Embryonic Explants from Xenopus
laevis as an Assay System to Study
Differentiation of Multipotent
|FIGURE 1 Schematic representation of the "Umhfillungsversuch"
(coating test) and "Auflagerungsversuch" (bedding test) by
Johannes Holtfreter, 1933. (a) Two pieces of ectoderm were isolated
using a self-made cutting tool (eyebrow). (b) The mortified implant
was placed between ectodermal tissues. (c) A small piece of animal
ectoderm of a gastrula stage embryo was excised (c) and placed on
the mortified sample (d). Adapted from Spemann, 1936.
A mayor question in developmental biology is how
cells or groups of cells are committed to their distinct
fate during the elaboration of the body plan. An experimental
system, which has been used to investigate
the nature of inductive signals and therefore has led to
the identification of components and interactions of
various signal transduction pathways, is the so-called
animal cap explant system of amphibian embryos. The
animal cap consists of the ectodermal roof of a blastula
stage embryo that can be kept in culture under the simplest
conditions. An early use of this explant system
was described by Johannes Holtfreter in an attempt to
investigate the nature of inductive signals. In 1933, he
established experimental tissue combination systems,
referred to as "Umhfillungsversuch" (coating test) and
"Auflagerungsversuch" (bedding test), to study the
influence of factors emanating from mortified tissues
on explanted ectoderm (Fig. 1) (Holtfreter, 1933).
Holtfreter showed that the mortified dorsal lip of a
gastrula stage amphibian embryo led to the induction
of neural tissue (neurale Blasen) in conjugated
ectodermal explants (Fig. lb). In 1971, Sudarwati
and Nieuwkoop demonstrated that vegetal tissue is
capable of inducing mesoderm in combined animal
cap explants using the South African clawed frog Xenopus laevis
as a source for animal cap explants
(mesoderm induction assay). More recently, the animal
cap system has been utilized to tackle a broad variety
of developmental problems, including the specification
of all germ layers, organ formation, and expression
screenings for secreted proteins (Sudarwati and
Nieuwkoop, 1971; Gurdon et al.
, 1985; Slack, 1990;
Smith et al.
, 1990; Green et al.
, 1992; Moriya et al.
II. MATERIALS AND
Adult X. laevis
frogs are from NASCO (Wisconsin).
The reagents used are agarose (Cat. No. 15510-027,
GibcoBRL); albumin bovine (BSA, Cat. No. A-8806,
Sigma); chorionic gonadotropin (HCG, Cat. No. CG-
10, Sigma); L-15 (Leibovitz, GibcoBRL); L-cysteine
hydrochloride monohydrate (Cat. No. 30129, Fluka);
HEPES (Cat. No. H-3375, Sigma); and penicillin/
streptomycin solution (with 10,000 units penicillin and
10mg streptomycin/ml, Cat. No. P-0781, Sigma). All
other chemicals used are from Merck.
The gastromaster and replacement microsurgery
tips are from XENOTEK Engineering (Belleville, IL).
The microinjector for RNA injection is from Eppendorf
(Microinjector 5242, Eppendorf).
III. BASIC PROCEDURES
The basic principles of the procedure have been
described previously (Grunz and Tacke, 1989; Hollemann et al.
, 1998). In addition to an update of the
basic protocol, this article describes several readout
systems that can be applied.
Modified Barth's solution (MBS)
2.4 mM NaHCO3
, 1.0 mM
KCI, 10 mM
, and 0.33mM
, pH 7.4
NaCl, 2.4mM NaHCO3
KCl, and 10mM
HEPES, pH 7.4
: 65% L-15 and 0.1% BSA, pH 7.4
PCR mix (one reaction; 25/µl)
: 2.5µl 10× polymerase
chain reaction (PCR) buffer containing 15mM
(Roche), 0.2µl 25mM
0.375 µl 10 µM
forward primer, 0.375 µl 10 µM
primer, 21.425 µl PCR grade H2
O and 0.125 µl Ampli
Taq DNA polymerase (5U/µl) (Roche)
Histone H4 primer
: H4-F: 5'-CGGGATAACATTC
AGGGTATCACT-3' and H4-R: 5'-ATCCATGGCG
RT mix (one reaction; 5µl)
: 1 µl 25 mM
0.5 µl 10× PCR buffer II, 2 µl 2.5 mM
dNTPs, 0.25 µl
RNase inhibitor (20U/µl), 0.25µl MuLV reverse
transcriptase (RT) (50U/µl), 0.25µl 50µM
hexamers, and 0.75µl (35 ng) RNA sample
PCR(RT) mix (one reaction; 20ml)
: 0.5 ml 25 mM
solution, 2ml 10× PCR buffer II, 16.625 ml RNasefree
O, 0.375ml 10mM
forward primer, 0.375 ml
reverse primer, and 0.125 ml Ampli Taq
polymerase (5 U/ml)
A. Preparation of Animal Cap Explants
At the late blastula stage (stage 8.5-9), Xenopus
embryos show a relatively defined segregation of presumptive
ectoderm (animal pole), mesoderm (equatorial
area), and endoderm (vegetal pole); the blastocoel
has attained its full size, and the inner surface of the
blastocoel becomes smooth. The roof of the blastocoel,
the animal cap (very top of the animal pole), consists
of a single outer layer and two inner layers of cells. The
pigmentation pattern and the cell size differences
make it possible to roughly distinguish the presumptive
ectoderm, mesoderm, and endoderm from outside
of the embryo. However, the easiest way to identify the
animal pole (animal cap) is to look at the embryos
directly. What you see from the top is the animal pole,
as the embryos are naturally floating inside the
vitelline membrane with the animal pole up and the
vegetal pole down due to gravity.
Preparation of Xenopus Embryos
Eggs from adult Xenopus
are obtained from
females 6-8h after injection with human chorionic
gonadotropin (500-1000 U/frog) and fertilized with
minced testis in 0.1× MBS. Forty minutes after fertilization,
dejelly the embryos in 2% cysteine (pH 8.0) and
wash extensively in 0.1× MBS.
Isolation (Cutting) of Animal Cap Explants
As there is no marker to clearly delineate the
boundary of presumptive ectoderm and mesoderm in
a living embryo, it is difficult to isolate animal caps
without presumptive mesodermal cell contamination.
Many tools, including forceps, hair loops, fine glass
needles, and tungsten needles, have been developed
for cutting animal caps. The most advanced tool is
the gastromaster. No matter which tool is used, the
first step is to manually remove the vitelline membrane
of stage 8.5-9 embryos (the best stage for
capping) with a pair of watchmaker's forceps in a
petri dish coated with 1% agarose. Although the
manufacturer of the gastromaster demonstrates that it
is possible to directly isolate animal caps without
removing the vitelline membrane (see video clips at
http://www.gastromaster.com/), we strongly recommend
removing the membrane manually. The forceps
do not have to be very sharp, but the two tips should
be well matching. Digging a small pit into the surface
of the coated agarose is helpful for fixing the embryo
during manipulation. It is recommended to hold and
remove the vitelline membrane with forceps from the
equatorial area or from the vegetal pole, thus avoiding
damage to the animal pole cells. Once the embryos are
released from the vitelline membrane, they can no longer rotate freely according to gravity. Therefore,
in order to isolate a clean animal cap from the right
animal pole region, it is extremely important to properly
orientate the nude embryo animal pole up and
vegetal pole down (Fig. 2a). Dissect the animal cap
(very top region of the animal pole, illustrated by the
dashed circle in Fig. 2a) in 0.5× MBS using a
gastromaster equipped with a yellow microsurgery tip
of 400-500 mm in width (Figs. 2b-2e). This step is quite
easy compared to the removal of vitelline membrane.
We encourage visiting the gastromaster manufacturer's
website for video clips that nicely demonstrate
the dissecting process. Culture animal caps in 0.5× MBS containing penicillin (100U/ml) and streptomycin
(100µg/ml) and harvest until control siblings
have reached desired developmental stages for further
|FIGURE 2 Cutting an animal cap with the gastromaster. (a) Animal pole view of a demembranated stage
9 Xenopus embryo. The dashed circle demarcates the very top region of the animal pole (the animal cap
or presumptive ectoderm). (b) An animal cap freshly dissected with the gastromaster, still sitting on the
mother embryo. (c) Wound after the isolation of the animal cap. (d) Outer layer view of an isolated animal
cap. (e) Inner layer view of an isolated animal cap. Bars: 100µm.
B. Manipulation of Animal Cap Cells
RNA Injection Techniques
One approach to investigate the activity of individual
proteins is to introduce the corresponding mRNAs
into the animal pole of early cleavage stage embryos
followed by the isolation of animal caps for further
analysis. Depending on the gene of interest, 5-2000 pg,
e.g., FGF or XCYP26 (Isaacs et al.
, 1992; Hollemann et al.
, 1998), of in vitro-synthesized capped mRNA
(Mega Message Machine, Ambion, USA) in 10nl
RNase-free water can be microinjected into the animal
pole of both blastomeres of two-cell stage embryos,
which are transferred into l× MBS and kept at 13°C to
slow down development during injection. Two hours
after injection, culture embryos again in 0.1× MBS.
Application of Growth Factors
An alternative approach to monitoring the inductive
activities of growth factors is to directly apply the
active form of a given growth factor protein to the
isolated animal caps with or without dissociation.
The advantage of dissociation is that each cell is
exposed to a uniform factor concentration.
Dissect animal caps from uninjected embryos as
described earlier and keep in l× Ca2+
in petri dishes coated with 0.7% agarose (about 50
caps per 60-mm petri dish). All the solutions used
during disaggregation and reaggregation include
penicillin (100 U/ml) and streptomycin (100µg/ml).
After approximately 15min, cells start to dissociate.
Mechanical sucking and blowing of the caps with a
yellow pipette tip facilitates the disaggregation process.
Caution should be taken to avoid damaging
the cells. When the caps are completely disaggregated,
replace the Ca2+
-free MBS first with l× MBS and then with the L-15/BSA solution. After the treatment,
wash and reaggregate cells in l× MBS by gentle
shaking of the petri dish. To reaggregate the cells,
remove most of the solution from the petri dish with
a vacuum pump and then swirl the cells together to
the center of the dish by applying l× MBS from one
edge of the dish with the special apparatus invented
by Horst Grunz that can supply continuous flow over
a regulated tube connected to a glass bottle (for details
of the apparatus, please visit Horst Grunz's website at
aggregation can be done by shaking the petri
dish gently. After one hour, cut the reaggregated cell
mass (cake-like) into several pieces with a glass needle
and transfer to sterilized larger glass culture bottles
(100ml) coated with 0.7% agarose. Freeze the tissues
with liquid nitrogen when control siblings have developed
to the desired stage of development.
C. Analysis (Readout Systems)
Effects on gene transcription can be analyzed by RTPCR
and whole mount in situ hybridization.
The animal cap system can be used to elucidate
which factors and signal transduction pathways regulate
genes of interest or, vice-versa, which genes are
downstream targets. Effects on gene transcription in
explants can be analysed either by RNase protection or
RT-PCR. We use a nonradioactive RT-PCR assay. It is
normally sufficient to use this simple, semiquantitative
approach to analyse strong effects on gene expression.
A variety of other RT-PCR methods have also been
described for Xenopus: quantitative, radioactive RTPCR
(Rupp and Weintraub, 1991) and quantitative,
competitive RT-PCR (Onate et al.
, 1992). The most
sensitive and most reproducible technique based on
fluorescence kinetics is quantitative real-time RT-PCR
(Xanthos et al.
, 2002), but it requires a special
RNA Isolation from Animal Cap Explants
|FIGURE 3 Induction of neuronal gene expression in animal caps
as analyzed by RT-PCR. One hundred picograms X-Ngnr-1 mRNA
(Ngn) or 300 pg Notch ICD mRNA (NICD) was injected in both cells
of two-cell stage embryos, animal caps were dissected at early stage
9, cultured to stage 15, and then analyzed by RT-PCR. RNA isolated
from uninjected total embryos of stage 15 was used as a positive
control. X-Ngnr-1 induces the neuronal differentiation marker
N-tubulin, as well as the Notch target gene ESR7 in animal caps.
Histone H4-specific primers were used to control RNA input and
quality. As shown by the absence of transcripts for cardiac actin, the
isolated explants were not contaminated by mesodermal tissue
(S61ter, et al., unpublished results).
We use the RNeasy minikit (Qiagen) to isolate RNA
from animal caps and whole embryos for RT-PCR
analysis following the RNeasy miniprotocol for the
isolation of total RNA from animal tissues (see manufacturer's
instructions with modifications detailed
later). Digest genomic DNA using DNase I (Qiagen)
for on column treatment according to the manufacturer's
Collect 10-12 animal cap explants (or 5 embryos)
per sample at the appropriate time in a microcentrifuge tube, remove excess buffer, and freeze animal
cap explants (embryos) with liquid nitrogen for
storage at -80°C. For RNA isolation, add 350 µl (600 µl)
buffer RLT/βME to each tube directly on the frozen
sample. Carry out all further steps at room temperature.
Homogenize animal cap explants (embryos)
immediately by passing the lysate five times through
a 24-gauge needle fitted to an RNase-free syringe.
Elute RNA by 30 µl (70 µl) RNase-free water (60°C) and
place on ice. After measuring the RNA concentration,
adjust all samples to the same concentration with
RNase-free water and store at -80°C. Control RNA
integrity by use of a 1.5% TBE agarose gel; 75-100ng
of total RNA is normally obtained from a single animal
Analysis of Genomic DNA Contamination
The exon-intron structure of most Xenopus genes is
unknown. Therefore, primers used for the analysis that
can target the same exon and genomic DNA contamination
of isolated RNA would thus result in falsepositive
signals. In order to control for genomic DNA
contamination, a regular PCR reaction for histone H4
genomic DNA is performed. Add 1 µl RNA (50ng/µl)
to 25µl PCR mix containing the histone H4-specific
primer. Use genomic DNA (5 ng) as a positive control
O as a negative control, with the following
cycling parameters: 94°C 2 min (94°C 45 s, 58°C 45 s,
72°C 30s) × 35, 72°C 5 min. Analyze one-half of the
PCR on a 2% TBE agarose gel.
|FIGURE 4 Induction of endodermal gene expression in
cap explants as analysed by whole mount in situ
Expression analysis of the activation of an
animal cap explants. Cy118 encodes a
peptidase that demarcates the developing embryonic
intestine. VegT (500pg/embryo) and β-catenin
mRNAs were injected into the animal
pole of both blastomeres of
two-cell stage embryos.
Animal caps were isolated from stage 9
and VegT/β-catenin-injected embryos and
control siblings had reached stage 34. (a) Cy118
expression is activated in a subset of cells of an elongated
cap injected with VegT/β-catenin. (b) Cy118 is
not expressed in the
control cap. (b) Cy118 is expressed
exclusively in the presumptive
intestine of stage 34
embryos (Chen et al., 2003).
Carry out the reverse transcriptase reaction in 5-µl
reactions (or a corresponding multiple of 5µl) using
the Gene Amp RNA PCR core kit (Roche) according
to the manufacturer's instructions in a TRIO Thermoblock
(Biometra). Use total RNA isolated from
whole embryos collected at corresponding developmental
stages and diluted to the same concentration as
the animal cap explant RNA preparations as a positive
control. As a negative control, perform the same reaction
without adding reverse transcriptase (H2
for the RT reaction are 22°C 10min, 42°C 30 min, 99°C 5 min, 5°C 5 min. Mix a 5-µl aliquot of the
RT reaction with 20~tl PCR(RT) mix. We use the following
cycling parameters: 94°C 2min (94°C 45s, X°C 45 s, 72°C 45 s) × Y cycles, 72°C 1 min. Analyze 10µl of
each RT-PCR on a 2% TBE agarose gel. The annealing
) and cycle number (Y
) have to be
optimized empirically for each primer pair. For that
purpose, remove aliquots from a RT-PCR reaction
using cDNA made from whole embryo RNA every
second cycle over a 12-cycle range, usually starting at 20 cycles. The optimal cycle number is within the
linear range of product accumulation. To control RNA
quality, we perform histone H4-specific PCR; X = 58°C Y
= 24 cycles. Control contamination of animal cap
explants by mesodermal tissue with primers for
brachyury (early ACs) and muscle actin (later ACs).
Sequences for these and other primers are available
Whole Mount in Situ Hybridization
Gene expression on the RNA level in animal cap
explants can also be analyzed by whole mount in situ
hybridization, originally adapted for Xenopus
by Harland (1991). The advantage of this method in
comparison to RT-PCR is that individual explants can
be controlled for homogeneity of gene expression on
the level of individual cells. The disadvantage is that
substantial numbers of explants are required if several
genes are to be analyzed in parallel. We use the basic
protocol for whole mount in situ
described previously (Hollemann et al.
, 1999) with
the following minor modifications. In case of several
samples, a transfer of fixed animal caps from glass
vials to 24-well tissue culture plates before rehydration
facilitates handling. Proteinase K treatment should be
shortened to 5-8 min at room temperature.
RT-PCR Analysis of Animal Cap Explants
Many different genes are involved in the activation
of the neuronal differentiation program, which drives
naive ectodermal cells to become postmitotic neurons.
In animal cap explants, X-ngnr-1 can also promote
neurogenesis, as monitored by the activation of the
neural determination marker N-tubulin. The density
of cells expressing endogenous X-ngnr-1 is controlled
by lateral inhibition mediated through Notch signalling
(Ma et al.
, 1996). In response to misexpression
of the intracellular domain of Notch (ICD) that mimics
active Notch signalling, the expression of target genes,
such as Esr7, is induced (Fig. 3).
Whole Mount in Situ Hybridization in Animal
|FIGURE 5 Elongation of animal caps mimics gastrulation
Animal cap explants of Xenopus laevis were
isolated at stage
8 and treated with activin for 2h. Treated
caps elongate until stage
18 (left). Arrows indicate
examples for elongation of the caps. In this
animal caps shown have indications of elongation.
Untreated control caps remain spherical (right).
In addition to neural and mesodermal tissue,
animal cap explants can also be converted into endoderm.
For instance, VegT can synergize with β-catenin
to induce a number of liver- and intestine-specific
genes in animal cap explants. Figure 4 shows an
example of whole mount in situ hybridization analysis
for the induction of an endoderm marker gene in an
animal cap upon VegT/β-catenin injection.
The Animal Cap to Study Cell Migration
Upon treatment with activin or FGF, the animal cap
is not only induced to become mesoderm (see above),
but as a consequence of mesoderm induction, the
animal cap also changes its form (Fig. 5). Whereas
untreated animal caps round up and look like spherical
balls, induced animal caps elongate as a consequence
of cell movements within the animal cap (Kuhl et al.
, 2001). It is widely accepted that this kind of cell
movement within the cap mimics mesodermal cell
movements normally occurring during gastrulation of
the Xenopus embryo. During gastrulation, cells from
a more ventral position migrate towards the dorsal
midline. The forces generated by this process in vitro
not only push cells during gastrulation over the dorsal
lip, but also lead to an elongation of the embryo along
its anterior-posterior axis (for a review on cell movements
during gastrulation, see Keller, 1986). With
respect to gastrulation movements, the animal cap
thus serves as an excellent system to study the molecular
basis of cell migration. The elongation behaviour
of injected or growth factor-treated caps can be studied
and conclusions can be drawn on the function of a gene of interest with respect to cell migration. As an
example, we show the effect of activin on animal caps
Recapitulation of Organ Formation in Animal Caps
a. Cement Gland Formation in Whole Animal Cap
|FIGURE 6 Cement gland formation in animal cap explants. (a)
Anterior to the left, sloped lateral view of a head of a noninjected
Xenopus larvae at NF stage 40. The cement gland is positioned
immediately adjacent to the mouth opening. (b) Example
an animal cap explant that had been injected with Xpitxl into
cell at the two-cell stage and cultured equivalent to stage 40.
50µm. cg, cement gland; cgl, cement gland like; ey; eye; mo,
mouth opening; np, nasal pit.
. The homeobox-containing transcription
factor PITX1 is expressed during all stages of cement
gland formation (Hollemann and Pieler, 1999). The
cement gland of young amphibian tadpoles is a specialised
transient organ, which arises from the outer
ectodermal layer (Fig. 6a). The function of the gland is
to secrete sticky mucus that helps larvae adhere to
solid surfaces, preventing larvae from being carried
along by the water flow. To investigate the question if Xpitxl
alone is able to induce cement gland formation,
100pg of the corresponding synthetic messenger RNA
was injected into one cell of a two-cell stage embryo.
At the equivalent of embryonic stage 8, the animal cap
was excised with the help of a gastromaster (yellow tip
with 400µm) and cultured in 0.5× MBS+P/S until
nontreated siblings had reached stage 40. Whereas
noninjected animal caps formed so-called artificial epidermis,
a high number of animal caps injected with
Xpitxl developed groups of cells, which could be identified
easily as typical cement gland cells based on
their morphology (Fig. 6b).
b. Notochord from Dispersed Animal Cap Explant
. In response to different concentrations of
activin, dispersed animal cap cells have been shown to
exhibit sharp thresholds in respect to target gene activation.
Activin A is added to the cells at a final concentration
of 4 ng/ml for notochord differentiation and 8 ng/ml for endoderm differentiation and incubated at
room temperature for 1 h
. In most cases, more than
90% of the reaggregated tissue mass from each batch
of preparation differentiated into notochords. Some
neural cells were formed concomitantly.
c. Inductive Processes
|FIGURE 7 Histological staining of notochord tissues generated
from animal cap cells treated with activin. In order to judge the
inductive activities, one piece of the tissues from each batch of
was cultured further and finally fixed in Bouin's
when control embryos reached stage 42.
Differentiation of the tissues
is assayed by histological
analysis, nt, notochord cells; n, neural cells.
: A Proteomics Approach.
The animal cap assay has widely been used to study the inductive properties of growth factors such as
activin or FGE Molecular analysis of inductive events
in general was on the level of mRNA expression.
Changes in gene expression were analyzed by Northern
blot or RT-PCR studies. This procedure resulted
in the identification of a plethora of early and late
response genes in the course of mesoderm or neural
induction. However, posttranscriptional and posttranslational
aspects of cellular regulation in this
context (like protein translation, protein phosphorylation,
and differential splicing events) were neglected.
Two-dimensional (2D) protein analysis in conjunction
with MALDI-TOF-based identification of proteins will
be a useful tool to explore these processes (Shevchenko et al.
, 1996). Figure 8 provides a 2D protein gel example
of activin-treated animal caps in comparison to
untreated animal cap explants, indicating that this
rationale can be applied on the animal cap system in Xenopus
. Thus, it can be expected that embryonic
inductive processes will also be analyzed on the level
of the proteome in the near future.
|FIGURE 8 Two-dimensional gel analysis of proteins expressed in untreated and activin-treated animal
caps. Embryos were injected at the two-cell stage with activin mRNA. Animal caps were isolated at stage 8
and cultured until stage 19. Equivalents of four animal caps were used for 2D protein gels. Circles indicate
spots of different intensity upon treatment. Whereas spots I to 3 most likely represent proteins that are upregulated
through activin treatment, spots 4 and 5 indicate two spots that are weakly affected and might also
represent artefacts. Two-dimensional gels with a broad IP range were used. For a more detailed analysis of
inductive processes, small range IP gels are required to increase the resolution. See detailed literature on 2D
gel analysis for methods and detailed interpretation of the 2D gel. (Shevchenko et al., 1996).
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