Wellcome Trust/Cancer Research UK, Gurdon Institute of Cancer and Developmental Biology and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
* Author for correspondence (e-mail: jim{at}gurdon.cam.ac.uk)
Accepted 23 June 2004
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SUMMARY |
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Key words: Xenopus, Mesoderm induction, TGFß family, Activin
![]() |
Introduction |
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The role of activin in the early embryo has, however, remained unclear
because other candidate inducing factors have been isolated, including
Vg1 (Thomsen and Melton,
1993; Weeks and Melton,
1987
), the nodal-related genes
(Jones et al., 1995
;
Joseph and Melton, 1997
;
Onuma et al., 2002
;
Takahashi et al., 2000
) and
Derrière (Sun et al.,
1999
; White et al.,
2002
), and because attempts to inhibit its function in a specific
manner have produced contradictory results. It is not clear, for example,
whether the activin-binding protein follistatin does
(Marchant et al., 1998
) or
does not (Schulte-Merker et al.,
1994
) inhibit mesoderm formation. The most recent view on the role
of activin in early Xenopus development has been articulated by
Green, who says `although activin was and still is an excellent model for
morphogen action, it may not be important in early vertebrate patterning'
(Green, 2002
).
In this paper, we first reinvestigate the temporal expression pattern of
activin B and demonstrate that zygotic expression of activin B
precedes that of one of its putative target genes, Xbra
(Smith et al., 1991). We then
use antisense morpholino oligonucleotides to inhibit the function of activin B
in the early Xenopus embryo. Our results indicate that activin B is
required for normal mesoderm formation in Xenopus in a
concentration-dependent manner. We also demonstrate, serendipitously, that in
performing antisense experiments of this sort in Xenopus one must
beware of polymorphisms in the 5' untranslated region of the gene(s) of
interest.
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Materials and methods |
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Antisense morpholino oligonucleotides
These were purchased from GeneTools. Sequences were as follows: MO1,
5'-GCAGAGGCAGTAACAGGAGAGCCAT-3'; mMO1,
5'-GCAGACGCACTAACATGAGAACCAT-3'; MO2,
5'-CCCGGCGAGGGTCTCCGAGCGGAAA-3'; MO3;
5'-CGAGGGTCTCCAAGCGGAGAGAGAA-3'.
The gross phenotypes obtained with these antisense morpholino oligonucleotides, described in Figs 3A-C and 6A-D, were observed in experiments involving at least 100 embryos for each morpholino.
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Whole-mount antibody staining
Whole-mount antibody staining using the monoclonal antibodies MZ15
(Smith and Watt, 1985) and
12/101 (Kintner and Brockes,
1984
), specific for notochord and muscle respectively, was carried
out as described (Smith,
1993
).
Whole-mount in situ hybridisation
In situ hybridisation was carried out essentially as described
(Harland, 1994), except that
BM purple was used as a substrate. Probes used were for Chordin
(Sasai et al., 1994
),
Derrière (Sun et al.,
1999
), Goosecoid (Cho
et al., 1991
), Xbra
(Smith et al., 1991
),
Xnot (von Dassow et al.,
1993
), Xnr2 (Jones et
al., 1995
), Xvent-1
(Gawantka et al., 1995
) and
Xwnt-8 (Christian et al.,
1991
; Smith and Harland,
1991
). The open reading frame of Xnr2 was amplified using the
polymerase chain reaction and the primers
5'-TCTGAATTCATGGCAAGCCTAGGAGTCATC-3' and
5'-ATTTCTAGAGTTACATCCACACTCATCCAC-3'. It was cloned into pCS2+,
linearised with EcoRI and transcribed with T7 RNA polymerase. Each
experiment shown was carried at least twice, with at least 20 embryos per
treatment.
RNA isolation and real-time RT-PCR
Total RNA was prepared from five pooled embryos using the TriPure reagent
(Roche), followed by DNAseI digestion, proteinaseK treatment,
phenol/chloroform extraction and ethanol precipitation. RNA was dissolved in
water and used as a template for real-time RT-PCR. Reactions were performed in
the LightCycler instrument (Roche) using a SYBR GreenI-based one-step RNA
amplification kit (Roche). A standard curve was generated from diluted RNA
derived from control embryos and experimental results were quantified
accordingly. PCR primers are listed in
Table 1. Each experiment was
carried out at least twice and usually three times.
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Results |
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The results that follow indicate that the mutated morpholino oligonucleotide mMO1 has no effect on Xenopus development. To confirm that the oligonucleotide is capable, given the opportunity, of inhibiting translation, we changed four nucleotides in the open reading frame of activin B to match the sequence of the mutated morpholino oligonucleotide. Misexpression of the mutated activin B was still capable of disrupting Xenopus development (Fig. 2K), and this disruption was prevented by the mutated oligonucleotide mMO1 (Fig. 2M) but not by the original version (Fig. 2L). These observations show that the mutated antisense morpholino oligonucleotide is stable and functional.
An antisense morpholino oligonucleotide directed against activin B disrupts Xenopus development in a dose-dependent fashion
Xenopus embryos were injected with increasing concentrations of
specific or mutated antisense morpholino oligonucleotide and then allowed to
develop to tadpole stage 32. The mutated oligonucleotide had no effect on
development, but the specific oligonucleotide MO1 caused severe disruption to
dorsal axial development, with both head and tail being affected
(Fig. 3A-C). Disruption to the
dorsal axis was presaged by slow passage through gastrulation (see
Fig. 5G-I and
Fig. 7H,I), and was confirmed
by in situ hybridisation, which showed that expression of Xnot
(Fig. 3D,E) persists around the
closing blastopore and that expression of chordin is more diffuse
than in control embryos (Fig.
3F,G). These observations suggest that disruption of activin B
function may cause a disruption of convergent extension (see Discussion).
|
|
To examine the concentration-dependent effects of the antisense morpholino
oligonucleotide, and to investigate its effects on early development, we
assayed the expression of a panel of genes expressed in mesoderm and
mesendoderm using RT-PCR (Fig.
3L). Dorsally expressed genes such as Goosecoid
(Cho et al., 1991),
chordin (Sasai et al.,
1994
) and Xhex (Jones
et al., 1999
; Newman et al.,
1997
) were most severely affected by low concentrations of
oligonucleotide, while expression of the pan-mesodermal marker Xbra
was reduced only by the highest concentration. These observations are
consistent with the suggestion that patterning of the mesoderm in the
Xenopus embryo occurs in response to different concentrations of
activin (Green et al.,
1992
).
In situ hybridisation confirmed that expression of Goosecoid and Xbra is decreased in embryos in which activin B function is inhibited. We note that downregulation of Goosecoid is not accompanied by a significant restriction of its expression domain but by a general decrease in levels of transcription (Fig. 4A,B), whereas downregulation of Xbra tends to occur not throughout the marginal zone but in a more restricted domain (Fig. 4C,D). This may correspond to the dorsal marginal zone, but it is also possible that it reflects the region where the concentration of MO1 is highest (Fig. 4C,D). The expression domains of other genes, including Xwnt8 and Xvent1, which are expressed laterally and ventrally, are, like that of Goosecoid, little affected by the antisense morpholino oligonucleotide (Fig. 4E-H), suggesting that the upregulation of Xvent1 observed in Fig. 6 is due to elevated levels of transcription within its normal expression domain.
|
We next attempted to `rescue' the MO1 phenotype by co-injecting the mutated
form of activin B RNA that is not affected by the antisense oligonucleotide
(see Fig. 2K-M). One difficulty
with experiments of this sort is that injected RNA diffuses less well than the
morpholino itself, so that the distribution of the two will differ
(Nutt et al., 2001;
Saka and Smith, 2004
). Another
is that activin causes a severe phenotype on its own, so that one has to
inject enough activin RNA to rescue, but not so much so as to cause defects
that are due to overexpression. In our attempts to address these concerns, we
injected constructs into one cell of the four cell stage embryo rather than
into the newly-fertilised egg, and we varied the concentration of injected RNA
in an effort to find a dose that would rescue the MO1 phenotype but not cause
defects through overexpression. The best results were obtained in an
experiment in which 2 pg of mutated activin BHA RNA was used in conjunction
with 20 ng MO1 (Fig. 5G-J). In
this experiment, 2 pg activin RNA caused abnormal development in 36% of
embryos (n=28; Fig.
5H), and a MO1 phenotype was observed in 64% of embryos
(n=28; Fig. 5I).
Injection of both mutated activin B-HA RNA and MO1 reduced the incidence of
abnormal embryos to 24% (n=25;
Fig. 5I).
2
analysis indicates that the observed rescue is significant at
P<0.01.
As a final control, we designed an alternative antisense oligonucleotide
positioned 5' of the original target sequence (MO2;
Fig. 2A). To our surprise this
oligonucleotide proved to have little or no effect on development (see
Fig. 6). To explore this
observation, we isolated and sequenced the 5' untranslated region of
activin B from members of our own colony of Xenopus laevis (see
Materials and methods). The sequence of this region proved to differ from the
published sequence, resulting in a two nucleotide mismatch with our second
antisense oligonucleotide (Fig.
2A). We note that a four nucleotide mismatch is sufficient to
render an oligonucleotide completely ineffective (see
Fig. 2A), and indeed a single
mismatch can lead to a significant reduction in potency
(Khokha et al., 2002), so it
is likely that the efficacy of this alternative antisense morpholino
oligonucleotide will be significantly compromised.
A third antisense morpholino oligonucleotide, MO3, was therefore designed to hybridise with the 5' untranslated region of activin B derived from our own colony of Xenopus laevis. This reagent proved to have similar effects to our original activin B morpholino, but to be even more effective (Fig. 6A-D).
These conclusions were confirmed at the molecular level by comparing the
expression of Goosecoid (Cho et
al., 1991), chordin
(Sasai et al., 1994
) and
Xvent1 (Gawantka et al.,
1995
) in embryos injected with different antisense morpholino
oligonucleotides. In this experiment, embryos were isolated at different
stages to investigate any temporal effects of inhibition of activin function.
MO1 and MO3 gave similar results, with a significant downregulation of
Goosecoid (particularly at mid-gastrula stage 11 for MO3) and of
chordin, and a strong upregulation of Xvent1
(Fig. 6E-J), indicating that in
the absence of activin function embryos acquire more ventral
characteristics.
Depletion of activin B changes the expression of other inducing factors
The effects of activin depletion might be exacerbated if other
mesoderm-inducing factors, such as the nodal-related proteins or
Derrière, are downregulated, or they might be reduced if the expression
of such factors is enhanced. To explore this issue, we investigated the
expression of these inducing factors in embryos injected with mMO1, MO1 or MO3
at early gastrula stage 10.5. Expression of Xnr1, Xnr4, Xnr5 and
Xnr6 was little affected by MO1 or MO3, but expression of
Xnr2 was substantially increased and expression of
Derrière was reduced. As observed with the expression of genes
such as Goosecoid and Xvent1, the change in expression
levels of these genes was not accompanied by changes in their expression
domains (Fig. 7B-J). This is
discussed below.
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Discussion |
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The requirement for zygotic activin B function in normal mesoderm formation
in Xenopus contrasts with a requirement for maternal activin in
Medaka (Wittbrodt and Rosa,
1994) and with the absence of a requirement for zygotic
activin A and activin B expression in mesoderm formation in
the mouse (Matzuk et al.,
1995
). It is clear, however, that in all these species members of
the TGFß family, and particularly the nodal proteins, play significant
roles in mesoderm formation (Schier,
2003
).
Other attempts to interfere with activin function
Our experiments do not represent the first attempt to investigate the role
of activin in Xenopus development, but they may use the most specific
tool to inhibit activin function. Dominant-negative activin receptors, for
example, disrupt normal development
(Hemmati-Brivanlou and Melton,
1992; New et al.,
1997
), but they are as likely to inhibit the functions of other
members of the TGFß family, including BMPs and nodal-related proteins, as
they are to inhibit activin (Hawley et
al., 1995
; Schulte-Merker et
al., 1994
; Wilson and
Hemmati-Brivanlou, 1995
;
Yamashita et al., 1995
). Even
a secreted version of the type II activin receptor, which displays
significantly greater specificity for activin
(Dyson and Gurdon, 1997
), may
also inhibit other members of the TGFß family, although we note that the
phenotype of embryos expressing such a construct resembles, at least
superficially, the phenotypes of embryos injected with MO1 or MO3
(Dyson and Gurdon, 1997
).
Other attempts to inhibit activin function have employed the
activin-binding protein follistatin. Experiments by Schulte-Merker and
colleagues were unable to demonstrate a role for activin following injection
of RNA encoding rat follistatin
(Schulte-Merker et al., 1994),
although more recent experiments, using higher concentrations of RNA encoding
the Xenopus protein, suggest that follistatin does inhibit mesoderm
formation (Marchant et al.,
1998
). Interpretation of these experiments is further complicated
by the observation that the inhibitory effects of follistatin are not
restricted to activin, and that it also binds to members of the BMP family
(Iemura et al., 1998
).
A final approach has involved the use of dominant-negative `cleavage
mutants', where expression in the embryo of a TGFß construct in which the
proteolytic cleavage site is mutated prevents the release of active dimers
(Lopez et al., 1992). In
Xenopus laevis, however, activin cleavage mutants prove to have
little effect on development (Hawley et
al., 1995
; Osada and Wright,
1999
), and indeed similar results have been obtained in Medaka,
although in this species it is maternal activin that appears to be required
for proper mesoderm formation (Wittbrodt
and Rosa, 1994
). Potential pitfalls concerning the use of cleavage
mutant constructs have been discussed by Eimon and Harland
(Eimon and Harland, 2002
), but
explanations for inappropriate lack of activity of a construct are few. One
possibility is that endogenous and exogenous activin B RNAs are
processed in different compartments of the cell. Another is that endogenous
activin can employ an alternative cleavage site; although activin is cleaved
at a single cleavage site in oocyte expression studies
(Hawley et al., 1995
), an
additional site may be employed after the mid-blastula transition. The
inability of activin cleavage mutants to affect early Xenopus
development requires further investigation.
Post-translational regulation of activin B
Analysis of the spatial expression pattern of activin B in the early
Xenopus embryo is hampered by its low expression level, but
dissection of embryos indicates that transcripts are distributed ubiquitously
(Dohrmann et al., 1993). This
observation suggests that there is translational or post-translational control
of activin function, as also occurs with BMP family members and the
nodal-related genes (Agius et al.,
2000
; Bouwmeester et al.,
1996
; Cheng et al.,
2000
; Dale and Wardle,
1999
; Glinka et al.,
1997
; Jones and Smith,
1998
; Smith,
1999
). The spatial control of effective activin concentration is
likely to be very complicated; for example, one known activin antagonist,
Xantivin (Cheng et al., 2000
),
is more effective in marginal zone tissue than in the animal cap
(Tanegashima et al., 2000
),
while experiments involving injection of activin into the blastocoels of
Xenopus embryos suggest that there is in addition an
intrablastocoelic inhibitor of activin function
(Cooke et al., 1987
).
The complicated regulation of activin function may help explain why it is
so difficult to `rescue' MO1- and MO3-injected embryos to normality by
introducing activin B RNA. The problem is exacerbated by the facts that
endogenous activin B expression levels are so low and that injected RNA
diffuses less well in the embryo than does injected morpholino oligonucleotide
(Nutt et al., 2001;
Saka and Smith, 2004
). These
problems notwithstanding, we have achieved partial rescue of the phenotype
caused by MO1 by injecting a quantity of activin B RNA that is just
sufficient, in 36% of embryos, to cause defects through overexpression
(Fig. 5G-J). These results
confirm the specificity of the observed phenotype and reinforce the conclusion
that activin B is required for normal development in Xenopus.
Loss of activin function is accompanied by an upregulation of Xnr2 and a downregulation of Derrière
Activin is but one of several mesoderm-inducing factors in the early
Xenopus embryo; there are, in addition, five nodal-related
genes (Takahashi et al., 2000;
Thomas et al., 1997
) as well
as Derrière (Sun et al.,
1999
) and Vg1 (Dale
et al., 1993
; Thomsen and
Melton, 1993
; Weeks and
Melton, 1987
). It is remarkable that the abolition of just one of
these, activin, should cause such a dramatic phenotype, especially as the
embryo seems to make some attempt to compensate for the loss of activin
activity; although the inhibition of activin function is accompanied by a
downregulation of Derrière, there is a marked upregulation of
Xnr2 (Fig. 7). The
first of these results is consistent with the observation that a
dominant-negative Derrière construct inhibits Xnr2 expression
in the Xenopus embryo, indicating that mesoderm-inducing factors
might positively regulate their own expression
(Eimon and Harland, 2002
). The
upregulation of Xnr2 in embryos injected with MO1 or MO3 does not
accord with this idea, however, and it may be necessary in the future to
conduct a systematic analysis of the effects of ablating candidate inducing
factors and to ask how they regulate each other's expression. We note that
inhibition of all mesoderm-inducing Xenopus nodal-related genes, by
expression of the C-terminal region of Cerberus
(Bouwmeester et al., 1996
),
causes severe defects in mesoderm formation. Such embryos form just a small
tail-like structure, and expression of
-actin and
-globin is
severely reduced (Wessely et al.,
2001
).
Changes in gene expression levels caused by MO1 and MO3 are not associated with changes in expression domains
The downregulation of Goosecoid expression in response to MO1 and
MO3, and the upregulation of Xvent1, appear to occur without
significant changes in the expression domains of these genes (Figs
4,
7). This suggests that during
normal development the spatial expression patterns of regionally expressed
genes are defined by the combined effects of members of the TGFß family,
including the nodal-related genes and derrière as
well as activin B. Loss of just one member of this network, such as
activin, may not disrupt spatial expression patterns to a significant extent,
but may affect expression levels such that development is severely
perturbed.
Polymorphism and the design of antisense morpholino oligonucleotides
The final point to be made from the results described is that one should
not rely solely on sequences derived from GenBank when designing antisense
morpholino oligonucleotides. Polymorphisms, particularly in the 5'
untranslated regions of Xenopus mRNAs, are likely to be frequent, and
even a single nucleotide difference between oligonucleotide and target RNA may
produce a significant reduction in potency
(Khokha et al., 2002). It may
be impractical to confirm the sequence of the mRNA of interest in every
experiment one does, but a failure to obtain a phenotype in one egg batch does
not necessarily invalidate the rest of one's results.
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ACKNOWLEDGMENTS |
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