Advances in Bioscience and Biotechnology, 2012, 3, 751-769 ABB Published Online October 2012 (
Maternally-preset program of apoptosis and caspases
involved in execution of the apoptosis at midblastula
transition (MBT) but not before in Xenopus laevis
Koichiro Shiokawa
Postgraduate School of Judo Therapy, Faculty of Medical Technology, Department of Biosciences, School of Science and Engineer-
ing, Teikyo University, Utsunomiya-City, Japan
Received 21 August 2012; revised 23 September 2012; accepted 4 October 2012
To study gene control mechanisms in Xenopus em-
bryos, we analyzed polyamines, cloned SAMDC (S-
adenosylmethionine decarboxylase), a key enzyme of
polyamine metabolism, and microinjected its mRNA
into Xenopus fertilized eggs. The microinjection in-
duced a large increase in SAMDC activity, exhaustion
of the substrate SAM (S-adenosylmethionine), and
execution of apoptosis at the stage called midblastula
transition (MBT). By tracing GFP (green fluores-
cence protein)-marked apoptotic cells, we reached a
conclusion that the apoptosis provides pre-blastula
embryos with a fail-safe mechanism of early devel-
opment. We analyzed caspase mRNAs and found that
caspase-9 and -3 mRNAs are maternal mRNA and
activation of caspase-9 is one of the key steps for the
execution of the apoptosis. We also found that over-
expression of caspase-8, and in addition p53, a tumor
suppressor protein, also induces apoptosis at MBT,
just like the overexpression of SAMDC and caspase-9
does. The apoptosis induced by p53 was suppressed
by Xdm-2, a negative regulator of p53, and by a pep-
tide inhibitor and a dominant-negative type mutant of
caspase-9, but not by those of caspase-8. By contrast,
apoptosis induced by SAMDC was suppressed by
peptide inhibitors and dominant-negative mutants of
both caspase-9 and caspase-8, but not by Xdm-2.
Unlike caspase-9 mRNA, caspase-8 mRNA was not a
maternal mRNA, but newly expressed during cleav-
age stage (pre-MBT stage) only in embryos overex-
pressed with SAMDC. In SAMDC-induced apoptotic
embryos activities to process procaspase-8 and pro-
caspase-9 appeared, whereas in p53-induced apop-
totic embryos only activity to process procaspase-9
appeared. Thus, Xenopus embryos have at least two
pathways to execute the maternal program of apop-
tosis: One induced by SAMDC overexpression th-
rough activation of caspase-9 and do novo expres-
sion of caspase-8 gene, and the other induced by p53
overexpression through activation of caspase-9 but
not caspase-8. In Xenopus embryos, it has long been
believed that zygotic genes are silent until MBT, but
results obtained with caspase-8 may provide a novel
example of gene expression before MBT.
Keywords: Maternal Program of Apoptosis; Midblastula
Transition (MBT); Polyamines; S-Adenosylmethionine
Decaroboxylase (SAMDC); Xenopus laevis Embryos,
Caspases; p53; pre-MBT Transcription
The normal table of development for Xenopus laevis has
been established by Nieuwkoop and Faber [1], in which
the development is divided into 66 stages from fertiliza-
tion to metamorphic climax. The fertilization which is
the fusion of two different genome sets is the start of the
development of a new life, and the metamorphosis which
is characterised by spectacular tail regression is the end
of a tadpole to make a juvenile. The development can be
roughly divided into four steps: cleavage (segmentation),
organogenesis, growth period and metamorphosis [2]
(Figure 1). Once the Xenopus egg is fertilized, it begins
to divide in about 1.5 hr, producing two similar embry-
onic cells (blastomeres), and after several divisions, be-
comes a morula. In Xenopus, such rapid cell divisions
(cleavage) continue for 12 rounds, and an egg becomes a
blastula consisting of ca. 4000 cells, which now has a
large internal cavity called blastocoel. After the 12
rounds of cell division, the embryo reaches the stage of
midblastula transition (MBT), when the cell cycle
aquires G1 and G2 phases. Active cross-talk of morpho-
genetic factors starts and the embryo reaches gastrula
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
Figure 1. Synoptic view of main developmental stages in Xenopus laevis. MBT takes place between cleavage
and organogenesis in this picture. From Estabel et al. [2].
stage (stage 10 to 12.5). The morphogenetic movements
called gastrulation consist of invagination of Spemann’s
organizer followed by embryonic induction. On the dor-
sal part of the embryo, the neuroectoderm is induced by
the invaginated dorsal mesoderm, and this phase is the
onset of neurulation (stage 13). At this stage, the neural
tube is formed, which becomes the origin of brain, neural
crest and spinal cord. In the neurula the axial mesoderm
differentiate to form origins of skeleton, muscles, kid-
neys, heart and circulatory system, and gonads. The en-
doderm whose cells still have a large amount of yolk
granules in their cytoplasm becomes the origin of endo-
dermal organs such as digestive tract and lungs. Along
with these changes, the embryo is elongated, reaches the
tailbud stage, and after hatching, becomes a tadpole.
In vertebrates, Glucksman [3] first reported cell death
during the development. Cell death was observed at
blastocyst stages in mammals, as well as during gastrula-
tion in the urodelan amphibian Cynops pyrrhogaster [4].
Apoptosis was detected in most vertebrates as pro-
grammed cell death in later embryos particularally dur-
ing the development of the nervous system, although as
described below, recent studies were devoted to the de-
tection of apoptosis at very early stages of development
[5-12]. In Xenopus nervous system, Hensey and Gautier
[8] detected apoptotic cells in brain, eyes, spinal cord,
and developing tail at the beginning of tail-bud stages
(stages 21 to 28). Apoptotic cells were also observed in
the central nervous system, particularly in the telen-
cephalon, diencephalon and mesencephalon from stages
29 to 35, possibly related to the acquisition of the defini-
tive form and structure of the brain, [13]. In Xenopus
early neurulae, a gene called XHR1 has been cloned
which defines later the region where apoptosis or pro-
grammed cell death occur for segmentation in brains [14].
At around stage 52, tadpoles start metamorphosis. At the
onset of metamorphosis, the spinal cord contains nu-
merous apoptotic cells, principally in the interneuron
area, and many cells of the caudal spinal ganglia also
undergo apoptosis [15]. During climax, caspase-3 activ-
ity increases in the spinal cord [16]. The frequency of
apoptotic cells becomes peak at stage 58 tadpole stage in
the spinal cord [15], and muscles of the tail regress to-
tally by programmed cell death [17]. Caspase-3 activity
increased in muscle cells during the climax stages [18].
In the early embryonic development of Xenopus laevis
midblastula transition (MBT) occurs after 12 cycles of
cleavage in [19,20], which is a time when embryos shift
from the phase of low (per embryo) transcription [21-24],
or the phase of development driven mainly by maternal
stockpiles, to the phase of high (per embryo) transcript-
tion or the phase driven mainly by gene products newly
produced based on the zygotic nuclei. During the pre-
MBT stage or the cleavage stage, cell divisions are rapid
without G1 and G2 phases in the cell cycle. This is be-
cause the Xenopus egg is a huge cell provided in its cy-
toplasm with an extremely large amounts of maternal
stockpiles and the egg can keep dividing without synthe-
sizing materials necessary for cell divisions. It is true that
maternal mRNAs in the oocyte provide most of the in-
formation necessary for harmonious development from
cleavage to MBT and to gastrula stage. However, this
does not necessarily implies that transcription should be
turned off during the cleavage. Thus, during this period,
cells produce a few RNAs [21-24], although cells in this
cleavage stage embryo have long been believed to be
transcriptionally totally silent [20]. The reason for the
difficulty to detect the synthesis of mRNA and other
small RNAs during the cleavage stage is mainly the
small number of cells constituting the embryo: For ex-
ample, a late blastula in which transcription could easily
be detected consists of 4000 to 8000 cells (nuclei),
whereas a 64-celled embryo at the cleavage stage con-
sists of only 64 cells (nuclei).
After the MBT stage (4096 cells/embryo), when G1
and G2 phases reappear in the cell cycle, various changes
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769 753
in cellular activities take place [25]. Thus, cell division
becomes asynchronous [19,20,26], and cells acquire mo-
tility [20,27], and cell cycles shift from checkpoint-un-
regulated to checkpoint-regulated ones [28,29]. At MBT,
transcriptional activity from zygotic nuclei is reportedly
very high. In fact, a strong activation of transcription
from zygotic nuclei takes place at the MBT: Quantitative
measurements revealed that the transcriptional activity to
form heterogeneous mRNA-like RNA at MBT is ca.
100-folds (on a per-nucleus basis) of the activity of the
cells of the later stage embryos (e.g. gastrula and neurula
cells). On the other hand, rRNA transcription does not
occur during the cleavage stage. Instead, it starts only at
and after MBT, and once it starts its rate per nucleus is
constant in later stage embryos [30,31]. As the cytology-
cal manifestation of the rRNA synthesis, nucleoli start to
be formed from the MBT stage [32]. The transcriptional
activation during the cleavage stage (pre-MBT stage) is
also proved for transcription from exogenously-intro-
duced genes like bacterial CAT (chloramphenicole ace-
tyltransferase) genes [33], although this was once also
reported and accepted to be expressed only at and after
MBT, but not before [34]. The elongation of the cell cy-
cle after the MBT which is due to the appearance of G1
and G2 phases may be regulated by the activation of
Xchk1 kinase [28], which is essential in remodelling the
cell cycle after MBT [28,35,36].
In this review article, I describe our studies on the
unique polyamine composition followed by cDNA clon-
ing of S-adenosylmethionine decarboxylase (SAMDC),
and the results derived from the microinjection of its
mRNA into Xenopus fertilized eggs, which quite unex-
pectedly induced massive cell dissociation, which turned
out to be due to the execution of the maternally-preset
program of apoptosis. I then proceed to describe our ex-
periments to microinject the SAMDC mRNA into only
one blastomere at 8- to 32-cell stage embryos, and to
trace the fate of the injected cells. The results obtained
lead us to conclude that the apoptosis which is executed
so early in the development is a kind of “fail-safe”
mechanism to check and eliminate damaged cells before
embryos enter the morphogenic phase after the MBT
stage. In the latter half of this article I describe the results
of analyses on mRNAs of caspases 9 and 8 which were
found to be involved in the maternal program of apop-
tosis. It appeared here that in SAMDC-overexpressed
embryos, de novo synthesis of caspase-8 mRNA takes
place at cleavage stage. Based on the results obtained, we
conclude that Xenopus embryos have at least two path-
ways to execute the maternal program of apoptosis; one
executed by SAMDC-overexpression, and the other
executed by p53-overexpression, and while the former is
executed through activation of caspase-8 before the acti-
vation of caspase-9, the latter is activated not through
caspase-8 but directly through caspase-9.
2.1. Polyamines and SAMDC
(S-Adenosylmethionine Decarboxylase)
Cloning in Xenopus Embryos
Natural polyamines (putrescine, spermidine, and sper-
mine) occur ubiquitously in both prokaryotic and eu-
karyotic cells [37], and polyamine biosynthesis is regu-
lated also by polyamines themselves [38]. Prokaryotic
cells like Escherichia coli have a high content of putre-
scine and spermidine, with no detectable amount of
spermine. The absence of spermine in E. coli cells is due
to the lack of spermine synthase, an enzyme which con-
verts spermidine into spermine [39]. Eukaryotic cells, by
contrast, have the enzyme spermine synthase, and hence
have a relatively high content of spermine [40].
Various studied have been performed on polyamines
in oocytes and embryos of mouse [41], chick [42], sea
urchin [43], poychete [44], and Drosophila [45]. In am-
phibians, especially in Xenopus laevis, amounts of
polyamines have been determined during oogenesis [46,
47], oocyte maturation [47-49], and early embryogenesis
[47,50-52]. In Xenopus oogenesis, levels of putrescine,
spermidine, and spermine keeps increasing, and at the
end of the oogenesis, the level of spermine decreases
abruptly, and further decreases during maturation (from
4 hr after the administration of progesterone). As a result,
the level of spermine reaches a very low level in the end
of maturation, and also from fertilization through tadpole
stage, the level of spermine remains low (less than 0.1
nmole per embryo) [47,50,51]. On the other hand, levels
of putrescine and spermidine are much higher from
oogenesis through early embryogenesis [47,53]; the
amount of putrescine being more than twice that of
spermidine throughout stages. Thus, polyamine compo-
sition in Xenopus early embryos is apparently similar to
that of bacteria.
Both spermidine synthase and spermine synthase are
constitutively synthesized [54], and therefore, ornithine
decarboxylase (ODC) and S-adenosylmethionine decar-
boxylase (SAMDC) are key enzymes which are rate-
limiting in polyamine biosynthesis. ODC produces pu-
trescine from ornithine by eliminating its carboxyl group,
whereas SAMDC decarboxylates S-adenosylmethionine
(SAM ) and produces decarboxylated SAM (dcSAM),
which provides cells with the aminopropyl group neces-
sary to form spermidine and spermine from putrescine
and spermidine, respectively.
We screened a Xenopus tailbud cDNA library using
the human SAMDC cDNA as a probe and isolated
Xenopus SAMDC cDNA (1030 bp). We examined the
changing level of SAMDC mRNA by Northern blot
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
analysis in growing oocytes, oocytes during maturation,
and developing embryos at various stages, and found that
the signal for SAMDC mRNA (3.5 Kb) was detected at
all the stages examined. The level of SAMDC mRNA
was relatively high from the earliest stage of oogenesis,
but after fertilization, decreases until the early neurula
stage, and again increases after tailbud stage [47,52]. The
level of SAMDC activity was quite low both at the
cleavage and early neurula stage, and it starts to increase
only at the early tadpole stage, although another key en-
zyme, ODC increases in its level shortly after MBT [51].
2.2. Overexpression of SAMDC mRNA in
Xenopus Embryos and Discovery of Sudden
Cell Dissociation at MBT
To test if the SAMDC mRNA obtained from the cDNA
is functional, we in vitro-transcribed mRNA from the
cDNA, and microinjected the mRNA (1 ng/egg) into
Xenopus fertilized eggs. The mRNA-injected embryos
cleaved and developed quite normally up to the blastula
stage, but at the mid to late blastula stage (shortly after
MBT), all the embryos suddenly underwent cell disso-
ciation (Figure 2). These embryos were soon dissolved
completely due to osmotic shock [7]. When we co-in-
jected beta-galactosidase mRNA and SAMDC mRNA
into fertilized eggs, only cells which expressed beta-ga-
lactosidase were dissociated (Figure 3). When we co-
injected mRNAs for GFP (green fluorescent protein) and
SAMDC into only one blastomere of 2-celled embryos,
only a half portion of embryos which expressed GFP was
dissociated at the late blastula stage (Figure 4). Then, it
Figure 2. Apoptosis induced by SAMDC
overexoression. Fertilized eggs were injected
with Xenopus SAMDC mRNA (100 pg), and
cultured in isotonic 1× Steinberg’s solution to
protect dissociated cells from a osmotic shock.
Embryos were filmed at late blastula stage.
Dissociated cells appeared in the mRNA-in-
jected region. From Shibata et al. [7].
Figure 3. Beta-Galactosidase marking of dis-
sociated cells. Fertilized eggs were injected
with 0.5 ng/egg each of beta-galactosidase
mRNA and SAMDC mRNA. Only cells stained
with X-gal were dissociated. From Shibata et
al. [7].
Figure 4. Apoptosis is induced not at early
blastula stage but at late blastula stage, or after
MBT. Only one blastomere of a 2-celled em-
bryo was co-injected with SAMDC mRNA (1
ng/egg) and GFP mRNA (100 pg/egg), and
embryos were filmed at early blastula (top two)
and early gastrula (bottom two) stages using
the visible light (left two) and UV light (right
two) [Kuroyanagi S, Shiokawa K, unpub-
was apparent that the injection of SAMDC mRNA was
the cause of the sudden cell dissociation.
When sectioned materials of these SAMDC-overex-
pressed embryos were examined at early blastula stage,
there was no difference in the appearance of blastoceol
and arrangement of cells between the control and the
SAMDC-injected embryos. At late blastula stage, how-
ever, a large number of dissociated cells were found in
the blastoceol. The percentage of embryos which under-
went the cell dissociation was dosage-dependently changed
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769 755
between 0.01 ng/egg and 10 ng/egg, and in this wide
range of the dosage, the timing of the cell dissociation
was constant: The cell dissociation took place always at
shortly after MBT, but not before. Injection of mRNAs
other than SAMDC mRNA, such as Xenopus type IIA
activin receptor, a Xenopus RNA-binding protein (nrp-1),
and Xenopus initiation factor eIF4E had no effect, and
SAMDC mRNA (1 ng/egg) without a cap structure was
also not effective, indicating that the cell autonomous
dissociation observed was due to the specific function of
the injected SAMDC mRNA.
Using 3H-thymidine, 3H-uridine and 14C-leucine to la-
bel DNA, RNA and protein, respectively, we found that
protein synthesis was the first to be inhibited in the
SAMDC-overexpressed embryos and the inhibition was
detected at early blastula stage before the initiation of
cell dissociation. Since the endogenous SAMDC mRNA
was estimated as 0.005 ng or less per embryo, injection
of 1 ng/egg of SAMDC mRNA resulted in overexpres-
sion of SAMDC mRNA at least 200-folds, and this re-
sulted in ca. 400-folds of the increase in SAMDC en-
zyme activity. In the SAMDC mRNA-injected embryos,
the high level of SAMDC mRNA was maintained until
the blastula stage, but it was reduced to the background
level by the gastrula stage. This SAMDC overexpression
exerted little influence on the polyamine composition
within the embryo [47], probably because we did not
simultaneously overexpress the spermidine synthase and
spermine synthase. The cell dissociating effect of
SAMDC mRNA was completely abolished by co-inject-
tion of EGBG (ethylglyoxal-bis-guanylhydrazone) (20
pmoles/egg), a specific inhibitor of SAMDC [38,55]. We
found here that the intracellular level of SAM was re-
duced by more than 80% by the SAMDC mRNA inject-
tion, and when SAM was co-injected with SAMDC
mRNA, embryos were completely rescued (Figure 5).
Therefore, we concluded that injection of SAMDC
mRNA induces SAM deficiency and this was the pri-
mary cause of the induction of the massive cell dissocia-
2.3. The Cell Dissociation Is Due to Apoptosis
The evidence that the cell dissociation observed was due
to execution of apoptosisis is as follows. First, electron
microscopic analyses revealed that nuclei of SAMDC
mRNA-induced dissociated cells were fragmented into
two or three portions and then further fragmented [9]
(Figure 6). In such SAMDC mRNA-injected embryos, a
large number of cells became TUNEL-positive (Figure
7), and furthermore, DNA extracted therefrom formed
“ladders” on agarose gels [9]. We first injected SAMDC
mRNA into uncleaved fertilized eggs, and at the 2-cell
stage further injected into only one of the blastomeres a
Figure 5. Rescue of embryos from cell dissociation by SAM.
Fertilized eggs were injected with either SAMDC mRNA (0.1
ng/egg) alone or SAMDC mRNA (0.1 ng/egg) plus SAM (200
pmoles/egg) and percentages of normally developing embryos
were plotted. From Shibata et al. [7].
Figure 6. Electron microscopic pictures of dissociated cells.
Fertilized eggs were injected with SAMDC mRNA (1 ng/egg)
and cultured in 1× Steinberg’ solution to protect from osmotic
shock and examined at early gastrula stage (A), (B) or midges-
trula stage (C), (D). Scale bars are 2 μm. N, nucleus; LD, lipid
droplet; M, mitochondria. Kai et al. [9].
mixture of GFP mRNA and Xenopus Bcl-2 mRNA, as an
anti-apoptotic factor that suppresses the release of cyto-
chrome c from mitochondria [56,58]. We found here that
cell dissociation was suppressed only in the GFP-positive
and hence Bcl-2-expressing half embryo [9]. When we
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
Figure 7. TUNEL staining of SAMDC mRNA-
injected embryos. Embryos were injected with
SAMDC mRNA (1 ng) and examined for
TUNEL reaction at early gastrula stage. Kai et
al. [9].
injected Bcl-2 mRNA and SAMDC mRNA together into
uncleaved fertilized eggs, all the embryos were found to
escape the cell dissociation and about half of such res-
cued embryos became normal tadpoles (Figure 8). These
results indicated that the dissociation observed was due
to execution of apoptosis.
While we were analyzing the SAMDC-induced apop-
tosis, various toxic agents, such as gamma-ray [6,12],
hydroxyurea [59], cycloheximide [6,59], and alpha-
amanitin [5,6] were reported to induce similar apoptosis
in Xenopus blastulae. Sible et al. [5] and Hensey and
Gautier [6] reported that injection of Bcl-2 mRNA re-
tarded the onset of apoptosis by 2 - 3 hr. However, such
rescued embryos were then dissociated and died. When
we injected 5-aza-2’ deoxycytidine (5-Aza-CdR) which
induces hypomethylation of DNA and 5-methyl-2’-de-
oxycitidine-triphosphate (5-methyl dCTP) which induces
hypermethylation of DNA [60], embryos developed
normally up to MBT, but they were dissociated and died
due to apoptosis. When Bcl-2 mRNA was co-injected
with these inhibitors of methylation, Bcl-2 here again
only postponed the cell dissociation by 2 - 3 hr. There-
fore, the complete rescue which was realized by Bcl-2 in
the SAMDC-induced embryos is interesting. Probably,
the reason for the complete rescue is due to the disap-
pearance of the overexpressed SAMDC due to its meta-
bolic instability [61].
2.4. The Apoptosis May Be a Fail-Safe
Mechanism of Early Development for
Embryos to Proceed beyond MBT
To obtain some idea about the developmental signifi-
cance of the apoptosis executed at such an early stage of
development, we examined embryos that had been in-
jected with either wild-type SAMDC mRNA or artifi-
Figure 8. Rescue of SAMDC mRNA-injected embryos by
Bcl-2 mRNA. Fertilized eggs were injected with 0.5 ng of
SAMDC mRNA and cultured in 1× Steinberg’s solution. Con-
trol cells were dissociated at early gastrula stage (Top, left),
and remained dissociated (Top, right) when rescued embryos
reached tailbud stage. Fertilized eggs co-injected with 0.5 ng of
SAMDC mRNA and 2 ng of Bcl-2 mRNA were not dissociated
at early gastrula stage (Bottom, left) and reached the tailbud
stage (Bottom, right). From Kai et al. [9].
cially mutated, non-effective (defective), SAMDC mRNA
into only one blastomere of embryos at 16 - 32 cell
stages. In this experiment both types of mRNAs were
mixed with GFP mRNA as a lineage tracer, so that the
descendant cells of the injected blastomere could be
traced. All the embryos injected with wild-type SAMDC
mRNA into one of their blastomeres at these stages be-
came tadpoles, without showing any sign of cell disso-
ciation (apoptosis) at MBT at least in their outer appear-
ance [61]. At late blastula stage, however, luminescent
cells which had been recognized on the surface of the
embryo disappeared from the wild-type mRNA-injected
embryos, whereas widely-spread luminescent cells re-
mained on the surface of embryos injected with the de-
fective SAMDC mRNA [61]. To trace the luminrscent
cells we dissected the embryos and examined the inside
of the embryo. Here, we found many dissociated lumi-
nescent cells within the blastocoel in the wild-type
mRNA-injected embryos, but not in the defective mRNA-
injected embryos. The occurrence of dissociated cells
inside the blastocoel was also confirmed in the sec-
tioned materials. Thus, it appears that the blastocoel pro-
vides not only the space into which mesodermal cells
invaginate, but also the space for apoptotic cells to be
separated and to undergo apoptosis [61].
We examined these embryos also at the tadpole stage.
We found that whole body of the mutant mRNA-injected
tadpoles were green in the UV light due to the presence
within the embryo of many luminescent cells throughout
the body. By a sharp contrast, no luminescent cells re-
mained in the wild-type mRNA-injected tadpoles. Inter-
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
Copyright © 2012 SciRes.
Similar apoptotic cell death has been reported in ze-
bra-fish embryos [63].
esting here was that tadpoles derived from wild-type
mRNA-injected embryos were shorter in body length,
and sometimes even abnormal, having small head (some-
times acephaly), small trunk and tail, and body axis-
bending. The execution of apoptosis at MBT and the size
of the tadpoles which survived and developed beyond
MBT in these experiments can schematically shown as in
Figure 9. As Hensey and Gautier [8] pointed out, cells in
Xenopus early embryos may check themselves at MBT
to see if they are capable of continuing further develop-
ment. If some cells find themselves physiologically ab-
errant, they disappear from the embryo by executing the
apoptotic program (Figure 10). The cellular activities to
be checked here seem to include intactness of DNA
structure, occurrence of DNA replication, and DNA me-
thylation, normal RNA transcription, and translation, or
protein synthesis, and the level of SAM, as expected
from the results summarized above. We assume that this
apoptosis constitutes a surveillance or a “fail-safe”
mechanism for normal development to check and elimi-
nate damaged cells at MBT when G1 phase first appears
in the cell cycle in order to save the rest of the embryo
and permit them to continue the development [61,62].
2.5. Possible Mechanism of the Execution of the
Apoptosis at MBT, but Not before
One question which arises here is the reason why the
embryonic apoptosis is executed at MBT but not before.
There have been several experimental challenges about
this [2]. One possible approach could be related to DNA
methylation control. It has been reported that DNA me-
thylation supported by methyltransferase cDnmt1P might
control the switching on-off of apoptosis in cleavage
stage [64]. This is because the depletion of this enzyme
appears to activate Xenopus p53 system which is related
to apoptosis. Another pathway may involve a signal pro-
vided by members of BMP (bone morphogenetic protein)
family. The Xenopus protein Smad8 (xSmad8) is a in-
tracellular mediator of BMP signalling, carrying the sig-
nal from the cytoplasm to the target DNA within the nu-
cleus. Depletion of xSmad8 in embryos causes apoptosis
[65]. Segmentation was observed in xSmad8-depleted
embryos, but these embryos could not deveop beyond
gastrulation and died. Another molecule, Bix3, is also
Figure 9. Experimental design and results of injection of SAMDC into only one of the blastomeres at differ-
ent stages. SAMDC mRNA was injected into only one blastomere at different stages and cultured. The
amount of SAMDC mRNA was adjusted so that the concentration of the mRNA in the cytoplasm of the in-
jected cell was approximately the same (0.5 ng/1 micro liter; 1 micro liter is the approximate volume of an
egg). Red marks indicate the cell dissociation which takes place at MBT. From Kai et al. [61].
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
Figure 10. A model which shows how early development proceeds. This model suggests possible occurrence of
apoptotic check point which functions as a surveillance or “fail-safe” mechanism in Xenopus early embryonic de-
velopment. Fertilized eggs cleave rapidly until the early blastula stage. At MBT, the “first developmental check-
point” comes when G1 phase first appears. We assume that this check mechanism determines cell-autonomously
if the cell continues development. However, even when apoptosis was executed, embryos follow two different
courses. If the number of apoptotic cells was large, the whole embryo stops development and dies. If the number
of apoptotic cells was small, apoptotic cells are confined within the blastocoel and the embryo itself continues on
development. From Kai et al. [61].
expected to regulate cell death. The overexpression of
this molecule in the vegetal hemispheres of early em-
bryos triggered the apoptosis of embryonic cells [66]. In
oocytes and in early embryos up to stage 8 (midblastula),
the maternal survivin mRNAs are strongly expressed
[67]. This molecule may be a member of the IAP (in-
hibitors of apoptosis) family, and could be one of the
proteins that control programmed cell death in such a
way that its execution is not permitted until MBT. After
the increase in zygotic transcription at MBT, the amount
of maternal survivin mRNAs is reported to decrease
quickly, and this decline may be necessary for the execu-
tion of the apoptosis. If this is correct, survivin could be a
maternal inhibitor of apoptosis, which suppresses the
execution of the apoptosis before MBT.
2.6. Involvement of Caspase 9 in SAMDC
mRNA-Induced Apoptosis
The unique enzymes involved in the execution of apop-
tosis are caspases which constitute a cysteine protease
gene family [68]. Previous studies showed that cell lys-
ates of hydroxyurea-treated or gamma-ray-irradiated
Xenopus embryos have activity to cleave poly-ADP-
ribose polymerase (PARP) [6,59], a substrate of most of
caspases, including caspases-3 and 7 [68]. Also, it has
been reported that the synthetic peptide, which possibly
inhibits caspase-9 and caspase-3, partially blocks or de-
lays the onset of apoptosis induced by gamma ray-rra-
diation or cycloheximide treatment [6,59].
We first tested if caspase-9 is involved in the apoptosis
which is activated so early in the development in
SAMDC-overexpressing Xenopus embryos. Here, we
microinjected different doses of synthetic peptide-in-
hibitors for caspase-9 (Ac-LEHD-CHO) and caspase-1
(Ac-YVAD-CHO) into Xenopus fertilized eggs together
with SAMDC mRNA (200 pg/egg). The peptide-inhibit-
tor for caspase-9 suppressed the SAMDC mRNA-in-
duced apoptosis at 2000 and 200 pmoles/egg, and res-
cued embryos developed to neurula and even to swim-
ming tadpole stage (Figure 11, left). By contrast, the
peptide inhibitor for caspase-1 suppressed the induction
of apoptosis only slightly at 2000 pmoles/egg, but all the
embryos partially rescued here died before they reached
the neurula stage.
It is known that caspase-9 without the active site binds
to Apaf-1 and functions as dominant-negative caspase-9
[69]. It is also known that caspase-1 whose cysteine in
the active site was converted to glycine can work as a
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769 759
Figure 11. Inhibition of execution of SAMDC mRNA-
injected apoptosis by peptide inhibitors or dominant-
negative type caspase-9. Left: Different amounts of
synthetic peptide inhibitor for caspase-9 (Ac-LEHD-
CHO) (a) or caspase-1 (Ac-YVAD-CHO) (b) were
co-injected with 100 pg/egg of SAMDC mRNA into
Xenopus lavies fertilized eggs. Right: Effects of co-
injection (each 1000 pg/egg) of dominant-negative type
mutant of caspase-9 (dnCaspase-9) or caspase-1
(dnCaspase-1) together with SAMDC mRNA. From
Takayama et al. [71].
dominant-negative type caspase-1 [70]. We then gene-
rated “dominant-negative type” mutants (dn-caspases) of
caspase-9 [69], in which the cysteine residue at the active
site was replaced by phenylalanine, and we injected the
mutated mRNA into fertilized eggs together with
SAMDC mRNA [71]. We also generated here a domi-
nant-negative type caspase-1 [69,72] by modifying the
active site of wild type cDNAs [73,74], and injected
them into fertilized eggs together with SAMDC-mRNA
[71]. We found here that dominant-negative type cas-
pase-9 mRNA, but not dominant-negative type caspase-1
mRNA, suppressed SMDC-induced apoptosis (Figure
11, right). These results suggest that activation of cas-
pase-9, but not caspase-1, is the key step for the execu-
tion of the apoptosis in SAMDC-mRNA injected em-
It is well known that caspases are provided as inactive
procaspases and such precursor molecules of caspases
(procapsases) are cleaved to give rise to active form [68].
We then tested if there appears the caspase-activating
activity in the cytoplasm of the SAMDC mRNA-injected
embryos before the execution of the apoptosis. We in-
jected here SAMDC mRNA (200 pg/egg) into fertilized
eggs, and prepared cell-free lysates from the injected
embryos at stages 6.5 (still normally cleaving) and at
10.5 (already in the apoptotic process, and embryonic
cells stop cleaving, but they were still not lyzed due to
osmotic protection) (Figure 12(a)). On the other hand,
we prepared 35S-procaspases by in vitro-translation of
mRNAs, and then incubated the radioactive procaspases
in the embryo lysates. Analysis of the reaction products
by gel electrophoresis revealed that the lysate of
SAMDC-induced apoptotic embryos at stage at 10.5, but
not at stage 6.5, cleaved 35S-procaspase-9 into two pep-
tides (Figure 12(b)). By contrast, both of the cell lysates
of SAMDC-induced apoptotic embryos prepared at stage
6.5 embryos and stage 10.5 did not cleave 35S-procas-
pase-1 (Figure 12(c)). These results suggested that in
SAMDC mRNA-injected embryos, the activity to cleave
procaspase-9 but not procaspase-1, appeared at post-
MBT stage. Based on these results, we concluded that
caspase-9, but not caspase-1, is involved in the apoptosis
executed at this very early stage.
2.7. Maternal Nature of Caspase-9 in Xenopus
We carried out Northern blot analysis using RNAs ex-
tracted from SAMDC mRNA-injected embryos and un-
injected control embryos (Figure 13). In uninjected con-
trol embryos, we detected approximately 2.7 and 2.0 kb
signals for caspase-9 and caspase-1, respectively. mRNA
for caspase-9 occurred abundantly already in unfertilized
eggs (stage 1) as a maternal mRNA. The level was main-
tained until the midblastula stage (stage 8), and started to
decrease (at stage 10), reached the minimum level (at
stage 12), then increased toward the late neurula stage
(stage 22). This time-course of the changing level of
caspase-9 mRNA was just as those observed with other
maternal mRNAs such as aldolase A and C mRNAs [75].
On the other hand, mRNA for caspase-1 was detected
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
Copyright © 2012 SciRes.
(a) (b) (c)
Figure 12. Induction of procaspase-9-cleaving activity in SAMDC mRNA-injected Xenopus embryos. (a) Embryos were injected
with 100 pg/egg of SAMDC mRNA (closed circles) or beta-globin (open circles) into both of the blastmeres at the 2-cell stage (200
pg/embryo) and cultured in 1× Steinberg’s solution. Cell-lysate was prepared at stage 6.5 or stage 10.5. 35S-labelled procaspase-9 (b)
and 35S-labelled procaspase-1 (c) were incubated with the cell lyzates prepared at stage 6.5 or 10.5 from embryos injected with either
SAMDC mRNA (+) or b-globin mRNA (). Reaction mixtures were subjected to gel electrophoresis under reducing conditions.
From Takayama et al. [71].
Figure 13. Northen blot analysis of caspase mRNAs in Xenopus embryos. Fer-
tilized eggs were injected with SAMDC mRNA (100 pg/egg) or distilled water
(uninjected control embryos) and cultured in 1× Steinberg’s solution. RNAs
isolated were subjected to Northern bot analysis with 32P-labelled probes spe-
cific for Xenopus caspase-9 or caspase-1. 18S rRNA wasstained as a loading
marker with ethidium bromide. Experiments were not performed in SAMDC
mRNA-injected embryos after stage 12, because of the embryo death. From
Takayama et al. [7].
only after embryos developed to the late gastrula stage
(stage 12), and the level increased thereafter toward the
late neurula stage (stage 22). Thus, caspase-1 mRNA is
not a maternal mRNA and is activated only zygotically
after the gastrula stage. The appearance of the mRNA for
caspase-1 in post-gastrular embryos may be correlated to
the fact that this enzyme is involved probably in pro-
grammed cell death in neural tissues [76]. These expres-
sion profiles of two mRNAs were not appreciably af-
fected by SAMDC mRNA-injection throughout early
stages (from stage 4 to stage 12).
In this series of experiments, we also analyzed the
mRNA for caspase-3, which is known to be activated by
caspase-9 [68,69]. Caspase 3 mRNA (1.6 Kb) also oc-
curred as a maternal mRNA, but the level was much
lower as compared with that of caspase-9 throughout
early stages. We then co-injected SAMDC mRNA (0.5
ng/egg) and a specific peptide inhibitor for caspase 3
(Z-D(OMe)GMD(OMe)FMK) (0.5 ng/egg) into Xenopus
fertilized eggs. In this experiment, ca. 70% of the co-
injected embryos did not undergo cell dissociation and
developed beyond neurula stage (Kuroyanagi and Shio-
kawa, unpublished). We, therefore, assumed that cas-
pase-3 is also involved as an executive caspase in the
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769 761
SAMDC-induced apoptosis, probably at a step down-
stream of caspase-9 [77,79].
We in vitro-transcribed mRNAs for caspase-9 or cas-
pase-1 and then performed experiments to inject different
doses of these mRNAs into fertilized eggs. Injection of
mRNAs of both caspase-9 and caspase-1 at 100 pg/egg
induced cell dissociation shortly after MBT. We con-
firmed here that the DNAs extracted from the caspase-
overexpressed embryos formed DNA ladder for both
caspase-9 and caspase-1. When we injected these mRNAs
together with Bcl-2 mRNA (100 pg/egg), we found that
onset of cell dissociation was delayed for only 2 - 3 hr.
Therefore, these caspases induce apoptosis when they are
overexpressed in early embryos.
2.8. Involvement of Caspase 8 in the SAMDC
mRNA-Induced Apoptosis in Xenopus
We extended our studies on the possible involvement of
caspase-8. We first co-injected a synthetic peptide in-
hibitor for caspase-8 (Ac-IETD-CHO) and SAMDC
mRNA into Xenopus fertilized eggs. We found here that
the inhibitor of caspase-8 suppressed the SAMDC-in-
duced apoptosis dosage-dependently [80]. We then pre-
pared a dominant-negative type mutant of caspase-8, and
injected it into fertilized eggs together with SAMDC
mRNA. Here again, apoptosis executed by SAMDC-
overexpression was suppressed. These results suggested
that SAMDC induces apoptosis via steps that involve
activation of caspase-8 [80].
We prepared lysates of SAMDC-overexpressed em-
bryos at stage 6.5 (apoptosis is not yet executed) and at
stage 10.5 (embryos are already apoptotic but are not
lyzed because of the protection from the osmotic shock),
and incubated the lysates with in vitro synthesized 35S-
labelled procaspase-8. We found here that the lysate
from SAMDC mRNA-injected embryos at the stage 10.5,
but not stage 6.5, cleaved procaspase-8 [80], suggesting
that procaspase-8 is converted into active caspase-8 in
SAMDC-overexpressed embryos by the time of the exe-
cution of the apoptosis. We assume that the activation of
caspase-8 occurs prior to the activation of caspase-9 as in
other systems [77,78].
2.9. Comparison of SAMDC-Induced Apoptosis
and p53-Induced Apoptosis
p53 is a tumor suppressor protein and is highly expressed
in Xenopus early embryos, and essential for normal de-
velopment [81-84], yet its overexpression in Xenopus
early embryos induces cell dissociation and develop-
mental arrest at the gastrula stage [83]. We performed
experiment to overexpress p53 mRNA in Xenopus em-
bryos, and compared the effects of the overexpression of
p53 with that of SAMDC on Xenopus development. For
this purpose, we injected different amounts (10, 100, and
1000 pg/egg) of mRNAs for SAMDC, caspase-8, p53,
beta-galactosidase, caspase-9, and alpha-globin into
Xenopus fertilized eggs and examined the development
of the injected embryos. As in embryos injected with
mRNAs for SAMDC and caspase-9, cell dissociation
took place in embryos injected with mRNA for caspase-8
and p53 at the early gastrula stage, although injection of
mRNA for beta-galactosidase and alpha-globin did not
induce such effects even at 1000 pg/egg. The apoptosis-
inducing effects were dosage-dependent for both cas-
pase-8 mRNA and p53 mRNA. DNAs extracted from
embryos injected with mRNA for caspase-8, p53 or
SAMDC were all found to form DNA ladder in their
fast-moving regions, whereas DNA from embryos in-
jected with alpha-globin mRNA migrated as high-mo-
lecular-weight DNA at the top of the gel. When we in-
jected mRNA of an anti-apoptotic factor, Bcl-2 [56,57],
together with mRNA for p53 or caspase-8, cell dissocia-
tion was delayed by about 3 hours but all the embryos
died after the 3 hr. We then concluded that injection of
caspase-8 or p53 mRNA induces cell dissociation due to
the execution of apoptosis.
Xdm-2, a Xenopus homologue of mouse Mdm-2,
which directly binds p53 and inhibits p53-mediated
transactivation [85], is expressed in early Xenopus em-
bryos, and is important for the control of p53 activities
[86]. When we injected mRNA for Xdm-2 together with
mRNA for either p53 or SAMDC into Xenopus fertilized
eggs, p53-induced apoptosis, but not SAMDC-induced
apoptosis, was suppressed. Also, these results suggest
that SAMDC-induced apoptosis is not executed through
the p53-mediated pathway. We then tested effects of
co-injection of a synthetic peptide-inhibitor for caspase-9
(Ac-LEHD-CHO) and caspase-1 (Ac-YVAD-CHO) on
the apoptosis to be executed by overexpression of p53
mRNA and SAMDC mRNA. The peptide inhibitor for
caspase-9 inhibited both p53-induced and SAMDC-in-
duced apoptosis, whereas the peptide inhibitor for cas-
pase-1 did not show such effects. We further tested ef-
fects of co-injection of dominant-negative type caspase-9
mRNA and dominant-negative type caspase-1 mRNA
[69] on the apoptosis-inducing effects of SAMDC
mRNA and p53 mRNA. Dominant-negative type cas-
pase-9 suppressed both p53-induced and SAMDC-in-
duced apoptosis, whereas dominant-negative type cas-
pase-1 did not show such effects. These results indicated
that both SAMDC and p53 execute apoptosis through the
activity of caspase-9, but not through the activity of cas-
We then injected a peptide-inhibitor for caspase-8
(Ac-IETD-CHO) or caspase-1 (Ac-YVAD-CHO) to-
gether with mRNA for p53 or SAMDC into fertilized
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
eggs. Both the peptide inhibitors for caspase-8 and cas-
pase-1 did not inhibit the p53-induced apoptosis, whe-
reas the peptide inhibitor for caspase-8, but not cas-
pase-1, inhibited the SAMDC-induced apoptosis (Figure
14). In this case, SAMDC-overexpressed embryos res-
cued by the co-injection of the peptide inhibitor devel-
oped beyond the neurula stage (stage 22). We also found
that dominant-negative type caspase-8 mRNA did not
suppress p53-induced apoptosis, although it suppressed
the execution of SAMDC-induced apoptosis. These re-
sults suggest that while p53 induces apoptosis without
passing the step that involves caspase-8, SAMDC-in-
duced apoptosis is mediated through the step that in-
volves caspase-8.
2.10. In p53 mRNA-Induced Apoptotic Embryos
Activity to Process Procaspase-8 Does Not
We injected either p53 mRNA or SAMDC mRNA into
Xenopus fertilized eggs, and after culturing the injected
(a) (b)
(c) (d)
Figure 14. Effects of coinjection of peptide inhibitors and
dominant-negative type mutants of caspase-8 and caspase-1
into p53 or SAMDC mRNA-injected embryos. Fertilized eggs
were co-injected with 1000 pmoles of peptide-inhibitor for
caspase-8 or caspase-1 together with mRNA for p53 (a) or
SAMDC (b). Also, fertilized eggs were co-injected with 1000
pg/egg of mRNA for dominant-negative type mutant of cas-
pase-8 (dnCaspase-8) or caspase-1 (dnCaspase-1) together with
mRNA for p53 (c) or SAMDC (d). From Shiokawa et al. [80].
embryos in 1× Steinberg’ solution, prepared their lys-
ates at stages 6.5 (still normally cleaving) and 10.5 (al-
ready in the apoptotic process). We incubated 35S-la-
belled procaspase-8 in the embryo lysate, and analyzed
the reaction products by gel electrophoresis. It was found
here that while the lysate from p53 mRNA-induced
apoptotic embryos cleaved procaspase-9, it does not
cleave procaspase-8, inspite of the fact that the lysate of
SAMDC mRNA-injected apoptotic embryos cleaved
both procaspase-9 and procaspase-8 (Figure 15). It is
then concluded that in p53 mRNA-injected embryos the
activity to cleave procaspase-9, but not procaspase-8,
appears, although in SAMDC mRNA-injected embryos,
activities to cleave both procaspase-8 and -9 appear be-
fore the execution of apoptosis.
2.11. Caspase-8 mRNA Is Newly Synthesized in
SAMDC-Overexpressed Cleavage Stage
Since caspase-8 appeared to be involved in the SAMDC-
induced apoptosis, we injected mRNA of either p53 or
SAMDC into fertilized eggs and analyzed the RNAs
extracted from embryos at stage 6.5 (pre-MBT stage) or
8.5 (MBT stage). Northern blot analyses revealed that
caspase-8 mRNA does not occur as a maternal mRNA in
the untreated control embryos (Figure 16). To our sur-
prise, however, the caspase-8 mRNA (3.0 Kb) appeared
in SAMDC mRNA-injected embryos both at non-apop-
totic (stage 6.5) and apoptotic (stage 8.5) stages [8]. In
p53 mRNA-injected embryos, caspase-8 mRNA was not
detected at both stages, although caspase-9 mRNA was
detected throughout stages. The level of caspase-9 mRNA
was not significantly affected by p53 mRNA injection at
both stages. By RT-PCR analysis we obtained the paral-
lel results (Figure 17).
These results indicate that caspase-8 mRNA was de-
tected as early as at stage 6.5. For many years, most
Xenopus researchers believed that pre-MBT embryos are
transcriptionally totally silent. It is true that rRNA syn-
thesis is absent during pre-MBT stage until it starts right
after MBT [30,31]. However, several RNAs have been
reported to be synthesized during the pre-MBT stage.
Such RNAs include heterogeneous mRNA-like RNA
labeled with 3H-uridine [21], and RNAs of nodal-related
TGF-beta superfamily member genes, Xnr5 and Xnr6
[24]. Thus, caspase-8 mRNA is the third species of po-
lymerase II-transcribed RNA that has been reported to be
expressed in pre-MBT stage. It is worth pointing out here
that the activity to cleave pro-caspase-8 was detected
only at stage 10.5 but not 6.5. We, therefore, suggest that
while transcription of caspase-8 is activated at stage 6.5,
a factor that converts procaspase-8 to active caspase-8 is
induced at stages later than stage 6.5.
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
Copyright © 2012 SciRes.
(a) (b)
Figure 15. Appearance of the activity to cleave pro-caspase-8 and procaspase-9 in
the lysates of p53 mRNA- and SAMDC mRNA-overexpressed apoptotic embryos.
35S-methionine-labelled enzymatically-defective pro-caspases were prepared and
incubated with lysates prepared from embryos injected with mRNA (+) (1000
pg/embryo) for p53 or SAMDC. Lysates were also prepared from embryos in-
jected with beta-globin mRNA alone () (1000 pg/embryo) as control experiments.
Reaction products were analyzed on SDS-PAGE under the reducing condition and
autoradiographed. (a) Lysate of p53-overexpressed embryos. (b) Lysate of
SAMDC-overexpressed embryos. Molecular size markers, locations of pro-cas-
pase (open arrow head), and cleaved products (closed arrow heads) are shown.
From Shiokawa et al. [80].
Figure 17. RT-PCR analyses for caspase-8 and
caspase-9 mRNAs in p53- or SAMDC-overex-
pressed embryos. RNA analyzed in the experi-
ment in Fig. 18 was subjected to RT-PCR. The
signal obtained for caspase-8 mRNA was 396 bp,
and that for caspase-9 mRNA was 539bp. 28S
and 18S rRNAs were stained with ethidium bro-
mide. From Shiokawa et al., [80].
Figure 16. Northern blot analyses for caspase-8 and
caspase-9 mRNAs in p53- or SAMDC-overexpressed
embryos. Fertilized eggs were injected (+), or not ()
injected, with p53 or SAMDC mRNA (1000 pg/em-
bryo) and cultured in 1× Steinberg’s solution. RNAs
were isolated from embryos at different stages. (A)
RNAs were separated on a 1% agarose gel containing
formaldehyde, transferred to a nylon membrane, and
hybridized with 32P-labelled DNA probes for Xenopus
caspase-8 and 9. From Shiokawa et al. [80].
and this might cause the inhibition of mRNA cap methyl-
lation, which may in turn induce the inhibition of protein
synthesis [7]. In our early experiment to label Xenopus
embryonic cells with (methyl-3H)methionine at cleavage,
blastula, gastrula, and neurula stages, a very high activity
was detected in mRNA cap methylation in cleavage
stage embryos [30] (Figure 18). At this stage there is no
ribosomal RNA synthesis as detected by almost negligi-
ble 2’-O-methylation in high-molecular-weight RNA
species [30,31]. It may be assumed that SAMDC over-
expression suppresses this active mRNA cap methylation.
2.12. A Possible Correlation between
SAMDC-Induced Apoptosis and a High
Activity of mRNA Cap Methylation in
pre-MBT Stage
SAMDC overexpression depletes SAM from cells [7],
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
Fraction number
Figure 18. Sucrose density gradient profiles of (methyl-3H)
methionine-labeled RNA and DEAE-Sephadex A25 chroma-
tographic profiles of nuclease digests of the high-molecular
weight RNA fractions recovered from sucrose density gradients.
Embryos were dissociated and their cells were labeled with
(methyl-3H)methionine. Numbers indicated are positions of
marker oligonucleotides of the indicated charge values. The
number of embryos used were 3000 morulae (a), 300 early
blastulae (b), 50 gastrulae (c) in order to obtain RNA from ca.
106 cells at all the stages. In (a), experiments were carried out
using 300 morulae at a time and the whole procedure was per-
formed with 10 separate samples and nuclease digests obtained
were pooled and used at a time. Two yellow components (3
and 4 components) are for m
NN and mm
NNN, both derived
only from rRNA and the green peak (5 component) is for type
I cap structure (m
pp p
m7G Np
N). From Shiokawa et al. [30].
In this connection, we co-injected SAMDC mRNA and
cap analogue (m7GpppG) into Xenopus fertilized eggs.
We found here that ca. 50% of the co-injected embryos
were rescued from SAMDC-induced apoptotic arrest and
developed into tadpoles. By contrast, the apoptosis exe-
cuted by p53 overexpression was not suppressed by the
cap analogue [87] (Figure 19). Therefore, the exoge-
nously-added cap analog might have rescued the
SAMDC-induced inhibition of protein synthesis by sup-
plying the methylated CAP analogue.
Xenopus embros have unique polyamine composition.
From the studies of the polyamine metabolism, we
cloned cDNA of SAMDC, one of the key enzyme in the
polyamine regulation, and then the present series of ex-
periments started when we overexpressed the in vitoro-
transcribed SAMDC mRNA in Xenopus fertilized eggs.
In SAMDC-overexpressed embryos, SAM, a substrate of
SAMDC, was exhausted, and protein synthesis was
greatly inhibited, and at midblastula stage, or MBT,
SAMDC-overexpressed embryos suddenly underwent
massive cell dissociation and died, although they devel-
oped quite normally up to early blastula stage. The
SAMDC-induced cell dissociation turned out to be due
to apoptosis, and the caspase-9 which seems to be a key
caspase in this system was found to exist in the egg cy-
toplasm from the beginning as a maternal mRNA. Thus,
we came across quite unexpectedly the phenomenon of
the execution of the maternal program of apoptosis. This
apoptosis was completely suppressed by co-injection of
Bcl-2 mRNA, EGBG, an inhibitor of SAMDC, or SAM,
and rescued embryos developed beyond tadpole stage.
We microinjected SAMDC mRNA into only one blas-
tomere at 8- to 32-cell stage embryos, and after analyz-
ing the behavior of its descendant cells, reached a con-
clusion that the apoptosis which is executed so early in
the development is a kind of “fail-safe” mechanism to
check and eliminate damaged cells before they enter the
morphogenic phase which comes after the MBT stage. In
SAMDC-overexpressed embryos, it appeared that cas-
pase-8 is also involved. Interestingly, de novo synthesis
of caspase-8 mRNA takes place at cleavage stage and it
appeared that the activation of caspase-8 is followed by
activation of caspase-9. Caspase-8 mRNA is not a ma-
ternal mRNA, but is newly synthesized during cleavage
stage (pre-MBT stage) as a result of the overexpression
of SAMDC, but not of p53. Based on the results, we
conclude that Xenopus embryos have at least two path-
ways to execute the maternal program of apoptosis; one
executed by SAMDC-overexpression, and the other
executed by p53-overexpression, and while the former is
executed through activation of caspase-8 before the acti-
vation of caspase-9, the latter is activated not through
Copyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
Copyright © 2012 SciRes.
Figure 19. Partial rescue of embryos from SAMDC mRNA-induced, but not p53 mRNA-induced, apoptosis by
co-injection of a cap analogue. Embryos were co-injected with either SAMDC mRNA and cap analogue or p53
mRNA and cap analogue as indicated in the figures, and percentage of normal developing embryos were plotted.
From Shiokawa et al. [87].
Figure 20. A model which shows sequence of events in
activetion of apoptosis in SAMDC- and p53-overex-
pressed Xenopus embryos. From Shiokawa et al. [88].
caspase-8 but directly through caspase-9 [88] (Figure
20). We assume that further experiment to clarify the
control mechanism of these unique apoptotic systems in
Xenopus early embryos would provide important insights
not only for the studies of apoptosis itself but also for the
elucidation of the developmental control mechanism.
[1] Nieuwkoop, P.D. and Faber, J. (1967) Normal Table of
Xenopus laevis (Daudin). North Holland, Amsterdam.
[2] Estabel, J., Koenig, N., Shiokawa, K. and Exbrayat, J.-M.
(2005) Apoptosis, research signpost. In: Scovassi, A.I.
Ed., Kerala, 145-167.
[3] Glucksman, A. (1951) Cell deaths in normal vertebrate
ontogeny. Biological Review, 26, 59-86.
[4] Imoh, H. (1986) Cell death during normal gastrulation in
the newt, Cynops pyrrhogaster. Cell Differentiation, 19,
35-42. doi:10.1016/0045-6039(86)90023-0
[5] Sible, J.C., Anderson, J.A., Lewelly, A.L. and Maller, J.
L. (1997) Zygotic transcription is required to block a ma-
ternal program of apoptosis in Xenopus embryos. Devel-
opmental Biology, 189, 335-346.
[6] Hensey, C. and Gautier, J. (1997) A developmental timer
that regulates apoptosis at the onset of gastrulation.
Mechanism of Development, 69, 183-195.
[7] Shibata, M., Shing, J., Yasuhiko, Y., Kai, M., Miura, K.,
Shimogori, T., Kashiwagi, K., Igarashi, K. and Shiokawa,
K. (1998) Overexpression of S-adenosylmethionine de-
carboxylase (SAMDC) in early Xenopus embryos induces
cell dissociation and inhibits transition from the blastula
to gastrula stage. International Journal of Developmental
Biology, 42, 675-686.
[8] Hensey, C. and Gautier, J. (1998) Programmed cell death
during Xenopus development: A spatial-temporal analysis.
Developmental Biology, 203, 36-48.
[9] Kai, M., Higo, T., Yokoska, J., Kaito, C., Kajita, E., Fu-
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
kamachi, H., Takayama, E., Igarashi, K. and Shiokawa, K.
(2000) Overexpression of S-adenosylmethionine decar-
boxylase (SAMDC) activates the maternal program of
apoptosis shortly after MBT in Xenopus embryos. Inter-
nationa Journal of Developmental Biology, 44, 507-510.
[10] Hensey, C. and Gautier, J. (1999) Developmental regula-
tion of induced and programmed cell death in Xenopus
embryos. Annals of the New York Academy of Sciences,
887, 105-119. doi:10.1111/j.1749-6632.1999.tb07926.x
[11] Finkielstein, C.V., Lewellyn, A.L. and Maller, J.L. (2001)
The midblastula transition in Xenipus embryos activates
nultiple pathways to prevent apoptosis in response to
DNA damage. Proceedings of National Academy of Sci-
ence of USA, 98, 1006-1011. doi:10.1073/pnas.98.3.1006
[12] Anderson, J.A., Lewellyn, A.L., and Maller, J.L. (1997)
Ionizing radiation induces apoptosis and elevates cyclin
A1-Cdk2 activity before but not after the midblastula
transition in Xenopus. Molecular Biology of Cell, 8,
[13] Tribulo, C., Aybar, M. Sanchez, S.S. and Mayor, R.
(2004) A balance between the anti-apoptotic activity of
Slug and the apoptotic activity of msx1 is required for the
proper development of the neural crest. Developmental
Biology, 275, 325-342. doi:10.1016/j.ydbio.2004.07.041
[14] Shinga, J., Itoh, M., Shiokawa, K., Taira, S. and Taira, M.
(2001) Early patterning of the prospective midbrain-
hinfdbrain boundary by the HES-related gene XHR1 in
Xenopus embryos. Mechanisms of Development, 109,
225-239. doi:10.1016/S0925-4773(01)00528-7
[15] Estabel, J., Mercer. A., König, N. and Exbrayat, J.-M.
(2003) Programmed cell death in Xenopus laevis spinal
cord, tail and other tissues, prior to, and during, meta-
morphosis. Life Science, 73, 3297-3306.
[16] Rowe, I., Coen, L., Le Blay, K., Le Mével, S. and Deme-
neix, B. (2002) Autonomous regulation of muscle fibre
fate during metamorphosis in Xenopus tropicalis. Devel-
opmental Dynnamics, 224, 381-390.
[17] Shi, Y.-B. and Brown, D.D. (1993) The earliest changes
in gene expression in tadpole intestine induced by thyroid
hormone. Journal of Biological Chemistry, 268, 20312-
[18] Das, B., Schreider, A.M., Huang, H. and Brown, D.D.
(2002) Multiple thyroid hormone-induced muscle growth
and death programs during metamorphosis in Xenopus
laevis. Proceedings of National Academy of Science of
USA, 99, 12230-12235. doi:10.1073/pnas.182430599
[19] Signoret, J. and Lefresne, J. (1973) Contribution à l’étude
de la segmentation de l’oeuf d’axolotl. II. Influence de
modifications du noyau et du cytoplasme sur les modali-
tés de la segmentation. Annuals of Embryology and
Morphology, 6, 299-307.
[20] Newport, J. and Kirschner, M. (1982) A major develop-
mental transition in early Xenopus embryos: I. Charac-
terization and timing of cellular changes at the midblas-
tula stage. Cell, 30, 675-686.
[21] Nakakura, N., Miura, T., Yamana, K., Ito, A. and Shio-
kawa, K. (1987). Synthesis of heterogeneous mRNA-like
RNA and low-molecular-weight RNA before the mid-
blastula transition in embryos of Xenopus laevis. Devel-
opmental Biology, 123, 421-429.
[22] Shiokawa, K., Kurashima, R. and Shinga, J. (1994)
Temporal control of gene expression from endogenous
and exogenously-introduced DNAs in early embryogene-
sis of Xenopus laevis. International Journal of Develop-
mental Biology, 38, 249-255.
[23] Shiokawa, K., Misumi, Y. Tashiro, K. and Yaman, K.
(1989) Changes in the patterns of RNA synthesis in early
embryogenesis of Xenopus laevis. Cell Differentiaion, 28,
17-25. doi:10.1016/0922-3371(89)90019-1
[24] Yang, J., Tan, C., Darken, R.S., Wilson, P.A. and Klein
P.S. (2002) β-Catenin/Tcf regulated transcription prior to
the midblastula transition. Development, 129, 5743-5752.
[25] Heasman, J. (2006) Patterning the early Xenopus embryo.
Development, 133, 1205-1217. doi:10.1242/dev.02304
[26] Graham, C.F. and Morgan, R.W. (1966) Changes in the
cell cycle during early amphibian development. Devel-
opmental Biology, 14, 439-460.
[27] Minoura, I., Nakamura, H., Tashiro, K. and Shiokawa, K.
(1995) Stimulation of circus movement by activin, bFGF
and TGF-beta 2 in isolated animal cap cells of Xenopus
laevis. Mechanism of Development, 49, 65-69.
[28] Carter, A.D. and Sible, J.C. (2003). Loss of XChk1 func-
tion triggers apoptosis after the midblastula transition in
Xenopus embryos. Mechanism of Development, 120, 315-
323. doi:10.1016/S0925-4773(02)00443-4
[29] Wroble, B.N. and Sible, J.C. (2005) Chk2/Cds1 protein
kinase blocks apoptosis during early development of
Xenopus laevis. Developmental Dynamics, 233, 1359-
1365. doi:10.1002/dvdy.20449
[30] Shiokawa, K., Misumi, Y. and Yamana, K. (1981) Dem-
onstration of rRNA synthesis in pre-gastrular embryos of
Xenopus laevis. Development Growth and Differentiation,
23, 579-587. doi:10.1111/j.1440-169X.1981.00579.x
[31] Shiokawa, K., Tashiro, K., Misumi, Y. and Yamana, K.
(1981) Non-coordinated synthesis of RNA’s in pre-gas-
trular embryos of Xenopus laevis. Development, Growth
and Differentiation, 23, 589-597.
[32] Nakahashi, T. and Yamana, K. (1976) Biochemical and
cytological examination of the initiation of ribosomal
RNA synthesis during gastrulation of Xenopus laevis.
Development, Growth and Differentiation, 18, 329-339.
[33] Shiokawa, K., Yamana, K., Fu, Y., Atsuchi, Y. and Ho-
sokawa, K. (1990) Expression of exogenously introduced
bacterial chloramphenicol acetyltransferase gene in Xeno-
pus laevis embryos before the midblastula transition.
Rouxs Archive of Developmental Biology, 198, 322-329.
[34] Etkin, L.D. and Balcells, S. (1985) Transformed Xenopus
opyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769 767
embryos as a transient expression system to analyze gene
expression at the midblastula transition. Developmental
Biology, 108, 173-178.
[35] Kappas, N.C., Savage, P., Chen, K.C., Walls, A.T. and
Sible, J.C. (2000) Dissection of the XCHk1 signaling
pathway in Xenopus laevis embryos. Molecular Biology
of the Cell, 11, 3101-3108.
[36] Petrus, M.J., Wilhem, D.E., Murakami, M., Kappas, M.C.,
Carter, A.D., Wroble, B.N. and Sible, J.C. (2004) Altered
expression of Chk1 disrupts cell cycle remnodeling at the
midblastula transition in Xenopus laevis embryos. Cell
Cycle, 3, 212-217. doi:10.4161/cc.3.2.647
[37] Taber, C.W. and Taber, H. (1984) Polyamines. Annual
Review of Biochemistry, 53, 749-790.
[38] Pegg, A.E. (1986) Recent advances in the biochemistry of
polyamines in eukaryotes. Biochemical Journal, 234,
[39] Davis, R.H., Morris, D.R. and Coffino, P. (1992) Se-
questered end products and enzyme regulation: The case
of ornithine decarboxylase. Microbiological Review, 56,
[40] Guirard, B.M. and Snell, E.E. (1964) Effect of polyamine
structure on growth stimulation and spermine and sper-
midine content of lactic acid bacteria. Journal of Bacteri-
ology, 88, 72-80. doi:10.1126/science.6768132
[41] Fozard, J.R., Part, M., Prakash, N.J., Grove, J., Schechter,
P.J., Sjoerdsma, A. and Koch-Weser, J. (1980) L-Orni-
thine decarboxylase: An essential role in early mammal-
ian embryogenesis. Science, 208, 505-508.
[42] Loewkvist, B., Emanuelsson, H. and Heby, O. (1985)
Changes in polyamine synthesis and concentrations dur-
ing chick embryo development. Journal of Experimental
Zoology, 234, 375-382. doi:10.1002/jez.1402340307
[43] Kusunoki, S. and Yasumasu, I. (1978) Inhibitory effect of
alpha-hydrazinoornithine on egg cleavage in sea urchin
eggs. Developmental Biology, 67, 336-345.
[44] Emanuelsson, H. and Heby, O. (1978) Inhibition of pu-
trescine synthesis blocks development of the polychete
Ophryotrocha labronica at gastrulation. Proceedings of
National Academy of Science of USA, 75, 1039-1042.
[45] Dion, A.S. and Herbst, E.J. (1970) Polyamine changes
during development of Drosophila melanogaster. Annual
of New York Academy of Science, 171, 723-734.
[46] Osborne, H.B., Mulner-Lorillon, O., Marot, J. and Belle,
R. (1989) Polyamine levels during Xenopus laevis oo-
genesis: A role in oocyte competence to meiotic resump-
tion. Biochemical and Biophysical Research Communica-
tions, 158, 520-526.
[47] Shinga, J., Kashiwagi, K., Toshiro, K., Igarashi, K. and
Shiokawa, K. (1996) Maternal and zygotic expression of
mRNA for S-adenosylmethionine decarboxylase and its
relevance to the unique polyamine composition in Xeno-
pus oocytes and embryos. Biochimica et Biophysica Acta,
1308, 31-40. doi:10.1016/0167-4781(96)00020-6
[48] Sunkara, P.S., Wright, D.A. and Nishioka, K. (1981) An
essential role for putrescine biosynthesis during meiotic
maturation of amphibian oocytrd. Developmental Biology,
87, 351-355. doi:10.1016/0012-1606(81)90158-5
[49] Younglai, E.V., Godeau, F., Mester, J. and Baulieu, E.E.
(1980) Increased ornithine decarboxylase activity during
meiotic maturation in Xenopus laevis oocytes. Biochemi-
cal and Biophysical Research Communications, 96, 1274-
1281. doi:10.1016/0006-291X(80)90089-3
[50] Osborne, H.B., Duval, C., Ghoda, L., Omilli, F., Bassez,
T. and Coffino, P. (1991) Expression and post-ytansla-
tional regulation of ornithine decarboxylase during early
Xenopus development. European Journal of Biochemistry,
202, 575-581. doi:10.1111/j.1432-1033.1991.tb16410.x
[51] Osborne, H.B., Cormier, P., Lorillon, O., Maniey, D. and
Belle, R. (1993) Anappraisal of the devekiomental im-
portance of polyamine changes in early Xenopus embryos.
International Journal of Developmental Biology, 37, 615-
[52] Rosander, U., Holm, I., Grahn, B., Lovtrup-Rein, H.,
Mattsson, M. and Heby, O. (1995) Down-regulation of
ornithine decarboxylase by an increased degradation of
the enzyme during gastrulation of Xenopus laevis. Bio-
chimica et Biophysica Acta, 1264, 121-128.
[53] Russell, D.H. (1971) Putrescine and spermidine biosyn-
thesis in the development of normal and anucleokate mu-
tants of Xenopus laevis. Proceedings of National Acad-
emy of Science of USA, 68, 523-527.
[54] Heby, O. and Persson, L. (1990) Molecular genetics of
polyamine synthesis in eukaryotic cells. Trends in Bio-
chemical Science, 15, 153-158.
[55] Suzuki, T., Sadakata, Y., Kashiwagi, K., Hoshino, K.,
Kakinuma, Y., Shirahata, A. and Igarashi, K. (1993)
Overproduction of S-adenosylmethionine decarboxylase
in ethylglyoxal-bis(guanylhydrazone)-resistant mouse FM3A
cells. European Journal of Biochemistry, 215, 247-253.
[56] Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M.,
Cai, J., Peng, T.-I., Jones, D.P. and Wang, X. (1997)
Prevention of apoptosis by Bcl-2: Release of cytochrome
c from mitochondria blocked. Science, 275, 1129-1132.
[57] Kluck, R.M., Bossy-Wetzel, E., Green, D.R. and New-
meyer, D.D. (1997). The release of cytochrome c from
mitochondria: A primary site for Bcl-2 regulation of
apoptosis. Science, 275, 1132-1136.
[58] Curz-Reyes, J. and Tata, J.R. (1995) Cloning, characteri-
zation and expression of two Xenopus bcl-2-like cell-
survival genes. Gene, 158, 171-179.
[59] Stack, J.H. and Newport, J.W. (1997) Developmentally
regulated activation of apoptosis early in Xenopus gas-
trulation results in cyclin A degradation during interphase
opyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
of the cell cycle. Development, 124, 3185-3195.
[60] Kaito, C., Kai, M., Higo, T., Takayama, E., Fukamachi,
H., Sekimizu, K. and Shiokawa, K. (2001) Activation of
the maternally preset program of apoptosis by microin-
jection of 5-aza-2’-deoxycytidine and 5-methyl-2’-de-
oxycytidine-5’-triphosphate in Xenopus laevis embryos.
Development, Growth and Differentiation, 43, 383-390.
[61] Kai, M., Kaito, C., Fukamachi, H., Higo, T., Takayama,
E., Hara, H., Ohya, Y., Igarashi, K. and Shiokawa, K.
(2003) Overexpression of S-adenosylmethionine decar-
boxylase (SAMDC) in Xenopus embryos activates ma-
ternal program of apoptosis as a “fail-safe” mechanism of
early embryogenesis. Cell Research, 13, 147-158.
[62] Shiokawa, K., Kai, M., Higo, T., Kaito, C., Fukamachi,
H., Yaoita, Y. and Igarashi, K. (2000) Maternal program
of apoptosis activated shortly after midblastula transition
by overexpression of S-adenosylmethionine decarboxy-
lase in Xenopus early embryos. Comparative Biochemis-
try and Physiology B, 126, 149-155.
[63] Ikegami, R., Hunter, P. and Yager, T.D. (1999) Devel-
opmental activation of the capability to undergo check-
point-induced apoptosis in the early zebrafish embryo.
Developmental Biology, 209, 409-433.
[64] Stancheva, I, EI-Maarri, O., Walter, J., Niveleau A. and
Meehan, R.R. (2002) DNA methylation at promoter re-
gions regulates the timing of gene activation in Xenopus
laevis embryis. Developmental Biology, 243, 155-165.
[65] Miyanaga, Y., Torregroza, I. and Evans, T. (2002) A
maternal Smad protein regulates early embryonic apop-
tosis in Xenopus laevis. Molecular and Cellular Biology,
22, 1317-1328. doi:10.1128/MCB.22.5.1317-1328.2002
[66] Trindade, M., Messenger, N., Papin, C., Grimmer, D.,
Fairclough, L., Tada, M. and Smith, J.C. (2003) Regula-
tion of apoptosis in the Xenopus embryo by Bix3. Devel-
opment, 130, 4611-4622. doi:10.1242/dev.00489
[67] Murphy, C.R., Sabel, J.L., Sandler, A.D. and Dagle, J.M.
(2002) Survivin mRNA is downregulated during early
Xenopus laevis embryogenesis. Developmental Dynamics,
225, 597-601. doi:10.1002/dvdy.10194
[68] Salvesen, G.S. and Dixit, V.M. (1997) Caspases: Intra-
cellular signaling by proteolysis. Cell, 91, 443-446.
[69] Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M.,
Ahmad, M., Alnemri, E.S. and Wang, S. (1997) Cyto-
chrome C and dATP-dependent formation of Apaf-1/cas-
pase-9 complex initiates an apoptotic cascade. Cell, 91,
479-489. doi:10.1016/S0092-8674(00)80434-1
[70] Ona, V.O., Li, M., Vonsattel, J.P.G., Andrews, L.J., Khan,
S.Q., Chung, W.M., Frey, A.S., Menon, A.S. and Fried-
lander, R.M. (1999) Inhibition of caspase-1 slows disease
progression in a mouse model of Huntington’s disease.
Nature, 399, 263-267. doi:10.1038/20446
[71] Takayama, K., Higo, T., Kai, M., Fukasawa, M., Naka-
jima, K., Hara, H., Tadakuma, T., Igarashi, K., Yaoita, Y.
and Shiokawa, K. (2004) Involvement of caspase-9 in
execution of the maternal program of apoptosis in
Xenopus late blastulae overexpressed with S-adenosy-
lmethionine decdarboxylase. Biochemical and Biophysi-
cal Research Communications, 325, 1367-1375.
[72] Friedlander, R.M., Gagliardini, V., Hara, H., Fink, K.B.,
Li, W., MacDonald, G., Fishman, M.C., Greenberg, A.H.,
Moskowitz, M.A. and Yuan, J. (1997) Expression of a
dominant negative mutant of interleukin-1beta converting
enzyme in transgenic mice prevents neuronal cell death
induced by trophic factor withdrawal and ischemic brain
injury. Journal of Experimental Medicine, 185, 933-940.
[73] Nakajima, K., Takahashi, A. and Yaoita, Y. (2000) Struc-
ture, expression, and function of the Xenopus laevis cas-
pase family. Journal of Biological Chemistry, 275, 10484-
10491. doi:10.1074/jbc.275.14.10484
[74] Yaoita, Y. and Nakajima, K. (1997) Induction of apop-
tosis and CPP32 expression by thyroid hormone in a
myoblastic cell line derived from tadpole tail. Journal of
Biological Chemistry, 272, 5122-5127.
[75] Kajita, E., Wakiyama, M., Miura, K., Mizumoto, K., Oka,
T., Komuro, I., Miyata, T., Yatsuki, H., Hori, K. and
Shiokawa, K. (2000) Isolation and characterization of
Xenopus laevis aldolase B cDNA and expression patterns
of aldolase A, B, and C genes in adult tissues, oocytes,
and embryos of Xenopus laevis. Biochimica et Biophysica
Acta, 1493, 101-118.
[76] Andreassen, O.A., Ferrante, R.J., Hughes, D.B., Klivenyi,
P., Dedeoglu, A., Ona, V.O., Friedlander, R.M. and Beal,
M.F. (2000) Malonate and 3-nitropropionic acid neuro-
toxicity are reduced in transgenic mice expressing a cas-
pase-1 dominant-negative mutant. Journal of Neuroche-
mistry, 75, 847-852.
[77] Slee, E.A., Adrain, C. and Martin, S.J. (1999) Serial kill-
ers: Ordering caspase activation events in apoptosis. Cell
Death and Differentiation, 6, 1067-1074.
[78] Slee, E.A., Harte, M.T., Kluck, R.M., Wolf, B.B., Ca-
siano, C.A., Newmeyer, D.D., Wang, H.G., Reed, J.C.,
Nicholson, D.W., Alnemri, E.S., Green, D.R. and Martin,
S.J. (1999) Ordering the cytochrome c-initiated caspase
cascade: Hierarchial activation of caspase-2, -3, -6, -7, -8,
and -10 in a caspase-9 dependent manner. Journal of
Cellular Biology, 144, 281-292.
[79] Thornberry, N.A., Rano, T.A., Peterson, E.P., Rasper,
D.M., Timkey, T., Garcia-Calvo, M., Houtzager, V.M.,
Novdstrom, P.A., Roy, S., Vaillancourt, J.P., Chapman,
K.T. and Nicholson, D.W. (1997) A combinatorial ap-
proach defines specificities of members of the caspase
family and granzyme, B. Functional relationsip estab-
lished for key mediators of apoptosis. Journal of Bio-
logical Chemistry, 272, 17907-l7911.
[80] Shiokawa, K., Takayama, E., Higo, T., Kuroyanagi, S.,
opyright © 2012 SciRes. OPEN ACCESS
K. Shiokawa / Advances in Bioscience and Biotechnology 3 (2012) 751-769
Copyright © 2012 SciRes.
Kaito, C., Hara, H., Kajitani, M., Sekimizu, K., Tada-
kuma, T., Miura, K.-I., Igarashi, K. and Yaoita, Y. (2005)
Occurrence of pre-MBT synthesis of caspase-8 mRNA
and activation of caspase-8 prior to execution of SAMDC
(S-adenosylmethionine decarboxylase)-induced, but not
p53-induced, apoptosis in Xenopus late blastulae. Bio-
chemical and Biophysical Research Communications,
336, 682-691. doi:10.1016/j.bbrc.2005.08.144
[81] Wallingford, J.B., Seufert, D.W., Virta, V.C. and Vize,
P.D. (1997) p53 activity is essential for normal develop-
ment in Xenopus. Current Biology, 7, 747-757.
[82] Tchang, F., Gusse, M., Soussi, T. and Mechali, M. (1993)
Stabilization and expression of high levels of p53 during
early development in Xenopus laevis. Developmental Bi-
ology, 159, 163-172. doi:10.1006/dbio.1993.1230
[83] Hoever, M., Clement, J.H., Wedlich, D., Montenarh, M.
and Knochel, W. (1994) Overexpression of wild-type p53
interferes with normal development in Xenopus laevis
embryos. Oncogene, 9, 109-120.
[84] Soussi, T., Caron de Fromentel, C., Mechali, M., May, P.
and Kress, M. (1987) Cloning and characterization of a
cDNA from Xenopus laevis coding for a protein ho-
mologous to human and murine p53. Oncogene, 1, 71-78.
[85] Momand, J., Zambetti, G.P., Olson, D.C., George, D. and
Levine, A.J. (1992) The mdm-2 oncogene product forms
a complex with the p53 protein and inhibits p53-mediated
transactivation. Cell, 69, 1237-1245.
[86] Marechal, V., Elenbaas, B., Taneyhill, L., Piette, J.,
Mechali, M., Nicolas, J.C., Levine, A.J. and Moreau, J.
(1997) Conservation of structural domains and bioche-
mical activities of the MDM2 protein from Xenopus
laevis. Oncogene, 14, 1427-1433.
[87] Shiokawa, K., Aso, M., Kondo, T., Takai, J.-I., Tashiro,
K. and Igarashi, K. (2009) Polyamines and S-adenosy-
lmethionine decarboxylase (SAMDC) in Xenopus em-
bryos and effects of overexpression of SAMDC on
Xenopus early embryogenesis. In: Dandrifosse, G., Ed.,
Biological Aspects of Biogenic Amines, Polyamines and
Conjugates, Transworld Research Network, Kerala, 113-
[88] Shiokawa, K., Aso, M., Kondo, T., Uchiyama, H., Ku-
royanagi, S., Takai, J.-I., Takahashi, S., Kajitani, M.,
Kaito, C., Sekimizu, K., Takayama, E., Igarashi, K. and
Hara, H. (2008) Gene expression in pre-MBT embryos
and activation of maternally-inherited program of apop-
tosis to be executed at around MBT as a fail-safe mecha-
nism in Xenopus early embryogenesis. Gene Regulation
and Systems Biology, 2, 1-19.