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Identification of a Primary Target of Thalidomide
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Identification of a Primary Target of
Takumi Ito,1* Hideki Ando,2* Takayuki Suzuki,3,4Toshihiko Ogura,3Kentaro Hotta,2
Yoshimasa Imamura,5Yuki Yamaguchi,2Hiroshi Handa1,2†
Half a century ago, thalidomide was widely prescribed to pregnant women as a sedative but was found
to be teratogenic, causing multiple birth defects. Today, thalidomide is still used in the treatment of
leprosy and multiple myeloma, although how it causes limb malformation and other developmental
defects is unknown. Here, we identified cereblon (CRBN) as a thalidomide-binding protein. CRBN forms
an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1) and Cul4A that is
important for limb outgrowth and expression of the fibroblast growth factor Fgf8 in zebrafish and chicks.
Thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting the associated ubiquitin
ligase activity. This study reveals a basis for thalidomide teratogenicity and may contribute to the
development of new thalidomide derivatives without teratogenic activity.
scribed to pregnant women as a treatment for
morning sickness. Before its teratogenic activity
came to light and its use was discontinued,
~10,000 affected children were born from
women taking thalidomide during pregnancy
(1–3). Use of thalidomide during weeks 3 to 8
of gestation causes multiple birth defects such as
limb, ear, cardiac, and gastrointestinal malfor-
mations (1–3). The limb malformations, known
as phocomelia and amelia, are characterized,
respectively, by severe shortening or complete
absence of legs and/or arms, whereas the ear
malformations lead to anotia, microtia, and
hearing loss. Despite considerable effort, little
is known about how these developmental
defects are caused. Previous studies have
suggested thalidomide-induced oxidative stress
and its antiangiogenic action as a possible cause
of teratogenicity (4, 5). However, several impor-
tant questions remain unanswered, such as what
are direct targets of thalidomide and how the
target molecules mediate its teratogenic effects.
Recently, thalidomide use has increased for
the treatment of multiple myeloma and erythe-
ma nodosum leprosum, a painful complication
of leprosy (2, 3, 6, 7). Owing to its teratoge-
nicity, however, thalidomide is used under
strict control (8), and removal of its side ef-
uring the late 1950s and early 1960s,
thalidomide was sold as a sedative in
over 40 countries and was often pre-
fects is desirable for wider applications of this
potentially useful drug. It is important to
elucidate the molecular mechanism of thalid-
omide teratogenicity, especially to identify its
molecular target(s), because such knowledge
might allow rapid screening for potentially
useful related compounds devoid of teratogenic
activity. In this regard, we have been developing
high-performance affinity beads that allow
single-step affinity purification of drug target
proteins from crude cell extracts (9). Here we
show that cereblon (CRBN), a protein en-
coded by a candidate gene for mild mental
retardation, is a primary target of thalidomide
Binding of thalidomide to CRBN and DDB1.
To purify thalidomide-binding proteins, we
performed affinity purification using ferrite-
glycidyl methacrylate (FG) beads (9). The
carboxylic thalidomide derivative FR259625
was covalently conjugated to the beads (fig.
S1) and incubated with human HeLa cell
extracts (10). After extensive washing, bound
proteins were eluted with free thalidomide, and
the eluate fractions were subjected to SDS gel
electrophoresis and silver staining. Two poly-
peptides were specifically eluted (Fig. 1A, lane
3). When free thalidomide was added to extracts
before incubation with the beads, the yields of
these proteins were reduced (Fig. 1A, lane 4),
which suggested that these proteins specifically
interact with thalidomide. The 127- and 55-kD
proteins were therefore subjected to proteolytic
digestion and tandem mass spectrometry and
were identified as CRBN and damaged DNA
binding protein 1 (DDB1), respectively (table
S1). Identities of these proteins were confirmed
by immunoblotting (Fig. 1A). CRBN and
DDB1 were isolated similarly as thalidomide-
binding proteins from various cell types (fig. S2).
To determine whether this interaction is direct, we
used purified recombinant proteins. FLAG-tagged
CRBN, but not V5 (GKPIPNPLLGLDST) (11)
epitope- and histidine (His)–tagged DDB1,
bound to thalidomide beads (Fig. 1B). We
therefore asked whether DDB1 binds to thalid-
omide beads through its interaction with CRBN.
As expected, DDB1 was coprecipitated with
FLAG- and hemagglutinin (HA) epitope–tagged
(FH-) CRBN (Fig. 1C) and was not affinity-
purified from CRBN-depleted 293T cells (fig.
1Integrated Research Institute, Tokyo Institute of Technology,
Yokohama 226-8503, Japan.2Graduate School of Bioscience
and Biotechnology, Tokyo Institute of Technology, Yokohama
Cancer, Tohoku University, Sendai 980-8575, Japan.
cursory Research for Embryonic Science and Technology,
Japan Science and Technology Agency (JST), Saitama 332-
0012, Japan.5Drug Discovery Research, Astellas Pharma Inc.,
Ibaraki 305-8585, Japan.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
3Institute of Development, Aging and
Fig. 1. Thalidomide
binds to CRBN and
DDB1. (A) Thalidomide
were purified from HeLa
cell extracts by using
(+) or control (–) beads.
Where indicated, bound
proteins were eluted
with free thalidomide.
As indicated, 0.3 mM
thalidomide was added
to extracts before incu-
bation with the beads.
Eluted proteins were
analyzed by silver stain-
ing (top) or immuno-
blotting (IB) (bottom).
Asterisk indicates non-
specific signal. (B) Purified
and DDB1-V5-His were,
with thalidomide beads. Input materials used for affinity purification
(AP) and bound materials were immunoblotted. (C) FH-CRBN was
immunoprecipitated (IP) from 293T cells stably expressing FH-CRBN or from control cells, followed by
SDS gel electrophoresis and silver staining.
VOL 32712 MARCH 2010
on November 6, 2010
S3A), which led us to conclude that thalidomide
interacts directly with CRBN and indirectly with
DDB1 through its interaction with CRBN. The
equilibrium dissociation constant of the CRBN-
thalidomide interaction was calculated to be 8.5
nM (10). Moreover, CRBN did not bind to
phthalimide, a nonteratogenic analog of thalid-
omide (12), which substantiated the high affinity
and specificity of the CRBN-thalidomide inter-
action (fig. S3B).
Formation of an E3 complex by CRBN, DDB1,
and Cul4A. Human CRBN was originally iden-
tified as a candidate gene for autosomal
recessive mild mental retardation and encodes
a 442–amino acid protein that is highly con-
served from plants to humans (13). Although
CRBN was reported to interact with DDB1 in a
recent proteomic analysis (14), the functional
relevance of this interaction remains unclear.
Consistent with the apparently stoichiometric
interaction of CRBN and DDB1 (Fig. 1C), these
proteins are colocalized mainly in the nucleus,
but also in the cytoplasm (Fig. 2A). DDB1 is a
component of E3 ubiquitin ligase complexes
containing Cullin 4 (Cul4A or Cul4B), regulator
In principle, the function of E3 ubiquitin ligases
is to direct the polyubiquitination of substrate
proteins by a specifically interacting ubiquitin-
conjugating enzyme (E2) (17, 18). Cul4 is
thought to play a scaffold function, whereas
Roc1 has a RING finger domain that associates
with the E2 ubiquitin-conjugating enzyme.
Substrate receptors, such as DDB2, CSA, and
CDT2, directly bind to specific substrates and
mediate their ubiquitination (15, 19, 20). We
examined whether CRBN interacts with other
components of the E3 complex and found that
Cul4A and Roc1 were indeed coprecipitated with
FH-CRBN (Fig. 2B). If CRBN functions as a
substrate receptor of a Cul4-DDB1 E3 complex,
it would be expected to compete for binding to
DDB1 with other substrate receptor subunits, such
as DDB2. Consistent with this, the amount of
DDB1 coprecipitated with FH-CRBN was re-
duced in the presence of increasing amounts of
coexpressed DDB2 (Fig. 2C). Although thalid-
omide can induce oxidative DNA damage (4),
CRBN is likely to function independently of the
DDB2-mediated DNA damage response path-
way [see supportingonline material(SOM)text].
We then examined whether the CRBN com-
plex actually has E3 ubiquitin ligase activity.
Because substrate receptors and Cul4 are known
to undergo autoubiquitination in vitro in the
absence of their specific substrates (15, 16), in
vitro ubiquitination assays were performed using
purified protein components. Indeed, intrinsic
ubiquitination activity was observed in the pres-
ence of the CRBN complex (fig. S4). We then
examined whether CRBN is autoubiquitinated in
cells. For this, CRBN was affinity-purified from
293Tcells expressing FH-CRBN in the presence
or absence of the proteasome inhibitor MG132.
Autoubiquitination of FH-CRBN was detected
in the presence of MG132, and its ubiquitina-
tion was abrogated by small interfering RNA
(siRNA)–mediated depletion (knockdown) of
Cul4A (Fig. 2D, fig. S5A, and table S2). Knock-
down of DDB1 led to a substantial reduction of
the CRBN protein level (fig. S5B), and it was
not possible to determine the effect of DDB1
knockdown on CRBN ubiquitination. Neverthe-
less, this finding suggests that DDB1 and CRBN
are functionally linked.
To further investigate the role of DDB1 in
CRBN function, we obtained a CRBN mutant
deficient in DDB1 binding. Mutational analysis
revealed that deletion of amino acids 187 to 260
of CRBN (DMid) abolishes its interaction with
DDB1 (fig. S6). DMid was therefore stably
expressed in 293T cells and examined for its
ubiquitination after MG132 treatment. Ubiquiti-
nation of DMid was reduced compared with
wild-type CRBN (Fig. 2E). Collectively, these
findings suggest that CRBN is a subunit of a
functional E3 ubiquitin ligase complex and
undergoes autoubiquitination in a Cul4A- and
Inhibition of CRBN function by thalidomide.
To investigate the structural basis of the CRBN-
thalidomide interaction and its functional signif-
icance, we wished to obtain a CRBN point mutant
that does not bind to thalidomide but is assem-
bled into a functional E3 complex. Using a series
of deletion mutants, we mapped its thalidomide-
binding region to the C-terminal 104 amino acids,
which corresponds to the most highly conserved
region of the protein (figs. S7 and S8). Assum-
ing that evolutionarily conserved residues may
be important for thalidomide binding, we con-
structed a series of point mutants, and two point
Fig. 2. CRBN forms an E3 complex with DDB1 and Cul4A. (A)
FH-CRBN and DDB1-V5-His were coexpressed in HeLa cells and
immunostained. DAPI, 4′,6′-diamidino-2-phenylindole. (B)
CRBN-containing complexes were immunoprecipitated (IP) from
293T cells stably expressing FH-CRBN or from control cells
(mock) by using FLAG-specific antibody. Lysates (input) and
immunoprecipitates were immunoblotted (IB). (C) 293T cells
were cotransfected with the indicated amounts of FH-CRBN and
DDB2 expression vectors. Lysates and FLAG-specific immuno-
precipitates were immunoblotted. (D) 293T cells stably ex-
pressing FH-CRBN were transfected with Cul4A or control siRNA
and treated with vehicle or MG132. FH-CRBN was immunopre-
cipitated under stringent conditions and immunoblotted. (E)
293T cells stably expressing wild-type FH-CRBN or DMid were
incubated with vehicle or MG132 and processed as in (D).
12 MARCH 2010VOL 327
on November 6, 2010
mutants, Y384A and W386A, were found to be
defective for thalidomide binding (Fig. 3A) (11).
Moreover, the double point mutant Y384A/
W386A (CRBNYW/AA) had extremely low
thalidomide-binding activity. We then asked
whether CRBNYW/AAis functionally active in
cells. The subcellular localization of the mutant
was indistinguishable from wild-type CRBN
(fig. S7C). Moreover, CRBNYW/AAwas copre-
cipitated with DDB1, Cul4A, and Roc1 (Fig.
3B) and was autoubiquitinated after MG132
treatment (Fig. 3C), which demonstrated that
CRBNYW/AAis assembled into a complete E3
ubiquitin ligase complex.
We examined possible effects of thalidomide
on ubiquitination by treating 293T cells stably
expressing FH-CRBN or FH-CRBNYW/AAwith
MG132 and thalidomide at similar or higher
concentrations relative to the therapeutic doses
used in humans (21). Autoubiquitination of
wild-type CRBN was inhibited by thalidomide
in a concentration-dependent manner, whereas
autoubiquitination of CRBNYW/AAwas not
affected by thalidomide even at the highest
concentration used (Fig. 3D). Together, these
results suggest that thalidomide inhibits E3
function of the CRBN-containing complex by
directly binding to CRBN.
CRBN as an in vivo target of thalidomide.
Next, we investigated a possible role of CRBN
in thalidomide teratogenicity in animal models.
Thalidomide is teratogenic in rabbits and chicks,
but not in mice and rats (1–3). We first used
zebrafish as a model system because (i) the
rapid progress of development of zebrafish can
be monitored in real time because of the
transparency of the embryo, (ii) knockdown of
genes of interest can be carried out easily (22),
and (iii) zebrafish are suitable for pharmaco-
toxicological studies (23). Given that thalido-
mide was recently shown to inhibit angiogenesis
in zebrafish embryos (24), we reasoned that
zebrafish might be susceptible to other activities
To examine the effects of thalidomide on
zebrafish development, we transferred dechorio-
nated embryos to media containing different
concentrations of thalidomide at 2 hours post
fertilization (hpf) and allowed them to develop
for 3 days. It was immediately apparent that in
thalidomide-treated embryos, development of
pectoral fins and otic vesicles was disturbed,
whereas other aspects of development were not
generally affected (Fig. 4, A and B, and fig. S9).
More specifically, formation of the proximal
endoskeletal disc of the pectoral fin was
severely inhibited at 75 hpf (Fig. 4A), and otic
vesicle size was significantly reduced at 30 hpf
(Fig. 4B and fig. S11). Pectoral fin malforma-
tions were already apparent at 48 hpf (Fig. 5, C
and D). More detailed phenotypes induced by
thalidomide are described in the SOM text.
Recent studies have suggested that development
of pectoral fins and otic vesicles in teleosts share
common molecular pathways with that of
tetrapod limbs and ears (25–27).
Zebrafish have a CRBN orthologous gene
which we call zcrbn, whose product has ~70%
identity to human CRBN (fig. S8). We first
examined the expression pattern of zcrbn
mRNA and found that the gene is highly ex-
pressed in the brain, head vasculature, otic
vesicles, and developing pectoral fins at 30 and
48 hpf (fig. S12). zCrbn interacts with DDB1
and is affinity-purified from zebrafish embryos
as a major interactor with thalidomide (fig.
Fig. 4. Thalidomide
treatment or down-
regulation of the CRBN
complex causes similar
in zebrafish. (A and B)
Zebrafish embryos were
allowed to develop in
media containing the
of thalidomide. (A) Em-
bryos at 75 hpf were
fixed and stained with
Alcian blue. Pectoral fins
are indicated by arrow-
heads. (B) Close-up view
of otic vesicles of 30-hpf
live embryos. (C and D)
Where indicated, zcrbn
AMO was injected with
(rescued) or without
zcrbn mRNA into one-
cell stage embryos. (E
and F) Where indicated,
zcul4a AMO was injected
with or without zcul4a
mRNA into one-cell stage
views of pectoral fins of 72-hpf embryos. Pectoral fins are indicated by arrowheads.
(D and F) Otic vesicle size of 30-hpf embryos relative to the size of the embryo.
Representative raw data are shown in fig. S14. *P < 0.001. uninj, uninjected.
Fig. 3. Thalidomide inhibits
E3 ubiquitin ligase activity of
the CRBN-containing complex
in vitro. (A) Extracts prepared
from 293T cells overexpressing FH-CRBN or one of its mutants were
incubated with thalidomide-immobilized beads, and lysates (input) and
affinity-purified (AP) materials were immunoblotted (IB). (B) 293T cells
stably expressing FH-CRBNYW/AAwere subjected to FLAG-specific antibody
immunoprecipitation (IP) and immunoblotting. (C and D) 293T cells
stably expressing FH-CRBN or FH-CRBNYW/AAwere processed as in Fig.
2E. In (D), cells were treated with the indicated concentrations of
thalidomide for 4 hours before harvest.
VOL 327 12 MARCH 2010
on November 6, 2010
S13), which suggests that the findings of our
cell culture studies are valid in zebrafish. Hence,
the function of zCrbn during early development
was examined. Embryos injected with an anti-
sense morpholino oligonucleotide (AMO) for
zcrbn exhibited specific defects in fin and otic
vesicle development (Fig. 4, C and D, and figs.
S9 to S11 and S14), phenotypes similar to those
size of otic vesicles was reduced by half in the
knockdown embryos (Fig. 4D). These defects
were rescued by coinjection of zcrbn mRNA
(Fig. 4, C and D, and figs. S9 to S11 and S14).
The above findings suggested an interesting
possibility that thalidomide exerts teratogenic
effects by inhibiting zCrbn function. If so, its
teratogenic effects might be reversed by over-
expression of a functionally active, thalidomide
binding–defective form of zCrbn. To test this
idea, we used zCrbn carrying Y374A and
W376A mutations, which correspond to Y384A
and W386A mutations in human CRBN.
zCrbnYW/AAhad extremely low thalidomide-
had no discernible effect on fin and otic vesicle
development (Fig. 5 and figs. S9 to S11). As we
have already seen in Fig. 4, thalidomide treat-
ment significantly reduced otic vesicle size (P <
0.001, Mann-Whitney U test) (Fig. 5B and fig.
S11). Thalidomide treatment of embryos over-
expressing wild-type zCrbn similarly reduced
otic vesicle size (P < 0.001). However, thalido-
mide treatment of embryos overexpressing
zCrbnYW/AAdid not affect otic vesicle size
significantly (P = 0.59). Thalidomide-induced
pectoral fin malformations were also rescued by
overexpression of zCrbnYW/AA(Fig. 5A and fig.
S10), which demonstrated that thalidomide
exerts teratogenic effects by binding to CRBN
and inhibiting its function.
Molecular mechanism of thalidomide terato-
genicity. As the connection between thalidomide
and CRBN was established, we then examined
whether the CRBN-containing E3 complex is
and pectoral fins (fig. S12). As expected, micro-
injection of AMO for zcul4a caused similar de-
fects in otic vesicles and pectoral fins, and these
phenotypes were rescued by coinjection of zcul4a
mRNA (Fig. 4, E and F, and figs. S9 to S11 and
S14). Nevertheless, phenotypic similarities be-
tween zCrbn and zCul4A knockdown embryos
may be just coincidental. To rule out this pos-
sibility, we examined the importance of the phys-
thalidomide did not bind to this mutant, and
To obtain a clue to the pathway(s) downstream
of thalidomide and CRBN, we examined expres-
sion of key signaling molecules during pectoral
fin development. Sonic hedgehog (Shh) is ex-
pressed in the zone of polarizing activity (ZPA)
and is responsible for anteroposterior patterning
of limbs (28), whereas fibroblast growth factor
(fgf) 8 is expressed in the apical ectodermal
ridge (AER) of limbs and is responsible for limb
outgrowth along the proximodistal axis (29, 30).
In thalidomide-treated 48-hpf embryos, fgf8a
expression in the AER was severely reduced or
absent (Fig. 5C), whereas shh expression in the
ZPA was affected negligibly (Fig. 5D). In ad-
dition, fgf8a expression was restored by injec-
effect on shh expression in the ZPA (fig. S14).
downstream target of thalidomide and the
CRBN-containing E3 complex.
Conserved role for CRBN in zebrafish and
chicks. Finally, in order to validate our findings,
for studying thalidomide teratogenicity. As re-
resulted in the complete absence of a forelimb at a
sion of human CRBNYW/AA, but not wild-type
CRBN, in the forelimb field remarkably reduced
thalidomide sensitivity (Fig. 6A and fig. S16).
Expression of fgf8 and fgf10 was then examined.
Fgf10 is also an important regulator of proximo-
distal limb patterning and is normally expressed
in the mesoderm beneath the AER (Fig. 6B).
Thalidomide down-regulated fgf10 expression
in the mesoderm and, perhaps to a lesser extent,
fgf8 expression in the AER, and their expression
was restored by overexpression of CRBNYW/AA
(Fig. 6B). These results, together with the find-
ing that chick CRBN binds to thalidomide and
DDB1 (fig. S17), suggest that the developmental
role of CRBN is conserved in fins and limbs.
Discussion. The mechanism of action of tha-
lidomide appears to be multifaceted, but is not
fully understood. The immunomodulatory and
antiangiogenic activities of thalidomide have been
proposed to be partly responsible for its terato-
genic activity, as well as its therapeutic value in
the treatment of leprosy and multiple myeloma
(2, 3, 6, 7). In this respect, thalidomide is known
to inhibit the production of some cytokines such
as tumor necrosis factor–a and vascular endo-
thelial growth factor (32, 33). Thalidomide is
also capable of inducing apoptosis and produc-
ing reactive oxygen species (3, 4). Despite such
accumulating data, little is known about direct
Fig. 5. Expression of a drug binding–deficient form of CRBN suppresses thalidomide-induced
teratogenicity in zebrafish. After injection of zcrbn or zcrbnYW/AAmRNA, embryos were
allowed to develop in the presence or absence of thalidomide. (A) Dorsal views of pectoral
fins of 72-hpf embryos. Fins are indicated by arrowheads. (B) Otic vesicle size of 30-hpf embryos relative to the size of the embryo. *P < 0.001. (C and
D) Embryos at 48 hpf were subjected to hybridization with antisense probes for fgf8a or shh. Close-up views of fin buds are shown. uninj, uninjected.
12 MARCH 2010 VOL 327
on November 6, 2010
molecular targets of thalidomide. Here we pro-
vided several lines of evidence that CRBN is a
primary target of thalidomide teratogenicity. Be-
cause overexpression of the thalidomide-insensitive
form of CRBN rescued the effects of thalidomide
largely, if not entirely, in zebrafish and chicks,
CRBN is thought to play an important role as an
these species. Whereas CRBN is ubiquitously ex-
pressed in humans, thalidomide exerts tissue-
specific effects. Evidently, CRBN is necessary,
but not sufficient, for thalidomide teratogenicity,
and downstream components are likely to con-
tribute to the tissue-specific effects of thalidomide
(see SOM text).
thalidomide and CRBN fits well with a previous
sensitive species (34). In developing chick limb
sion of a subset of bone morphogenetic protein
(BMP) family genes and to induce apoptosis
(12). Coincidentally, mouse BMPs were shown
to inhibit fgf8 expression and to induce
apoptosis in the AER (35). Thus, CRBN appears
to be a missing link between thalidomide and
these key developmental regulators.
However, this study does not rule out other
mammals. Thalidomide-induced oxidative stress
is thought to occur through the direct formation
of reactive oxygen species (4) and is therefore
clearly a CRBN-independent process. Second, a
recent study suggested antiangiogenic activity of
thalidomide as a primary cause of chick limb
malformations, demonstrating that thalidomide-
induced inhibition of vasculogenesis precedes
inhibition of fgf8 expression and cell death in
in zebrafish, inhibition of vasculogenesis follows
thalidomide-induced morphological and transcrip-
tional changes in pectoral fin buds (fig. S18 and
SOM text), which implies that the sequence of
the common view of species differences in tha-
the species differences). Another point to consider
(see SOM text).
Our findings suggest that thalidomide exerts
teratogenic effects, at least in part, by binding to
CRBN and inhibiting the associated ubiquitin lig-
ubiquitin-dependent proteolysis by thalidomide
and CRBN leads to abnormal regulation of the
BMP and fgf8 signaling pathways and of devel-
opmental programs that require their normal func-
tions. Incidentally, many E3 ubiquitin ligases are
known to target developmental and/or transcrip-
grams (37, 38). There are, however, a number of
unanswered questions, such as: What are the sub-
strates of CRBN E3 ubiquitin ligase? How does
thalidomide inhibit the ubiquitination of CRBN in
the ligase complex? How might this pathway be
interconnected to the other pathways targeted by
thalidomide? These issues need to be addressed to
fully appreciate the model. Last, but not least,
because thalidomide is now used for the treatment
of multiple myeloma and leprosy, identification of
its direct target may allow rational design of more
effective thalidomide derivatives without terato-
genic activity (see SOM text).
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Fig. 6. Expression of a drug binding–deficient form of CRBN suppresses
thalidomide-induced limb malformations in chicks. FH-CRBN and EGFP
were electroporated as indicated into the forelimb field of stage 14
embryos. Thalidomide (+) or vehicle (-) was then directly applied onto
one of the forelimb buds, and embryos were analyzed at stage 36. (A)
Skeletal cartilages stained with Victoria blue. A, anterior; Pos, poste-
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fgf10 visualized by in situ hybridization. EGFP marks area of electroporation.
VOL 32712 MARCH 2010
on November 6, 2010
37. Y. Cang et al., Cell 127, 929 (2006). Download full-text
39. We thank T. Wada, S. Sakamoto, and S. Ishihara for
discussions; P. Raychaudhuri, T. Matsunaga, S. Krauss,
B. Thisse, A. Kawakami, S. Noji, J. Izpisua-Belmonte,
K. Kawakami, and J. Yamauchi for valuable reagents;
Y. Tsuboi for technical support; and P. Sharp and A. Berk for
comments on this manuscript. This work was supported by
Special Coordination Funds for Promoting Science and
Technology from JST, by the Global COE (Center of
Excellence) Program from the Japan Ministry of Education,
Culture, Sports, Science, and Technology (MEXT), and
by a grant for Research and Development Projects in
Cooperation with Academic Institutions from the New
Energy and Technology Development Organization (H.H.
and H.A.). This work was also supported by grants-in-aid for
Scientific Research (20370084 to T.O.) and for Young
Scientists (21770226 to T.S.) from MEXT and by the
Precursory Research for Embryonic Science and Technology
program from JST (T.S.). T.I. was a Japan Society for the
Promotion of Science Research Fellow. An application for a
patent has been filed in the Japan Patent Office.
Supporting Online Material
Materials and Methods
Figs. S1 to S19
Tables S1 and S2
5 June 2009; accepted 10 February 2010
Variations in the Sun’s Meridional
Flow over a Solar Cycle
David H. Hathaway1* and Lisa Rightmire2
The Sun’s meridional flow is an axisymmetric flow that is generally directed from its equator toward
its poles at the surface. The structure and strength of the meridional flow determine both the
strength of the Sun’s polar magnetic field and the intensity of sunspot cycles. We determine the
meridional flow speed of magnetic features on the Sun using data from the Solar and Heliospheric
Observatory. The average flow is poleward at all latitudes up to 75°, which suggests that it extends
to the poles. It was faster at sunspot cycle minimum than at maximum and substantially faster on
the approach to the current minimum than it was at the last solar minimum. This result may help to
explain why this solar activity minimum is so peculiar.
nitude weaker than that of the other major
flows on the surface of the Sun (granulation
~3000 m s−1, supergranulation ~300 m s−1, and
differential rotation ~170 m s−1). In the past, this
has led to reports of vastly different flow speeds
and directions (2–5). Despite its weakness, the
meridional flow plays a key role in the magnetic
evolution of the Sun’s surface. It transports mag-
netic elements that, when carried to the poles,
reverse the magnetic polarity of the poles and
build up polar fields of opposite polarity after
each sunspot cycle maximum. Models of this
magnetic flux transport process (6–8) have em-
ployed a variety of substantially different flow
profiles. The fidelity of these flux transport mod-
els is important because they are used in climate
change studies (9, 10) to estimate the total ir-
radiance of the Sun over the past century. The
meridional flow is also key to flux transport dy-
namo models that have been used to predict the
conflict between the surface flux transport mod-
els (6–10) and the flux transport dynamo models
he Sun’s meridional flow has been dif-
ficult to measure (1). Its amplitude (10 to
20 m s−1) is more than an order of mag-
of the meridional flow. A stronger meridional
flow produces weaker polar fields in the surface
flux transport models, whereas the same flow
cycles) in the flux transport dynamos. Solar
Cycle 23 (1996 to 2008) provides an interesting
problem for all of these models. The strength of
thepolarfields produced after cycle maximumin
2000–2001 was only about half that seen in the
previous three solar cycles (13). Furthermore,
start for cycle 24 has left behind a long quiet
minimum unlike any in the past 100 years.
We measured the Sun’s meridional flow to
determine its variability over Solar Cycle 23 by
following the motions of the small magnetic
elements that populate the entire surface of the
Sun. These are precisely the elements whose
motions are modeled in both the surface flux
transport models and the flux transport dynamo
models. Motions of sunspots, and even the plas-
ma at the surface, are known to differ from those
of the small magnetic elements (1–5). The data
we used have been acquired by the Michelson
Doppler Imager (MDI) on the European Space
Agency (ESA)/National Aeronautics and Space
Administration (NASA) Solar and Heliospheric
Observatory (SOHO). MDI produces images of
the line-of-sight magnetic field across the visible
solar disc every 96 min. This is done by measur-
ing differences in circular polarization on either
side of a spectral absorption line caused by traces
of nickel in the Sun’s atmosphere (14). We
measured the displacement of the magnetic
elements by comparing their positions at 8-hour
The 1024-by-1024 pixel magnetic images were
latitude and longitude from the central meridian.
angle of the Sun’s rotation axis relative to the
spacecraft’s vertical axis, changes in the tilt angle
spacecraft, and changes in perspective at different
distances from the Sun. Because sunspots have
very different proper motions (4) and produce
their immediate surroundings by masking all
pixels with measured absolute field strengths
greater than 500 Gauss and all contiguous pixels
of the same polarity with absolute field strengths
1NASA Marshall Space Flight Center, Huntsville, AL 35812,
USA.2University of Memphis, Memphis, TN 38152, USA.
*To whom correspondence should be addressed. E-mail:
Fig. 1. Magneticelementmotion.A
pair of masked magnetic maps from
5 June 2001 that were obtained 8
representing negative magnetic po-
larity and yellow representing posi-
tive magnetic polarity. The tick
marks around the borders are at
15° intervals in latitude and in
longitude from the central meridian.
The masked-out sunspot areas are
evident as white patches. The stron-
of pixels in the earlier map (left) is calculated to occur for a shift of 23.7 pixels in longitude and 0.4 pixels in
latitude for a similar strip in the later map (right).
2001/06/05 04:482001/06/05 12:48
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