ldHuttner, 2005; Miller and Gauthier, 2007). Thus, maintenance
of neural progenitors until later stages of development is essen-
tial for the generation of cells both in correct numbers and with
a full spectrum of cell types. It has been shown that Notch signal-
ing plays an important role in the maintenance of neural progen-
itors (Artavanis-Tsakonas et al., 1999; Gaiano and Fishell, 2002;
Honjo, 1996; Selkoe and Kopan, 2003). Upon activation of Notch
signaling by its ligands, such as Delta-like1 (Dll1), the intracellular
domain of the transmembrane protein Notch (NICD) is released
from the membrane region and transferred into the nucleus,
where the NICD converts RBP-J from a repressor to an activator
latory expression is induced by serum stimulation or Notch
activation and is regulated by negative feedback: Hes1 can re-
press its own expression by directly binding to its own promoter
(Takebayashi et al., 1994), and repression of the promoter leads
to rapid disappearance of both Hes1 mRNA and Hes1 protein,
because they are extremely unstable, which allows the next
round of expression. In this way, Hes1 autonomously starts
oscillatory expression (Hirata et al., 2002). However, it remains
to be determined whether Hes1 expression oscillates in neural
progenitors.
To address these questions, we examined the dynamics ofOscillations in Notch Signa
Maintenance of Neural Prog
Hiromi Shimojo,
1
Toshiyuki Ohtsuka,
1
and Ryoichiro Kageya
1
Institute for Virus Research, Kyoto University, and Japan Science an
*Correspondence: rkageyam@virus.kyoto-u.ac.jp
DOI 10.1016/j.neuron.2008.02.014
SUMMARY
Expression of the Notch effector gene Hes1 is re-
quired for maintenance of neural progenitors in the
embryonic brain, but persistent and high levels of
Hes1 expression inhibit proliferation and differentia-
tion of these cells. Here, by using a real-time imaging
method, we found that Hes1 expression dynamically
oscillates in neural progenitors. Furthermore, sus-
tained overexpression of Hes1 downregulates ex-
pression of proneural genes, Notch ligands, and
cell cycle regulators, suggesting that their proper
expression depends on Hes1 oscillation. Surpris-
ingly, the proneural gene Neurogenin2 (Ngn2) and
the Notch ligand Delta-like1 (Dll1) are also expressed
in an oscillatory manner by neural progenitors, and
inhibition of Notch signaling, a condition known to
induce neuronal differentiation, leads to downregula-
tion of Hes1 and sustained upregulation of Ngn2 and
Dll1. These results suggest that Hes1 oscillation reg-
ulates Ngn2 and Dll1 oscillations, which in turn lead
to maintenance of neural progenitors by mutual
activation of Notch signaling.
INTRODUCTION
Neural progenitors change their competency over time, giving
rise to distinct types of cells during development (Alvarez-Buylla
et al., 2001; Fishell and Kriegstein, 2003; Fujita, 2003; Go¨tz andby forming a complex with it (Honjo, 1996; Selkoe and Kopan,
2003). The complex of NICD and RBP-J activates expression
of the basic helix-loop-helix transcriptional repressors Hes1
and Hes5 (Ohtsuka et al., 1999), downregulates proneural gene
52 Neuron 58, 52–64, April 10, 2008 ª2008 Elsevier Inc.Neuron
Article
ing Regulate
enitors
ma
1,
*
Technology Agency, CREST, Kyoto 606-8507, Japan
expression, and inhibits neuronal differentiation (Bertrand
et al., 2002; Ross et al., 2003; Kageyama et al., 2007). Inactiva-
tion of Hes1 upregulates expression of proneural genes, acceler-
ating neuronal differentiation (Ishibashi et al., 1995; Tomita et al.,
1996; Hatakeyama et al., 2004), whereas misexpression of Hes1
inhibits neuronal differentiation (Ishibashi et al., 1994; Ohtsuka
et al., 2001), suggesting that Hes1 is one of essential effectors
of Notch signaling. It has been shown that postmitotic neurons
express Notch ligands and activate Notch signaling of neighbor-
ing neural progenitors (Henrique et al., 1995; Myat et al., 1996;
Dunwoodie et al., 1997). However, Notch ligands are already
expressed in the developing nervous system before overt neuro-
nal differentiation (Bettenhausen et al., 1995; Hatakeyama et al.,
2004), raising the possibility that Notch ligands are also ex-
pressed by dividing neural progenitors.
Another issue is the expression mode of Hes1 in the develop-
ing nervous system. We previously found that Hes1 protein
expression by neural progenitors that actively proliferate and dif-
ferentiate is variable, with high levels in some cells, but lower
levels or no expression in others (Baek et al., 2006). Furthermore,
sustained Hes1 expression inhibits proliferation of cultured neu-
ral progenitors by G1 phase retardation, suggesting that Hes1
expression should be downregulated at some points of the cell
cycle (Baek et al., 2006). There are at least two possible explana-
tions for such variable levels of Hes1 expression. One is that
Hes1 expression is initially high in neural progenitors but is grad-
ually downregulated and finally lost during neuronal differentia-
tion. Another possibility is that Hes1 expression is oscillatory in
neural progenitors. We previously found that Hes1 expression
oscillates with a period of about 2 hr in cultured cells, such as
fibroblasts (Hirata et al., 2002; Masamizu et al., 2006). This oscil-Hes1 expression in neural progenitors by taking advantage of
a real-time imaging method that we previously developed
(Masamizu et al., 2006). We found that Hes1 expression dynam-
ically oscillates in neural progenitors. Furthermore, we found that

the proneural gene Neurogenin2 (Ngn2) and the Notch ligand
Dll1 are expressed in an oscillatory manner by neural progeni-
tors, and that these oscillations are regulated by Hes1 oscillation.
In contrast, downregulation of Hes1 expression, which is known
to induce neuronal differentiation, leads to sustained upregula-
tion of Ngn2 and Dll1 expression. These results suggest that
oscillations in Notch signaling play an important role in mainte-
nance of neural progenitors.
RESULTS
Hes1 Is Expressed at Various Levels
by Neural Progenitors
We first examined Hes1 expression in neural progenitors in the
developing telencephalon. Hes1 protein was expressed in the
ventricular zone of the developing nervous system (Figures 1A,
1E, and 1I). Virtually all Hes1-expressing cells were found to be
positive for Ki67, a marker for mitotic cells (see Figures S1A–
S1D available online), indicating that Hes1 is expressed only by
dividing cells. To reveal the relationship between Hes1 expres-
sion and the cell cycle, we administered BrdU to mouse embryos
at embryonic day (E) 14.5 and examined brain sections 30 min,
90 min, 8 hr, and 14 hr later, which corresponds to the labeling,
were labeled by antiphosphorylated histone H3 (pH3) antibody.
In addition, the cell cycle phases were assessed according to
the location of cell bodies of neural progenitors. During S phase,
cell bodies are present at the outer region of the ventricular zone
but descend toward the ventricular surface during G2 phase
(Fujita, 2003; Takahashi et al., 1993). Cell division occurs at the
ventricular surface, and cell bodies ascend during G1 phase
(Fujita, 2003; Takahashi et al., 1993).
Hes1 protein was expressed in the nuclei of neural progenitors
in S and G2 phases (Figures 1J–1L, Hes1
+
BrdU
+
). Some cells
expressed Hes1 protein at high levels (Figures 1J–1L, arrows)
whereas others expressed it at low levels (Figures 1J–1L, arrow-
heads). During M phase (pH3
+
), Hes1 protein was not expressed
by some cells but was present in the cytoplasm of others (Fig-
ures S1E–S1H). However, in early G1 phase, when cell bodies
were located near the ventricular surface, Hes1 protein expres-
sion was mostly absent (Figures 1B–1D; many BrdU
+
cells near
the ventricular surface were negative for Hes1). After early G1
phase, various levels of Hes1 expression occurred again in about
50% of the BrdU-labeled cells, whereas no expression occurred
in other BrdU
+
cells (some Hes1
+
and Hes1

cells are indicated
by arrows and arrowheads, respectively, in Figures 1F–1H).
Because neural progenitors undergo asymmetric cell division
Figure 1. Hes1 Expression in Neural Progenitors
BrdU was administered to E14.5 mouse embryos, and the telencephalon was examined 90 min, 8 hr, and 14 hr later, which corresponds to labeling of cells in
S-G2 phase (I–L), early G1 phase (A–D), and late G1 phase (E–H) , respectively. Hes1 protein expression and BrdU incorporation were immunohistochemically
analyzed. Hes1 expression occurred at variable levels from S to G2. Cells expressing Hes1 protein at high and low levels are indicated by arrows and arrowheads,
respectively (J and L). In early G1 phase, when cell bodies were located near the ventricular surface, Hes1 protein expression was mostly absent (B–D), (some
of them are indicated by arrows). After early G1 phase, various levels of Hes1 expression occurred again in about 50% of the BrdU-labeled cells, whereasno
expression occurred in other BrdU
+
cells. Some Hes1
+
and Hes1

cells are indicated by arrows and arrowheads, respectively (H). Scale bars, 50 mm (A), (E),
and (I) and 10 mm (B–D), (F–H), and (J–L).Neuron
Notch Signaling Oscillations in Neural Progenitorsrespectively, of cells in S phase, S-G2 phase, early G1 phase,
and late G1 phase (Takahashi et al., 1995). Cells in M phaseat this stage, which produces a new neural progenitor and a
neuron or a neuronal precursor, it is likely that half of the
Neuron 58, 52–64, April 10, 2008 ª2008 Elsevier Inc. 53