Resource
Visualizing Spatiotemporal Dynamics
of Multicellular Cell-Cycle Progression
Asako Sakaue-Sawano,1,3 Hiroshi Kurokawa,1,4 Toshifumi Morimura,2 Aki Hanyu,5 Hiroshi Hama,1 Hatsuki Osawa,1
Saori Kashiwagi,2 Kiyoko Fukami,4 Takaki Miyata,6 Hiroyuki Miyoshi,7 Takeshi Imamura,5 Masaharu Ogawa,2
Hisao Masai,8 and Atsushi Miyawaki1,3,*
1Laboratory for Cell Function and Dynamics
2Laboratory for Cell Culture Development
Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan
3Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan
4School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
5Departments of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku,
Tokyo 135-8550, Japan
6Department of Anatomy and Cell Biology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Syowa-ku,
Nagoya, Aichi 466-8550, Japan
7Subteam for Manipulation of Cell Fate, BioResource Center, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
8Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan
*Correspondence: matsushi@brain.riken.jp
DOI 10.1016/j.cell.2007.12.033
SUMMARY
The cell-cycle transition from G1 to S phase has been
difficult to visualize. We have harnessed antiphase
oscillating proteins that mark cell-cycle transitions
in order to develop genetically encoded fluorescent
probes for this purpose. These probes effectively label
individual G1 phase nuclei red and those in S/G2/
M phases green. We were able to generate cultured
cells and transgenic mice constitutively expressing
the cell-cycle probes, in which every cell nucleus exhibits
either red or green fluorescence. We performed
time-lapse imaging to explore the spatiotemporal
patterns of cell-cycle dynamics during the
epithelial-mesenchymal transition of cultured cells,
the migration and differentiation of neural progenitors
in brain slices, and the development of tumors
across blood vessels in live mice. These mice and
cell lines will serve as model systems permitting unprecedented
spatial and temporal resolution to help
us better understand how the cell cycle is coordinated
with various biological events.
INTRODUCTION
Considerable progress has been made toward understanding
the mechanism of cell-cycle progression in individual cells
(Nurse et al., 1998; Nurse, 2000). However, the cell cycle is regulated
not only by intracellular signals, but also by extracellular
signals, and less is known about how the cell cycle is coordinated
with differentiation, morphogenesis, and cell death in a
multicellular context. While the transition from M to G1 phase—
namely cell division—can be monitored by morphological
changes, the transition from G1 to S is difficult to observe in
live samples. To date, the G1/S transition has mostly been observed
either after nuclear bromodeoxyuridine (BrdU) staining,
or by synchronizing the cell cycle by pharmacological means.
Recently, several cell-cycle markers that identify the S phase
and the subsequent transition to G2 in live cells have been developed
by fusing fluorescent proteins to proliferating cell nuclear
antigen (PCNA) (Leonhardt et al., 2000; Essers et al., 2005; Kisielewska
et al., 2005), DNA ligase I (Easwaran et al., 2005), or the
C terminus of helicase B (GE healthcare). However, since identification
of cell-cycle transitions requires the detection of subtle
and often minute changes in the distribution pattern and intensity
of fluorescence signals, these markers cannot track phase transitions
with high contrast.
In addition to being regulated at the transcriptional and posttranslational
levels, the cell cycle is controlled by ubiquitin
(Ub)-mediated proteolysis (Figure 1A) (Ang and Harper, 2004;
Nakayama and Nakayama, 2006). The APCCdh1
and SCFSkp2
complexes are E3 ligase activities that mark a variety of proteins
with Ub in a cell cycle-dependent manner (Vodermaier, 2004).
Because the SCFSkp2
complex is a direct substrate of the
APCCdh1
complex but also functions as a feedback inhibitor of
APCCdh1
(Wei et al., 2004; Benmaamar and Pagano, 2005), these
two ligase activities oscillate reciprocally during the cell cycle.
The APCCdh1
complex is active in the late M and G1 phases, while
the SCFSkp2
complex is active in the S and G2 phases.
Two direct substrates of the APCCdh1
and SCFSkp2
complexes,
Geminin and Cdt1, are involved in ‘‘licensing’’ of replication origins
(Nishitani et al., 2000). This carefully regulated process ensures
that replication occurs only once in a cell cycle. In higher
eukaryotes, proteolysis and Geminin-mediated inhibition of
the licensing factor Cdt1 are essential for preventing re-replication.
Due to cell cycle-dependent proteolysis, protein levels of
Geminin and Cdt1 oscillate inversely. Western blot analysis of
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synchronized cultured cells has shown that Cdt1 levels are highest
during G1, while Geminin levels are highest during the S, G2,
and M phases (Nishitani et al., 2004). In this study, we harnessed
the regulation of cell cycle-dependent ubiquitination in order to
develop two genetically encoded indicators for cell-cycle pro-
gression.
RESULTS AND DISCUSSION
Construction of Cell-Cycle Probes
We fused red- and green-emitting fluorescent proteins to E3 ligase
substrates, Cdt1 and Geminin, to develop dual-color fluorescent
probes that indicate whether individual live cells are in
G1 phase or S/G2/M phases (Figure 1B). First, a fast-folding variant
of mKO (monomeric version of Kusabira Orange) (Karasawa
et al., 2004) was generated (H.O.S. Karasawa and A.M., unpublished
data) and named mKO2. mKO2 was fused to full-length
human Cdt1 (hCdt1) (Figure 1C, [1–546]). When the chimeric
protein was expressed in HeLa cells under the control of the
ubiquitous CMV promoter, red fluorescence was observed in
the nuclei of a fraction of cells. Cells were then time-lapse imaged
using computer-assisted fluorescence microscopy (Olympus,
LCV100). Cell morphology was monitored by differential
interference contrast (DIC) to follow cell division. During the first
48 hr after transfection, we observed a sudden disappearance of
red fluorescence, suggesting that mKO2-hCdt1 protein was
being degraded by the SCFSkp2
complex at the onset of S phase.
However, after 48 more hours, we noticed that the transfected
cells failed to proceed to mitosis, whereas nontransfected cells
divided normally. This is consistent with the fact that overexpression
of hCdt1 causes re-replication of DNA (Vaziri et al., 2003;
Blow and Dutta, 2005). In addition, we were unable to obtain
any healthy stable transformants expressing the chimeric
protein.
To overcome the cell-cycle arrest, numerous hCdt1 deletion
mutants were constructed and fused to mKO2 (Figure 1C),
then evaluated for cell cycle-dependent red fluorescence in the
nucleus by time-lapse imaging. As expected, the Cy motif (amino
acids 68 – 70), which binds to the SCFSkp2
E3 ligase (Nishitani
et al., 2006), was required for proper function. Although the
N-terminus of hCdt1 (amino acids 1–10) binds to a different E3
ligase (Cul4) (Senga et al., 2006; Nishitani et al., 2006), removal
of this region appeared to be critical for the establishment of stable
transformants with normal cell division. Since Cdt1 degradation
by the SCFSkp2
complex has been shown to be independent
of its binding to Geminin (Lee et al., 2004; Nishitani et al., 2004;
Sugimoto et al., 2004), we also removed the Geminin-binding region.
The resulting truncated hCdt1 protein (amino acids 30–120)
is sufficient for marking cells in G1 phase [mKO2-hCdt1(30/120),
Figure 1C]. Interestingly, mKO2 in mKO2-hCdt1(30/120) could
not be replaced with mAG (the monomeric version of Azami
Green) (Karasawa et al., 2003), mEGFP, or mRFP1; the use of
these latter fluorescent proteins resulted in constant fluorescence
signal throughout the cell cycle (Figure 1C).
Like Cdt1, the cyclin-dependent kinase (Cdk) inhibitor p27 is
ubiquitinated by the SCFSkp2
complex. Since a p27-luciferase
(p27Luc) fusion protein can be used to monitor Cdk2 inhibitor
pharmacodynamics in vivo (Zhang et al., 2004), we attempted
to fuse fluorescent proteins to p27 or the p27 domains recognized
by ubiquitin ligase, but none of the fusions produced
bright, cell cycle-dependent fluorescence comparable to that
observed with mKO2-hCdt1(30/120) (data not shown).
Next, mKO2 was fused to truncated versions of human Geminin
(hGem) (Figure 1D) and tested as described for mKO2hCdt1.
A chimeric protein composed of mKO2 and the 110
amino acid N-terminus of hGem [mKO2-hGem(1/110)] showed
the best performance among the constructs tested. It should
be noted that mKO2-hGem(1/110) also lacks the Cdt1 binding
region (Lee et al., 2004). Unlike hCdt1(30/120), hGem(1/110)
could be fused to several other fluorescent proteins. mAG was
substituted to generate a green version, mAG-hGem(1/110)
(Figure 1D).
Cell Cycle Analysis of Cultured Cells Stably
Expressing the Cell-Cycle Probes
We next used lentiviral vectors for coexpression of the two constructs
in HeLa cells. Since this gene-transfer technique is highly
efficient, cotransduction allowed us to obtain stable transformants
expressing equivalent levels of mKO2-hCdt1(30/120) and
mAG-hGem(1/110). In each transformant, red fluorescence alternated
with green fluorescence in the nucleus (Movie S1 available
online). A typical time series is shown in Figure 1E. The cellcycle
period was variable, presumably due to differences in cell
density and serum concentration. Since the green fluorescence
disappeared rapidly in late M phase and the red fluorescence became
detectable in early G1 phase, a small gap in fluorescence
Figure 1. Development and Characterization of a Fluorescent Indicator for Cell-Cycle Progression
(A) Cell-cycle regulation by SCFSkp2
and APCCdh1
maintains bistability between G1 and S/G2/M phases.
(B) A fluorescent probe that labels individual G1 phase nuclei in red and S/G2/M phase nuclei green.
(C) Various constructs with concatenated mKO2 and deletion mutants of human Cdt1 for labeling nuclei in G1 phase. Grey box, QXRVTDF motif (amino acids
1–10); blue box, Cy motif (amino acids 68–70); cyan box, Geminin binding domain (data from mouse Cdt1). Symbols and abbreviations are as follows: B,
pass; 3, failure; N, nucleus; C, cytosol.
(D) Various constructs with concatenated mAG and deletion mutants of human Geminin for labeling nuclei in S, G2, and M phases. Pink box, D (destruction) box;
black box, NLS; yellow box, coiled coil domain (Cdt1 binding domain). Symbols and abbreviations are as follows: B, pass; 3, failure; D, marginal; —, not determined;
N, nucleus; C, cytosol.
(E) Cell cycle-dependent changes in fluorescence of mKO2-hCdt1(30/120) and mAG-hGem(1/110) in HeLa cells. Arrows indicate cells that were tracked. The
scale bar represents 10 mm.
(F) Typical fluorescence images of HeLa cells expressing mKO2-hCdt1(30/120) and mAG-hGem(1/110) and immunofluorescence for incorporated BrdU at G1,
G1/S, S, G2, and M phases. The scale bar represents 10 mm.
(G) HeLa cells showing red [mKO2(+)mAG(-)], yellow [mKO2(+)mAG(+)], and green [mKO2(-)mAG(+)] fluorescence were collected, and their DNA contents were
stained with Hoechst33342 and measured using a fluorescence-activated cell sorter.
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was observed in newborn daughter cells. By contrast, during
red-to-green conversion, red and green fluorescence always
overlapped to yield a yellow nucleus. To examine whether the
timing of the color conversion correlates with the onset of S
phase, transformants were pulse-labeled with BrdU for five minutes
and immediately immunostained for BrdU. Typical confocal
images of cells at the G1/S transition and in the G1, S, G2, and M
phases are shown in Figure 1F (see Figures S1A–S1D for a widefield
image). Since all of the cells with yellow nuclei showed BrdU
incorporation, the emergence of the green fluorescence is indicative
of the initiation of the S phase. Similar results were obtained
from a separate experiment in which we immunostained for
PCNA (Bravo and Macdonald-Bravo, 1987) (Figure S1E). Cells
with nuclei emitting pure green fluorescence were also observed.
These cells were either in the S or G2 phase, and were
distinguishable by nuclear BrdU or PCNA immunostaining.
These results are consistent with the fact that Cdt1 accumulates
in G1, while Geminin accumulates in S/G2/M phases.
We named this fluorescent, ubiquitination-based cell cycle indicator,
‘‘Fucci.’’ Analysis of DNA content by flow cytometry
revealed the same distribution between Fucci-expressing and
parental HeLa cells (Figure 1G, left). Cells expressing Fucci
were divided into red-, yellow-, and green-emitting populations
[mKO2(+)mAG(-), mKO2(+)mAG(+), mKO2(-)mAG(+), respectively]
(Figure 1G, middle), and their DNA contents were analyzed
following Hoechst33342 staining. Green and yellow cells had
fully- and partially-replicated complements of DNA, respectively
(Figure 1G, right). Thus, differential profiling of cells at G1 and
S/G2/M phases can be achieved by sorting a population of cells
into red, yellow, or green and examining various cellular functions,
such as gene expression and antigen surface expression.
Stable transformants constitutively expressing Fucci were obtained
using other cell lines, including normal murine mammary
gland (NMuMG) cells, PC12 cells, and COS7 cells.
Monitoring Structural and Behavioral Changes
and Cell-Cycle Dynamics of Cultured Cells
The epithelial-mesenchymal transition (EMT) is a fundamental
morphogenetic process by which mesenchymal cells are formed
from epithelia during embryonic development, wound repair,
and tumor progression in multicellular organisms (Thiery and
Sleeman, 2006). In vitro EMT is characterized by dissolution of
cell-cell junctions, cytoskeletal rearrangements, and increased
motility of cultured cells. It is possible that specific stages of
the cell cycle are involved in the process. Indeed, it was recently
reported that transforming growth factor b (TGFb) efficiently induced
EMT in AML-12 hepatocytes synchronized at the G1/S
phase, but not in cells synchronized at the G2/M phase (Yang
et al., 2006). Moreover, NMuMG cells undergo EMT in response
to TGFb (Piek et al., 1999; Tojo et al., 2005).
To examine cell-cycle progression during EMT, we examined
Fucci-expressing, stably transformed NMuMG cells. After cells
were plated on a glass coverslip, they proliferated as clusters
maintaining cell-cell adhesion with their neighbors (Figure 2A,
1 hr). The high proliferation rate of these cells was evidenced
by the large fraction of cells with green nuclei (Figure 2A, 25–
49 hr). However, at confluence, the green nuclei were replaced
with red nuclei (Figure 2A, 73 hr), indicating that cells remained
in G1 phase (Movie S2, left). When we introduced a wound in
the confluent monolayer (Figure 2C, 1 hr), cells at the edge of
the wound turned green (Figure 2C, 13 hr, arrows), indicating
that closure of the wound required proliferation of NMuMG cells.
Notably, the green nuclei appeared 9-13 hr after wound induction.
Such a time delay of more than 8 hr was reproducibly observed
in other similar wound healing experiments and is reminiscent
of the 8 hr required for NIH 3T3 cells to re-enter the
cycle from a state of quiescence (G0) after the onset of proliferation
stimuli (Zetterberg and Larsson, 1985). It is thus possible
that the confluent NMuMG cells (Figure 2A, 85 hr) remained in
G0 phase.
Next, we performed the same experiments in the presence of
1 ng/ml of TGFb. Within one day following TGFb treatment,
the number of cells with green nuclei increased (Figure 2B,
1–49 hr), indicating that this ligand induced a G1/S transition.
Subsequently, cells began to adopt a spindle-shaped, fibroblast-like
morphology and high motility (Figure 2B, 49 hr). After
two days of TGFb treatment, the number of cells with green nuclei
diminished, reflecting the G1 arrest effect of TGFb (Figure 2B,
49–85 hr) (Movie S2, right). Thus, TGFb-treated cells spread
without proliferation, in contrast with the untreated NMuMG
cells, which were densely packed in a confluent monolayer. In
addition, the introduction of a wound did not result in proliferation,
but rather a further expansion of cells (Figure 2D).
Cell-Cycle Progression of Tumor Cells in Live Mice
Whole-body and intravital cellular imaging of mice injected with
cultured tumor cells genetically labeled with fluorescent proteins
has proven to be a powerful technique for investigating tumor
development (Hoffman, 2005; Yamauchi et al., 2006). We tested
whether or not Fucci could be used to monitor tumor development
by subcutaneously injecting Fucci-expressing NMuMG
cells into the mammary fat pad of nude mice (Figure 3A). One
day after inoculation, both green and red cells were observed
(Figure 3B). After 16 days, however, only red cells were seen
(Figure 3C), indicating that NMuMG cells are nontumorigenic.
Next, Fucci-expressing HeLa cells were injected in a similar fashion
into nude mice (Figure 3D). The injected cells gradually grew
and emitted both green and red fluorescence for a month, suggesting
tumor progression (Figures 3E and 3F). The expanded
mass was observed through the skin under a microscope (Olympus,
IV100, 103, UplanFL N N.A. = 0.30) 27 days after injection
(Figure 3G). Well-developed tumor vessels were visualized by
loading AngioSense750, which emits far-red fluorescence. Although
triple-color live imaging identified HeLa cells in G1 and
S/G2 phases, their positions relative to the vessels were not clear
due to the low spatial resolution. The tumor was fixed, sectioned,
and stained with an antibody against CD31. Both the red and
green fluorescence of Fucci remained after conventional immunostaining
procedures, including fixation in 4% PFA. The cell-cycle
phase pattern of HeLa cells around blood vessels was clearly
visualized (Figure 3H). The pattern appeared to depend on several
factors, including the maturity of vessels and the degree of
necrosis in the surrounding tissues. A statistical analysis is underway
to investigate these relationships.
Next, cell-cycle progression of tumor cells was examined during
the initial steps of the classic metastatic cascade, such as
490 Cell 132, 487–498, February 8, 2008 ª2008 Elsevier Inc.
adhesion to endothelial cells and extravasation. Fucci-expressing
HeLa cells in a gel were injected into a skin vein (Figure S2),
and intravital cellular imaging was performed. Interestingly, at
early stages, nearly all of the cells attached to the inner wall of
the veins were in G1 phase (Figures 3I and 3J). We captured an
image of a cell in the process of extravasation (Figures 3K, 3L,
and 3M). Within a cluster of HeLa cells across a vein wall, an
elongated cell with a yellow, fragmented nucleus was observed
to pass through the wall. Four days postinjection, HeLa cells
were found to invade and proliferate over the veins (Figures 3N
and 3O), suggesting multiple occurrences of extravasation.
Previous work showed that cultured cells with differentially labeled
cytoplasm and nuclei that were injected into mice could be
used to image nuclear-cytoplasmic dynamics in order to monitor
Figure 2. Cell-Cycle Regulation of Cultured NMuMG Cells
(A and B) Fluorescence images of Fucci-expressing NMuMG cells in the absence (A) and presence (B) of TGFb (1 ng/ml) while reaching a confluent monolayer
state.
(C and D) Fluorescence images of Fucci-expressing NMuMG cells in the absence (C) and presence (D) of TGFb (1 ng/ml) after the monolayer was scratched. Two
nuclei entering S phase at 13 hr are indicated by arrows in (C). The scale bar represents 50 mm.
Cell 132, 487–498, February 8, 2008 ª2008 Elsevier Inc. 491
Figure 3. Cell-Cycle Progression of Cultured Cells Injected into Live Mice
(A–H) Observation of Fucci-expressing cultured cells subcutaneously inoculated into the mammary fat pad of nude mice. (A–C) Fucci-expressing NMuMG cells.
(D–H) Fucci-expressing HeLa cells. (A–F) Whole-body images were acquired using the Olympus OV100 Imaging System with a 0.143 objective lens and F-View II
camera (Soft Imaging System). The scale bar represents 1 cm. (A, B, D, and E) 1 day postinoculation. (C and F) 16 days postinoculation. (A and D) bright-field
images. (B, C, E, and F) Red and green fluorescence images were merged. The yellow mass (B, E, and F) consisted of both red and green nuclei, while the red
mass (C) consisted predominantly of red nuclei. (G) A triple-color intravital cellular image of the expanded mass of HeLa cells 27 days post inoculation. Tumor
vessels were visualized (blue) after loading the mouse with AngioSense750. Image acquisition was performed using an IV100 intravital laser scanning microscope
(Olympus). The scale bar represents 100 mm. (H) A triple-color image of a section of the fixed mass. Vessels were stained for CD31 and displayed in blue. Image
acquisition was performed using an FV1000 confocal microscope system. The scale bar represents 100 mm.
(I–O) Observation of Fucci-expressing HeLa cells after injection into an epigastrica cranialis vein. Fluorescence images were acquired using the Olympus OV100;
red and green images were merged and superimposed on DIC images. Vessels are delineated with white dotted lines. The scale bar represents 100 mm. (I and J)
492 Cell 132, 487–498, February 8, 2008 ª2008 Elsevier Inc.
cancer cell trafficking, deformation, extravasation, mitosis, and
cell death in live mice (Yang et al., 2003). In combination with
these cytoplasmic labeling techniques, fluorescence imaging
of stably transformed Fucci-expressing cells injected into live
animals will provide reliable pharmacodynamic readouts for
the growth and metastatic behavior of tumors.
Cell Cycle Analysis of Developing Neural Tissue
in Fucci Transgenic Mice
One major advantage of our genetically encoded probe is that it
need not depend on transcriptional regulation; its transcription
can be driven using constitutive promoters. Thus, we can easily
generate transgenic organisms for cell cycle analysis. Using the
CAG promoter (Niwa et al., 1991), we have made transgenic
mouse lines that ubiquitously express mKO2-hCdt1(30/120).
From 16 mouse lines emitting red fluorescence, #596 was selected
for further characterization. We also made eight green
fluorescent mAG-hGem(1/110) mouse lines from which #504
was chosen for further characterization. These mouse lines provide
us with an unprecedented model system with which to
study the coordination of the cell cycle and development. #504
is particularly useful because it provides in vivo information
about proliferation patterns. During early development of the
mammalian cerebral cortex, neural progenitors in the ventricular
zone (VZ) undergo expansion. To determine whether
mAG-hGem(1/110) green fluorescence is produced by neural
progenitors, we performed immunohistochemistry on telencephalic
sections of an embryonic day (E)14 #504 transgenic
embryo. Since the telencephalic cells with green nuclei were immunopositive
for Nestin but not MAP2 (Figure S3), these cells
were likely to be neural progenitors.
Next, we crossbred #596 and #504 transgenic mice to generate
a mouse line producing Fucci, in which every somatic cell nucleus
exhibited either red or green fluorescence. We fixed an E13
Fucci transgenic embryo and prepared coronal sections of the
brain. Red and green fluorescence was examined in every section
using confocal laser scanning microscopy. Fluorescence
images of three representative sections are shown in Figures
4A, 4E, and 4I. The red and green signals appear to be well balanced
at the embryonic stage, but the overall ratio of green-tored
signal decreases as the mice grow (data not shown).
In the developing cerebral cortex (Figures 4B, 4F, 4G, and 4J),
nuclei emitting red mKO2-hCdt1(30/120) fluorescence were
identified in two main cell populations: mitotic neural progenitors
in the VZ and postmitotic neurons destined to populate different
layers in the cortical plate (CP). The postmitotic neurons exhibited
much brighter red fluorescence, probably due to accumulation
of mKO2-hCdt1(30/120) after cell-cycle exit. The bright
red nuclei of blood vessels were also visible in the VZ of the dorsal
telencephalon (Figures 4B and 4F). It is interesting to note that
in the diencephalon there was a stripe of cells in G1 phase, which
corresponded to the zona limitans intrathalamica (zli). The dorsal
thalamus contained more green nuclei than the ventral thalamus
(Figures 4I and 4J), which suggests that cells in the ventral region
undergo cell-cycle exit for differentiation prior to those in the
dorsal region.
The differential intensity of red fluorescence between mitotic
and postmitotic cells was observed also in the developing neuroepithelia
of the olfactory and vomeronasal systems (Figures 4C
and 4D, respectively) and the retina (Figure 4H). The random distribution
of high- and low-intensity fluorescent nuclei may suggest
that the architecture of the olfactory and vomeronasal epithelia
is not yet established at E13. In contrast, bright red
nuclei were observed in the central apical region of the developing
retina (Figure 4H), whose developing retinal ganglion cells undergo
centrifugal differentiation (Neumann and Nuesslein-Volhard,
2000). The epithelial cells of the lens had also exited the
cell cycle by this stage. Other extra-neural tissues with bright
red fluorescence include the trigeminal ganglion (Figure 4K)
and pituitary gland (Figure 4L).
Coronal sections of mouse embryos (E13) were also examined
immunohistochemically. Proliferation was visualized by nuclear
immunostaining for BrdU (Figure S4) or PCNA (Figure S5).
PCNA images were merged with fluorescence images of
mKO2-hCdt1(30/120) to indicate neuronal differentiation. The
balance between proliferation and differentiation in the telencephalon,
diencephalon, olfactory vesicle, and retina (Figures
S4 and S5) was very similar to that observed through the green
and red signals of Fucci (Figure 4).
Geminin and Cdt1 were previously shown to be abundantly
expressed by neural progenitors during early mouse neurogenesis,
but transcriptionally downregulated at late developmental
stages (Spella et al., 2007). It should be again noticed that Fucci
signal is not affected by transcriptional regulation in our transgenic
mice.
In the developing cerebral cortex, some neural progenitors exit
the cell cycle and migrate beyond the VZ, where they differentiate
into neurons or, at later stages, into glial cells. Neural progenitors
also undergo a typical migration pattern within the VZ; their
nuclei undergo characteristic movements, known as interkinetic
nuclear movements (Sauer, 1935). M-phase nuclei are located
on the ventricular surface, while S-phase nuclei are farther
from the ventricle. In order to observe the spatial and temporal
regulation of proliferation, differentiation, and migration of neural
progenitors, we performed time-lapse imaging experiments using
slices of dorsal telencephalon prepared from an E13 Fucci
transgenic embryo (Figure 5A). Time-lapse imaging experiments
using acute cortical slices are usually acquired at >3 hr intervals.
With such long intervals, neither nuclear movements nor cell-cycle
progression can be adequately followed. However, the bright
Fucci fluorescence enables 3D time-lapse imaging with 10 min
intervals in the xyz-t mode using the FV1000 multiposition stage
system. At each time point, 20 confocal images along the z-axis
(2 mm step) were acquired. In addition, exposure of slices to 40%
oxygen, instead of the usual 20%, has significantly improved cell
proliferation, differentiation, and migration during imaging experiments
(Miyata et al., 2002, 2004). As mentioned earlier, the red
nuclei of mitotic neural progenitors were much dimmer than
At an early stage, most of the cells remaining in vessels were in G1 phase. The box region in (I) is expanded and shown in (J). Two HeLa cells with red nuclei
attached to the inner surface of the vein. (K–M) An image of the process of extravasation. Red (L) and green (M) fluorescent images showing the presence of
a cell with an elongated, yellow nucleus. (N and O) 4 days postinjection. The box region in (N) is expanded and shown in (O).
Cell 132, 487–498, February 8, 2008 ª2008 Elsevier Inc. 493
Figure 4. A Survey of the Cell Cycle in the Developing Mouse Head
Coronal sections of an E13 Fucci transgenic embryo. Red and green fluorescence signals are merged. The scale bar represents 100 mm.
(A–D) The section containing the brain, olfactory system, and vomeronasal system. The box regions in (A) are expanded in (B), dorsal telencephalon, (C), olfactory
vesicle, and (D), vomeronasal organ.
(E–H) Section containing the brain, hippocampus, and eye. The boxed regions in (E) are expanded in (F), dorsal telencephalon, (G), hippocampus, and (H), eye.
(I–L) Section containing the brain, trigeminal ganglion, and pituitary gland. The box regions in (I) are expanded in (J), the dorsal/ventral boundary of diencephalon,
(K), trigeminal ganglion, and (L), pituitary gland.
494 Cell 132, 487–498, February 8, 2008 ª2008 Elsevier Inc.
those of postmitotic ones. To visualize migration of the nuclei in
the cell cycle within the VZ, we increased the photomultiplier
tube (PMT) sensitivity for red fluorescence. While nuclei in the
CP showed saturated red fluorescence, nuclei in the VZ exhibited
equivalent levels of either green or red fluorescence
(Figure 5B). Under these conditions, the change in color between
green and red during cell-cycle progression and the migration of
cells could be clearly followed. First, we followed the trajectories
of VZ neural progenitor nuclei corresponding to interkinetic nuclear
movements. The trajectory of a migrating cell along with
its cell-cycle progression from S/G2 to G1 and cell division at
the ventricular surface is illustrated in Figure 5C (left). A green nucleus
descended in the VZ and completed mitosis upon reaching
the ventricular surface. Then, the two daughter nuclei turned red
and started migrating away from the surface. Another nucleus
that progressed from G1 to M (Figure 5C, middle) underwent
the G1/S transition while making a hairpin turn near the intermediate
zone (IZ). In addition, we observed nuclear migration, which
might be involved in nonsurface mitoses (Haubensak et al., 2004;
Noctor et al., 2004); a green nucleus was observed to wander
about in the IZ until it entered M phase (Figure 5C, right). Finally,
we observed bright red nuclei traveling quickly in the IZ from the
ventral to dorsal part of the telencephalon (Figure 5C, top). These
nuclei are likely to belong to cortical GABA (g-amino-butyric
acid) neurons, which are born in the subpallial telencephalon
and migrate tangentially to reach their final destination (Marı´n
and Rubenstein, 2001).
Figure 5. Cell Cycle-Related Migration of Nuclei in the
Dorsal Telencephalon of an E13 Fucci Transgenic
Mouse Embryo
Abbreviations are as follows: CP, cortical plate; IZ, intermediate
zone; VZ, ventricular zone. The scale bar represents
100 mm.
(A) A cultured slice for 3D time-lapse imaging.
(B) An expanded image of the IZ and VZ with red fluorescence
detection sensitivity increased.
(C) A schematic diagram showing migration of neural progenitors
(bottom) and an interneuron (top) in the cultured slice. Abbreviation
is as follows: M, M phase. Entry into prometaphase
can be detected by the spread of green fluorescence throughout
the cell due to breakdown of the nuclear envelope.
Fucci Is Compatible with Established
Imaging Protocols
The nuclear localization of Fucci is advantageous in
the following respects: additional far-red fluorescent
proteins that are spectrally distinct from both
mAG and mKO2, such as mCherry (Shaner et al.,
2004) and mKeima (Kogure et al., 2006), can be expressed
in the cytoplasm by tagging them with Nuclear
Export Signal (NES), in order to identify cell
types and observe cell morphology. The third color
fluorescence signal can also be provided by chemical
dyes. In the experiment shown in Figure 5, we
placed fine DiD crystals on the pial surface of the
brain slice to sparsely label pia-connected progenitors.
The bipolar morphology of a progenitor with
a green nucleus whose movement was tracked
(Figure 5C, left) could be identified. The DiD image at a particular
time point (indicated by an arrow in Figure 5C) is shown in
Figure S6. The excitation and emission spectra of Fucci and possible
complementary dyes are presented in Figure S7.
Many genetically encoded indicators that utilize Green Fluorescent
Protein (GFP)-based Fluorescence Resonance Energy
Transfer (FRET), including cameleon (Miyawaki et al., 1997)
and Raichu-Ras (Mochizuki et al., 2001), are distributed in the cytoplasm.
Thus, cell-cycle phase can be monitored in parallel with
signaling events taking place in the cytoplasm. For instance, we
transfected Raichu-Ras (Raichu 124X) into Fucci-expressing
COS7 cells, and observed that K-ras was more active in G1
phase than in S/G2 phase in response to epidermal growth factor
signaling (Figure S8). Thus, the cell-cycle dependency of numerous
cellular events can be elucidated without using cell-cycle
synchronization techniques. Multicolor imaging in combination
with these fluorescent probes and proteins will further expand
the applications of the Fucci technology.
Future Perspectives of the Fucci Technology
The Fucci technology allows dual-color imaging, which can distinguish
between live cells in the G1 and the S/G2/M phases. This
technology allows for in vivo analysis of spatial and temporal patterns
of cell-cycle dynamics, owing to the brightness of the fluorescence
and the high contrast between the two colors (red and
green). Although Fucci is composed of mKO2-hCdt1(30/120)
and mAG-hGem(1/110), single transfection of either would
Cell 132, 487–498, February 8, 2008 ª2008 Elsevier Inc. 495
suffice in conferring the cell-cycle indicator function; for instance,
the transgenic mouse line #504 just produces
mAG-hGem(1/110) but nevertheless provides in vivo information
about proliferation patterns. However, coexpression of both
constructs is still considerably more useful because it highlights
the G1/S transition with a yellow signal, and because it permits us
to continuously track migrating cells or nuclei in the cell cycle. In
this regard, reliable gene-transfer techniques which control the
stoichiometry of two constructs will be required.
Future challenges involve further developing the Fucci derivatives
(1) with different colors so that coexpressed GFP or RFP
can be spectrally distinguished, (2) that highlight cell-cycle transitions
other than G1/S, and (3) that function in nonmammalian
cell types. Such research will benefit from exploration of the molecular
mechanisms underlying both cell-cycle progression and
ubiquitin-mediated protein degradation. Regarding the last challenge,
it should be noted that the primary structures of Cdt1 and
Geminin vary among species. By tagging certain domains of the
lower eukaryotic homologs of these two proteins to mKO2 or
mAG, we have developed a version of Fucci that functions in
fish and insect cells (data not shown). We have also generated
transgenic zebrafish and Drosophila lines expressing the nonmammalian
Fucci in an effort to investigate the spatial and temporal
regulation of cell-cycle progression during major morphogenetic
events such as gastrulation and metamorphosis, and
during basic morphogenetic processes such as invagination, involution,
and branching.
EXPERIMENTAL PROCEDURES
Gene Construction
mKO2 was developed by introducing eight mutations (K49E, P70V, F176M,
K185E, K188E, S192D, S196G, and L210Q) into mKO (Karasawa et al.,
2004). mKO2 absorbs light maximally at 551 nm (molar extinction coefficient,
63,800 M-1
cm-1
) and emits fluorescence at 565 nm (fluorescence quantum
yield, 0.57). mKO2 and mAG cDNAs (Medical Biological Laboratory, Amalgaam)
were amplified using primers containing 50
-EcoRI and 30
-EcoRV sites,
and digested products were cloned in-frame into the EcoRI/EcoRV sites of
pcDNA3 (Invitrogen) vector to generate pcDNA3/mKO2 and pcDNA3/mAG,
respectively. The entire or numerous partial cDNA species of human Cdt1
(GenBank: NM_030928) or human Geminin (GenBank: NM_015895) were amplified
using primers containing 50
-XhoI and 30
-XbaI sites, and digested products
were cloned in-frame into the XhoI/XbaI sites of pcDNA3/mKO2 or
pcDNA3/mAG.
Cell Culture
HeLa cells and COS7 cells were grown in DMEM supplemented with 10% fetal
bovine serum and penicillin/streptomycin. Mouse NMuMG breast epithelial
cells were grown in DMEM (high glucose) medium supplemented with 10%
fetal bovine serum, penicillin/streptomycin, and 10 mg/ml Insulin (Sigma).
EGF and TGFb1 were purchased from R&D.
Imaging of Cultured Cells
Cells were grown on a 35 mm glass-bottom dish in phenol red-free Dulbecco’s
modified Eagle’s medium containing 10% fetal bovine serum (FBS). Cells were
transiently or stably transfected with cDNA using Lipofectin (Invitrogen) and
subjected to long-term, time-lapse imaging using a computer-assisted fluorescence
microscope (Olympus, LCV100) equipped with an objective lens
(Olympus, UAPO 403/340 N.A. = 0.90), a halogen lamp, a red LED (620 nm),
a CCD camera (Olympus, DP30), differential interference contrast (DIC) optical
components, and interference filters. For fluorescence imaging, the halogen
lamp was used with two filter cubes, one with excitation (BP520-540HQ)
and emission (BP555-600HQ) filters for observing mKO2 fluorescence, and
the other with excitation (470DF35) and emission (510WB40) filters for observing
mAG fluorescence. For DIC imaging, the red LED was used with a filter
cube containing an analyzer. Image acquisition and analysis were performed
by using MetaMorph 6.13 software (Universal Imaging, Media, PA).
Lentivirus Construction and Production
Replication-defective, self-inactivating lentivirus vectors were used (Miyoshi
et al., 1997, 1998). cDNA encoding mKO2-hCdt1(30/120) or mAG-hGem(1/
110) was cloned into a CSII-EF-MCS vector. The plasmid was cotransfected
with the packaging plasmid (pCAG-HIVgp) and the VSV-G- and Rev-expressing
plasmid (pCMV-VSV-G-RSV-Rev) into 293T cells. High-titer viral solutions
for mKO2-hCdt1(30/120) and mAG-hGem(1/110) were prepared and used for
cotransduction into several cell lines: HeLa, COS7, NMuMG, and PC12 cells.
Immunocytochemical Cell Cycle Analysis
Fucci-expressing HeLa cells grown on a coverslip were treated with BrdU
(Sigma) for 5 min at 37
C. After being washed with PBS(À), cells were fixed
with 4% PFA for 10 min at 4
C and then with 0.1% Triton X-100/PBS(À) for
5 min at room temperature. The antibodies used were: mouse anti-BrdU
mAb (ImmunologicalsDirect), mouse anti-PCNA mAb (DAKO), and goat antimouse
IgG conjugated with Alexa Fluor 633 (Molecular Probes). Image acquisition
was performed using an FV500 (Olympus) confocal microscope system
equipped with 488 nm (argon), 543 nm (He/Ne), and 633 nm (He/Ne) laser lines.
Flow Cytometry
Hoechst 33342 solution (56 ml of 1 mg/ml stock) (DOJINDO) was added to a
10 cm dish containing parental or Fucci-expressing HeLa cells. After incubation
for 30 min, cells were harvested and analyzed using BD LSR (Becton Dickinson).
Both mKO2 and mAG were excited by a 488 nm laser line (argon), and
Hoechst 33342 was excited by a 325 nm laser line (HeCd). Fluorescence signals
were collected at 530 nm (530/28 BP)(FL1) for mAG, at 575 nm (575/
26 BP)(FL2) for mKO2, and at 400 nm (380 LP) (FL5) for Hoechst33342. The
data were analyzed using FlowJo software (Tree Star).
Generation of Transgenic Mice
cDNA encoding mKO2-hCdt1(30/120) or mAG-hGem(1/110) was cloned into
a pCAGGS vector (Niwa et al., 1991). The transgenic insert, devoid of vector
sequences, was gel-purified and microinjected into the pronuclei of zygotes
of BDF1 inbred mice. Screening for fluorescent founders was performed by illumination
with a blue LED (for mAG) and a green LED (for mKO2). 16 lines expressing
mKO2-hCdt1(30/120) and eight lines expressing mAG-hGem(1/110)
were obtained. The experimental procedures and housing conditions for animals
were approved by the Institute’s Animal Experimental Committee, and
all animals were cared for and treated humanely in accordance with the Institutional
Guidelines for Experiments using Animals.
Whole-Body Imaging of Mice
Subcutaneous and intravenous injection of cultured cells, and whole-body
imaging with OV100 (Olympus) were performed as described elsewhere
(Hoffman and Yang, 2006). To visualize blood vessels, Angio Sense-IVM750
(VisEn medical) was injected, or endothelial cells were stained using antiCD31
mAb (Chemicon).
Histological Observation of Tissue Sections
E13 Fucci (#596/#504) embryos were perfused transcardially with fixative (4%
PFA), placed in ice-cold fixative for 2 hr, cryoprotected in PBS containing 20%
sucrose, and embedded in OCT compound. Coronal head sections (15 mm
thick) were imaged using FV1000 equipped with two laser diodes (473 nm
and 559 nm). The images were tiled to create wide-field pictures. Brain sections
from an E14 #504 embryo were fixed, incubated with mouse antiMAP2
mAb (Chemicon) or mouse anti-Nestin mAb (PharMingen), followed
by goat anti-mouse IgG conjugated with AlexaFluor 546X (Molecular Probes).
Imaging of Cultured Brain Slices
Brain slices were prepared from Fucci-expressing mice (#596/#504) at E13,
and cultured in collagen gel as previously described (Miyata et al., 2002). Slices
496 Cell 132, 487–498, February 8, 2008 ª2008 Elsevier Inc.
were exposed to 5% CO2 and 40% O2. Time-lapse 3D imaging was performed
in the xyz-t mode using the FV1000 multiposition stage system. The recording
interval was 10 min. At each time point, 20 confocal images along the z-axis
(2 mm step) were acquired. To avoid crossdetection of green and red signals,
images were acquired sequentially at 488 nm (Argon) and 543 nm (He/Ne).
Green and red images were merged for each confocal image. Proper alignment
and correct image registration of FV1000 with the two laser lines and detection
channels were verified using double-labeled fluorescent beads (TetraSpeck
Fluorescent Microsphere Standards, 0.5 mm in diameter, Molecular
Probes). Data analysis was performed using Volocity software (Improvision)
and METAMORPF software (Universal Imaging, Media, PA).
Distribution of Materials
DNA constructs such as mKO2-hCdt1(30/120) and mAG-hGem(1/110), their
stable transformant cell lines, and transgenic mouse lines reported in this paper
will be distributed with concomitant purchase of cDNA for mKO2 or mAG
from MBL International (Amalgaam) (http://www.mblintl.com/mbli/index.asp).
Supplemental Data
Supplemental Data include eight figures and three movies and can be found
with this article online at http://www.cell.com/cgi/content/full/132/3/487/
DC1/.
ACKNOWLEDGMENTS
The authors would like to thank Mr. Kenji Ohtawa for technical assistance with
FACS analysis; Dr. Mika Tanaka for technical assistance with development of
transgenic mouse lines; Mr. Katashi Ishihara and Mr. Atsuhiro Tsuchiya for
technical assistance with microscopy; Ms. Naoko Kakusho for technical assistance
with blotting analysis; Drs. Satoshi Karasawa and Tomomi Shimogori for
valuable advice; Drs. Masao Ito and Shun-ichi Amari for support; and Mr. David
Mou for critically reading the manuscript. This work was partly supported
by grants from Japan MEXT Grant-in-Aid for Scientific Research on priority
areas and HFSP (the Human Frontier Science Program). A.M. is a member
of the scientific advisory board of Amalgaam, which will be handling the distribution
of mAG and mKO2.
Received: September 11, 2007
Revised: November 19, 2007
Accepted: December 18, 2007
Published: February 7, 2008
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Accession Numbers
The sequences reported in this paper have been deposited in the DDBJ database
[AB370332, mKO2-hCdt1(30/120); AB370333, mAG-hGem(1/110)].
498 Cell 132, 487–498, February 8, 2008 ª2008 Elsevier Inc.