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Disruption of actin cytoskeleton and anchorage-dependent cell spreading induces apoptotic death of mouse neural crest cells cultured in vitro.

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Disruption of Actin Cytoskeleton and
Anchorage-Dependent Cell Spreading
Induces Apoptotic Death of Mouse
Neural Crest Cells Cultured In Vitro
Department of Anatomy and Developmental Biology, Kyoto University Graduate
School of Medicine, Kyoto, Japan
Department of Plastic and Reconstructive Surgery, Kyoto University Graduate
School of Medicine, Kyoto, Japan
Congenital Anomaly Research Center, Kyoto University Graduate School of
Medicine, Kyoto, Japan
In vertebrate embryos, neural crest cells emigrate out of the neural tube
and contribute to the formation of a variety of neural and nonneural tissues.
Some neural crest cells undergo apoptotic death during migration, but its
biological significance and the underlying mechanism are not well understood.
We carried out an in vitro study to examine how the morphology and survival
of cranial neural crest (CNC) cells of the mouse embryo are affected when their
actin cytoskeleton or anchorage-dependent cell spreading is perturbed. Disruption of actin fiber organization by cytochalasin D (1 ␮g/ml) and inhibition of cell
attachment by matrix metalloproteinase-2 (MMP-2; 2.0 units/ml) were followed by morphologic changes and apoptotic death of cultured CNC cells.
When the actin cytoskeleton was disrupted by cytochalasin D, the morphologic
changes of cultured CNC cells preceded DNA fragmentation. These results
indicate that the maintenance of cytoskeleton and anchorage-dependent cell
spreading are required for survival of CNC cells. The spatially and temporally
regulated expression of proteinases may be essential for the differentiation and
migration of neural crest cells. © 2005 Wiley-Liss, Inc.
Key words: cranial neural crest; apoptotic cell death; anoikis;
actin cytoskeleton; anchorage dependency; mouse
During the development of vertebrate embryos, neural
crest cells develop at the junction between the neural
plate and the surface ectoderm. They migrate along spatially and temporally distinct pathways and differentiate
into a variety of neuronal and nonneuronal cell types that
are important in the formation of craniofacial structures,
the cardiovascular system, and much of the peripheral
nervous system (Le Douarin and Kalcheim, 1999). It has
been shown that some cells in the cranial and trunk neural crests die by apoptosis during migration and that such
cell death may play crucial roles in differentiation and
migration of neural crest cells (Jeffs and Osmond, 1992;
Jeffs et al., 1992; Hirata and Hall, 2000). Jeffs et al. (1992)
observed cell death in specific populations of cranial neural crest (CNC) cells and postulated that it may contribute
to the patterning of early CNC migration. In the trunk of
embryos, Wakamatsu et al. (1998) demonstrated that
Grant sponsor: the Japanese Ministry of Education, Culture, Sports,
Science and Technology; Grant number: 12670016 and 13470003.
*Correspondence to: Kohei Shiota, Department of Anatomy and
Developmental Biology, Kyoto University Graduate School of
Medicine, Kyoto 606-8501, Japan. Fax: 81-75-751-7529.
Received 21 June 2004; Accepted 20 October 2004
DOI 10.1002/ar.a.20150
Published online 2 January 2005 in Wiley InterScience
some neural crest cells undergo apoptosis on the lateral
migration pathway, and they argued that such cell death
may help neural crest cells find the right migration pathway and remove “ectopic” cells by a “proofreading mechanism.” They suggested that some environmental cues regulate the survival and death of neural crest cells.
Developmental neuronal cell death has been reported to
occur in both the central and peripheral nervous systems
and is assumed to contribute to eliminating excessive and
inappropriately located cells (Jacobson, 1991; Wakamatsu
et al., 1998; Maynard et al., 2000). Various mechanisms
have been proposed for developmental neuronal death,
and it seems that the mechanism of cell death is not the
same at different locations and at various stages in development. Since apoptotic death of neural crest cells often
occurs after they leave the neural tube and become nonepithelial, we postulated that the survival of neural crest
cells may depend on anchorage-dependent cell spreading
and the maintenance of normal cytoarchitecture. Such
anchorage-dependent survival has been noted in some
epithelial cell types (Boudreau et al., 1995a, 1995b) but
has not been demonstrated so far in neural crest cells.
To investigate whether anchorage-dependent cell
spreading and maintenance of cytoarchitecture are required for survival of CNC cells, we undertook an in vitro
study where their actin cytoskeleton and attachment to
the substratum were disrupted by chemical agents. Cultured CNC cells emigrating out of the explanted mouse
embryonic neural tube were treated with cytochalasin D
and matrix metalloproteinase-2 (MMP-2) to disrupt their
actin cytoskeleton and adhesion to the underlying substratum, respectively. Cytochalasin D blocks the formation of microfilament structures by abrogating actin polymerization and cytochalasin D-treated cells cannot
maintain normal morphology (Korn, 1982). MMP-2 degrades extracellular matrix (ECM) components such as
type IV and V collagens, laminin-5, elastin, fibronectin,
and proteoglycans, thereby disrupting cell-ECM interactions (Okada et al., 1990; Giannelli et al., 1997; Zuo et al.,
1998). It has been shown that MMP-2 has an important
functional role in epithelial-mesenchymal transformation
(EMT) and migration of neural crest cells and that perturbation of MMP activity may lead to neural crest-related congenital defects (Cai et al., 2000; Cai and Brauer,
2002; Duong and Erickson, 2004). We analyzed the effects
of cytochlasin D and MMP-2 on the morphology and survival of cultured CNC cells by cytochemical staining for
actin microfilaments and by in situ labeling of DNA fragmentation (TUNEL method). The disruption of actin fiber
organization and adhesion to the substratum was followed
by apoptotic death of cultured CNC cells, suggesting that
properly organized cytoarchitecture and anchorage-dependent cell spreading are essential for survival of CNC
ICR strain mice (SLC Japan, Shizuoka, Japan) were
maintained in a temperature- and humidity-controlled animal facility with a 12-hr light/12-hr dark cycle. Animals
were given laboratory chow and tap water ad libitum.
Virgin female mice were mated overnight with a male
mouse, and the noon of the day on which a vaginal plug
was found was taken as embryonic day 0.5 (E0.5). On
E8.5, embryos were obtained by Caesarean section and
transferred to sterile Tyrode buffer solution. The experimental protocol was approved by the Committee for Animal Experimentation of Kyoto University Graduate
School of Medicine.
Neural Crest Cell Explant Culture
Primary culture of mouse CNC cells was carried out
according to the method originally described by Ito and
Takeuchi (1984) and Murphy et al. (1991). Each mouse
embryo (E8.5) was treated with 0.25% trypsin/PBS for 15
min and the hindbrain neural folds were isolated. The
dorsal ridge of the neural folds was surgically removed
free from the surrounding tissues and cultured on a 35
mm culture dish coated with fibronectin (Becton Dickinson). The culture medium contained 90% ␣-MEM medium
(Gibco-BRL), 10% fetal bovine serum (Gibco-BRL), 4%
chick embryo extract (Gibco-BRL), 50 U/ml penicillin, 50
␮g/ml streptomycin (Gibco-BRL), and 1% insulin-transferrin-selenium-X (Gibco-BRL). The cultures were maintained for 48 hr at 37°C in a CO2 incubator under a
humidified atmosphere composed of 95% air and 5% CO2.
At 48 hr after culture, the neural folds in the colonies were
scraped away with syringe needles, and the remaining
CNC cells, which had migrated out of the neuroepithelium, were used for the following experiments.
Cranial neural crest cells were identified by immunohistochemical staining with a rat antimouse monoclonal
antibody 4E9R (Kubota et al., 1996), which was kindly
provided by Dr. Kazuo Ito (Osaka University Graduate
School of Science, Toyonaka, Japan). The 4E9R antibody
has been shown to identify migratory mouse neural crest
cells (Kubota et al., 1996).
Treatment With Cytochalasin D and MMP-2
After preparation of cultured CNC cells as described
above, the culture medium was replaced by a serum-free
medium to eliminate the influence of substances contained in fetal bovine serum. Then, either cytochalasin D
(1 ␮g/ml; Sigma, St. Louis, MO) or MMP-2 (2.0 unit/ml;
Cosmo Bio, Tokyo, Japan) was added to the medium for
experimental groups. For stock solutions, cytochalasin D
and MMP-2 were dissolved in DMSO and PBS, respectively. The final concentration of cytochalasin-D (1 ␮g/ml)
was determined according to previous studies in which
microtubule assembly and cell shape of cultured cells were
disrupted (Nishi et al., 2002; Nemeth et al., 2004). The
concentration of MMP-2 (2.0 unit/ml) was used because
the concentration 1.0 unit/ml or below had no observable
effects on cultured CNC cells. In control groups, an equivalent volume of the vehicle was added to the culture medium. After adding the chemicals, each culture dish was
further incubated in a CO2 incubator and was observed
and photographed at every hour using a Nikon Diaphoto
phase-contrast microscope (Nikon, Tokyo, Japan). At 12
hr after treatment, cells in culture dishes were fixed with
4% paraformaldehyde for further stainings and the
TUNEL reaction.
Dual Staining for Actin Fibers and Nuclear
The fixed CNC cells were washed three times with PBS,
immersed in 0.1% Triton X-100/PBS, then incubated for
10 min at room temperature with 33 ␮M phalloidin-
rhodamin (Sigma) and 0.025% Hoechst33342 (Sigma) dissolved in PBS. After incubation, the cells in the dishes
were washed three times with PBS and coverslipped. The
cells were observed using an Axioplan 2 fluorescence microscope (Carl Zeiss, Germany) equipped with fluorescein
filters and were photographed using a CoolSNAP CCD
camera (Photometrics).
In Situ Labeling of DNA Fragmentation by
TUNEL Method
The in situ visualization technique for DNA fragmentation was carried out according to the TUNEL method
(Gavrieli et al., 1992; Mori et al., 1995). Fixed CNC cells
were washed three times with double-distilled water, airdried, and treated with 5 ␮g/ml proteinase K (Wako Pure
Chemical, Tokyo, Japan). After proteinase K treatment,
the cells were incubated in TdT buffer containing 12.5 ␮M
biotinylated-dUTP (Boehringer Mannheim) and 0.15
unit/␮l TdT (Takara, Kyoto, Japan) at 37°C for 70 min.
Then, they were reacted with rhodamine-avidin (Sigma)
for visualization of fragmented DNA. After being rinsed
several times with PBS, the samples were coverslipped
and observed. The samples were photographed using a
Zeiss Axioplan 2 fluorescence microscope and a CoolSNAP
CCD camera (Photometrics). For each sample, the ratio of
TUNEL-positive cells to the total cells was calculated. Cell
counts were executed on the monitor of a Power Macintosh
computer (Apple) using a counting tool of the National
Institutes of Health Image program (
Morphologic Changes of Cultured CNC Cells
Treated With Cytochalasin D and MMP-2
In control cultures, numerous cells began to emigrate
out of the explanted neural tube within 4 hr after culture
and their emigration and mitotic divisions continued for
over 72 hr. Migrating cells at the peripheral part of the
colonies showed the bipolar or multipolar morphology,
which is typical for neural crest cells, as has been described by Ito and Takeuchi (1984) (Fig. 1A). Immunohistochemical staining with a monoclonal antibody against
neural crest cells (4E9R) (Kubota et al., 1996) revealed
that over 90% of the cells emigrating out of the explanted
neural tube were 4E9R-positive, indicating that they were
CNC cells (Fig. 1B). By 60 hr in culture, many of those
emigrated cells began to extend axon-like protrusions and
appeared to undergo morphologic differentiation (data not
shown). Therefore, the cells cultured for 48 hr were used
for the following experiments.
When CNC cells were cultured with cytochalasin D (1
␮g/ml), obvious morphologic changes were observed
within 1 hr. Many cells showed a blebbing appearance,
which is characteristic to apoptotic cells. By 12 hr after
culture, the cells retracted their cell protrusions (filopodia)
and became shrunken and rounded (Fig. 1C). When
MMP-2 (2.0 units/ml) was added to the culture medium,
cultured cells showed morphologic changes such as membrane blebbing and retraction of filopodia, and the cell
area was reduced as compared with controls (Fig. 1D).
Some cells became rounded.
Morphologic Changes of Nucleus and Actin
Fiber Organization of Cultured CNC Cells
When the cells in control cultures were subjected to dual
staining for actin fibers and nuclear DNA, the morphology
of cell nuclei and the arrangement of actin stress fibers in
the cytoplasm appeared normal (Fig. 2A and B). Actin
fibers were well organized and arranged orderly in their
cytoplasm. The cells were spread well and had numerous
When the cultured CNC cells were treated with cytochalasin D, they became remarkably shrunken within 1 hr
and their actin fibers became disorganized and condensed
around nuclei (Fig. 2C). Most of the cells showed nuclear
shrinkage and chromatin condensation (Fig. 2D), which
are often observed in apoptotic cells. When cultured cells
were treated with MMP-2, many of the cells appeared to
retract filopodia and the cell area was reduced by 12 hr
after treatment, indicating that the cell-matrix attachment was disrupted to some extent (Fig. 2E). Some cells
with cytoplasmic shrinkage were associated with actin
fiber condensation around the nuclei, and condensed nuclei were intensely stained with Hoechst 33342, indicative
of possible apoptotic bodies (Fig. 2F).
Apototic Death of Cultured CNC Cells
In each culture, apoptotic cells were identified by the
TUNEL method, which detects DNA fragmentation, and
the number of TUNEL-positive cells was counted.
TUNEL-positive cells often had condensed nuclei and
small apoptotic bodies (Fig. 3A–C). The ratio of TUNELpositive cells to the total number of cells was calculated by
counting at least 200 cells in each culture and the data
were compared between the groups (n ⫽ 5 for each group).
The frequencies of apoptotic CNC cells in cultures treated
with cytochalasin D and MMP-2 were 13.4% ⫾ 2.0%
(mean ⫾ SE) and 20.1% ⫾ 2.0%, respectively, which were
significantly higher than the control value (5.4%% ⫾ 0.5%;
Fig. 3C). The frequency of apoptotic cells was higher in the
MMP-2-treated group than in the cytochalasin D-treated
group, although the morphologic change was less severe in
the former group (Figs. 1 and 2).
By using in vitro culture of migrating CNC cells, we
have demonstrated that the survival of CNC cells is dependent on their cytoarchitecture maintained by actin
stress fibers as well as on anchorage-dependent cell
spreading. When the intercellular actin fiber organization
of cultured CNC cells was disrupted by cytochalasin D,
they rapidly became rounded and underwent apoptotic
morphologic changes. It is interesting to note that the
dying cells showed such morphologic alterations before
they became TUNEL-positive. Since cytochalasin D abrogates actin polymerizaion and disrupts actin filaments
(Walling et al., 1988), it is likely that malfunction of actin
fibers and/or disruption of normal cytoarchitecture can
induce cell death without directly affecting nuclear DNA.
Although DNA fragmentation is a major molecular landmark of apoptotic cell death, it has been shown that apoptosis is a process involving some cytoplasmic alterations
and does not necessarily require nuclear DNA fragmentation or other changes in the nucleus at its initial step
(Cohen et al., 1992; Falcieri et al., 1993; Oberhammer et
al., 1993). It has been well accepted that the maintenance
Fig. 1. Cultured CNC cells in the control and experimental groups. A:
A phase-contrast image of CNC cells in the control group cultured for 48
hr. The cells are spread well and have numerous filopodia. B: Immunohistochemical staining of a control culture with the 4E9R monoclonal
antibody, which identifies neural crest cells. Most of the cells are 4E9Rpositive (green). Nuclei are stained with Hoechst 33342 (purple). C: CNC
cells cultured for 12 hr with cytochalasin D (1 ␮g/ml). Most of the cells
are shrunk and rounded and have retracted filopodia. D: CNC cells
cultured for 12 hr with MMP-2 (2.0 unit/ml). Some cells show membrane
blebbing and have retracted cell protrusions (arrowheads). The cell area
is reduced as compared with that in controls (A). Scale bar ⫽ 100 ␮m.
of cell morphology and differentiation is dependent on the
actin cytoskeleton (Hay, 1993). In addition, recent studies
suggest that the cytoskeleton and cortical actin network
play critical roles in various intercellular signaling (Aplin
and Juliano, 1999; Zoubiane et al., 2004). Therefore, disruption of the actin cytoskeleton by cytochalasin D may
not only have affected the cytoarchitecture but also have
precluded effective signaling via the cytoskeleton, resulting in cell death.
MMP-2 degrades various ECM proteins including fibronectin, type IV and V collagens, laminin, and elastin
(Vassalli and Pepper, 1994; Werb and Chin, 1998). Recently, Duong and Erickson (2004) demonstrated that
MMP-2 is expressed as neural crest cells detach from the
Fig. 2. Dual staining for actin fibers (red) and nuclear DNA (blue; A, C,
and E) and nuclear staining with Hoechst 33342 in the same fields (B, D, and
F). A and B: CNC cells in a control culture. The arrangement of actin fibers
is well organized (A) and their nuclei are round and appear healthy (B). C and
D: CNC cells at 12 hr after cytochalasin D treatment. Actin fibers aggregate
around nuclei (C), and nuclei are clearly condensed (D). E and F: CNC cells
at 12 hr after MMP-2 treatment. Cells are not so well expanded as in the
control culture (A), and actin fibers are not well organized and appear
aggregated around nuclei in some cells (stained heavily in red; E). Condensation of nuclear chromatin is recognized (arrowheads in F). Scale bar ⫽
100 ␮m.
Fig. 3. Apoptotic cell death of cultured CNC cells visualized by the
TUNEL method. A: Control culture. B: Cells treated with cytochalasin D
for 12 hr. C: Cells treated with MMP-2 for 12 hr. TUNEL-positive nuclei
and apoptotic bodies are visualized in orange yellow. Scale bar ⫽ 100
␮m. D: The frequency of TUNEL-positive cells in cultured CNC cells. The
percentage of TUNEL-positive cells increased significantly in both
treated groups as compared with controls (asterisk, P ⬍ 0.05; double
asterisk, P ⬍ 0.01 vs. control). Error bars show SEM (n ⫽ 5).
neural epithelium during EMT but is rapidly extinguished
as they disperse. They also showed that MMP inhibitors
and knockdown of MMP-2 expression perturb EMT that
generates neural crest cells but do not affect migration of
neural crest cells, suggesting that MMP-2 plays a crucial
role at some steps in EMT of neural crest cells but is not
required for the later migration process. Cai et al. (2000)
observed the expression of MMP-2 mRNA in the craniofacial region of avian embryos and found that early migrating CNC cells do not synthesize MMP-2 mRNA but can
interact with extracellular MMP-2 protein synthesized by
the mesoderm. Furthermore, Cai and Brauer (2002)
showed that neural crest migration is decreased when the
MMP activity is perturbed. Thus, it seems that proteolytic
degradation of ECM by MMP-2 is required at some stages
of neural crest development but its expression may be
finely regulated spatially and temporally so that the differentiation and migration of neural crest cells can take
place properly. Patch mutant mice, which have a deficit in
MMP-2 and membrane-type MMP expression and decreased migratory capacity of craniofacial mesenchyme
(Robbins et al., 1999), exhibit neural crest-related craniofacial and cardiac defects (Morrison-Graham et al., 1992;
Schatteman et al., 1995).
We showed in the present study that treatment of cultured CNC cells with MMP-2 disrupts cell-matrix interaction and induces apoptotic death. When treated continuously with MMP-2, cultured CNC cells retracted their
protrusions (filopodia) and appeared to detach from the
substratum, increasing TUNEL-positive cells by 12 hr.
Thus, adhesion of CNC cells to ECM seems essential not
only for maintaining their normal cell shape but also for
their survival. This finding is consistent with the previous
finding that some epithelial cells undergo apoptosis when
they are separated from the basement membrane
(Schmidt et al., 1993; Frisch and Francis, 1994). We have
confirmed that degradation of ECM components by
MMP-1 (type I collagenase) also resulted in apoptotic
death of cultured mouse CNC cells (data not shown). In
the present study, it was noted that the proportion of
TUNEL-positive cells, which is indicative of DNA fragmentation, was higher in MMP-2-treated cells than in
cytochalasin D-treated cells, although the morphologic effect appeared more severe in the latter (Figs. 2 and 3).
When the CNC cells were treated with 1 ␮g/ml cytochalasin D, many cells became rounded and their nuclei appeared condensed. However, their nuclei became rodlike
(Fig. 2D), which were different from typically pycnotic
nuclei, as were seen in MMP-2-treated cells (Fig. 2F).
Thus, actin fiber disorganization induced by cytochalasin
D may not instantly result in apoptotic death of CNC cells.
It has been reported that various types of epithelial cells
need anchorage-dependent cell spreading for survival and
that they undergo apoptotic death when their cell-matrix
attachment is disrupted (Boudreau et al., 1995a, 1995b;
Roberts et al., 2002). Frisch and Francis (1994) coined the
term “anoikis” for such a kind of apoptotic cell death. It is
likely that adhesion-dependent regulation of cell survival
is mediated by integrin signaling and that efficient cellECM adhesion is required for forming focal adhesion
plaques by clustering ECM-receptor integrins. It has been
shown that some signaling pathways of tyrosine phosphorylation mediated by focal adhesion kinase (FAK) are
required for organization and maintenance of actin stress
fibers to assemble cytoskeleton-associated proteins, such
as paxillin, vinculin, talin, tenascin, and ␣-actinin, at the
cytoplasmic domains of integrins (Clark and Brugge,
1995; Miyamoto et al., 1995). Frisch et al. (1996) showed
that the interaction of integrins with ECM proteins can
activate FAK and thereby suppress apoptosis in endothelial and other epithelial cells.
In the case of endothelial cells, not only cell-ECM adhesion but also cell spreading are important for preventing
them from entering the process of apoptosis. Re et al.
(1994) demonstrated that endothelial cells cultured under
low concentrations of fibronectin or vitronectin became
rounded and underwent rapid cell death, while the cells
became flattened and remained viable under high substrate concentrations. They concluded that cell-matrix attachment is not sufficient for sustaining cell viability but
cells need to achieve some shape changes to survive. Chen
et al. (1997) directly examined the effects of cell spreading
on growth and viability and showed that growth and survival of cultured endothelial cells increased as the extent
of cell spreading increased but were independent of the
total area of cell-ECM contact. Furthermore, it was shown
that DNA synthesis is tightly coupled with the cell shape
(Folkman and Moscona, 1978) and that stretch stimuli
activate DNA synthesis and cell proliferation (Lansman et
al., 1987; Olesen et al., 1988). These results support the
hypothesis that unfavorable alterations of cell architecture and cell spreading can affect the survival and differentiation of some cells and trigger their apoptosis. During
the migration of neural crest cells, their interaction with
ECM may be crucial. It is likely that some proteinases
including MMPs synthesized by mesenchymal cells help
the EMT and migration of neural crest cells but their
expression needs to be temporally and spatially regulated
not only to facilitate their differentiation but also to keep
them viable.
Recent molecular studies have demonstrated that a cystein protease family caspase, which is an ICE/CED-3 gene
product, is involved in the onset of apoptosis (Miura et al.,
1993; Nicholson et al., 1995; Kuida et al., 1996; Mashima
et al., 1997; Miller, 1997). Caspase-1 (ICE) and caspase-3
(CPP-32), which cleave the existing actin fibers as a substrate, are activated following the disruption of cell-ECM
adhesion (Boudreau et al., 1995b; Mashima et al., 1997).
Actin is an inhibitor of deoxyribonuclease I (DNase I),
which is a candidate endonuclease responsible for apoptotic DNA fragmentation (Peitsch et al., 1993), and the actin
cleaved by ICE/CPP-32 loses the ability to inhibit DNase I
(Kayalar et al., 1996). These data are consistent with the
assumption that the disruption of actin fiber organization
is a critical event for commencement of apoptosis. It is
possible that the perturbation of actin microfilaments and
the disruption of cell adhesion to ECM or adjacent cells
can induce cell shape changes and perturb some signaling
pathways in the cell.
As for the roles of caspases in developmental apoptosis,
Umpierre et al. (2001) demonstrated that activated
caspase-3 and DNA fragmentation were colocalized in the
mesenchymal cells of branchial arches and in neuroepithelial cells of day 9 mouse embryos. The cells expressing
caspase-3 were found to be abundant in the mesenchyme
of the first and second branchial arches, which coincided
with the CNC cell migratory areas in E8.5 mouse embryos
(Kubota et al., 1996). It was also shown that caspase-3deficient mice exhibit embryonic or early postnatal lethality and their central nervous system development was
severely impaired (Kuida et al., 1996). Further, caspase-3
deficiency in mice resulted in decreased embryonic neuroblast apoptosis and neoplastic growth of the brain (Pompeiano et al., 2000). Thus, caspase-3 seems to play a critical role in the induction of morphogenetic apoptosis in
embryos, especially in the nervous system. These reports
warrant further investigation for elucidating the mechanisms of differentiation and migration of CNC cells as well
as the significance of their apoptotic death observed in
craniofacial morphogenesis.
The authors are grateful to Dr. Kazuo Ito, Osaka University Graduate School of Science, for providing the 4E9R
antibody and Dr. Shigehito Yamada for his technical assistance.
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