The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2014) 56, 460–468

doi: 10.1111/dgd.12145

Original Article

Sirtuin inhibitor Ex-527 causes neural tube defects, ventral
edema formations, and gastrointestinal malformations in
Xenopus laevis embryos
Yoshihisa Ohata, 1 Shinya Matsukawa, 2 Yuki Moriyama, 3 Tatsuo Michiue, 2 Kenta
Morimoto, 4 Yuka Sato 4 and Hiroki Kuroda 4 *1Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529,
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku,
Tokyo 153-8902, Japan; 3Department of Biological Chemistry, Howard Hughes Medical Institute, University of California,
Los Angeles, California 90095-1662, USA; and 4Faculty of Environment and Information Studies, Keio University, 5322
Endo, Fujisawa-shi, Kanagawa 252-0882, Japan
2

Chemical reagent Ex-527 is widely used as a major inhibitor of Sirtuin enzymes, which are a family of highly
conserved protein deacetylases and have been linked with caloric restriction and aging by modulating energy
metabolism, genomic stability, and stress resistance. However, the extent to which Ex-527 controls early developmental events of vertebrate embryos remains to be understood. Here, we report an examination of Ex-527
effects during Xenopus early development, followed by a confirmation of expressions of xSirt1 and xSirt2 inembryonic stages and enhancement of acetylation by Ex-527. First, we found that reductions in size of neuralplate at neurula stages were induced by Ex-527 treatment. Second, tadpoles with short body length and large
edematous swellings in the ventral side were frequently observed. Moreover, Ex-527-treated embryos showed
severe gastrointestinal malformations in late tadpole stages. Taken together with these results, we conclude that
the Sirtuin family start functioning at early embryonic stages and is required for various developmental events.
Key words: Ex-527, Sirtuin, teratogenesis, Xenopus.

Introduction
The Sirtuins are highly conserved NAD+-dependent
protein deacetylases and are able to extend the lifespan of several model organisms such as yeast,
worm, flies, and some vertebrates (Tissenbaum &Guarente 2001; Rogina & Helfand 2004; Sommer
et al. 2006). In mammals, the Sirtuins are composedof seven Sir2 orthologues (Sirt1-7) and have a conserved deacetylase core that uses NAD+ as a cofactor
at the central region of proteins (Frye 2000). Functional
specificity on Sirt1-7 is thought to be regulated by the
N- or C-terminal domains. For instance, a 25 amino*Author to whom all correspondence should be addressed.
Email: hkuroda@sfc.keio.ac.jp
Received 4 December 2013; revised 22 May 2014;
accepted 25 May 2014.
ª 2014 The Authors
Development, Growth & Differentiation ª 2014 JapaneseSociety of Developmental Biologists

acids sequence at the C-terminal domain of Sirt1,
called ESA (essential for Sirt1 activity), interacts withand functions as an “on switch” for the deacetylasecore (Kang et al. 2011). Sirt1 orchestrates various
biological events, including cell differentiation (Fulco
et al. 2008), apoptosis (Luo et al. 2001), autophagy(Lee et al. 2008), cancer (Kim et al. 2008), metabolism(Picard et al. 2004; Li et al. 2007), and circadianrhythms (Asher et al. 2008). However, little is known
about the role of Sirt1 on the early developmental
stage in vertebrates, although Sirt1 is widely expressed
during early embryogenesis (McBurney et al. 2003). In
the study using Sirt1-deficient mice, notable developmental defects of the retina and heart are observed,
and embryos of null mice become significantly smaller
than normal embryos, leading to infrequent postnatal
survival (Cheng et al. 2003).
The chemical reagent Ex-527 is a potent Sirt1
inhibitor, 200–500-fold more selectively binds to Sirt1
than to Sirt2 or Sirt3, and does not bind to Sirt4-7(Peck et al. 2010). Treatment with Ex-527 canTeratogenic effects by Ex-527

increase acetylation of p53 in human mammary epithelial cells and several cell lines (Solomon et al.
2006). Interestingly, developmental delay of cyst
growth in mouse autosomal dominant polycystic kidney and apoptosis in human leukemia cells arestrongly induced by treatment of Ex-527 (Cea et al.
2011; Zhou et al. 2013). In Xenopus, p53 activity isessential for normal development by linking to transforming growth factor (TGF)-b signaling (Wallingford
et al. 1997; Cordenonsi et al. 2003; Takebayashi-Suzuki et al. 2003). Recently, we found that developmental delay was caused by inhibition of the TOR
kinase in Xenopus embryos (Moriyama et al. 2011),
which is thought to be a co-regulator of the Sirtuins
for the autophagy network to prolong lifespan in several species (Medvedik et al. 2007; Ghosh et al.
2010; Zhang et al. 2013). These current discoveries
suggest that the Sirtuins play important roles atembryonic stages of vertebrate. In this study, we first
confirm expressions of the Sirtuins and acetylatedlysine level on Ex-527-treated Xenopus embryos, andthen introduce severe phenotypical effects due to Ex527 treatments on early Xenopus embryogenesis.

Materials and methods
Embryology and histology
Xenopus laevis eggs were obtained from females
injected with 400 units of human chorionic gonadotropin (HCG; Fuji Pharma Co., Japan), fertilized in vitro
with minced testis, and then cultured in 0.19 Steinberg’s solution (SS). 19 SS contains 58 mmol/L NaCl,
0.67 mmol/L KCl, 0.34 mmol/L Ca(NO3)2, 0.83 mmol/
L MgSO4, 100 mg/L kanamycin sulfate, and 5 mmol/L
Tris-HCl (pH 7.4). Frog embryos were cultured at 22°C
at all times. The jelly coats were removed with thioglycolic acid solution (1% of thioglycolic acid in 0.19 SS,
pH 8.0). Embryos were fixed with PBSFA for 2 h,dehydrated with ethanol, embedded in paraffin wax,
sectioned at 8 lm, and stained with hematoxylin andeosin (HE).
Ex-527 treatment
Ten milligrams of Ex-527 (S1541; Selleck Chemicals,
Houston, TX, USA) was dissolved in 500 lL of dimethylsulfoxide (DMSO), and each 20-lL aliquot (80 mmol/L) was stored at 20°C. In times of usage, aliquots
were diluted with 0.19 SS into the proper concentration. Ex-527 (200 lmol/L), which is the maximum concentration used in this study, includes 0.25% DMSO.
No toxic effects were observed in the solutions including 0.5% of DMSO. Embryos were exposed to Ex-527

461

solutions from the 2-cell stage to the stages designed
for observation.
Nicotinamide treatment
12.5 mg of nicotinamide (N0636-100G; Sigma-Aldrich)
was dissolved in 10 mL 0.19 SS (20 mmol/L). Embryos
were exposed to nicotinamide solutions from the 2-cellstage to the stages designed for observation.
RT-PCR
The following primers were used for reverse transcription–polymerase chain reaction (RT–PCR):
xSirtuin1-RT-fw, 50 -CTTAATGATGGGAACGGATCCC
C-30 , xSirtuin1-RT-rv, 50 -CGGCCTTGGATCTTTCCTGA
AG-30 , xSirtuin2-RT-fw, 50 -TCGAGCGAACCAGCGGAT
CT-30 , xSirtuin2-RT-rv, 50 -GAAATAGTTCTCGAGCCAG
TGC-30 , xSirtuin3-RT-fw, 50 -AGCCTTTCTTCCACCTG
GCC-30 , xSirtuin3-RT-rv, 50 -CTTGGGAAAGTCCTGGT
AAGCTT-30 , xSirtuin4-RT-fw, 50 -ACACACCAAGGCTG
GGCAGT-30 , xSirtuin4-RT-rv, 50 -AGTGCAAACCGATAACCAGAATAAACT-30 , xSirtuin5-RT-fw, 50 -GCACTGGAAATGACACGCCC-30 , xSirtuin5-RT-rv, 50 -AGCCTGCC
TTGCGATGTAACT-30 , xSirtuin6-RT-fw, 50 -ACGGGAG
CCGGGATCAGTA-30 , xSirtuin6-RT-rv, 50 -GCCTCGCA
CCTTTGGCACAT-30 , xSirtuin7-RT-fw, 50 -GAGACGGAAGTTGCACAGCG-30 , xSirtuin7-RT-fw, 50 -GCTTCTCTTGGTAATCCACTCC-30 , noggin-RT-fw, 50 -ACATCAGACCGGCTCCTAGT-30 , noggin-RT-rv, 50 -CTGCGTTGACATCTCCACCT-30 , six3-RT-fw, 50 -GCAACTTCAGGG
AGCTCTAC-30 , six3-RT-rv, 50 -TAGGGATCCTGCAAGT
ACCA-30 , sox17-RT-fw, 50 -AACAATGTCATGGTAGGAGAGAAC-30 , sox17-RT-rv, 50 -GCCATCTGTTTAGCCATCACTG-30 , EF1alpha-RT-fw, 50 -GCAAGTTTGCTGAGCTCAAGG-30 , EF1alpha-RT-rv, 50 -AATGGTCTCAAATTT
GGTGACAGA-30 , ODC-RT-fw, 50 -GCAACTGATGCATGATATTAAAGAAC-30 , ODC-RT-rv, 50 -GAACTTTTATTTGTAAAACTGGTCAA-30 .
The annealing temperatures were 55°C, 30 cycles
for xSirtuin1, xSirtuin2, xSirtuin3, xSirtuin4, xSirtuin5,
xSirtuin6, xSirtuin7, noggin, ODC, and EF1alpha, and35cycles for six3 and sox17 were performed for amplifications.
Western blot analysis
Embryos were lysed in RIPA buffer containing a complete protease inhibitor cocktail (04693159001, Roche,
Germany), and lipids were removed by an equal volume of trichloroethylene (208-02486, Wako, Japan).
Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)
gels and transferred onto polyvinylidene fluoride (PVDF)ª 2014 The Authors
Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

462

Y. Ohata et al.

membranes. Western blots were performed using antiacetylated-lysine (1:1000; 9441S, Cell Signaling Technology, USA) as primary antibodies, anti-Rabbit IgG as
secondary antibody (1:2000; 31460, Thermo Scientific,
MA, US), and Coomassie Brilliant Blue (178-0051;
Wako).

Results
All Sitruin family genes were expressed fromunfertilized egg to tadpole
If the Sirtuins have functions at early embryogenesis,
transcripts should be expressed at the early stage ofembryos. Therefore, in order to examine the expression patterns of Sirt1-7, RT–PCR was carried out inembryos at various developmental stages. Then, we
found that high amounts of transcripts in all cases
were expressed in all early embryonic stages of Xenopus in addition to unfertilized eggs (Fig. 1A). The
expressions of Sirt1-7 were also confirmed in the database created from RNAseq analysis that we originallyperformed in order to list all transcripts in gastrula
embryos (data not shown). On the other hand, noggin,
famous for its function as a dorsal factor of vertebrate(De Robertis & Kuroda 2004), was only observed zygotically as expected (Fig. 1A), supporting the fact that
Sirt1-7 are maternal expressed genes. We also carriedout in situ hybridization to see specific expression of
Sirt1. The expression of Sirt1 was ubiquitous in all
stages that we examined (Stages 1–42, data notshown).

(A)

Deacetylations in Xenopus embryos were inhibited by
Ex-527
Acetylation is one of the major post-translational modifications for cellular proteins, and the Sirtuins are alarge family of deacetylation enzymes that negatively
regulate the acetylation status of several proteins suchas histone and p53. Therefore, if chemical reagent
Ex-527 can work for Xenopus embryogenesis, it
should be changing the acetylation status of embryos.
In order to validate activity of Ex-527 for Xenopus
embryos, we examined acetylation level of Ex527-treated embryos (Fig. 1B). Previous studies haveshown that the acetylation of lysine 382 of p53 iseffectively modulated by Sirt1 (Vaziri et al. 2001). As aresult, Ex-527 clearly showed a concentration-dependent increase in the amount of acetylated proteins,with a maximum effect at 200 lmol/L (Fig. 1B). To
eliminate the possibility that Ex-527 might influence
total amount of proteins, we verified that the amountof protein was not changed by CBB staining (Fig. 1C).
Neural plate in Ex-527-treated embryos became lessdifferentiated than normal
In order to examine whether Sirt1 was working in early
embryogenesis, loss-of-functional experiment was performed. Ex-527 (100 and 200 lmol/L) was used onlive Xenopus embryos beginning at the 2-cell stage up
to stage 45. The results were not what we expected,
in that the developmental delay occurred by rapamycin, which was a famous mTOR inhibitor treatment in

(B)

(C)

Fig. 1. Expression of xSirtuins and inhibition of xSirtuin1 activity by Ex-527 treatment. (A) Reverse transcription–polymerase chain reaction (RT–PCR)analyses for xSirtuin1 to 7 mRNA expressions from unfertilized egg to tadpole stage. All xSirtuins are consecutively
expressed in Xenopus embryos. (B) Western blot analysis of acetylated-lysine in Ex-527-treated embryos. Embryos were treated at the
2-cell stage with 100 and 200 lmol/L of Ex-527, and extracts were collected 12 h later. Predictable band size of p53 is 53 kDa.
(C) CBB staining for SDS-PAGE gel. Total amounts of proteins were not changed by any treatments.
ª 2014 The Authors
Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

Teratogenic effects by Ex-527

our previous study (Moriyama et al. 2011), and that
Sirt1 might be working as an upstream factor of
mTOR. All embryos treated with Ex-527 formed dorsal
blastopore, which means stage 10, at almost thesame time as control embryos, at later stages we
could not observe any remarkable difference on developmental speed between treated and control embryos(data not shown). It was relevant to note that morethan 400 lmol/L concentration was too high for
Ex-527 treatment to observe effects because leaks ofyolk occurred in all embryos under these conditions
before stage 8 (data not shown). However, focusing
on morphogenesis, obvious effects were recognized atneurula stages. At late neurula stages, a pigmented
midline of neural plate was observed on the dorsalside of normal embryos, while a part of the anterior
midline and pigmentation of presumptive cement glandregion were confirmed on the ventral side (Fig. 2A).
Almost the same results were obtained from DMSOtreated embryos (Fig. 2B). On the other hand, pigmented midlines on Ex-527-treated embryos wereundoubtedly shorter than on control embryos and did
not reach the ventral side (Fig. 2C,D). The mild blanching region, which is the feature of neural plate and
(A)

463

observed in control and DMSO-treated embryos(Fig. 2A,B), was not detected in Ex-527-treatedembryos (Fig. 2C,D). The facts suggest that Ex-527
has inhibitory activity on neural induction. On the other
hand, slight swellings on the ventral side weredetected in most embryos treated with 200 lmol/Lof
Ex-527 (Fig. 2D). Furthermore, we performed analysis
to detect the influence of inhibition of xSirtuin1 by
Ex-527 for neural and endomesoderm formation. Dorsal lips treated with Ex-527 were elongated the same
as control and DMSO-treated dorsal lips (Fig. 2E–G).
The expression of Sox17 in the dorsal lip, which was
the marker for endoderm, was not affected by treatment of Ex-527, while the expression of neural marker
in dorsal lips cut from Ex-527-treated embryos was
slightly decreased (Fig. 2H). These facts suggest that
Ex-527 has an inhibitory action on neural induction
independent of dorsal endomesoderm formation.
Ex-527 treatment caused anterior defects, shorter
body length, and large edematous swelling
At late stages, the effects of Ex-527 on morphogenesis
appeared drastically. As described above, most

(B)
(H)

(C)

(D)

(E)

(F)

(G)

Fig. 2. Ex-527 treatments reduced the size of neural plate region. (A) Control embryo at stage 17. (B) 0.25% of dimethylsulfoxide(DMSO)-treated embryo. In both cases, the pigmented midlines of neural plate were observed from the ventral side (15/15), and no
edema formation was observed. (C) Embryo treated with 100 lmol/L of Ex-527. The pigmented midlines were located at the dorsal side
(15/15). No obvious edema formation was detected. (D) Embryo treated with 200 lmol/L of Ex-527. The pigmented midlines wererestricted at the dorsal side (15/15). Ventral edema formations were detected (12/15). White arrowhead shows the anterior end of thepigmented midline of neural plate and black arrowhead shows the posterior end of the side. (E) Dorsal lip explants cut from control
embryos were cultured in 19SS. Elongations were detected in 18/20 of explants. (F) Dorsal lip explants cut from control embryos weretreated with 0.25% of DMSO. Elongations were detected in 17/20 of explants. (G) Dorsal lip explants cut from control embryos weretreated with 100 lmol/L of Ex-527. Elongations were detected in 17/20 of explants. (H) reverse transcription–polymerase chain reaction(RT–PCR) analyses for the expression of pan-neural marker six3 and endodermal marker sox17 in dorsal lip explants. Six3 expression
was reduced by treatment of Ex-527, while sox17 expression was not.
ª 2014 The Authors
Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

464

Y. Ohata et al.

embryos treated with 200 lmol/L of Ex-527 showed aslight edematous swelling on the ventral side at theneurula stages. Moreover, at the tailbud stages thisswelling was clearly observed in all of treated embryos.
Unfortunately, all embryos treated with 200 lmol/L of
Ex-527 died before reaching the tailbud stage. Thismay be a result of explosion of these swelling structures (data not shown). Therefore, our observations
after stage 38 were performed only for the embryos
treated with 50 or 100 lmol/L of Ex-527. At stage 38,
obvious pigmentations of the optic region wereobserved at the anterior region of normal and DMSOtreated embryos. Also, a body length, which is the distance from anterior to posterior end, was reached toapproximately 6.0 mm (Fig. 3A,B). However, optic formations were clearly damaged in embryos treated with
50 lmol/L of Ex-527 (Fig. 3C) and almost diminishedin embryos treated with 100 lmol/L (Fig. 3D). Body
lengths of embryos treated with 50 and 100 lmol/L of
Ex-527 became shorter than those of normal embryos,
scoring around 4.6 and 3.9 mm, respectively (Fig. 3C,
D). Remarkably, edematous swelling at the ventral
anterior region was frequently formed in both cases(Fig. 3C,D), and in some cases of embryos treated with100 lmol/L of Ex-527, edematous swelling was obviously detected in the posterior side (Fig. 3D). Interestingly, the same results were observed by nicotinamide
treatment, which was a sirtuin inhibitor. These features
(A)

(F)

(B)

observed at stage 38 became pronounced over time
(Fig. 3F–I). Especially, the distance of left and right eyesin embryos treated with Ex-527 were clearly shorter
than in control embryos (compare dorsal panels of
Fig. 3F–I). Also, compared to stage 38, body lengths of
Ex-527-treated embryos did not elongate well (Fig. 3C,
D,H,I), while body lengths of control and DMSO-treatedembryos reached 7.0 mm (Fig. 3F,G).
Ex-527 exerts a specific effect on the digestive tract
We additionally noticed mild phenotypic effects aroundyolk in Ex-527-treated embryos at stage 41, meaningthat the sizes of yolk, which includes future gastrointestinal tract, were slightly larger than normal (Fig. 3F–I). At
stage 45, as we expected, large effects of Ex-527 weredetected at the gastrointestinal tract. Normally at thisstage the intestine is forming a double-coiled structure,and both external and internal coils have two loops(Chalmers & Slack 1998). This type of structure wasfound in control and DMSO-treated embryos (Fig. 4A,
B,E,F), while embryos treated with 50 lmol/L of Ex-527
had single-coiled and much thicker intestine (Fig. 4C,
G). Moreover, in the case of 100 lmol/L, no-longer
coiled structure of intestine was found (Fig. 4D,H). We
next tried to determine the details of the inside
structures of Ex-527-treated embryos by histologicalapproach. All tissue and organs were actually

(C)

(G)

(D)

(H)

(E)

(I)

Fig. 3. Ex-527 treatment induced edematous swelling, short body axis, and small head. (A,F) Control embryos at stage 38 and 41. (B,G) Dimethylsulfoxide (DMSO)-treated embryos. (C,H) Embryos treated with 50 lmol/L of Ex-527. Ventral edema formations (11/15) andeye malformations (12/15) were detected at stage 38 and 41. (D,I) Embryos treated with 100 lmol/L of Ex-527. Ventral edema formations (12/12) and eye malformations (12/12) were detected at stage 38 and 41. (E) Embryos treated with 20 mmol/L of nicotinamide.
Ventral edema formations (16/18) and eye malformations (18/18) were detected at stage 38. Average lengths of embryos weredecreased in dose-dependent manner. Scale bars of A to E represent 1 mm.
ª 2014 The Authors
Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

Teratogenic effects by Ex-527

465

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

Fig. 4. Ex-527 treatment induced severe gastrointestinal malformations. (A) Control embryo at stage 45. (B) Dimethylsulfoxide (DMSO)treated embryo. (C) Embryo treated with 50 lmol/L of Ex-527. Ventral edema formations (11/12) and eye (11/12) and gastrointestinal
malformation (12/12) were detected. (D) Embryo treated with 100 lmol/L of Ex-527. Ventral edema formations (11/11) and eye (11/11)
and gastrointestinal malformation (11/11) were detected. (E–H) Surgically resected guts from embryos shown in A–D. The diagram ofgut structure was shown on the left. These panels respectively indicate ventral and lateral views. Secondary coiled structure of gut was
not detected in Ex-527-treated embryos. Scale bars of A to D represent 2 mm, and E to H 0.5 mm.

confirmed in DMSO-treated embryos (Fig. 5A–B), whilein the case of embryos treated with 50 lmol/L of Ex527, forebrain was not detected (panel k in Fig. 5C),
and in the case of 100 lmol/L no brain structure wasobserved (panel p in Fig. 5D). We also found that left
and right eyes were fused in the case of 100 lmol/L(panel m in Fig. 5D), which resembles the phenotype
called Cyclops, which resulted from interference with€hle et al.
dorsal mesoderm (Hatta et al. 1991; Stra
1993; Goudevenou et al. 2011). There was no significant difference between embryos treated with 50 lmol/
L and control embryos as well as DMSO-treatedembryos from the aspect of the size of notochord andsomite region (Fig. 5 panel c in A, panel h in B, panel min C). However, it was obviously smaller in treatment
with 100 lmol/L (Fig. 5 panel r in D). Additionally, aslight reduction in the size of pronephros was observedin embryos treated with 50 and 100 lmol/L of Ex-527(Fig. 5 panel m in C, panel r in D). This deficiency of
dorsal and paraxial structures may have induced inadequate convergent extension of dorsal mesoderm,
resulting in the phenotype of Cyclops (Hatta et al.
€hle et al. 1993; Goudevenou et al. 2011).
1991; Stra
Focusing on gastrointestinal formations, distinct malformations or deletions of several organs had been
observed in embryos treated with 100 lmol/L of Ex-

527. It was impossible to distinguish between small andlarge intestines and find liver formation (Fig. 5 panel r inD). Moreover, reduction in the size of heart wasobserved in embryos treated with 50 and 100 lmol/Lof Ex-527 (Fig. 5 panel l in C, panel q in D). From theseresults, we conclude that Sirt1 plays important roles inthe early stages of Xenopus development.

Discussion
In this study, we have investigated the loss-of-functional effects of Sirtuin1 by using chemical reagent
Ex-527 on Xenopus early development and confirmedthat Ex-527 had a far greater influence on early
embryogenesis than previously expected. Deficienciesof neural tube and anterior neural formations, frequentappearances of ectopic edematous swelling in ventral
side, maldevelopment of dorsal and paraxial tissues,
gastrointestinal organs, and heart formation were
defined as the typical features by loss-of-functional
effects of Sitruin1 on Xenopus early embryogenesis.
Edematous swelling induced by Ex-527 treatments
The reason why Edematous swelling appeared insirtuin inhibitor treated Xenopus embryos has to beª 2014 The Authors
Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

466

Y. Ohata et al.

(A)

b

c

d

l

e

k

(C)

m

n

f

(B)

a

g

h

i

(D)

o

q

j

p

r

s

t

Fig. 5. Several gastrointestinal tissue, brain, and liver formations were damaged by Ex-527-treated embryos. (A) Histological section ofcontrol embryo at stage 45. (B) Dimethylsulfoxide (DMSO)-treated embryo. (C) Embryo treated with 50 lmol/L of Ex-527. The size ofheart and pronephros were slightly reduced. No forebrain structure was detected. (D) Embryos treated with 100 lmol/L of Ex-527. The
size of heart and pronephros were clearly reduced, while proctodeum regions were expanded. It was impossible to distinguish the large
and small intestine. No brain and liver structure was detected. CNS, central nervous system; fb, forebrain; ht, heart; i, intestine; li, large
intestine; lu, lung; lv, liver; mb, midbrain; no, notochord; nt, neural tube; oe, esophagus; pa, pancreas; ph, pharynx; pr, proctodeum; pn,
pronephros; si, small intestine; so, somite; st, stomach.

discussed (Figs 3–5). Removal of the pronephrosregion from the amphibian embryos highly frequently
induced the edema formation (Howland 1916). Interestingly, major pronephros markers are diminished byantisense morpholino oligomers for golph2, and theedema formations are frequently observed in thesemorphants (Li et al. 2012). In our study even thoughpronephros formed in Ex-527-treated embryos, thesize of pronephros was certainly reduced (Fig. 5), suggesting that edematous swelling observed in our study
was caused by nonfunctional or less-functional pronephros. Edematous swelling is also observed inembryos treated with SU5402, which is generally used
as an inhibitor for fibroblast growth factor (FGF) receptor 1 (Doherty et al. 2010). SU5402-treated Xenopus
embryos also have short axis formations and reductionof heart formation identical to Ex-527-treated embryos(Doherty et al. 2010; Deimling & Drysdale 2011).
Therefore, crosstalk of Sirtuin1 and FGF signals canbe an alternative possibility.

The roles of Sirtuins in early embryogensis
Studies on expression of the Sirtuin family revealed
that Sirt1-7 existed both maternally and zygotically(Fig. 1A). The Sirtuin family is originally recognized asanti-aging genes in various organisms such as yeast,
Caenorhabditis elegans, Drosophila melanogaster,mouse, and human (Guarente 2007; Tang 2011).
However, our study clearly revealed additional understanding towards the function of the Sirtuin family. In
Sirt1 null mouse, lethal or sublethal effects werecaused (Li et al. 2008; Boily et al. 2009). TOR kinase,which is another famous anti-aging factor, is known tofunction under regulations of the Sirtuin family (Tucci
2012; Lionaki et al. 2013) and starts working at early
developmental stages (Murakami et al. 2004; Guertin
et al. 2006; Moriyama et al. 2011). Interestingly, thechemical reagent rapamycin is a potent TOR inhibitor,
which causes malformations in Xenopus early development (Moriyama et al. 2011). In these phenotypes,ª 2014 The Authors
Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

Teratogenic effects by Ex-527

short body length, edematous swelling, and gastrointestinal malformation were observed identically to
Ex-527-treated-embryos (Fig. 5C,D), suggesting that
the Sirtuin signal may have a crosstalk with the TOR
signal. Moreover, we showed that Ex-527 inhibited
deacetylation activity of xSirtuin1 for p53 and the othertarget proteins (Fig. 1B). p53 regulates cellular differentiation by modulating the signaling of TGF-b family during Xenopus embryogenesis (Cordenonsi et al. 2003;
Takebayashi-Suzuki et al. 2003). However, Ex-527
does not seem to regulate mesoderm formation. The
activation of p53 requires phosphorylation of serine
392 in addition to acetylation of lysine 382 (Shahar
et al. 2013), so only inhibition of deacetylation by
Ex-527 might not be enough to lead activation of p53.
These reports and our present findings strikingly suggest that the Sirtuin family is not only the key factor
required for adult stage, but also has fundamentalroles from the beginning of the life cycle.

Acknowledgments
We thank Professor N. Shiojiri, M. Shibutani, S. Mori,
and T. Furukawa for valuable suggestions on this manuscript and R. Watanabe and K. Asami for preliminary
experiments of this study.

References
Asher, G., Gatfield, D., Stratmann, M., Reinke, H., Dibner, C.,
Kreppel, F., Mostoslavsky, R., Alt, F. W. & Schibler, U.
2008. SIRT1 regulates circadian clock gene expression
through PER2 deacetylation. Cell 134, 317–328.
Boily, G., He, X. H., Pearce, B., Jardine, K. & McBurney, M. W.
2009. SirT1-null mice develop tumors at normal rates but
are poorly protected by resveratrol. Oncogene 28, 2882–
2893.
Cea, M., Soncini, D., Fruscione, F., Raffaghello, L., Garuti, A.,
Emionite, L., Moran, E., Magnone, M., Zoppoli, G., Reverberi, D., Caffa, I., Salis, A., Cagnetta, A., Bergamaschi, M.,
Casciaro, S., Pierri, I., Damonte, G., Ansaldi, F., Gobbi, M.,
Pistoia, V., Ballestrero, A., Patrone, F., Bruzzone, S. &Nencioni, A. 2011. Synergistic interactions between HDAC
and sirtuin inhibitors in human leukemia cells. PLoS ONE 6,
e22739.
Chalmers, A. D. & Slack, J. M. 1998. Development of the gut in
Xenopus laevis. Dev. Dyn. 212, 509–521.
Cheng, H. L., Mostoslavsky, R., Saito, S., Manis, J. P., Gu, Y.,
Patel, P., Bronson, R., Appella, E., Alt, F. W. & Chua, K. F.
2003. Developmental defects and p53 hyperacetylation in
Sir2 homolog (SIRT1)-deficient mice. Proc. Natl Acad. Sci.
USA 100, 10794–10799.
Cordenonsi, M., Dupont, S., Maretto, S., Insinga, A., Imbriano,
C. & Piccolo, S. 2003. Links between tumor suppressors:
p53 is required for TGF-beta gene responses by cooperatingwith Smads. Cell 113, 301–314.
De Robertis, E. M. & Kuroda, H. 2004. Dorsal-ventral patterningand neural induction in Xenopus embryos. Annu. Rev. Cell
Dev. Biol. 20, 285–308.

467

Deimling, S. J. & Drysdale, T. A. 2011. Fgf is required to regulate
anterior-posterior patterning in the Xenopus lateral plate
mesoderm. Mech. Dev. 128, 327–341.
Doherty, J. T., Conlon, F. L., Mack, C. P. & Taylor, J. M. 2010.
Focal adhesion kinase is essential for cardiac looping andmultichamber heart formation. Genesis 48, 492–504.
Frye, R. A. 2000. Phylogenetic classification of prokaryotic andeukaryotic Sir2-like proteins. Biochem. Biophys. Res. Commun. 273, 793–798.
Fulco, M., Cen, Y., Zhao, P., Hoffman, E. P., McBurney, M. W.,
Sauve, A. A. & Sartorelli, V. 2008. Glucose restriction inhibits
skeletal myoblast differentiation by activating SIRT1 through
AMPK-mediated regulation of Nampt. Dev. Cell 14, 661–
673.
Ghosh, H. S., McBurney, M. & Robbins, P. D. 2010. SIRT1 negatively regulates the mammalian target of rapamycin. PLoS
ONE 15, e9199.
Goudevenou, K., Martin, P., Yeh, Y. J., Jones, P. & Sablitzky, F.
2011. Def6 is required for convergent extension movements
during zebrafish gastrulation downstream of Wnt5b signaling. PLoS ONE 6, e26548.
Guarente, L. 2007. Sirtuins in aging and disease. Cold Spring
Harb. Symp. Quant. Biol. 72, 483–488.
Guertin, D. A., Stevens, D. M., Thoreen, C. C., Burds, A. A., Kalaany, N. Y., Moffat, J., Brown, M., Fitzgerald, K. J. & Sabatini, D. M. 2006. Ablation in mice of the mTORC
components raptor, rictor, or mLST8 reveals that mTORC2
is required for signaling to Akt-FOXO and PKCalpha, but not
S6K1. Dev. Cell 11, 859–871.
Hatta, K., Kimmel, C. B., Ho, R. K. & Walker, C. 1991. The
cyclops mutation blocks specification of the floor plate of
the zebrafish central nervous system. Nature 350, 339–
341.
Howland, R. B. 1916. On the effect of removal of the pronephrosof the amphibian embryo. Proc. Natl Acad. Sci. USA 2,
231–234.
Kang, H., Suh, J. Y., Jung, Y. S., Jung, J. W., Kim, M. K. &Chung, J. H. 2011. Peptide switch is essential for Sirt1
deacetylase activity. Mol. Cell 44, 203–213.
Kim, J. E., Chen, J. & Lou, Z. 2008. DBC1 is a negative regulator of SIRT1. Nature 451, 583–586.
Lee, I. H., Cao, L., Mostoslavsky, R., Lombard, D. B., Liu, J.,
Bruns, N. E., Tsokos, M., Alt, F. W. & Finkel, T. 2008. A role
for the NAD-dependent deacetylase Sirt1 in the regulation of
autophagy. Proc. Natl Acad. Sci. USA 105, 3374–3379.
Li, L., Wen, L., Gong, Y., Mei, G., Liu, J., Chen, Y. & Peng, T.
2012. Xenopus as a model system for the study of GOLPH2/GP73 function: Xenopus GOLPH2 is required for pronephros development. PLoS ONE 7, e38939.
Li, X., Zhang, S., Blander, G., Tse, J. G., Krieger, M. & Guarente,
L. 2007. SIRT1 deacetylates and positively regulates thenuclear receptor LXR. Mol. Cell 28, 91–106.
Li, Y., Xu, W., McBurney, M. W. & Longo, V. D. 2008. SirT1 inhibition reduces IGF-I/IRS-2/Ras/ERK1/2 signaling and protects neurons. Cell Metab. 8, 38–48.
Lionaki, E., Markaki, M. & Tavernarakis, N. 2013. Autophagy andageing: insights from invertebrate model organisms. Ageing
Res. Rev. 12, 413–428.
Luo, J., Nikolaev, A. Y., Imai, S., Chen, D., Su, F., Shiloh, A.,
Guarente, L. & Gu, W. 2001. Negative control of p53 by
Sir2alpha promotes cell survival under stress. Cell 107, 137–
148.
McBurney, M. W., Yang, X., Jardine, K., Hixon, M., Boekelheide,K., Webb, J. R., Lansdorp, P. M. & Lemieux, M. 2003. Theª 2014 The Authors
Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists

468

Y. Ohata et al.

mammalian SIR2alpha protein has a role in embryogenesis
and gametogenesis. Mol. Cell. Biol. 23, 38–54.
Medvedik, O., Lamming, D. W., Kim, K. D. & Sinclair, D. A.
2007. MSN2 and MSN4 link calorie restriction and TOR tosirtuin-mediated lifespan extension in Saccharomyces cerevisiae. PLoS Biol. 5, e261.
Moriyama, Y., Ohata, Y., Mori, S., Matsukawa, S., Michiue, T.,
Asashima, M. & Kuroda, H. 2011. Rapamycin treatment
causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis. Biochem. Biophys. Res. Commun. 404, 974–978.
Murakami, M., Ichisaka, T., Maeda, M., Oshiro, N., Hara, K.,
Edenhofer, F., Kiyama, H., Yonezawa, K. & Yamanaka, S.
2004. mTOR is essential for growth and proliferation in earlymouse embryos and embryonic stem cells. Mol. Cell. Biol.
24, 6710–6718.
Peck, B., Chen, C. Y., Ho, K. K., Di Fruscia, P., Myatt, S. S.,
Coombes, R. C., Fuchter, M. J., Hsiao, C. D. & Lam, E. W.
2010. SIRT inhibitors induce cell death and p53 acetylation
through targeting both SIRT1 and SIRT2. Mol. Cancer Ther.
9, 844–855.
Picard, F., Kurtev, M., Chung, N., Topark-Ngarm, A., Senawong,T., Machado De Oliveira, R., Leid, M., McBurney, M. W. &Guarente, L. 2004. Sirt1 promotes fat mobilization in white
adipocytes by repressing PPAR-gamma. Nature 429, 771–
776.
Rogina, B. & Helfand, S. L. 2004. Sir2 mediates longevity in the
fly through a pathway related to calorie restriction. Proc. Natl
Acad. Sci. USA 101, 15998–16003.
Shahar, O. D., Gabizon, R., Feine, O., Alhadeff, R., Ganoth, A.,
Argaman, L., Shimshoni, E., Friedler, A. & Goldberg, M.
2013. Acetylation of lysine 382 and phosphorylation of serine
392 in p53 modulate the interaction between p53 and
MDC1 in vitro. PLoS ONE 8, e78472.
Solomon, J. M., Pasupuleti, R., Xu, L., McDonagh, T., Curtis, R.,
DiStefano, P. S. & Huber, L. J. 2006. Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell
survival following DNA damage. Mol. Cell. Biol. 26, 28–38.

Sommer, M., Poliak, N., Upadhyay, S., Ratovitski, E., Nelkin, B.
D., Donehower, L. A. & Sidransky, D. 2006. DeltaNp63alphaoverexpression induces downregulation of Sirt1 and an
accelerated aging phenotype in the mouse. Cell Cycle 5,2005–2011.
€hle, U., Blader, P., Henrique, D. & Ingham, P. W. 1993. Axial,
Straa zebrafish gene expressed along the developing body axis,
shows altered expression in cyclops mutant embryos. Genes
Dev. 7, 1436–1446.
Takebayashi-Suzuki, K., Funami, J., Tokumori, D., Saito, A., Watabe, T., Miyazono, K., Kanda, A. & Suzuki, A. 2003. Interplay between the tumor suppressor p53 and TGF beta
signaling shapes embryonic body axes in Xenopus. Development 130, 3929–3939.
Tang, B. L. 2011. Sirt1’s systemic protective roles and its promise as a target in antiaging medicine. Transl. Res. 157, 276–
284.
Tissenbaum, H. A. & Guarente, L. 2001. Increased dosage of asir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410, 227–230.
Tucci, P. 2012. Caloric restriction: is mammalian life extension
linked to p53? Aging 4, 525–534.
Vaziri, H., Dessain, S. K., Ng Eaton, E., Imai, S. I., Frye, R. A.,
Pandita, T. K., Guarente, L. & Weinberg, R. A. 2001. hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase.
Cell 107, 149–159.
Wallingford, J. B., Seufert, D. W., Virta, V. C. & Vize, P. D. 1997.
p53 activity is essential for normal development in Xenopus.
Curr. Biol. 7, 747–757.
Zhang, X. M., Li, L., Xu, J. J., Wang, N., Liu, W. J., Lin, X. H.,
Fu, Y. C. & Luo, L. L. 2013. Rapamycin preserves the follicle
pool reserve and prolongs the ovarian lifespan of female rats
via modulating mTOR activation and sirtuin expression.
Genes 523, 82–87.
Zhou, X., Fan, L. X., Sweeney, W. E. Jr, Denu, J. M., Avner, E.
D. & Li, X. 2013. Sirtuin 1 inhibition delays cyst formation in
autosomal-dominant polycystic kidney disease. J. Clin.
Invest. 123, 3084–3098.

ª 2014 The Authors
Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists