Expert Opinion on Drug Discovery

ISSN: 1746-0441 (Print) 1746-045X (Online) Journal homepage: http://www.tandfonline.com/loi/iedc20

Advances in the use of Xenopus for successful

drug screening

Lorena A. Maia, Ian Velloso & Jose G. Abreu

To cite this article: Lorena A. Maia, Ian Velloso & Jose G. Abreu (2017): Advances in the
use of Xenopus for successful drug screening, Expert Opinion on Drug Discovery, DOI:
10.1080/17460441.2017.1367281

To link to this article: http://dx.doi.org/10.1080/17460441.2017.1367281

Published online: 28 Aug 2017.

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 EXPERT OPINION ON DRUG DISCOVERY, 2017
https://doi.org/10.1080/17460441.2017.1367281

REVIEW

Advances in the use of Xenopus for successful drug screening

Lorena A. Maia*, Ian Velloso* and Jose G. Abreu
Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

ABSTRACT ARTICLE HISTORY

Introduction: Understanding embryogenesis currently relies largely on the control of gene expression Received 12 May 2017
via several signaling pathways. Many of the embryonic signaling pathways guiding embryological Accepted 10 August 2017
events are implicated in diseases that lack effective cure or treatment. Because of the large number KEYWORDS
and size of the eggs, the rapid development of the embryos and the fact they are amenable to Amphibian embryo;
pharmacological, surgical and genetic techniques, Xenopus laevis has been successfully used in search- embryonic axis formation;
ing for compounds that target embryonic signaling pathways. Wnt; translational
Areas covered: Here, the authors address the use of amphibian eggs/embryos in successful chemical developmental biology;
Downloaded by [Australian Catholic University] at 11:05 28 August 2017

screenings; egg extracts as well as embryo phenotypes have been assayed to reveal drug toxicology chemical screening; FETAX;
effects and novel compounds acting in the Wnt/β-catenin signaling pathway. They do not discuss the natural compounds;
flavonoids
use of Xenopus oocyte two-electrode voltage clamps or genome editing tools as approaches for drug
discovery because they have been discussed elsewhere.
Expert opinion: While high-throughput screening is commonly performed in egg extracts, the embryo
axes perturbation system is more suited to the refinement and/or the validation of drug discovery
targeting embryonic signaling (particularly the Wnt/β-catenin pathway). In addition, Xenopus has also
been used in FETAX (frog embryo teratogenesis assay: Xenopus) to address chemical toxic/teratogenic
effects. However, further studies are necessary.

1. Introduction (1.3 mm) develop into remarkably robust embryos for explant/
transplant procedures [4].
It is accurate to say that the involvement of amphibian embryo in
In 1968, Gurdon used Xenopus oocytes as an undifferen-
drug screening is a still fresh addition to the set of scientific
tiated host environment for a transplanted somatic nucleus
approaches included in the theme ‘developmental biology of
and amused the scientific community by revealing that this
amphibians’ since the beginning of the 20th century. At that
chimera was able to develop until the blastula stage [5]. This
time, Spemann began to build the concept of ‘primary embryo-
was a scientific hallmark not only from a cloning perspective
nic induction’ by studying the embryogenesis of salamanders. In
but also from a ‘developmental biology of amphibians’ per-
1903, he showed that the dorsal cell, but not the ventral cell, of a
spective, since it proved the major role played by cytoplasmic
two-cell embryo could develop into a whole organism [1]. Then,
molecules in nuclear regulation throughout development [6]
together with Mangold, he transplanted the dorsal marginal
and established Xenopus laevis as an amenable model for
zone of a pre-gastrulae embryo into the ventral side of another
genetic approaches.
embryo and observed a secondary axis being formed [2].
The evolution of embryology into developmental biology
Whatever induced the dorsal cell of the salamander’s embryo
brought practical changes in the way one could experimen-
to develop into a whole organism was also present in the so-
tally handle the clawed frog embryo; it became possible to
called ‘organizer’ just before gastrulation, and the behavior trig-
microinject dominant-negative constructs, plasmids, mRNAs or
gered by this, so far, mysterious molecule(s) became known as
even proteins into the uncommonly large embryo of Xenopus
‘primary embryonic induction’ [3].
at early stages to disrupt gastrulation morphogenesis less than
While most discoveries were coming from salamander
15 hours later [7–10]. By the early 1990s, many key genes for
embryos, Xenopus laevis had already become a laboratory spe-
Xenopus embryogenesis had already been discovered and
cies worldwide because of its usage for pregnancy tests, and it
molecular studies aiming to describe their signaling activity
was just a matter of time for Nieuwkoop to realize, in the 1940s,
became a hot topic in developmental biology [11–14].
its potential for embryological experiments [4]. Although all
Many of the insights into the signaling pathways important
amphibians have their whole development outside the mater-
for the embryo morphological transformation have come from
nal organism and many frog species pass through embryologi-
the genetic modulation of key components of development
cal phases as fast as clawed frogs do, Xenopus laevis stands out
and also from their chemical modulation. This second
because the female lays eggs all year long and these large eggs
approach uses small organic molecules that potentially alter

CONTACT Jose G. Abreu garciajr@icb.ufrj.br

*These authors contributed equally to this work.
© 2017 Informa UK Limited, trading as Taylor & Francis Group
 2 L. A. MAIA ET AL.
Article highlights
● Xenopus oocyte/embryo is a bright spot within the field of drug
discovery;
● Amphibians share some important apomorphies with mammals, such
as development of limbs, lungs, a complex heart and immune
system;
● Xenopus oocyte/embryo is a robust system for drug bath, microinjec-
tion, microsurgery and tissue transplant experiments;
● FETAX is a classical protocol to identify drugs that pose potential
teratogenic hazards.
● The Xenopus egg extract system allows the analysis of a large
number of compounds and therefore is an alternative to the in
vitro assays;
● Wnt/β-catenin pathway perturbation provides straightforward and
reproducible phenotypes;
● A flow chart is presented as basis to design functional chemical
screenings aiming to find new modulators of Wnt/β-catenin signaling
pathway in Xenopus embryo.
This box summarizes key points contained in the article.
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the function of one or more gene(s), and therefore is consid-
ered complementary to the first [15]. Now, chemical interfer-
ence of embryogenesis may be performed to answer
fundamental questions of developmental biology and also as
a refinement phase of drug screening tests [16].
Although zebrafish is the pioneer model for drug screening
tests against vertebrate embryos [17–19], Xenopus laevis has a
major advantage over zebrafish. In a phylogenetic tree that
draws mammals as the most derivate vertebrate group,
amphibians branched off 90–100 million years later than fish.
Being evolutionarily closer to mammals may not be that rele-
vant when early development is the focus, but at the organo-
genesis stage, quite a few apomorphies must be taken into
account: development of limbs (amphibians, but not fish, are
tetrapods), lungs, a complex heart and an immune system
[15]. Importantly, non-tetrapods possess two copies of many
mammalian genes and such redundancy might lead to erro-
neous conclusions regarding gene function. Superior verte-
brate models for developmental biology (chick and mouse)
are not suitable for drug screening tests because of the low
quantity of their breeding and their internal development (in
utero or in ovo). The first matter makes it very difficult for one
to go through all the necessary statistical data and the second
is a barrier for microinjection and transplantation experiments.
Although tests will need to be carried out on mouse at some
point, leading investigations are more suitable to be per-
formed in lower vertebrate embryos. One should keep in
mind that in the aquatic animal epidermis, the selectivity is
distinct from that of the mammal epidermis. When performing
a bath-like treatment against Xenopus laevis (and also Danio
rerio), special care needs to be taken regarding the penetrance
of the compound into the organism. Hydrophilic compounds
are often not absorbed by the embryonic epidermis but can
be microinjected into the cytoplasm of fertilized eggs or the
blood circulation of the larvae [15]. For microinjections into
the cytoplasm of early embryos, a practical procedure should
be highlighted: one can conduct an experiment that interferes
with half of the embryo and the other half can be considered a
control condition (possible for Xenopus, but not Zebrafish)
(please see xenbase.org; zenfin.org).
2. Xenopus usage in chemical screenings
Xenopus embryogenesis has been much explored since early
1980s, when Dumont and coworkers designed a protocol named
frog embryo teratogenesis assay: Xenopus (FETAX) to identify
drugs that pose potential teratogenic hazards. They proposed
that the embryo should be treated with the drug of interest
through a bath assay for 96 hours [20], and two parameters should
be quantified and analyzed: mortality and malformation [20]. The
same features mentioned earlier as the deciding experimental
advantages for fundamental discoveries in embryonic induction
helped FETAX to become an interesting alternative to mammalian
models when testing drugs for toxicity toward embryogenesis
[21]. One of the main uses for which the FETAX approach has
provided important results is a medical one: establishing if drugs in
development can or cannot be consumed by pregnant women
[22,23]. It is worth mentioning that long after the thalidomide
tragedy, it was demonstrated that thalidomide induces limb
abnormalities during Xenopus development [24]. FETAX should
be employed to reinforce in vitro genotoxic screens and as a
bottleneck phase for tests against mammalian embryos [25].
Ecological risk assessments (ERAs) are also an important appli-
cation of FETAX; it is not uncommon for environmental contami-
nants to present a teratogenic effect, and laboratory experiments
should be conducted to determine if they could be endangering
the local fauna [21]. Although it is quite common that a study
supported by FETAX is conducted in a screening methodology, the
molecular and physiological effects (not only the embryological
effects) started to be taken into account only when the oocyte
extract system began to be explored along with chemical genetics.
Extracting the cytoplasm from Xenopus eggs or oocytes is an
old procedure, very widely used to understand microtubule
assembly, as well as its regulation through meiosis cell cycle
phases. It was useful at the time to fill the gap between in vivo
and in vitro studies of microtubule assembly [26]. The same expla-
nation applies to the ‘chemical genetics’ theme, only now foreign
small molecules play a major role. For instance, 200 out of 26,000
tested compounds were found to inhibit net actin assembly when
applied to egg extracts [27] and another 15 out of the 1,561
screened were able to destabilize microtubule arrangement [28].
Furthermore, ubistatin inhibitors were found to be capable of
arresting cell cycle progression through the use of the egg extract
system [29]. The egg extract system stands out as a powerful
system because it mimics many cellular processes in vitro and
also preserves the full machinery responsible for cell division
cycle. Those features make it possible for one to partially manip-
ulate cell cycling dynamic according to their interest.
In 2005, Wheeler’s group started to look to Xenopus laevis late
embryogenesis as a screening system for potential therapeutic
compounds [30]. The phenotypes acquired were compared to
and judged compatible with previous phenotypes found in zebra-
fish embryos exposed to the same compounds [30]. Brandli and
Wheeler ended up publishing together, in 2009, a broad review on
drug screening assays in vivo, with special attention paid to
Xenopus. Two successful papers that had been released by

Wheeler’s group in the same year guided their train of thought;
one reported a 2,990 compound screening project against the
Xenopus embryo [31] and the other highlighted the compound
that, out of those 2,990 screened, promoted the most dramatic
pigmentation phenotype. This compound is a matrix metallopro-
teinase (MMP) inhibitor, NSC 84093. Using several tools, including
Xenopus embryos, it was shown that NSC 84093’s molecular target
(s) act early in melanophore development to generate a segmen-
ted phenotype and also act slightly later to inhibit melanophore
migration into the ventral tail region. The tests were always per-
formed after gastrulation had finished (St. 15 and later), since the
pigmentation pattern begins to reveal itself only around stage 26.
The results provided have potential implications for human health
because human melanomas are known to express a number of
MMPs [32].
Interestingly, Xenopus larvae behavioral analysis can be
performed using automated system assays. Such analyses
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have been recently employed to quantify cognitive develop-
ment processes, which are often linked to the visual nervous
system [33]. This platform allows high-throughput assays and
will certainly increase reliance on experiments aiming at
potential behavior-interfering drugs. As Xenopus laevis has
been proved applicable for screening tasks, it is only natural
that systems employed to answer fundamental questions of
developmental biology came to be explored with ‘chemical
genetics.’ Below we present a promising new system: ‘the
Xenopus embryo body axis perturbation system’.
3. Xenopus body axis perturbation as a system for
drug screening: targeting Wnt/β-catenin signaling
In respect to early embryonic development, it is essential to
take into account that any morphological transformation relies
on a complex fine-orchestrated molecular arrangement.
Perhaps the biggest transformation of all for a vertebrate
embryo such as Xenopus, is passing through gastrulation/neur-
ulation to assume a bilaterian body plan. In addition, if the
revelation of lateral symmetry comes as a startling feature,
gastrulation tells a story of asymmetric migratory behavior
when specifying primary axes: the dorsal marginal zone
holds all the migratory and inductive power to trigger the
formation of the head and dorsal structures, such as the
nervous system, whereas the lateral and ventral marginal
zones come to fill in posterior (tail) and ventral positions
such as blood in a coordinated migratory pattern [34,35].
Nowadays, it is widely considered that this asymmetric
behavior, described first by Spemann and Mangold, is a nat-
ural consequence of a much earlier (molecular) asymmetry: β-
catenin accumulation only at the side opposite to sperm entry.
Wnt/β-catenin pathway activation at this moment is com-
monly known as the maternal wave and, although temporally
distant from gastrulation, its spatial range is fundamental to
specifying the dorsal dominium range as well (Figure 1(A)).
Interestingly, the mid-blastula transition coincides with a turn-
around in the Wnt/β-catenin pathway story [36]. As the
Spemann organizer becomes palpable, a plethora of Wnt
antagonists begin to be expressed right on the prospective
head mesendoderm and Wnt8 starts to be expressed through-
out the lateral and ventral marginal zones (Figure 1(A)). This
EXPERT OPINION ON DRUG DISCOVERY 3
new pattern is called Wnt/β-catenin zygotic wave and pro-
vides a fine-tuned gradient responsible for regulating anterior-
posterior equilibrium within the dorsal domain; Wnt/β-catenin
activation is necessary for the formation of posterior struc-
tures, while its inhibition allows the formation of the embryo-
nic head [37].
Quite a few signaling pathways impact the molecular equili-
brium arranged during early Xenopus embryogenesis, but the Wnt/
β-catenin pathway is the one that when disturbed entails the most
straightforward and reproducible phenotypes. With regard to the
maternal wave, enlarging or shrinking the area of β-catenin accu-
mulation in the early embryo results in a spatially disturbed
Spemann organizer and therefore the development of a dorsalized
or ventralized embryo, respectively (Figure 1(C,D)) [38,39]. Also
with regard to the maternal wave, if a pathway activator such as
Wnt8 or β-catenin is carefully injected right on the ventral side of a
4-cell embryo, an ectopic Spemann organizer will emerge and the
formation of a secondary axis will be induced (Figure 1(B)) [7]. For
the zygotic wave perturbations, a detuned gradient will be trans-
lated in an anteriorized or posteriorized embryo (Figure 1(E,F)) [41].
A secure methodology to test new drugs is challenging
these classical phenotypes. For instance, one could choose to
upregulate or downregulate the maternal wave with a known
modulator of the Wnt/β-catenin pathway and then add an
unknown molecule to determine any change or reversibility
of the phenotype. Alternatively, the methodology could chal-
lenge the formation of a secondary axis by adding a drug that
has been identified as inhibiting the Wnt/β-catenin pathway
(Figure 1) [41–43].
The reverse train of thought should be explored as well;
growing embryos could be treated with the drug of interest in
one condition and with the drug of interest plus a known
modulator of the pathway in another condition. The pheno-
types scored in the first and second conditions, regardless of
the assay, will respectively show the penetrance of the drug
into the embryo and also its specificity toward the Wnt/β-
catenin pathway.
The inhibitors XAV939, IWR-1, WntC59, and the activator
BIO are examples of chemical compounds designed over the
last few years to specifically modulate the Wnt/β-catenin sig-
naling pathway. Since their effectiveness has been evaluated
in embryonic systems [44], they have become very much used
as positive controls for the kinds of assays listed above.
Another important example is lithium chloride (LiCl), which
has become a solid interfering drug because it is capable of
causing dorsal domain expansion [45]. Later, LiCl was found to
specifically target GSK-3 [38], an important component of the
β-catenin destruction complex, which must be disrupted at
the dorsal pole for β-catenin accumulation to occur [46].
Importantly, the dorso-anteriorized embryonic phenotypes
induced by LiCl can be qualitatively and quantitatively scored
in a dorsal anterior index (DAI) [47]. One could challenge these
DAIs induced by LiCl with a given set of compounds as a
chemical screening strategy [41,42].
Taking advantage of the body axes perturbation system,
Thorne and coworkers were able to identify pyrvinium and 3,6-
dihydroxyflavone as robust modulators of the Wnt/β-catenin
pathway [48]. This, along with deeper investigations in which
they specified pyrvinium targets [49], represented a
 4 L. A. MAIA ET AL.
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Figure 1. (A-F) Possible phenotypes obtained with the manipulation of the Wnt/β-catenin pathway at different stages of the development of Xenopus laevis. Red
represents the Wnt signaling during the maternal wave of Xenopus development and Purple represents the Wnt/β-catenin signaling in the zygotic wave. The
interference in the Wnt/β-catenin pathway occurs through injection or bathing with Wnt inhibitors or activators. The resulting phenotypes are found in the last
column. Detailed explanation of the figure is presented in the text. V, ventral; D, dorsal; A, anterior; P, posterior; DAI, dorsal anterior index; LiCl, lithium chloride. Full
color available online.

refinement phase of a huge screening task. By monitoring β-
catenin and axin degradation in egg extracts treated with a
diversity of compounds taken from three different libraries
(ChemBridge/ChemDiv, NINDS bioactives, and Natural pro-
duct), they were able to perform a high-throughput and
highly reliable assay. The initial 8,500 compounds were
narrowed down to 80 potential modulators, and pyrvinium
turned out to be the most potent inhibitor of the Wnt/β-
catenin pathway among these 80 [48].
It was not until 2012 that the body axis perturbation sys-
tem began to be fully explored for chemical genetics analysis,
when our group started to investigate the effects of natural
 EXPERT OPINION ON DRUG DISCOVERY 5

compounds on Xenopus embryogenesis. Two flavonoids (quer- 4. Conclusion

cetin and rutin) were chosen to be assessed through a combina-
We present a brief history of how the Xenopus oocyte/embryo
tion of approaches connected to axis establishment in Xenopus
has come to occupy an important place within the theme of
laevis development. Quercetin, but not rutin, induced axial
‘drug discovery’ and how along the way, it filled the gaps
defects as well as inhibited a Wnt8-specific reporter, and it also
between the narrow-down phase, which usually employs cell
inhibited secondary axis formation induced by Wnt8 [41]. In
culture reporter assays, and the clinical phase. The egg extract
2014, we showed that the flavonoid isoquercitrin is a Wnt inhi-
system allows the analysis of a large number of compounds
bitor through microinjections into Xenopus embryos, followed by
and therefore is an alternative to the in vitro assay. We want to
histological analyses and gene expression of the anterior region
focus, though, on the ‘Xenopus axes perturbation system’ and
of the embryo. It was shown that isoquercitrin interference with
its reliability when searching for compounds that target the
Xenopus development had the power to cause microcephalic
Wnt/β-catenin signaling pathway.
and headless embryo phenotypes and that these were linked
A flowchart to test possible modulators of Wnt/β-catenin is
to the disturbance of the expression of anterior markers.
presented in Figure 2. Starting from a set of compounds, one
Furthermore, isoquercitrin inhibited ectopic secondary-axis for-
should first perform luciferase reporter assays, which allow
mation and rescued lithium chloride-dorsalized embryos [42].
high-throughput screening. This first analysis will narrow
More recently, derricin and derricidin (from the chalcone
down the candidates and identify the best hits (Figure 2(A)).
subclass) were shown to be capable of inhibiting double axis
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Next, the elected compounds must be challenged through a

formation in Xenopus embryos and suppressing Wnt/β-catenin
battery of Xenopus analyses: gene reporter assay, double axis
luciferase reporter activity in vivo. These results agreed with
assay, animal cap assay, ectopic expression and anterior mar-
previous cell culture experiments that identified these chal-
ker analyses (Figure 2(B)). As an outcome, the researcher will
cones as novel negative modulators of the Wnt/β-catenin
be able to measure the drug penetrance toward an organism
pathway and of colon cancer cell growth in vitro [43].
and gain insights into where in the pathway each compound

Figure 2. Flowchart to test possible modulators of Wnt/β-catenin using Xenopus. (A) The drug screening begins with in vitro assays using specific reporter lines for
the Wnt/β-catenin pathway. (B) Xenopus laevis embryos are used for in vivo assays. These assays are well characterized in the Xenopus model and are specific for the
Wnt/β-catenin pathway analysis, providing known and expected phenotypes according to the inhibition or activation of the pathway compounds. (C) Toxicological
test performed using FETAX protocols before moving to clinical phases.
 6 L. A. MAIA ET AL.
is acting. Furthermore, we propose a novel role for the FETAX
protocol: taking advantage of its bona fide toxic/teratogenic
contributions for several commercial drugs, we recommend
that as an additional control phase before moving into clinical
phases (Figure 2(C)).
5. Expert opinion
In light of the revolution in genetics in the past century,
Xenopus has played a unique role as a firm life table after
the discovery of DNA permanently merged embryology, cell
and molecular biology and evolution into a highly turbulent
field. This is due to the vast set of approaches that have been
emerging, making Xenopus an adaptable model that is
responsive to a very solid and purely embryological para-
meter: phenotype scoring.
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Here, we have presented studies that allow us to attest that
Xenopus laevis is a robust model that can be applied in screen-
ing projects. Although the cell culture system may be seen as
a mandatory phase for narrowing down in screening projects,
the oocyte extract system might be much more useful if
experiments are likely to be conducted against an embryolo-
gical system at a later point. This is due to the preservation of
the statistical force and the closer physiological similarity with
the next (refinement) phase, thereby reducing the chances of
a contradiction arising between the ‘narrow down phase’ and
the ‘embryological phase’ if the culture cell system is replaced
by the oocyte extract system.
As for the embryological phase, the two systems must be
clearly distinguished: ‘axis establishment system’ and ‘organo-
genesis system.’ The second has been fully explored, mainly by
Wheeler and Brandli, to answer physiological questions and
toxicity issues, such as imperfections in kidney formation and
angiogenesis. The ‘body axis perturbation’ system, on the other
hand, has already contributed enormously to fundamental ques-
tions of developmental biology and we have been putting a lot
of effort toward this system to solidify it as a screening approach.
We strongly recommend the flowchart presented here as a basis
to design functional chemical screenings that aim to find new
modulators of the Wnt/β-catenin signaling pathway in the
Xenopus embryo. Following this approach, we have identified
novel inhibitors and their mechanisms of action. However,
further preclinical and clinical testing remains to be performed.
Acknowledgments
The authors gratefully acknowledge S Rodrigues for their frog technical
support.
Funding
This work was supported by Conselho Nacional de Desenvolvimento
Científico e Tecnológico [Grant No. 462073/2014-9] from the Ministério
da Ciência, Tecnologia e Inovação, CNPQ, Fundação CAPES and Fundação
Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro
[FAPERJ Grant No. #E-26/202.964/2015].
Declaration of interest
Lorena Agostini Maia is working on her PhD via the Graduate Program in
Morphological Sciences at the Federal University of Rio de Janeiro and is
supported by a CNPq fellowship while Ian Randolph Velloso is a master’s
student of the Graduate Program in Morphological Sciences at the Federal
University of Rio de Janeiro and is supported by a CAPES fellowship. The
authors have no other relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manuscript apart
from those disclosed.
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