
cells. J. Cell Sci. 119,4293
-4304.
Astrom, K. E. and Webster, H. D. (1991). The early development of the neopallial wall and area choroidea in fetal rats. A light and electron microscopic study. Adv. Anat. Embryol. Cell Biol. 123,1 -76.[Medline]
Betschinger, J. and Knoblich, J. A. (2004). Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates. Curr. Biol. 14,R674 -R685.[CrossRef][Medline]
Cappello, S., Attardo, A., Wu, X., Iwasato, T., Itohara, S., Wilsch-Brauninger, M., Eilken, H. M., Rieger, M. A., Schroeder, T. T., Huttner, W. B. et al. (2006). The Rho-GTPase cdc42 regulates neural progenitor fate at the apical surface. Nat. Neurosci. 9,1099 -1107.[CrossRef][Medline]
Castoria, G., Migliaccio, A., Di Domenico, M., Lombardi, M., de
Falco, A., Varricchio, L., Bilancio, A., Barone, M. V. and Auricchio, F.
(2004). Role of atypical protein kinase C in estradiol-triggered
G1/S progression of MCF-7 cells. Mol. Cell. Biol.
24,7643
-7653.
Chenn, A. and McConnell, S. K. (1995). Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis. Cell 82,631 -641.[CrossRef][Medline]
Clayton, E., Doupe, D. P., Klein, A. M., Winton, D. J., Simons, B. D. and Jones, P. H. (2007). A single type of progenitor cell maintains normal epidermis. Nature 446,185 -189.[CrossRef][Medline]
Cline, E. G. and Nelson, W. J. (2007).
Characterization of Mammalian par 6 as a dual-location protein.
Mol. Cell. Biol. 27,4431
-4443.
Das, T., Payer, B., Cayouette, M. and Harris, W. A. (2003). In vivo time-lapse imaging of cell divisions during neurogenesis in the developing zebrafish retina. Neuron 37,597 -609.[CrossRef][Medline]
Dehay, C. and Kennedy, H. (2007). Cell-cycle control and cortical development. Nat. Rev. Neurosci. 8, 438-450.[CrossRef][Medline]
Desai, A. R. and McConnell, S. K. (2000). Progressive restriction in fate potential by neural progenitors during cerebral cortical development. Development 127,2863 -2872.[Abstract]
Englund, C., Fink, A., Lau, C., Pham, D., Daza, R. A., Bulfone,
A., Kowalczyk, T. and Hevner, R. F. (2005). Pax6, Tbr2, and
Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells,
and postmitotic neurons in developing neocortex. J.
Neurosci. 25,247
-251.
Götz, M. and Huttner, W. B. (2005). The cell biology of neurogenesis. Nat. Rev. Mol. Cell Biol. 6,777 -788.[CrossRef][Medline]
Götz, M., Stoykova, A. and Gruss, P. (1998). Pax6 controls radial glia differentiation in the cerebral cortex. Neuron 21,1031 -1044.[CrossRef][Medline]
Hack, M. A., Sugimori, M., Lundberg, C., Nakafuku, M. and Gotz, M. (2004). Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6. Mol. Cell. Neurosci. 25,664 -678.[CrossRef][Medline]
Haubensak, W., Attardo, A., Denk, W. and Huttner, W. B.
(2004). Neurons arise in the basal neuroepithelium of the early
mammalian telencephalon: a major site of neurogenesis. Proc. Natl.
Acad. Sci. USA 101,3196
-3201.
Haubst, N., Berger, J., Radjendirane, V., Graw, J., Favor, J.,
Saunders, G. F., Stoykova, A. and Gotz, M. (2004). Molecular
dissection of Pax6 function: the specific roles of the paired domain and
homeodomain in brain development. Development
131,6131
-6140.
Haydar, T. F., Ang, E., Jr and Rakic, P.
(2003). Mitotic spindle rotation and mode of cell division in the
developing telencephalon. Proc. Natl. Acad. Sci. USA
100,2890
-2895.
Heins, N., Cremisi, F., Malatesta, P., Gangemi, R. M., Corte, G., Price, J., Goudreau, G., Gruss, P. and Gotz, M. (2001). Emx2 promotes symmetric cell divisions and a multipotential fate in precursors from the cerebral cortex. Mol. Cell. Neurosci. 18,485 -502.[CrossRef][Medline]
Heins, N., Malatesta, P., Cecconi, F., Nakafuku, M., Tucker, K. L., Hack, M. A., Chapouton, P., Barde, Y. A. and Gotz, M. (2002). Glial cells generate neurons: the role of the transcription factor Pax6. Nat. Neurosci. 5, 308-315.[CrossRef][Medline]
Horne-Badovinac, S., Lin, D., Waldron, S., Schwarz, M., Mbamalu, G., Pawson, T., Jan, Y., Stainier, D. Y. and Abdelilah-Seyfried, S. (2001). Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis. Curr. Biol. 11,1492 -1502.[CrossRef][Medline]
Imai, F., Hirai, S., Akimoto, K., Koyama, H., Miyata, T., Ogawa,
M., Noguchi, S., Sasaoka, T., Noda, T. and Ohno, S. (2006).
Inactivation of aPKClambda results in the loss of adherens junctions in
neuroepithelial cells without affecting neurogenesis in mouse neocortex.
Development 133,1735
-1744.
Joberty, G., Petersen, C., Gao, L. and Macara, I. G. (2000). The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat. Cell Biol. 2, 531-539.[CrossRef][Medline]
Kawaguchi, A., Ogawa, M., Saito, K., Matsuzaki, F., Okano, H. and Miyata, T. (2004). Differential expression of Pax6 and Ngn2 between pair-generated cortical neurons. J. Neurosci. Res. 78,784 -795.[CrossRef][Medline]
Kosodo, Y., Roper, K., Haubensak, W., Marzesco, A. M., Corbeil, D. and Huttner, W. B. (2004). Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells. EMBO J. 23,2314 -2324.[CrossRef][Medline]
Kovac, J., Oster, H. and Leitges, M. (2007). Expression of the atypical protein kinase C (aPKC) isoforms iota/lambda and zeta during mouse embryogenesis. Gene Expr. Patterns 7, 187-196.[CrossRef][Medline]
Kuchinke, U., Grawe, F. and Knust, E. (1998). Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka. Curr. Biol. 8,1357 -1365.[CrossRef][Medline]
Lavado, A. and Oliver, G. (2007). Prox1 expression patterns in the developing and adult murine brain. Dev. Dyn. 236,518 -524.[CrossRef][Medline]
Lukaszewicz, A., Savatier, P., Cortay, V., Giroud, P., Huissoud, C., Berland, M., Kennedy, H. and Dehay, C. (2005). G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex. Neuron 47,353 -364.[CrossRef][Medline]
Lyons, D. A., Guy, A. T. and Clarke, J. D.
(2003). Monitoring neural progenitor fate through multiple rounds
of division in an intact vertebrate brain. Development
130,3427
-3436.
Manabe, N., Hirai, S., Imai, F., Nakanishi, H., Takai, Y. and Ohno, S. (2002). Association of ASIP/mPAR-3 with adherens junctions of mouse neuroepithelial cells. Dev. Dyn. 225, 61-69.[CrossRef][Medline]
Miyata, T., Kawaguchi, A., Okano, H. and Ogawa, M. (2001). Asymmetric inheritance of radial glial fibers by cortical neurons. Neuron 31,727 -741.[CrossRef][Medline]
Miyata, T., Kawaguchi, A., Saito, K., Kawano, M., Muto, T. and
Ogawa, M. (2004). Asymmetric production of surface-dividing
and non-surface-dividing cortical progenitor cells.
Development 131,3133
-3145.
Mizuguchi, R., Sugimori, M., Takebayashi, H., Kosako, H., Nagao, M., Yoshida, S., Nabeshima, Y., Shimamura, K. and Nakafuku, M. (2001). Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Neuron 31,757 -771.[CrossRef][Medline]
Mollgard, K., Balslev, Y., Lauritzen, B. and Saunders, N. R. (1987). Cell junctions and membrane specializations in the ventricular zone (germinal matrix) of the developing sheep brain: a CSF-brain barrier. J. Neurocytol. 16,433 -444.[CrossRef][Medline]
Nieto, M., Monuki, E. S., Tang, H., Imitola, J., Haubst, N., Khoury, S. J., Cunningham, J., Gotz, M. and Walsh, C. A. (2004). Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex. J. Comp. Neurol. 479,168 -180.[CrossRef][Medline]
Noctor, S. C., Flint, A. C., Weissman, T. A., Dammerman, R. S. and Kriegstein, A. R. (2001). Neurons derived from radial glial cells establish radial units in neocortex. Nature 409,714 -720.[CrossRef][Medline]
Noctor, S. C., Martinez-Cerdeno, V., Ivic, L. and Kriegstein, A. R. (2004). Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat. Neurosci. 7,136 -144.[CrossRef][Medline]
Petronczki, M. and Knoblich, J. A. (2001). DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3, 43-49.[CrossRef][Medline]
Plusa, B., Frankenberg, S., Chalmers, A., Hadjantonakis, A. K.,
Moore, C. A., Papalopulu, N., Papaioannou, V. E., Glover, D. M. and
Zernicka-Goetz, M. (2005). Downregulation of Par3 and aPKC
function directs cells towards the ICM in the preimplantation mouse embryo.
J. Cell Sci. 118,505
-515.
Price, J. and Thurlow, L. (1988). Cell lineage
in the rat cerebral cortex: a study using retroviral-mediated gene transfer.
Development 104,473
-482.
Qian, X., Goderie, S. K., Shen, Q., Stern, J. H. and Temple, S. (1998). Intrinsic programs of patterned cell lineages in isolated vertebrate CNS ventricular zone cells. Development 125,3143 -3152.[Abstract]
Quinn, J. C., Molinek, M., Martynoga, B. S., Zaki, P. A., Faedo, A., Bulfone, A., Hevner, R. F., West, J. D. and Price, D. J. (2007). Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous mechanism. Dev. Biol. 302, 50-65.[CrossRef][Medline]
Rakic, P. (1988). Specification of cerebral
cortical areas. Science
241,170
-176.
Rolls, M. M., Albertson, R., Shih, H. P., Lee, C. Y. and Doe, C.
Q. (2003). Drosophila aPKC regulates cell polarity and cell
proliferation in neuroblasts and epithelia. J. Cell
Biol. 163,1089
-1098.
Schuurmans, C., Armant, O., Nieto, M., Stenman, J. M., Britz, O., Klenin, N., Brown, C., Langevin, L. M., Seibt, J., Tang, H. et al. (2004). Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways. EMBO J. 23,2892 -2902.[CrossRef][Medline]
Shen, Q., Zhong, W., Jan, Y. N. and Temple, S. (2002). Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts. Development 129,4843 -4853.[Medline]
Shen, Q., Wang, Y., Dimos, J. T., Fasano, C. A., Phoenix, T. N., Lemischka, I. R., Ivanova, N. B., Stifani, S., Morrisey, E. E. and Temple, S. (2006). The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat. Neurosci. 9,743 -751.[CrossRef][Medline]
Smart, I. H., Dehay, C., Giroud, P., Berland, M. and Kennedy,
H. (2002). Unique morphological features of the proliferative
zones and postmitotic compartments of the neural epithelium giving rise to
striate and extrastriate cortex in the monkey. Cereb.
Cortex 12,37
-53.
Solecki, D. J., Model, L., Gaetz, J., Kapoor, T. M. and Hatten, M. E. (2004). Par6alpha signaling controls glial-guided neuronal migration. Nat. Neurosci. 7,1195 -1203.[CrossRef][Medline]
Stricker, S. H., Meiri, K. and Gotz, M. (2006).
P-GAP-43 is enriched in horizontal cell divisions throughout rat cortical
development. Cereb. Cortex
16 Suppl. 1,i121
-i131.
