Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
Author for correspondence (e-mail:
tesaito{at}frontier.kyoto-u.ac.jp)
Accepted 5 January 2005
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SUMMARY |
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Key words: Notch, Electroporation, Neurogenesis, Cerebral cortex, Asymmetric division
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Introduction |
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Notch signaling plays a crucial role in cortical neurogenesis (for a
review, see Justice and Jan,
2002). Mutations in key components of the Notch signaling pathway
have revealed that Notch signaling is required for the maintenance of
self-renewal of progenitors (Nakamura et
al., 2000
; Hitoshi et al.,
2002
). Conversely, overexpression of a constitutively active form
of Notch (caNotch) inhibits neurogenesis in P19 cells
(Nye et al., 1994
) and
promotes the generation of radial glial cells (RGCs) in the embryonic brain
(Gaiano et al., 2000
). RGCs
had been thought to be specialized cells that provide a scaffold along which
newborn neurons migrate from the VZ to the cortical plate
(Rakic, 1972
). Recent reports
have uncovered another important role of RGCs, as progenitors that generate
neurons and glia (Malatesta et al.,
2000
; Noctor et al.,
2001
; Miyata et al.,
2001
). Although the majority of progenitors in the VZ are RGCs
(Noctor et al., 2002
), RGCs
exhibit different neurogenic and gliogenic features, depending on their
location and developmental stage
(Malatesta et al., 2003
;
Anthony et al., 2004
). Because
the activation of the Notch pathway leads to gilal differentiation in various
settings (for a review, see Gaiano and
Fishell, 2002
), cortical cells overexpressing caNotch may be fated
to become gliogenic. The in vivo features of caNotch-expressing cells and
their daughter cells remain to be defined. To clarify these issues, we used
forced expression of caNotch in a spatially restricted domain using
in vivo electroporation. The activity of caNotch was temporally
controlled by the Cre recombinase-loxP system.
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Materials and methods |
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BrdU labeling analysis
To evaluate the activity of cell proliferation, pregnant mice were injected
at E14.5 intraperitoneally with 125 µg/gm body weight of bromodeoxyuridine
(BrdU) (Sigma), and embryos were collected 2 hours after the injection. To
examine what percentage of transfected cells in the VZ were proliferating,
cumulative labeling was performed by injecting BrdU three times every 5.5
hours from E14.5, and embryos were collected 4 hours after the final
injection. To determine the birth dates of neurons, P5 brains were recovered
after single injection of 50 µg/gm body weight of BrdU at E14.5 or
E16.5.
Immunostaining
Immunohistochemistry was performed as described
(Saba et al., 2003). Fixed
embryos and brains were embedded in OCT compound and sliced at 20 µm using
a cryostat. Primary antibodies included mouse anti-BrdU (BD Pharmingen),
rabbit anti-GFP (Molecular Probes), rabbit anti-LH2A/B, rabbit anti-ER81, goat
anti-ß-galactosidase (Biogenesis), mouse monoclonal anti-glial fibrillary
acidic protein (GFAP, Sigma), mouse monoclonal M2 anti-FLAG (Sigma), and mouse
monoclonal RC2 (Developmental Studies Hybridoma Bank, University of Iowa). The
anti-LH2A/B (Liem et al.,
1997
) and anti-ER81 (Arber et
al., 2000
) antibodies were gifts from T. Jessell (Columbia
University). Secondary antibodies were donkey anti-rabbit IgG conjugated with
Alexa Fluor 488 or 555 (Molecular Probes), donkey anti-goat IgG conjugated
with Alexa Fluor 647 (Molecular Probes), and donkey anti-mouse IgG or IgM
conjugated with Cy3 (Jackson ImmunoResearch). Apoptosis was examined by using
an In Situ Cell Death Detection Kit, TMR Red (Roche). Fluorescent images were
analyzed using a confocal microscope LSM510 (Zeiss). Each image was the
Z-projection of at least 20 optical planes through a depth of 11 µm. To
quantify the intensity of immunofluorescent signals, they were recorded under
the same non-saturated conditions and measured using an LSM510 software
release 3.0 (Zeiss). The BrdU+ cells were classified into two
groups on the basis of their relative fluorescence intensity (RFI).
Populations of cells that exhibited the highest fluorescence
(RFI=100.0±4.3) were designated heavily labeled. Cells that showed
lower fluorescence (RFI=51.1±4.3, 27.5±3.9 and 14.7±3.6)
were designated lightly labeled. Each result of immunohistochemistry was
replicated using several brains derived from at least three operated pregnant
mice.
Transplantation
In utero transplantation was performed as described
(Desai and McConnell, 2000;
Wichterle et al., 2001
), with
modifications. pCAG-ExZ or pCAG-ExNotchZ was transfected at E13.5, and
EYFP+ tissue was dissected from the VZ of the somatosensory area 3
hours after transfection of Cre at E15.5. As a control, tissue was
dissected 3 hours after transfection of pCAG-ExZ at E13.5. Cells were
mechanically dissociated and rinsed twice in L-15 medium containing 10
µg/ml of DNase I (Worthington), and suspended with L-15 medium. Cell
suspension (
500,000 cells/µl) was injected into the dorsal
telencephalon. At P5, brains were recovered. A three-hour incubation between
transfection and dissection was necessary to obtain better survival of
dissociated cells. Cells prepared 3 hours after transfection showed
essentially the same laminar patterns as those prepared immediately after
transfection (data not shown). EYFP+ cells were detected in the
dorsal but not ventral telencephalon (number of examined brains=52).
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Results |
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Contrastingly, electroporation at E15.5 labeled cells of layers II and III,
which were positive for Lhx2 (Fig.
2E-H,O-Q). The laminar fate of
EYFP+/ß-gal+ neurons after electroporation at these
two stages was in agreement with previous birth date analyses of neurons using
nucleotide analogs (Takahashi et al.,
1999). These results indicate that early progenitors transfected
at E13.5 generate neurons of both low and upper layers, whereas late
progenitors transfected at E15.5 generate neurons of only layers II and
III.
Sequential generation of low- and upper-layer neurons
To examine whether progenitors transfected at an early stage were
transfectable again at later stages, we performed double electroporation using
DsRed and EYFP. Electroporation of DsRed at E13.5
labeled neurons of both low and upper layers
(Fig. 3A), in the same fashion
as that of EYFP (Fig.
2A). DsRed signals were distributed as dots showing mitochondrial
sites, because DsRed was fused with a mitochondrial-targeted peptide
(Saito and Nakatsuji, 2001).
Double electroporation of DsRed at E13.5 and EYFP at E15.5
demonstrated DsRed+ neurons in layers II to VI and EYFP+
neurons in layers II and III (Fig.
3C). The laminar positions of labeled neurons were the same as
those by single electroporation, showing that the laminar fate of neurons is
not affected by double electroporation. Many EYFP+ later-born
neurons were also positive for DsRed (Fig.
3D), indicating that early progenitors transfected at E13.5 are
transfectable again at E15.5.
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The caNotch+ cells exhibited morphologies typical of RGCs, which extended a long thin process to the pial surface (Fig. 4H). Almost all (94.7±1.3%) of the caNotch+ cells were also immunoreactive with the RC2 antibody, a molecular marker for RGCs (Fig. 4I,J), confirming that the caNotch+ cells were RGCs. EYFP+ RGCs were also observed after transfection of EYFP alone (data not shown), but the ratio of RC2+ cells in the EYFP+ population was lower (71.8±2.9%), consistent with the generation of differentiating EYFP+ cells from progenitors. Taken together with the above BrdU-labeling data, these findings further suggest that caNotch keeps progenitors proliferating, and suggest that the caNotch+ cells may divide symmetrically without generating neurons, although we can not completely rule out the possibility that the caNotch+ cells generate two types of progenitors.
To confirm that the increment of the BrdU+ and RC2+ ratios by caNotch was not caused by cell death of a subset of cells, we performed TUNEL assays (Fig. 4K-M). No significant increase in cell death was detected by caNotch transfection, confirming the maintenance of proliferative progenitors by caNotch.
Neurons are generated after the removal of caNotch activity
After caNotch transfection at E13.5, EYFP+ cells
remained in a thin area adjacent to the ventricle in the P5 cortex
(Fig. 5A). At P15,
EYFP+ cells were positive for GFAP in layers adjacent to the
ventricle (see Fig. S1 in the supplementary material), consistent with
previous results obtained by retroviral expression of caNotch
(Gaiano et al., 2000).
Persistent expression of caNotch in the EYFP+ cells was confirmed
by immunostaining using an antibody against the FLAG peptide, which was fused
to the caNotch protein (Fig.
5E-G). Subsequent transfection of a Cre expression vector
at E15.5 generated EYFP+/ß-gal+ neurons
accumulating in layers II and III (Fig.
5B,C). No ß-gal+ cells were detected in brains
transfected with pCAG-ExNotchZ alone (data not shown). The
EYFP+/ß-gal+ neurons were not stained with the
anti-FLAG antibody (Fig. 5H),
confirming that ß-gal was expressed in caNotch-negative
(caNotch-) cells after the excision of caNotch. These
results suggest that neurogenesis resumed after the removal of
caNotch. The EYFP+/ß-gal+ neurons were
positive for Lhx2 (Fig.
5D,I-K), but not for ER81 (data not shown), indicating that the
neurons acquired the specificity of layers II and III, with respect to not
only laminar positions but also molecular markers. Similarly, transfection of
pCAG-ExZ at E15.5 labeled Lhx2+ neurons of layers II and III
(Fig. 2). Therefore, these
results suggest that progenitors skipped the generation of early-born neurons
during Notch activation and started generating later-born neurons in
accordance with the embryonic stage after the removal of caNotch activity.
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Neurons of upper layers are born after Cre transfection
These results, taken together with the finding that the vast majority of
caNotch+ cells were proliferating progenitors, suggested that the
Lhx2+/ß-gal+ neurons were born after the removal of
caNotch activity. However, we could not rule out the possibility that a minor
population of the caNotch+ cells had exited the cell cycle but did
not migrate into the cortical plate until the removal of caNotch activity. To
eliminate this possibility, we analyzed birth dates of the
EYFP+/ß-gal+ neurons. Cells were pulse-labeled with
BrdU before and after Cre transfection and examined at P5.
One day before Cre transfection, BrdU was injected at E14.5.
BrdU+ cells were observed in layers II to V
(Fig. 7). Neurons that were
heavily-labeled with BrdU mostly occupied low layers, whereas many
lightly-labeled neurons were scattered in the upper layers, consistent with
the inside-out generation of neurons (Fig.
7A). Overall patterns of BrdU+ cell distribution were
not affected by caNotch (Fig.
7E), showing that corticogenesis was not severely perturbed by
caNotch, presumably due to its restricted expression. The degree of BrdU
labeling, which is revealed by fluorescence intensity, is indicative of birth
dates of BrdU+ cells. The cohort of cells that exit the cell cycle
after incorporating BrdU will show maximum levels of fluorescence. Because the
amount of BrdU incorporated in nuclei is reduced by half every cell division,
successively weaker fluorescence will be detected in cells that have executed
the cell cycle after BrdU labeling. Quantification of fluorescence
distinguished at least two groups of BrdU-labeled cells: heavily labeled
(RFI=100.0±4.3), and lightly labeled (RFI 51.1±4.3). After
EYFP transfection, many EYFP+ neurons of layer IV were
heavily labeled with BrdU (Fig.
7B-D), suggesting that these neurons were the first born cells
after BrdU labeling. The ratio of EYFP+ neurons that were lightly
labeled with BrdU increased in upper layers, reflecting that the neurons were
born after further rounds of cell divisions. By contrast, caNotch
transfection resulted in no EYFP+ neurons in layer IV, and many
EYFP+ neurons were lightly labeled with BrdU and localized in upper
layers (Fig. 7F-H). These
findings suggest that the EYFP+/ß-gal+ neurons of
layers II and III were not born from caNotch+ progenitors before
Cre transfection.
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Discussion |
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The laminar fate of cortical neurons is determined by intrinsic properties
of progenitors and extracellular signals. Previous transplantation experiments
have shown that early progenitors are competent to generate both low- and
upper-layer neurons, whereas late progenitors lose competence to generate
low-layer neurons (McConnell and
Kaznowski, 1991; Frantz and
McConnell, 1996
). Our results indicate that caNotch+
progenitors become competent to generate upper-layer neurons after the removal
of caNotch activity. Moreover, the use of molecular markers revealed that
these upper-layer neurons were correctly specified as neurons of layers II and
III. Although we focused on the primary somatosensory area in this study
because of the availability of markers, the generation of upper-layer neurons
instead of low-layer neurons after the removal of caNotch was also
observed in other areas of the cortex (data not shown). Thus, the effect of
caNotch and the resumption of neurogenesis were not specific to the primary
somatosensory area. These findings corroborate that specification of laminar
fate involves extracellular cues that depend on the developmental stage. In
Drosophila, different types of neurons are generated by asymmetric
divisions of neuroblasts, which alter the expression of intrinsic factors
successively every division. Recently, continuous expression of
Hunchback, which is expressed by neuroblasts at early stages, has
been shown to keep neuroblasts competent to generate early-born neurons
(Pearson and Doe, 2003
). In
early corticogenesis, Foxg1 controls production of low-layer neurons by
suppressing the generation of CR cells
(Hanashima et al., 2004
).
E15.5 progenitors lost the ability to generate low-layer neurons, suggesting
that intrinsic factors in progenitors may also be important for the
specification of neurons of layers II to VI.
Many cell fates are determined through asymmetric divisions, when two daughter cells with distinct fates are generated. Asymmetric distribution of cell fate determinants is crucial for proper differentiation of daughter cells. Asymmetric divisions may also affect the state of progenitors. In our present work, inhibition of neurogenesis and maintenance of proliferation of cortical progenitors by caNotch suggest that caNotch+ progenitors may have undergone symmetric divisions. Although it was not possible to directly determine that the two daughter cells from a caNotch+ progenitor shared the same molecular properties, neurogenesis was synchronous with embryonic development when it resumed. This also appeared to occur uniformly, further supporting symmetric divisions of caNotch+ progenitors. Therefore, these results suggest that temporal inhibition of asymmetric divisions may not affect subsequent neurogenesis of cortical progenitors.
The maintenance of cortical progenitors by caNotch is consistent with
previous results obtained from in vitro cell cultures using loss-of-function
mutants of Hes1 and RBP-J, which are downstream effectors of the Notch
receptor (Nakamura et al.,
2000
; Hitoshi et al.,
2002
). The Notch pathway is activated by extracellular ligands
such as Delta-like and Jagged (for a review, see
Lai, 2004
). Considering that a
large number of neurons are generated from many progenitors simultaneously in
the mammalian cerebral cortex, a flexible way to control the number of
generated neurons using the balance between symmetric and asymmetric divisions
of progenitors may be a more advantageous mechanism than the fixed sequential
generation of neurons in Drosophila. ß-Catenin signals have also
been shown to regulate the balance between symmetric and asymmetric divisions
of cortical progenitors (Chenn and Walsh,
2002
). However, some neurons still differentiate from progenitors
in which ß-catenin signaling is active. By contrast, few neurons were
generated from caNotch+ progenitors. This may suggest that the
ß-catenin signaling pathway is different from the Notch pathway, even if
they have similar effects on progenitors. Mice carrying gain-of-function
mutations in the ß-catenin signaling pathway exhibit severe malformation
in the cortex (Chenn and Walsh,
2002
). caNotch transfection showed milder effects on the
morphology of the cortex, presumably because of its spatiotemporally
restricted expression.
The Notch pathway controls many cellular differentiation programs depending
on the developmental context. One of the well-known roles of the Notch pathway
is to keep progenitors in the undifferentiated state (for a review, see
Artavanis-Tsakonas et al.,
1999). Injection of caNotch mRNA into Xenopus
embryos has suggested the maintenance of progenitors by caNotch
(Coffman et al., 1993
).
However, control of the timing of caNotch activity has been difficult. Our
experimental system using in vivo electroporation and Cre recombinase partly
overcame this difficulty and revealed the resumption of neurogenesis after the
removal of caNotch activity. Notch activation has been also implicated in
gliogenesis (for a review, see Gaiano and
Fishell, 2002
). Transient Notch activation irreversibly switches
neural crest stem cells to a Schwann cell fate
(Morrison et al., 2000
).
Retroviral expression of caNotch leads to the formation of RGCs
(Gaiano et al., 2000
), but it
was not clear whether RGCs generated by forced expression of caNotch have
neurogenic potential. Our findings indicate that cortical progenitors maintain
neurogenic potential even after being kept as RGCs by caNotch. This contrasts
with the irreversible switching of neural crest stem cells. These different
actions of the Notch pathway may be explained by the difference in cellular
contexts. In Drosophila, the Notch pathway regulates gcm,
which is crucial for glial differentiation, positively for subperineurial glia
(Udolph et al., 2001
) and
negatively for peripheral sensory organs
(Van De Bor and Giangrande,
2001
). It will be important to identify cellular factors that are
involved in different actions of the Notch pathway.
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Supplementary material |
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ACKNOWLEDGMENTS |
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Footnotes |
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