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First published online 28 February 2007
doi: 10.1242/dev.02809
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1 Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen,
Germany.
2 DFG, Center of Molecular Physiology of the Brain (CMPB), Göttingen,
Germany.
3 Dulbecco Telethon Institute at IRCCS Fondazione Santa Lucia, Rome,
Italy.
4 University of Colorado, Boulder, Colorado, USA.
* Author for correspondence (e-mail: astoyko{at}gwdg.de)
Accepted 17 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Corticogenesis, Cre/loxP, Overexpression, Pax6, Pax6-5a, Progenitor, Apoptosis, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), outlining the anlage of the future cortex, while
cross-repressive interactions between Pax6 and Gsh2
establish the pallial-subpallial boundary
(Toresson et al., 2000). In
developing cortex, Pax6 is expressed in a prominent
rostrolateral-high to mediolateral-low gradient, with expression lost
progressively after E15.5 (Muzio et al.,
2002a; Stoykova et al.,
1997; Stoykova et al.,
2000). In the Pax6 loss-of-function (LOF) mouse Small
eye (Sey) (Hill et al.,
1991), the cortical progenitors acquire ventral molecular identity
starting at the ventral pallium (VP) and lateral pallium (LP), and
subsequently extending dorsally and medially
(Kroll and O'Leary, 2005;
Muzio et al., 2002b;
Stoykova et al., 1996;
Stoykova et al., 2000;
Torresson et al., 2000; Yun et al.,
2001). Recent data indicate that the different levels of
Pax6 expression in cortical progenitors along the anterior-posterior
axis play a role in progenitor regionalization. Thus, in homozygous
Sey/Sey embryos, the rostrolateral regions of the cortex, where
Pax6 is normally expressed at the highest level, are reduced, whereas
the caudomedial cortical domains, which normally show low Pax6
expression, are expanded (Bishop et al.,
2000). Similarly, the high expression level of Pax6 in
the progenitors of the rostral VP and LP appears essential for the
specification of distinct amygdalar nuclei
(Tole et al., 2005).
The expression of Pax6 specifies the majority of the cortical
progenitors, namely the RC2-positive radial glial (RG) cells
(Götz et al., 1998).
These cells have been shown to act as pluripotent progenitors, generating both
neuronal and glial cells (Heins et al.,
2002; Malatesta et al.,
2000; Miyata et al.,
2001; Noctor et al.,
2004). At birth, the cortical plate (CP) of Sey/Sey mice
is hypocellular, with overgrown ventricular and subventricular zones (V.Z. and
S.V.Z., respectively) (Schmahl et al.,
1993; Stoykova et al.,
1996; Stoykova et al.,
2000), and the RG progenitors show defects in their mitotic cycle
(Estivill-Torrus et al., 2002;
Götz et al., 1998),
migratory and adhesive properties (Caric et
al., 1997; Hartfuss et al.,
2001; Nomura and Osumi,
2004; Stoykova et al.,
1997), boundary formation
(Hartfuss et al., 2001;
Stoykova et al., 1996) and
differentiation (Götz et al.,
1998; Warren et al.,
1999).
In vertebrates, alternative splicing generates two different Pax6 protein
isoforms, Pax6 and Pax6-5a, possibly having different sets of targets
(Czerny et al., 1993;
Epstein et al., 1994b;
Kozmik et al., 1997).
Dorsoventral patterning and boundary formation seem to be mediated exclusively
by the Pax6 isoform, whereas progenitor proliferation is influenced by both
isoforms (Haubst et al.,
2004). Retrovirus-mediated overexpression of Pax6 in RG
progenitors in vitro and cell lineage experiments indicated a neurogenic
activity of Pax6 (Hack et al.,
2004; Heins et al.,
2002; Haubst et al.,
2004).
Using a Cre/loxP-based recombination approach we have developed an in vivo system for conditional Pax6 gain-of-function (GOF) expression in transgenic mice and studied the effects of the activation of the two Pax6 isoforms in different progenitors during corticogenesis. We found that ectopic activation or overexpression of the two isoforms, Pax6 and Pax6-5a, inhibits the proliferation of cortical progenitors. Furthermore, activation of transgenic Pax6 in vivo causes misregulation of the mitotic cycle, premature neurogenesis, and massive apoptosis in different progenitor pools, which seems to depend on the distinct spatiotemporal sensitivities of the cortical progenitors containing different levels of endogenous Pax6.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) followed
by a loxP-(XhoI)-IRES-lacZ-polyA cassette.
The coding sequence of Pax6 or Pax6-5a was inserted into the
XhoI site resulting in pJoP6 and pJoP6-5a,
respectively. The plasmids were used to generate JoP6 and
JoP6-5a mice by pronuclear microinjection. Transgenic mice were
identified by GFP fluorescence. Genomic PCR was performed with the primers
JoP6F, JoP6R and JoP65aR (Table
1). pPD and pHD were generated by cloning four
PD- and five HD-consensus sequences into pGL3 (Promega)
(Epstein et al., 1994a).
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) and
in situ hybridization analysis was according to Moorman et al.
(Moorman et al., 2001). We
used mouse monoclonal antibodies against Pax6 (BAbCO), 5-bromo-2-deoxyuridine
(BrdU; Roche), nestin (Chemicon) and neuron-specific class III ß-tubulin
(Tuj1; Babco), and rabbit polyclonal antisera against pHH3 (Biomol),
ß-actin (Abcam), and activated caspase3 (Cell Signaling Technology) on 8
µm cryostat sections (5 µm for BrdU labeling). Secondary antibodies were
from Molecular Probes (1:500). For TUNEL labeling, the ApopTag kit was used
(Intergen, Purchase, NY). The BrdU labeling index was estimated as the
percentage of the total cell number of BrdU+ cells on DAPI-stained
sections in equally sized areas in medial pallium (MP) and dorsal pallium (DP)
from cortex of both genotypes. pHH3+ cells were counted and
compared over the whole cortex in equally sized units of control and
double-transgenic apical VZ surface. Data are shown as means±s.d.
P values were derived using Student's t-test.
Luciferase reporter assay
Transient transfection of SAOS2 (ATC# HTB-85) and HeLa cells were performed
using Lipofectamine2000 (Invitrogen) and 3 µg of the following plasmids:
pCMV and pPax6
(Maulbecker and Gruss, 1993),
p53Luc (Stuart et al.,
1995), pmRb-pJ3Omega115ROX-p
(Bernards et al., 1989), and
pJoP6-5a. For in vitro overexpression of Pax6, 16 µg of
pPax6 was used (indicated by Pax6++ in
Fig. 7). Cells were subjected
to the Dual-Luciferase Reporter Assay System (Promega) after 48 hours. The
assay was performed in triplicate and luciferase activities were normalized to
internal control activities (data shown as means±s.d.).
q-PCR analysis
Total RNA isolated from cortex was quantified by optical density and used
for q-PCR with the QuantiTect Rev. Transcription Kit and the QuantiTect SYBR
Green PCR Kit (Qiagen). Assays were performed in triplicate and normalized to
internal 18S RNA (data as means±s.d.). Primer sequences are listed in
Table 1.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), driving
ubiquitous expression of the gfp reporter gene
(Fig. 1). Upon Cre
recombination the gfp-stop cassette is excised, leading to
simultaneous expression of Pax6 and a second reporter, lacZ,
via an IRES sequence. The transgenic mouse line generated with this construct
was named JoP6.
In order to test the recombination of the integrated construct,
JoP6 males were crossed with female Emx1IREScre
mice directing recombination in most of the pallial progenitors
(Gorski et al., 2002).
Recombination in the JoP6;Emx1IREScre mice was
monitored by PCR with the primers JoP6F and JoP6R (arrows in
Fig. 1), which bind 5'
and 3' of the floxed gfp-stop cassette, respectively. Genomic
PCR with DNA isolated from the cortex of JoP6 brains resulted in a
1831 bp DNA fragment, whereas after successful recombination an additional 261
bp DNA fragment was detected using DNA isolated from
JoP6;Emx1IREScre double-transgenic cortex
(Fig. 2A). Sections of
JoP6 control cortex exhibited widespread GFP fluorescence in the
entire brain, which was switched off in most of the cortical cells in the VZ
and CP of JoP6;Emx1IREScre transgenic mice,
indicating successful excision of the gfp-stop cassette
(Fig. 2B,B'). Remaining
GFP-positive cells might correspond to GABAergic interneurons
(Gorski et al., 2002). The
function of the second reporter, lacZ, was tested with isolated
brains after whole-mount staining for ß-galactosidase (ß-gal). The
JoP6 brain showed only non-specific staining of the choroid plexus,
whereas the JoP6;Emx1IREScre cortex, which is
smaller than in the control, was intensively stained, indicating activation of
transgenic Pax6. We further followed the expression of the
lacZ reporter throughout brain development. At E12.5,
ß-gal+ aggregates as well as some individual cells were
detected in the VZ and in the thin CP (Fig.
2E). Later, at E15.5, lacZ expression was apparent in
both VZ progenitors and cells migrating throughout the intermediate zone (IZ)
towards the CP, invading its lower part
(Fig. 2F). At E18.5, the late
cortical progenitors showed strong ß-gal staining and the CP was
massively populated by ß-gal+ postmitotic cells
(Fig. 2G), whereas at P28 the
whole cortex was extensively stained for ß-gal
(Fig. 2H), similar to the
recombination pattern in the adult Emx1IREScre brain
(Gorski et al., 2002).
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).
Interestingly, however, the results from the q-PCR assay revealed that in the
cortex of the double-transgenic JoP6;Emx1IREScre
mice, only the level of the Pax6 transcript was elevated, whereas the
level of Pax6-5a was in fact diminished
(Fig. 2K). The functionality of
the transgenic Pax6 was indicated by co-transfection experiments demonstrating
the binding activity of Pax6 to its target consensus sequences
(Fig. 2L).
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During neurogenesis, proliferating nuclei follow interkinetic nuclear
migration, entering into S phase at the basal surface of VZ and progressively
moving to the apical VZ surface, where they enter into M phase
(Takahashi et al., 1993).
After 90 minutes of BrdU incorporation, most of the progenitors in the control
JoP6 cortex had intensively stained (S-phase) nuclei, which were
still located predominantly within the basal region of the VZ
(Fig. 3C). Some nuclei with
diluted BrdU content (that were at the very end of their S phase, when the
BrdU pulse started) were seen at or near to the apical surface of the control
VZ (arrows in Fig. 3C). In the
JoP6;Emx1IREScre cortex, the S-phase labeled
progenitor nuclei appeared to be distributed throughout the VZ, and only a few
cells with diluted BrdU content were seen at the apical VZ
(Fig. 3C'). In addition,
faintly stained aggregates of cells were visible in the mutant VZ (arrowheads
in Fig. 3C'). After 6
hours of BrdU labeling, the intensively stained progenitor nuclei of the
control VZ reached the apical VZ, whereas in the
JoP6;Emx1IREScre VZ these nuclei were retained at
the basal VZ (Fig. 3D and
arrows in D') indicating cell cycle arrest or extended S phase.
Quantitation of equally sized areas of VZ with phospho-histone H3 (pHH3) at
E14.5 revealed 42±6% fewer mitotic cells at the apical VZ surface of
JoP6;Emx1IREScre as compared with JoP6
mice (P<0.001, n=14;
Fig. 3E,E'). Taken
together, these results indicate that in vivo activation of transgenic
Pax6 in the early cortical progenitors leads to a defect of
interkinetic nuclear migration, a reduction in progenitor proliferation, and
cortical hypocellularity.
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;
Heins et al., 2002), we
studied the effect of Pax6 GOF on neuronal differentiation in vivo by
immunostaining with monoclonal antibody (Tuj1) against neuron-specific class
III ß-tubulin. Despite the 21±2% reduction in
JoP6;Emx1IREScre cortical thickness compared with
JoP6 (P<0.001, n=12) mice, the thickness of the
Tuj1+ mantle zone of LP at E13.5 showed no significant difference
between genotypes, suggesting enhanced neuronal differentiation in
JoP6;Emx1IREScre mice
(Fig. 4A,A').
Furthermore, ectopic Tuj1+ cells were detected in the apical VZ
regions of the MP at E11.5 (Fig.
4C,C'), as well as in the LP and VP at E14.5
(Fig. 4B,B'), suggesting
premature neurogenesis in subsets of cortical progenitors. In addition, the
neuronal bHLH transcription factor gene Nex (Neurod6 - Mouse
Genome Informatics) showed a stronger in situ hybridization signal in the VP
and LP of the E11.5 JoP6;Emx1IREScre cortex as
compared with the control (see Fig. S1D,D' in the supplementary
material). Together, these results suggest that upon Pax6 activation
in vivo, a subset of cortical progenitors, mainly those within the VP and LP,
undergoes premature differentiation. Because of the cell aggregation detected after activation of transgenic Pax6 (Fig. 2E), we further analyzed the expression of genes involved in cell adhesion and cell signaling. q-PCRs performed with RNA extracted from E12.5 JoP6;Emx1IREScre cortex showed an increase in the expression of genes encoding the cell adhesion molecules N-CAM (Ncam1 - Mouse Genome Informatics) and R-cadherin (Cdh4 - Mouse Genome Informatics) as compared with the controls, whereas the expression of genes involved in cell-cell interaction and signaling such as paxillin (Pxn), tenascin C (Tnc), integrin beta 3 (Itgb3) and integrin alpha 5 (Itga5) were reduced (Fig. 4D). The change in the expression of these genes strongly suggests their involvement in the aggregation of cortical cells after activation of transgenic Pax6. However, further experiments are necessary to elucidate their individual roles.
Activation of transgenic Pax6 in the developing cortex causes apoptosis
In order to study whether the cortical hypoplasia seen in Pax6 GOF
in vivo might also involve an increase in cell death, we performed TUNEL
reactions. At stage E11.5, massive apoptosis could be detected in the
proliferating VZ of the JoP6;Emx1IREScre cortex,
in contrast to the JoP6 control
(Fig. 5A,A'). Double
immunolabeling against nestin, a marker for VZ progenitors associated with the
cell membrane, and cytoplasmic activated caspase 3 (Casp3) showed
colocalization in many cases suggesting that the cell death is confined to
progenitors (Fig. 5D-D'').
At stage E14.5, apoptosis was still detectable in progenitors of the
JoP6;Emx1IREScre cortex, although at a lower
level (Fig. 4B,B').
Labeling with antibodies against activated Casp3 implicated the
caspase-dependent pathway as causing apoptosis
(Fig. 4C,C'). At stage
E18.5, the activation of transgenic Pax6 expression was still
maintained, as judged by the expression of the lacZ reporter in the
VZ and CP (see Fig. 2G);
however, TUNEL reaction revealed no further apoptosis
(Fig. 4E). Recent evidence has
indicated that overexpression of ephrin A5 in vivo causes progenitor apoptosis
in the embryonic cortex (Depaepe et al.,
2005). Interestingly, both aggregates and single TUNEL+
cells in the JoP6;Emx1IREScre cortex show
enhanced ephrin A5 expression, suggesting that Pax6 might act in an
ephrin-A5-dependent manner (Fig.
4F).
When we examined whether Pax6 overexpression affects the early patterning of the cortical primordium, thereby possibly initiating progenitor apoptosis, we found that neither the dorsoventral nor the mediolateral patterning in the E11.5 JoP6;Emx1IREScre cortex was affected, as indicated by the normal expression of corresponding markers (e.g. Dlx1, Wnt3a, Emx2; see Fig. S1 in the supplementary material).
Pax6 GOF induces apoptosis in specific cortical progenitor pools
Several types of progenitors contribute to neurogenesis in the vertebrate
cortex: neuroepithelial cells (at the beginning of neurogenesis, E11) and RG
cells (after E13), which can be divided into
Ngn2+/Pax6-, RC2+/Pax6+ and
RC2+/Pax6-
(Guillemot, 2005;
Hartfuss et al., 2001) (Ngn2
is also known as Neurog2, and RC2 as Ifaprc2 - Mouse Genome Informatics). By
utilizing mouse lines with distinct spatiotemporal activation of the Cre
recombinase, we analyzed the effect of transgenic Pax6 activation in
different subsets of progenitors.
Recombination directed by Emx1IREScre starts at E9.5
and initially proceeds at highest level in the MP progenitors
(Li et al., 2003). Because the
expression of Pax6 in MP at this early stage is at the in situ
hybridization detection limit (Muzio et
al., 2002a), the massive apoptosis detected in the
JoP6;Emx1IREScre MP at E11.0 involves a set of
progenitors that either express endogenous Pax6 at extremely low
level or are Pax6-negative (Fig.
6A,B). The results suggest a high sensitivity of the early
progenitors of the MP to ectopic expression or enhancement of the
Pax6 expression level.
To gain insight into the consequences of overexpression of Pax6
specifically in Pax6-positive RG progenitors, we used the hGFAP-cre
line (Zhuo et al., 2001). This
line promotes activation of Cre recombinase in the majority of the
RC2+/Pax6+ radial glial progenitors as early as E13.5
(Götz et al., 1998;
Heins et al., 2002). After
overexpression of Pax6 in the JoP6;hGFAP-cre
cortex, as indicated by ß-gal staining
(Fig. 6C), massive apoptosis
was detected in single progenitors as well as in cell aggregates
(Fig. 6D,D'). Therefore,
we conclude that overexpression of Pax6 in the midgestation cortical
progenitors expressing Pax6 at a moderate level also leads to cell
death.
Interestingly, in the VP of JoP6;hGFAP-cre, where
endogenous Pax6 and its target Ngn2 are expressed at a very
high levels, apoptosis is seen only rarely
(Fig. 6D'). To
specifically examine the effect of Pax6 overexpression in the VP and
LP progenitors, we crossed JoP6 mice with the E1-Ngn2/Cre
line, in which Cre recombinase is directed by the E1 enhancer element of the
gene encoding transcription factor Ngn2
(Berger et al., 2004). Despite
significant recombination in the VP (as detected by ß-gal staining),
neither significantly increased apoptosis nor cell aggregates were seen at
stage E11.5 or E14.5 in the JoP6;E1-Ngn2/Cre
double-transgenic cortex as compared with the JoP6 control
(Fig. 6E-F' and data not
shown). Together, these data indicate that at the onset of neurogenesis, early
cortical progenitors show differential sensitivity towards the elevation of
the Pax6 expression level, which correlates inversely with their
endogenous Pax6 expression level: the highly Pax6-positive
progenitors of the rostral VP appear to be more resistant to Pax6
GOF, whereas the Pax6-negative progenitors, or progenitors that are expressing
Pax6 at extremely low level (e.g. in the MP), undergo apoptosis.
Pax6 GOF in postmitotic cells has no effect on cell survival
Although recombination and thus activation of transgenic Pax6 is
still detectable at E18.5 in postmitotic cells of the CP of
JoP6;Emx1IREScre mice, these cells do not undergo
apoptosis (Fig. 5G). In order
to directly assess the specific effect of the activation of transgenic
Pax6 expression in newly born neurons, we used the Nex-Cre
mouse line, which induces Cre recombinase activity in postmitotic neurons
after their exit from the mitotic cycle
(Schwab et al., 2000). In the
double-transgenic JoP6;Nex-Cre mice cortex, where the level
of Pax6 expression is higher than in the controls (see
Fig. 2I,J), no enhancement of
apoptosis was detected at E14.5 and P21
(Fig. 6H,H' and data not
shown). Thus, transgenic activation of Pax6 in vivo specifically
induces cortical progenitor apoptosis, whereas the fate of the postmitotic
neurons is not affected.
Pax6-induced apoptosis does not involve transcriptional activation of p53
Deregulation of proliferation and apoptosis is assumed to involve both the
p53 (Trp53) and pRb (Rb1)-dependent
pathways, where pRb prevents the induction of apoptosis through
transcriptional repression of p53
(Ookawa et al., 1997).
Previous evidence indicated that Pax6 binds to the human P53
(TP53) promoter with low affinity, although the effect on
p53 gene activity has not been assessed so far
(Stuart et al., 1995).
Remarkably, Pax6 also binds directly to pRb
(Cvekl et al., 2004),
suggesting the existence of a possible relationship between Pax6-,
pRb- and p53-dependent apoptosis. In an attempt to address this
issue, we studied the effect of Pax6 on the activity of the
P53-promoter in a human osteosarcoma cell line (SAOS2) lacking
endogenous pRb and p53. The reporter plasmid p53Luc
containing the P53 promoter followed by the luciferase gene was
co-transfected with pPax6 or pPax6-5a expression plasmids in
the absence and presence of a mouse pRb expression plasmid. As
illustrated in Fig. 7,
expression of Pax6 barely influenced luciferase activation via the
P53 promoter, whereas upon strong overexpression of Pax6 the
P53 promoter activity decreased. pRB was not able to significantly
enhance the effect of Pax6. Similar results were obtained with the cell lines
NIH-2H3, HeLa and HelaTAT (data not shown). Pax6-5a expression showed
no effect on the P53 promoter. Together, these results suggest that
the apoptosis induced by transgenic Pax6 in vivo is due neither to
activation of the cell death pathway through a direct transcriptional
activation of p53, nor by abolishment of the pRb-dependent active
repression of p53 activity.
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). To
study the in vivo activation of Pax6-5a, we generated the transgenic
line JoP6-5a based on the same principles as JoP6 (see
Materials and methods). Upon crossing with the Emx1IREScre
line, the functionality of the JoP6-5a line was tested: PCR with
primers JoP6F and JoP65aR detected recombination of genomic DNA (1955 bp and
394 bp fragments); expression of the two reporter genes gfp and
lacZ was monitored by GFP fluorescence and ß-gal staining,
respectively; and Pax6 immunostaining indicated the expression of transgenic
Pax6-5a (Fig.
8A-D'). Co-transfection experiments of pJoP6-5arec
with pHD and pPD indicated the functionality of transgenic
Pax6-5a (Fig. 8E). q-PCR showed
enhancement of Pax6-5a expression in the
JoP6-5a;Emx1IREScre cortex, which was much weaker
compared with the Pax6 enhancement in the
JoP6;Emx1IREScre cortex
(Fig. 8F). Statistical analysis
of the apoptotic pattern of JoP6-5a;Emx1IREScre
and control cortices at stages E11.5 and E13.5 revealed no difference, and no
cell aggregates were formed (n=5;
Fig. 8G,G').
Nevertheless, estimation of the BrdU index in the E11.5 DP after 30 minutes
labeling revealed a mild but significant reduction (by 10±2%) in
progenitor proliferation (P<0.001, n=4;
Fig. 8D,D'). Taken
together, these results suggest a difference in the biological activity of the
two Pax6 isoforms in vivo. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Heins et al.,
2002; Cartier et al.,
2006). Given the enhanced progenitor-proliferation rate found in
the Pax6 LOF Sey/Sey cortex at E10.5
(Warren et al., 1999) and
E14.5-E16.5 (Götz et al.,
1998), these findings confirm the pivotal role of both
Pax6 and Pax6-5a in controlling cortical progenitor
proliferation. Remarkably, in different environmental contexts, the two Pax6
isoforms exert different control functions on progenitor proliferation.
Pax6 enhances proliferation in the neural retina of vertebrates
(Marquardt et al., 2001) and
Drosophila (Dominguez et al.,
2004), but reduces proliferation of human glioblastoma cells
(Zhou et al., 2005),
cultivated corneal epithelial cells
(Ouyang et al., 2006), and
cortical progenitors in primary cell cultures
(Haubst et al., 2004;
Heins et al., 2002) as well as
in vivo (this work). Similarly, although Pax6-5a seems to promote
proliferation in the developing eye (Singh
et al., 2002), it inhibits proliferation of cortical progenitors
in vitro (Haubst et al., 2004)
and in vivo (this study).
The majority of the S-phase and many of the M-phase progenitors express
Pax6, suggesting that Pax6 regulates cell cycle progression
(Haydar et al., 2000). During
the transition from S to M phase, the nuclei of the progenitors migrate from
the basal to the apical surface of the VZ, a process termed interkinetic
nuclear migration. Previous results from our laboratory
(Götz et al., 1998) and
from other groups (Warren et al.,
1999) indicated that in Pax6 LOF, the interkinetic
nuclear movement of cortical progenitors is impaired, with more cells found in
the S phase as a result of a shorter cell cycle
(Estivill-Torrus et al.,
2002). Here we provide evidence that overexpression of
Pax6 in vivo leads to defects of mitotic cycle progression such that
many cells seem to be stuck or prolonged in S phase and a significantly
smaller proportion of progenitors undergo mitosis. In support of this
conclusion, recent results from quantitative FACS analysis demonstrate that
activation of Pax6 in HeLa cells strongly reduces the number of cells
in S and G2-M phases, indicating cell cycle arrest
(Cartier et al., 2006).
Similarly, overexpression of Pax6 in corneal epithelial cell lines
and primary cell culture causes inhibition of cell proliferation and
retardation of the cell cycle (Ouyang et
al., 2006).
Experiments involving Pax6 transduction in RG cell cultures and
adult neurospheres (Hack et al.,
2004; Heins et al.,
2002) as well as in HeLa cells
(Cartier et al., 2006) have
revealed premature neuronal differentiation. Owing to the massive apoptosis in
the JoP6;Emx1IREScre mice during early
corticogenesis, a quantitative estimation of possible premature neurogenesis
is difficult. However, we found that although the
JoP6;Emx1IREScre cortical thickness at E13.5 is
significantly reduced (by 21±2%, as compared with the control), the
thickness of the Tuj1+ mantle layer appeared unchanged.
Furthermore, the ectopically located Tuj1+ cells in the VZ, and the
enhanced Nex in situ hybridization signal in the E11.5 CP of
JoP6;Emx1IREScre mice, suggest enhanced
neurogenesis in the Pax6 GOF condition in vivo. Thus, subpopulations
of early progenitors of the JoP6;Emx1IREScre
cortex seem to exit prematurely from the mitotic cycle and differentiate,
which would diminish the cortical progenitor pool early in development and
contribute to the severe hypocellularity of the adult
JoP6;Emx1IREScre cortex. It would also be of
interest to test the expression of the potential Pax6 downstream
target Fabp7, recently reported to be involved in maintenance of
proliferation versus neuronal differentiation in cortical progenitors
(Arai et al., 2005).
In addition to misregulation of the mitotic cycle, we found that transgenic
Pax6 activation causes aggregation of cortical progenitors.
Previously, we reported on changes in the adhesive properties of isolated
cortical progenitors from Sey/Sey cortex and reduced expression of
R-cadherin (Stoykova et al.,
1997). After Pax6 GOF in vivo we found strong enhancement
of the expression of N-CAM, which encodes a cell-cell adhesion molecule
positively regulated by Pax6 (Holst et
al., 1998; Yamaoka et al.,
2000), as well as increased expression of R-cadherin. The latter
result further supports the idea of R-cadherin mediating
Pax6-dependent function in cell adhesion
(Andrews and Mastick, 2003),
possibly by direct genetic interaction of these two genes. We also found
Pax6-mediated inhibition of the expression of integrin alpha 5, which contains
Pax6-binding sites in its promoter (Duncan
et al., 2000), as well as a decrease in the expression levels of
integrin beta 3, paxillin and tenascin C, molecules involved in cell-cell
interaction. Given the complex pathways in which these proteins participate,
further detailed analysis is required to dissect the specific role of
Pax6 in these processes. However, the system for conditional
activation of Pax6 described here seems to be a reliable tool for
such studies in vivo.
Pax6 GOF in vivo induces apoptosis in specific sets of cortical progenitors
TUNEL assays and double immunohistochemistry with antibodies against
activated Casp3 and nestin revealed abundant apoptosis in the cortical
progenitors of E11.5 JoP6;Emx1IREScre mice. When
Pax6 GOF was directed into postmitotic cells by the Nex-Cre
mouse line (Schwab et al.,
2000), no apoptosis could be detected. These results provide the
first evidence that Pax6 is involved in progenitor apoptosis during
mammalian corticogenesis.
|
), the
apoptosis at the beginning of neurogenesis in
JoP6;Emx1IREScre mice is massive. Although
extensive apoptosis is apparent at E11.5, it weakens by E14.5, and at E18.5 no
increased cell death appears to occur. In parallel, a limited number of
ß-gal+ cells, marking recombined survived progenitors, are
found at E12.5, whereas at E15.5 and E18.5 the ß-gal staining is
progressively spreading into the IZ and CP, respectively, and at P28 the
entire depth of the cortex is populated by ß-gal+ cells. We
assume, therefore, that a significant part of the early (E9.5-E14.5) cortical
progenitors, where transgenic Pax6 is induced, undergo rapid
apoptosis and are removed from the cortex before they have accumulated enough
ß-gal to be confidently registered on thin sections, whereas the later
progenitors are affected much less by Pax6 GOF.
Subpopulations of cortical progenitors survive Pax6 GOF and are
monitored as ß-gal+ cells. By using different mouse lines for
regionalized recombination, we provide evidence that cortical progenitors in
vivo have spatiotemporal differences in their sensitivity towards
Pax6 GOF. In the JoP6;Emx1IREScre
cortex, activation of transgenic Pax6 is initiated at E9.5
predominantly in the MP, where endogenous Pax6 is only barely
expressed, if at all (Muzio et al.,
2002b). Thereafter, Pax6 GOF progressively spreads to the
majority of the glutamatergic cortical progenitors
(Li et al., 2003). Therefore,
the observed massive apoptosis in the MP of
JoP6;Emx1IREScre mice appears to be the result of
either ectopic expression of Pax6 in the early Pax6-negative
progenitor pool (neuroepithelial cells at E9.5-E12 in MP and Pax6-
RG progenitors), or overexpression of Pax6 in RG cells expressing
Pax6 only faintly. Also, in the JoP6;hGFAP-cre cortex, where
transgenic Pax6 becomes activated after E13.5 exclusively in the
RC2+/Pax6+ radial glial cells
(Malatesta et al., 2003;
Zhuo et al., 2001), extensive
apoptosis was detected, indicating that the overexpression of Pax6 in
the Pax6+ midgestation cortical progenitors leads to apoptosis as
well. In order to study the effect of Pax6 overexpression in the
early VP progenitors, where the endogenous Pax6 expression level is
at its highest (Stoykova et al.,
1997), we used the E1-Ngn2/Cre line
(Berger et al., 2004). This
line drives recombination directed by the E1-Ngn2 enhancer that is
activated only by a high dosage of Pax6
(Marquardt et al., 2001;
Scardigli et al., 2003). No
significant enhancement of apoptosis was observed in the
JoP6;E1-Ngn2/Cre cortex, suggesting that the VP and LP
progenitors with the highest level of endogenous Pax6 are resistant to further
elevation of Pax6. Collectively, these in vivo results demonstrate
that the cortical progenitors have different sensitivity towards the
Pax6 GOF condition, which is probably dependent upon the endogenous
Pax6 expression level.
The tumor suppressor gene p53 is involved in the control of cell
cycle arrest and apoptosis, inducing cell death upon activation
(Hickman et al., 2002). In an
attempt to study the molecular mechanism of the induced apoptosis in the
JoP6;Emx1IREScre cortex, we tested the effect of
Pax6 expression on the p53 promoter, which contains Pax6
target sequences (Stuart et al.,
1995). Pax6 protein binds to hypophosphorylated pRb
(Cvekl et al., 2004), whereas
pRb allows the formation of pRb-E2F complexes, which actively repress the
transcription of E2F-responsive promoters, including the pro-apoptotic gene
p53 (Ookawa et al.,
1997; Sellers et al.,
1995). Therefore, we also tested whether, upon overexpression,
Pax6 could sequester pRb and abrogate pRb-E2F-dependent repression of the
p53 promoter, thereby inducing apoptotic fate. We found that Pax6 is
unable to trigger p53 activation in vitro, but, by contrast, inhibits
p53 transcription upon strong overexpression and independently of
pRB. Therefore, although the underlying molecular mechanism of the
Pax6-induced apoptosis in vivo is still unclear, our results indicate
that this phenomenon is not likely to be a consequence of p53 pathway
activation. It is interesting to note that after overexpression of
Pax6, we detected a strong enhancement of ephrin A5 expression in
apoptotic cortical progenitors. Most intriguingly, a similar apoptotic
phenotype of early cortical progenitors has recently been discovered in GOF
experiments for ephrin A5 in transgenic mice in vivo
(Depaepe et al., 2005),
raising the possibility of genetic interplay between the Pax6- and
ephrin-A5-dependent pathways in the control of cortical progenitor
cell death.
Accumulating evidence supports the view that Pax6 is involved in
tissue growth, not only by modulating progenitor proliferation and cell cycle
progression, but possibly also by the involvement of apoptosis. Evidence has
been presented that a high copy-number of transgenic Pax6 leads to
microphtalmia in mice (Schedl et al.,
1996). Pax6 overexpression in cultivated corneal
epithelial cells slows down cell cycle progression and causes apoptosis
(Ouyang et al., 2006). Ectopic
activation of Pax6 in undifferentiated and mature pancreatic
ß-cells of transgenic mice inhibits progenitor proliferation and leads to
apoptosis (Yamaoka et al.,
2000), and activation of Pax6 suppresses tumorigenicity
of glioblastoma cells inducing apoptosis as well
(Zhou et al., 2005). In
addition, the Casp3 target gene Parp acts as a regulator of
Pax6 expression in neuroretina
(Plaza et al., 1999) and, in
developing Xenopus, expression of Pax6 at the neural-fold
stage overlaps with TUNEL-positive cells
(Hensey and Gautier, 1998). In
the Pax6 LOF mutant Sey/Sey, the failure in the
transition of the nasal ectoderm into nasal placode has been attributed to
abnormal apoptosis (Fukuda et al.,
2000), but no enhanced apoptosis is detected in the developing
cortex (Grindley et al., 1995)
(data not shown). Therefore, further research is required to reveal the
biological significance of the apoptosis induced by Pax6 GOF in vivo
in different cellular and experimental contexts.
We were unable to detect apoptosis in the cortical progenitors of
JoP6-5a;Emx1IRESCre mice. It should be noted,
however, that in contrast to the substantial elevation of the level of
Pax6 transcripts in JoP6;Emx1IRESCre
mice (3.8-fold higher, compared with the controls), the increase in the level
of Pax6-5a in JoP6-5a;Emx1IRESCre mice
was much less evident (1.2-fold, as compared with the controls). The
possibility remains that the Pax6-5a level necessary to induce apoptosis was
not achieved in this assay. Therefore, presently, it cannot be stated whether
the detected progenitor apoptosis is a specific feature of the in vivo
elevation of the Pax6 isoform only. However, in agreement with results from
Pax6 and Pax6-5a GOF experiments in vitro
(Haubst et al., 2004), we find
that the two Pax6 isoforms successfully repress progenitor proliferation in
the developing cortex.
Taken together with all the evidence available so far, the results presented in this study support the view that during corticogenesis, the modulation of Pax6 expression levels is crucial for progenitor cell fate acquisition as this influences cell proliferation, differentiation and apoptosis. Using the Pax6 GOF approach described here, we provide new in vivo evidence for a complex role of the Pax6 gene during multiple phases of mammalian corticogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/7/1311/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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