Biochemical and Biophysical Research Communications 404 (2011) 974–978Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Rapamycin treatment causes developmental delay, pigmentation defects,
and gastrointestinal malformation on Xenopus embryogenesis
Yuki Moriyama a, Yoshihisa Ohata b, Shoko Mori a, Shinya Matsukawa b, Tatsuo Michiue c,
Makoto Asashima c,d, Hiroki Kuroda a,b,⇑aGraduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
Department of Education (Sciences), Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japand
Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Baien, Tsukuba, Ibaraki 305-8562, Japanbca r t i c l e

i n f oArticle history:
Received 14 December 2010
Available online 25 December 2010
Keywords:
TOR
Rapamycin
Xenopus
Pigmentation
Gastrointestinal malformationa b s t r a c t
Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway andinfluences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote.
However, the extent to which rapamycin controls early developmental events of amphibians remains to
be understood. Here we report an examination of rapamycin effects during Xenopus early development,
followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First,
we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black
pigment spots located at dorsal and lateral skin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treatedembryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes
enormous influences on early developmental period.
! 2010 Elsevier Inc. All rights reserved.

1. Introduction
The target of rapamycin, TOR, is an essential serine/threonine
kinase and functions in two distinct multiprotein complexes called
as TOR complex 1 (TORC1) and 2 (TORC2). The structure and function of these complexes have been conserved from yeast to vertebrates. As easily imagine from its name, rapamycin binds to TOR
kinase and inhibits TORC1 activity although TORC2 is principallyinsensitive [1].
Rapamycin is originally developed as an antifungal agent. However, this usage was abandoned when it was discovered that it had
potent immunosuppressive and antiproliferative properties in human cells. Since then, various roles of rapamycin have been reported. In budding yeast, rapamycin treatment causes a dramatic
down-regulation of cellular anabolic processes and correspondingly an up-regulation of catabolic and growth inhibitory processes[1]. Most cells treated with rapamycin abruptly arrest growth andenter a G0-like state. In mouse, regular consumption of moderateamounts of rapamycin since 600 days of age can extend lifespan;on the basis of age at 90% mortality, it leads to an increase of
approximately 10% [2]. In addition, the FKBP–FRB binding assay⇑ Corresponding author at: Department of Education (Sciences), Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. Fax: +81 54 287 6693.
E-mail address: ehkurod@ipc.shizuoka.ac.jp (H. Kuroda).
0006-291X/$ - see front matter ! 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.12.093

using rapamycin has been very common tool in cell biology since
first reported [3], and it will soon be adopted in other fields suchas medical research and developmental biology. However, regarding rapamycin effects for early developmental stage, much remains
to be understood although it should be required for the safety use
and might be important for thinking the risk during pregnancy andthe effect on natural environment. For the investigation to know
the effect of rapamycin on early development of vertebrate, aquaticexperimental animals must be the best candidates. Interestingly, it
is already reported that, in zebrafish, rapamycin treated embryos
have mild developmental delay up to 72 hpf (hours post-fertilization) and severe effects on digestive tract development [4]. In thisstudy, we sought to determine the effects of rapamycin during
early development of amphibians and found that, in addition tosame effects in the case of zebrafish, pigmentation was reducedby rapamycin treatments.
2. Materials and methods
2.1. Embryology and histology
Xenopus laevis eggs were obtained from females injected with
400 units of human chorionic gonadotropin (HCG, Fuji PharmaCo., Japan), fertilized in vitro with minced testis, and then cultured
in 0.1! Steinberg’s solution (SS). 1! SS contains 58 mM NaCl,

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0.67 mM KCl, 0.34 mM Ca(NO3)2, 0.83 mM MgSO4, 100 mg/L kanamycin sulfate, and 5 mM Tris–HCl (pH 7.4). Frog embryos werealways cultured at 22 "C. The jelly coats were removed with thioglycolic acid solution (1% of thioglycolic acid in 0.1! 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 withhematoxylin and eosin (HE).
2.2. Rapamycin treatment
25 mg of rapamycin (R-5000, LC Laboratories, Massachusetts,USA) was dissolved in 1250 ll of DMSO, and each 20 ll of aliquot(22 mM) was stored at "20 "C. For using, aliquot was diluted
with 0.1! SS into proper concentration. Rapamycin (80 lM), which
is maximum concentration used in this study, includes 0.36% of
DMSO. Note: We confirmed that 0.5% of DMSO was not toxic fordevelopment. Embryos were exposed to rapamycin solutions from
2-cell stage to the stages designed for observations.
2.3. mRNA synthesis and microinjection
To generate synthetic mRNA in vitro, pCS2-dn-Rheb was linearized with NotI and transcribed with SP6 RNA polymerase by
mMESSAGEmMACHINE kit (Ambion, USA). dn-Rheb mRNA microinjection into Xenopus embryo was performed at 2-cell stage.
2.4. Western blot
Cell lysates were prepared in Phospho Stay Solution (Novagen,USA), and lipids were removed by an equal volume of trichloroethylene (Wako, Japan). Proteins were separated by 5–20% gradientSDS–PAGE gels. Western blots were performed using polyclonal
rabbit antibodies against S6K (1:250; CHEMICON, California, USA)and phospho-S6K (1:2000; GeneTex, California, USA).
2.5. RT-PCR
The following primers were used for RT-PCR.
TOR: 50 -CACAGGCTCTAATTTACCC-30 and 50 -GGTCTCTACCATATG
CCT-30raptor: 50 -AAGGGGAGAAACTGGACTACTTCCA-30 and 50 -TTT
CATCTCTCGGTCTGTATCC-30
EF1a: 50 -CCTGAACCACCCAGGCCAGATTGGTG-30 and 50 -GAGGGTAGTCAGAGAAGCTCTCCACG-30
Chordin: 50 -CCTCCAATCCAAGACTCCAGCAG-30 and 50 -GGAGGAGGAGGAGCTTTGGGACAAG-30
Noggin: 50 -AGTTGCAGATGTGGCTCT-30 and 50 -AGTCCAAGAGTCTCAGCA-30
The annealing temperatures were 55 "C, and 25 cycles for EF1aand 30 cycles for TOR, raptor, Chordin, and Noggin were performed
for amplifications.

Fig. 1. Inhibition of Xenopus TOR activity by rapamycin treatment. (A) RT-PCR
analyses for TOR and raptor mRNA expressions from unfertilized egg to larva stage.
Both TORC1 components are consecutively expressed in Xenopus embryo. (B)Generally conserved model of TORC1 inhibition by rapamycin. Structure of the
FKBP12-rapamycin complex interacts with TORC1 in which TOR and raptor are
major components. S6 kinase (S6K) is the major output signal activated by TORC1.
(C) Western blot analysis of phospho-S6 kinase (p-S6K) in rapamycin-treatedembryos. Embryos were treated at 2-cell stage with 80 lM of rapamycin, andextract was collected at stage 42.

experiments (data not shown). We first analyzed mRNA expressions of TOR and raptor by RT-PCR (Fig. 1A). In Xenopus embryos,
zygotic gene transcription is activated at mid-blastula transition(MBT), which is around stage 8.5. Compared to the expression patterns of famous zygotically-expressed genes Chordin and Noggin,both TOR and raptor were clearly determined as maternallyexpressed genes and continuously expressed in late stages as sameas FKBP12 (Fig. 1A).
3.2. Rapamycin treatment on Xenopus can inhibit TORC1 activity
Ribosomal protein p70 S6 kinase (S6K) is a serine/threonine kinase and a well-established target of TORC1 [7]. We analyzed theeffect of rapamycin on S6K phosphorylation targeted by TORC1
by western blot of 3 dpf (days post-fertilization) embryo extracts(Fig. 1C). Using phosphorylated form-specific antibody, we corroborated a substantial decrease in response to rapamycin (Fig. 1C),
demonstrating that rapamycin treatment is effective to downregulate TORC1 activity. This reaction interval is somewhat long, but, inzebrafish, which is with almost twice the developmental velocity
compared to Xenopus, complete inhibition of S6K by rapamycin
takes 28 h [4], suggesting that the efficiency of rapamycin treatment for Xenopus is equivalent for the case of zebrafish.

3. Results and discussion
3.1. TOR and raptor are expressed in all stages of embryos

3.3. Rapamycin treatment sets back developmental program in
dose-dependent manner

The TOR protein is conserved from yeast to vertebrate andforms two distinct physical and functional complexes, termed
TORC1, which is sensitive to rapamycin, and TORC2, which isinsensitive [1]. This inactivation of TORC1 follows the formationof a ternary complex between TOR, raptor, rapamycin, and FKBP12(Fig. 1B) [5]. FKBP12, which was also called as FK506-binding protein, is expressed in all stages of early development in Xenopus [6],meaning that rapamycin does not really require addition of FKBP12
in order to block TORC1 activity. In fact, microinjection of FKBP12mRNA did not increase rapamycin activity in our preliminary

To evaluate the effects of rapamycin during development wefirst used rapamycin treatment (20 lM) on live Xenopus embryos
beginning at the 2-cell stage up to 3 dpf. Interestingly, developmental speed of embryos treated with rapamycin was clearly delayed, but not arrested (Fig. 2A). Particularly, in terms of optic
vesicle formation, distinct difference of developmental speed
was observed between control and rapamycin-treated embryos(Fig. 2A). Then, using different concentrations of rapamycin, we
tried to measure the relative effectiveness of rapamycin on developmental speed compared to control embryos (Fig. 2B). At 8.5 hpf

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Fig. 2. The effects of TOR signal by rapamycin treatment and dn-Rheb mRNA on developmental speed. (A) Simple comparison of control and rapamycin-treated embryos atsame time-course. In embryos treated with rapamycin (20 lM) from 2-cell stage, developmental speed was clearly delayed (100%, n = 15). (B) Time-course of developmental
delay following rapamycin treatment. In embryos exposed to each test solution (10, 20, 40, and 80 lM), developmental delays were dose-dependently increased (100%,n = 15, 100%, n = 15, 100%, n = 15, and 100%, n = 15). Same effect was observed in dn-Rheb mRNA (50 pg) injected embryo (100%, n = 15). Refer to the scores before stage 42, theestimated delayed times for stage 45 (95.3 hpf in control embryo) were calculated because structural differences induced by rapamycin made collect staging difficult at thisstage. Each stage was determined as follow. Stage 10 was determined when blastopore pigmentation is first detected at the dorsal side. Stage 13 when the blastopore wascompletely closed. Stage 22 when the neural fold was perfectly closed. Stage 26 when dorsal axis was straighten, and primary melanocyte and optic vesicle bump wereobserved. Stage 28 when fin structure was first observed. Stage 36 when eyes and cement gland were observed clearly and darkly. Stage 38 when fin region was expanded to
50% of trunk-tail. Stage 40 when boundary between posterior yolk and fin was vertically located. Stage 42 when a part of ventral fin was clearly appeared anterior to anus
regions. Stage 45 when coiled-coil structure was observed in gut region. (C) Table for B. Rapamycin treatments were performed after 2-cell stage before control embryos
reached scheduled stage. dn-Rheb mRNA was microinjected at 2-cell stage. Every 15 min we counted number of embryos that already reached scheduled stage. Using thesescores, each averaged arrival time for scheduled stages was calculated.

in our experimental condition, 50% of control embryo reachedstage 10. On this timing we found that average 0.4 h of delay
was observed in embryos treated with 80 lM (Fig. 2B and C). In
treatment with 10 lM of rapamycin, obvious difference was detected at 24.0 hpf, which means stage 22; average 0.8 h of delay
was turned up in treated (Fig. 2B and C). In all cases time-lag between control and treated embryos were almost directly proportional to incubation period, and in the case of 80 lM it expanded
to 5.0 h in stage 42, meaning that it took average 70.3 hpf to reachstage 42 in control embryos and 75.3 hpf (93.4% on developmental
speed) in treated (Fig. 2B and C). GTP-binding protein Rheb (Rashomolog enriched in brain) is known as a positive regulator of
TORC1 [1]. If rapamycin actually works on TORC1, inhibition ofRheb activity is supposed to make same effect of rapamycin treatment, so we also tried to examine whether inhibition of TORC1
activity by dominant negative form of Rheb (dn-Rheb) has same effect as rapamycin treatment. As a result of this experiment, compared to rapamycin treatment, microinjection of dn-Rheb mRNA
led almost same level of developmental delay until tailbud stage(Fig. 2B and C), meaning that developmental speed of Xenopus embryo was surely controlled by TORC1 pathway. Interestingly, after
tadpole stage retardative activity was not observed in the case ofmRNA injection (Fig. 2B and C). The reason for this difference happened is probably because mRNA can be targeted by endogenous
ribonuclease and completely digested until tailbud stage. As
reported, some molecules can arrest development; artificial
ERK activation by mRNA microinjection of constitutive active
mutant of MEK can immediately block cell cleavage [8]. However,rapamycin-dependent type of developmental delay has neverbeen reported in amphibian embryogenesis. Inhibition of TORC1
signaling has been shown to extend lifespan in yeast [9,10], fruit
fly [11], Caenorhabditis elegans [12], and mice [2]. Moreover, decrease in speed of larval development by inhibition of TORC1 isalso reported in studies using zebrafish and C. elegans [4,13]. Lifespan elongation by inhibition of TORC1 may be considered of value
from the beginning of life.
3.4. Rapamycin treatment inhibits pigmentation without a decrease in
number of melanocytes
In addition to time-course changes we reached a conviction thatsome structural differences were also occurred in rapamycin-treated embryos. However, in order to avoid regarding time delaydependent difference as structural change induced by rapamycin,
time-course of developmental delay caused by rapamycin shouldbe always taken into consideration in phenotypic evaluations.
Therefore, we always referred to table (Fig. 2C) to fix on reasonabletiming for rapamycin-treated embryos. For instances, on the condition that control embryo reaches stage 42 at 70.3 hpf, we thoughtthat embryos treated with 10, 20, 40, and 80 lM of rapamycin
respectively took 73.8 (+3.5), 74.3 (+4.0), 74.8 (+4.5), and 75.3(+5.0) hpfs and that dn-Rheb mRNA (50 pg) injected embryo took
71.9 (+1.6) hpf for reaching stage 42. Moreover, enormous structural differences caused by rapamycin treatment after stage 42 to
be described later made it difficult to do correct staging for rapamycin-treated embryos, so we adopted the way of estimated hpf
for late stages embryos. For examples, on the condition that control
embryo reaches stage 45 at 95.3 hpf, we regarded that embryos

Y. Moriyama et al. / Biochemical and Biophysical Research Communications 404 (2011) 974–978

Fig. 3. Pigmentation defects induced by rapamycin treatment. Major pigmentation
located at lateral and dorsal side of embryos at stage 42 was obviously detected incontrol embryos, but weakly in rapamycin-treated (20 lM) embryos (100%, n = 15).
PCNT, PNT, PY, and PS indicate pigmentation underlain by cephalic neural tube,
neural tube, yolk, and somites. Slightly bigger yolk was frequently observed intreated. Scale bars represent 1 mm.

treated with 10, 20, 40, and 80 lM of rapamycin respectively took
101.1 (+5.8), 101.7 (+6.4), 102.0 (+6.7), and 102.6 (+7.3) hpfs andthat dn-Rheb mRNA (50 pg) injected embryo took 97.0 (+1.7) hpf
for reaching stage 45. Using this way, pigmentation defects on
the skin was first come under observation (Fig. 3). Usually normal
embryos have strong pigmentations on skin underlain by neuraltube, yolk, and somites (Fig. 3). On the other hand, these pigmentation, but not retinal pigment epithelium, were much weaker in977

all rapamycin-treated embryos, and pigmentation underlain bycephalic neural tube was remarkably reduced (Fig. 3). These pigmentation defects were not observed in dn-Rheb mRNA injected
embryos (Fig. 4C) probably because microinjected mRNA has been
already digested when melanocyte formation is started at tailbud
stage. In fact, we started rapamycin treatment at stage 20, it actually induced same level of inhibition of pigmentation as normal
rapamycin treatment (data not shown). There was not so muchof a difference of number of melanocytes between control andtreated embryos, indicating that rapamycin may inhibit cell progression but not affect primary differentiation and proliferationof melanocytes. In vertebrate pigment cells found in epidermalskin are derived from neural crest, in contrast pigment cells in
the retinal pigmented epithelium originate from the neural epithelium of the optic cup [14]. Therefore, rapamycin treatment may affect somewhere in the step of the segregation of cell lineages
derived from neural crest.
3.5. Rapamycin exerts a specific effect on the digestive tract
At stage 42, other structures outside the gastrointestinal tractdeveloped relatively normally despite treatment with rapamycin.
In contrast, we found mild phenotypic effects around yolk in
rapamycin-treated embryo at stage 42. The size of yolk, which

Fig. 4. Gastrointestinal malformation induced by rapamycin treatment and dn-Rheb mRNA injection. (A) Control embryo at stage 45 (95.3 hpf) for B and C. (B) Rapamycintreated embryo at stage 45. Embryos were treated with 40 lM of rapamycin and cultured. Pigmentation defects were stronger than Stage 42. Both size and shape of gut wereaffected by rapamycin treatment (100%, n = 15). (C) dn-Rheb mRNA (200 pg) injected embryo at stage 45. Both size and shape of gut were also affected by dn-Rheb mRNA
injection (100%, n = 15), but pigmentations were observed like control. Dotted lines mean sectioned sites for histological analyses shown in G–I. (D–F) Surgically-resected guts
from embryos shown in A–C. Upper and under panels respectively indicate ventral and dorsal views. Secondary coiled structure of gut was not detected in rapamycin-treated
and dn-Rheb mRNA injected embryos. 1", primary coil; 2", secondary coil. The diagram of gut structure was shown on the bottom-left corner. (G–I) Histological sections
created from embryos shown in A–C. In both rapamycin-treated and dn-Rheb mRNA injected embryos, gut formations were disordered and thicker than the case of control
embryo. bd, bile duct; li, large intestine; lu, lungs; pa, pancreas; si, small intestine; st, stomach. (J) Long cultured embryos in 20 lM of rapamycin (176 hpf). All embryos wereswollen like balloon but survived until 200 hpf maximum. The scale bars of A, B, and C and of D, E, and F represent 2 mm and 0.5 mm, respectively.

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is including future gastrointestinal tract, is slightly bigger thannormal (Fig. 3). At stage 45, the effect of rapamycin became much
more pronounced at several points on treated embryos. Total
length of rapamycin-treated embryos was shorter (Fig. 4A and B).
Same results were observed in dn-Rheb mRNA injected embryos(Fig. 4C). Maturation of melanocytes was clearly observed in control and dn-Rheb mRNA injected embryo (Fig. 4A and C), while
not in treated (Fig. 4B). Total volume of eye was smaller in both
rapamycin treated and dn-Rheb mRNA injected embryos, while
crystal lens was not properly covered with optic cup only inrapamycin treated (Fig. 4A–C). The gap between skin and inside
structure (especially around head region) in treated embryo was
wider than in control and dn-Rheb mRNA injected (Fig. 4A–C). Particularly, the big effect of rapamycin was detected at the gastrointestinal tract. Normally at this stage the intestine is forming adouble-coiled structure, and both external and internal coils have
two loops [15]. It was actually found in control embryos (Fig. 4A,
D, and G), while rapamycin-treated embryos had single-coiled
and much thicker intestine (Fig. 4B, E, and H). Double-coiled structure was not observed even in 176 h cultured embryo with rapamycin (Fig. 4J), indicating that time delay effect is directly
attributed to this single-coil structure. Same result was also obtained in the case of dn-Rheb mRNA injection (Fig. 4C, F, and I).
Intestinal morphogenesis requires a series of changes in the size
and architecture of the epithelial cells that line the gut tube. Rapamycin treatment and dn-Rheb mRNA injection may give someramification on presumptive gut region before stage 20, resulting
in derangement of timing of ideal communications of each region
in gastrointestinal area at later stage. Bile duct, large intestine,and small intestine were not observed in dn-Rheb mRNA injected
embryo, while all organs were observed in rapamycin-treated embryo same as control embryo. This difference was derived fromthat TORC1 inhibition by dn-Rheb mRNA injection was occurredearlier than by rapamycin treatment (Fig. 4G, H, and I). Almost similar effect by rapamycin is reported in zebrafish and C. elegans[4,16]. TORC1 kinase activity might be required for gastrointestinal
formation widely in animal kingdom.
3.6. The effect of rapamycin in early development
In this study, we examined the effect of rapamycin on Xenopus
early development and found three tangible results such as developmental delay, pigmentation defects, and gastrointestinal malformation caused by rapamycin treatment. Moreover, we tried to
culture to know how long rapamycin-treated embryos were survived, resulting that all treated embryos were swollen at laterstage and kept having gastrointestinal malformation (Fig. 4J). In
the end, all died before 200 hpf (stage 47 in control embryo) probably because nutrient supply was interfered with gastrointestinal
malformation. Interestingly, mice deficient for raptor, which isindispensable component for TORC1, die early in development[17]. The reason why the effect of rapamycin treatment is different
from raptor-knocked out phenotype is probably because rapamycin treatment does not affect oocyte or because complete inhibition of TORC1 activity by rapamycin needs long incubations suchas 1 day in fish and 2 days in frogs. If faster-acting drug is discovered, it may induce the phenotype like null mutant of raptor inmice. It is not clear how TORC1 is communicating with each developmental program that we reported here. However, our data justify special attention to the effect of very popular drug
rapamycin in control of very early lifespan of vertebrates.
Acknowledgments
We thank Prof. Ryuichi Nishinakamura, Prof. Tatsuya Maeda,
Prof. Ushio Kikkawa, and Prof. Kenta Hara for DNA constructs,
Hideyuki Ishibashi and Yoshito Tajima for preliminary experiments
for this work, and Prof. Takashi Ushimaru for good suggestions on
our study. This research was supported by the grants-in-aid received from the Shizuoka Research Institute.
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