The Laboratory of Vertebrate Embryology, The Rockefeller University, NY 10021, New York, USA
Author for correspondence (e-mail:
brvnlou{at}rockefeller.edu)
Accepted 10 December 2002
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SUMMARY |
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Key words: Coco, BMP, Wnt, Nodal, Gastrula, Xenopus laevis
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INTRODUCTION |
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Although the cell-fate choice of ectodermal cells is traditionally thought
to involve a decision to become epidermal or neural, it is known that
ectodermal cells prior to gastrulation are pluripotent. In particular,
ectodermal cells can adopt mesodermal fates if exposed to mesoderm-inducing
signals during a defined window of time (`competence' window) that ends after
gastrulation in Xenopus. Therefore, in order for correct ectodermal
patterning to take place, cells in the prospective ectoderm must avoid
exposure to mesoderminducing signals. The endogenous mesoderm-inducers are
likely to be members of the TGFß superfamily, in particular the
nodal-related members (Harland and Gerhart,
1997; Schier and Shen,
2000
). It is also thought that expression of vegetally localized
maternal factors (VegT) act in the embryo to promote mesodermal gene
expression through the activation of TGFß signals
(Heasman, 1997
;
Xanthos et al., 2002
).
Therefore it is likely that a TGFß inhibitor would be expressed in the
animal region of the early embryo (and ectoderm) in order to restrict the
effect of diffusible nodal signals to the vegetal and equatorial regions of
the embryo.
We describe the identification and characterisation of a novel member of
the Cerberus/Dan/Gremlin superfamily of secreted BMP inhibitors
(Bouwmeester et al., 1996;
Hsu et al., 1998
;
Rodriguez Esteban et al.,
1999
; Stanley et al.,
1998
; Piccolo et al.,
1999
). This gene, which we have termed Coco, was
initially identified as a gene differentially regulated by Smad7, a neural
inducer, in ectodermal explants in a microarray-based screen
(Muñoz-Sanjuán et al.,
2002
). Coco is expressed maternally in an animal to
vegetal gradient, and later on is restricted to the animal region of the
embryo. Coco is expressed broadly within the ectoderm and this
expression declines rapidly following gastrulation. We show that Coco can
inhibit signaling mediated by BMP, TGFß and Wnt ligands, and can act to
inhibit mesoderm formation in vivo and in explants. In addition, expression of
Coco in ectodermal cells changes their responsiveness to mesoderm-inducing
signals. Based on these results, we propose that the expression and
bioactivities of Coco are consistent with it being a bone fide inhibitor of
mesodermal signals that acts within the animal region of the embryo to inhibit
TGFß signals.
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MATERIALS AND METHODS |
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Reverse transcriptase polymerase chain reaction (RT-PCR)
analysis
RT-PCR was performed on animal and DMZ/VMZ explants as has been described
previously (Wilson and Melton,
1994). Ornithine decarboxylase (ODC) was used as a loading
control.
Whole-mount in situ hybridisation
Whole-mount in situ hybridisations were carried out as described previously
(Harland, 1991). In situ probes
were made as described elsewhere: brachyury
(Smith et al., 1991
),
emx1 (Pannese et al.,
1995
), en (Hemmati-Brivanlou et al., 1991), Fgf8
(Christen and Slack, 1997
),
goosecoid (Cho et al.,
1991
), hb9 (Wright et
al., 1990
), nkx2.5
(Raffin et al., 2000
) and
rx (Mathers et al.,
1997
). Embryos were embedded in 20% gelatin/PBS and fixed
overnight in 4% PFA at 4°C. Sections were cut at 100 µm using a
vibratome.
Interaction of Coco with Xnr1, BMP4, Wnt8 at a biochemical level
Coco was flag-tagged in the C terminus by standard PCR methods.
Flag-tagged Coco was co-injected into embryos at the 2-cell stage
with BMP4-HA or Xnr1-HA. Protein extracts were made at stage 10-11,
immunoprecipitated with an anti-HA polyclonal antibody, and probed with an
anti-flag monoclonal antibody.
Inhibition of Wnt8 and BMP4 promoter activity by Coco
Injections were made in the animal pole of 4-cell stage embryos with 25 pg
of reporter gene DNA, 10 pg Wnt8 RNA
(Hoppler et al., 1996) or 100
pg Bmp4 RNA (Hata et al.,
2000
), with or without addition of 1 ng Coco RNA. Embryos
were recovered at stage 9 for TOP-FLASH (a Wnt-responsive promoter) activity,
and stage 10.5 for Bmp response element (BRE) activity. Luciferase
transcription assays were performed with the Luciferase Assay (Promega Corp.,
Madison, WI) as described (Vonica et al.,
2000
).
Competence assay
Embryos were injected at the 2-cell stage with Coco RNA and animal
cap explants were cut at stage 8. Activin-conditioned medium was added to
uninjected and Coco-injected explants at stages 8, 9, 10 and 11.
Explants that were beginning to heal were carefully reopened prior to addition
of activin. Explants were harvested at stage 12/13 and analyzed for the
induction of mesodermal markers by RT-PCR.
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RESULTS |
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Coco is the earliest expressed BMP/TGFß inhibitor in Xenopus
laevis
Based on the sequence homology between Coco and related members, we
postulated that Coco would be a BMP antagonist. However, based on sequence
alone, we could not predict whether Coco would interact with other signaling
factors of the Wnt and TGFß families. In order to evaluate whether Coco
could function in vivo in the context of BMP signaling, we analyzed its
expression during early embryogenesis, when BMP signaling plays a critical
role in dorsoventral patterning and neural induction
(Muñoz-Sanjuán and
Brivanlou, 2002).
As shown by in situ hybridisation and RT-PCR, Coco is strongly
expressed maternally and at the gastrula stage, however levels of
Coco sharply decline in the embryo after stage 12
(Fig. 2A-C). In the egg,
Coco mRNA is expressed in the animal pole and overlaps with that of
Bmp4 mRNA expression (Fig.
2A, top panels). We did not detect expression of Coco in
the vegetal pole, in contrast to VegT
(Zhang and King, 1996) mRNA,
which is most strongly expressed vegetally (bottom panel). This result
suggests that Coco mRNA is maternally localized to the animal pole,
and that a gradient of Coco message exists in the egg and early
embryo. In order to independently evaluate this observation, we compared, by
RT-PCR the expression of Coco and Vg1
(Weeks and Melton, 1987
), a
member of the TGFß family expressed vegetally. Vg1 is expressed
in the vegetal pole and to a lesser extent in the equatorial region. These
results collectively suggest that there are two opposing gradients of maternal
RNA localization in the egg, one animal to vegetal (exemplified by
Coco), and the second vegetal to animal (exemplified by VegT
and Vg1). Given Coco's biological activities, the animal-vegetal
gradient of Coco RNA might act to restrict the activities of
vegetally localized TGFß ligands to the vegetal pole and equatorial
region, where Coco expression is less pronounced.
|
At pre-gastrula embryonic stages, Coco is expressed in the animal pole
exclusively (Fig. 2B). At
gastrula, Coco mRNA transcripts are detected in both the dorsal
marginal zone (DMZ; including the organizer, see *) and the ventral marginal
zone (VMZ) and at very high levels in the animal cap ectoderm
(Fig. 2D,E). There are also
very low levels of expression in the vegetal pole
(Fig. 2E). To date, Coco is the
only known BMP inhibitor expressed maternally and ubiquitously within the
ectoderm prior to neural induction. By contrast, Cerberus is expressed
zygotically between stages 9 and 13 (Fig.
2C) (Bouwmeester et al.,
1996) and is restricted to the anterior endoderm of the organizer
at gastrula stages (Bouwmeester et al.,
1996
). The maternal expression of Coco, its widespread expression
within the ectoderm, and the rapid decline in Coco mRNA levels
following gastrulation prompted us to evaluate Coco's function in the context
of BMP and TGFß inhibition during ectodermal patterning.
Biological activities of Coco in early Xenopus
development
As a first test for Coco's biological activities, we injected its mRNA into
embryos at either the 2- or 4-cell stage. Our initial analysis was done at
gastrula stages where Coco is expressed throughout the ectoderm and
marginal zones. In the gastrula both brachyury and Fgf8 are
expressed in a ring of mesodermal cells around the vegetal pole
(Fig. 3A,B, top panels). After
injection of Coco in one of the two cells in the vegetal pole
(Fig. 2), we found that both
markers are repressed (Fig.
3A,B, lower panels), suggesting that Coco can inhibit mesoderm
formation in vivo. Coco expands the size of the endogenous organizer as judged
by the increase in expression of Otx2
(Fig. 3C) and Gsc
(Fig. 3D). In addition, the
endogenous ectodermal expression of Otx2 is increased
(Fig. 3C, lower panel) and
there are ectopic ectodermal patches of Otx2 expression on the
contralateral sides of the embryo (not shown), suggesting that Coco acts in
cell non-autonomous manner. The inhibition of pan-mesodermal gene expression
at gastrula stages suggests that Coco might inhibit mesodermal signals,
probably through an inhibition of Nodal/Activin pathways
(Schier and Shen, 2000), the
presumed endogeneous mesoderm inducers.
|
The embryological consequences of expressing BMP inhibitors include expansion of dorsoanterior structures. In order to test the effects of Coco misexpression on dorsoventral patterning and anterior neural development, we analyzed Coco-injected embryos at tadpole stages. Overexpression of Coco in the animal pole results in embryos with expanded anterior structures and ectopic cement glands (compare Fig. 3E with 3F). In contrast, overexpression of Coco ventrally results in posterior truncations and the induction of extra anterior structures (75% of injected embryos have this phenotype; Fig. 3G). Very infrequently these extra structures also contain a single eye (5% of cases; not shown). Molecular analysis of these ectopic structures shows that they contain forebrain and midbrain tissue, as shown by the ectopic expression of the forebrain markers Rx (Fig. 3H), Emx1 (Fig. 3I), Otx2 (Fig. 3J), and the midbrain marker En2 (Fig. 3K). En2 expression is detected where the ectopic head contacts the main dorsal axis of the embryo (see *, Fig. 3K, lower panel). By contrast, we failed to detect ectopic expression of Hoxb9, a marker of spinal cord (Fig. 3L). In addition, we have shown that there is no muscle tissue in the ectopic structures (Fig. 3N), although the heart marker Nkx2.5 was strongly induced around the extra cement gland (Fig. 3M). However, we never detected ectopic hearts in the Coco-injected embryos, possibly because of the lack of endoderm formation in Coco-injected embryos (not shown and Fig. 4).
|
These phenotypes, ectopic anterior tissue including head structures, are
consistent with an inhibitory activity of BMP and Wnt signaling by Coco
(Glinka et al., 1998;
Piccolo et al., 1999
). In
order to unravel the molecular mechanism underlying Coco's activity, we
analyzed fate changes in embryonic explants by RT-PCR for a variety of
molecular markers. When embryos were injected at the 2-cell stage in the
animal caps, we failed to detect markers for the organizer or endoderm in
gastrula-staged explants (not shown), but we observed a decrease in epidermal
markers, and an increase in the early neural marker, ß-tubulin at
mid-gastrulation (Fig. 4A).
This result suggests that Coco has the ability to inhibit BMP signaling. By
stage 21, the pan-neural markers (Ncam and nrp1) and
anterior-specific markers (Otx and XAG) are induced in
explants expressing Coco (Fig.
4B), suggesting that Coco can neuralize ectodermal explants,
consistent with an inhibition of BMP signaling
(Wilson and Hemmati-Brivanlou,
1995
). Similar to results seen with Cerberus overexpression
(Bouwmeester et al., 1996
),
Coco also induced Nkx2.5 in this assay
(Fig. 4B).
It has previously been shown that Cerberus can neuralize VMZ explants
(Bouwmeester et al., 1996). We
injected Coco mRNA into the VMZ at the 4-cell stage and analyzed its
effects on cell fate determination in VMZ explants isolated at gastrula stages
or for morphological changes at tadpole stages (stage 27) to test whether Coco
can also neuralize ventral tissue (Fig.
4C-E). At the gastrula stage, the organizer markers
chordin and goosecoid were weakly induced in the VMZ
expressing Coco, whereas the expression of brachyury was suppressed
(Fig. 4D), consistent with the
in vivo results that Coco blocks mesoderm formation and neutralizes the
embryo. At stage 27, the morphology of the VMZ+Coco explants were
similar to that of the DMZ explants (Fig.
4C), and the injected explants contained anterior neural tissue
and cement glands, but not dorsal mesodermal derivatives, such as muscle and
notochord (Fig. 4C). During
normal development at tailbud stages (stage 27), VMZ explants do not express
the neural markers Ncam, nrp1 and Otx2, or the cement gland
marker XAG. In VMZ explants expressing Coco all of these
markers are now induced (Fig.
4E), consistent with Coco blocking both mesoderm and ventral
ectoderm (epidermis) inducing signals mediated by BMPs. The neural fate
acquisition of VMZ explants expressing Coco and the absence of dorsal
mesodermal markers strongly suggests that Coco acts to neuralize the explants,
rather than having an effect on dorsalisation of the mesoderm. Therefore,
although Coco can block BMP signaling, the lack of dorsal mesodermal gene
expression highlights the notion that Coco efficiently blocks signaling by
mesodermal inducers and might act endogenously to inhibit mesodermal gene
expression in the ectoderm.
Coco can inhibit signaling by BMP, Nodal, Activin and Wnt
signaling
In order to test the inhibitory interactions of Coco with BMPs, TGFß
members and Wnts, we co-injected Coco for animal cap assays with RNAs
of BMP4, Xnr1 [nodal-related factor-1
(Hyde and Old, 2000)], Wnt8
(Sokol and Melton, 1991
) or
Activin (Fig. 5) (Smith et al., 1990
), and
monitored the expression of immediate response genes normally activated by
these signaling molecules in the ectoderm. For instance, it has been shown
that both Xbra and epidermal keratin expression are
upregulated in animal caps following overexpression of BMP4. In this assay,
Coco blocked induction of these markers
(Fig. 5A). In similar assays,
Coco could also block Wnt8 induction of Xnr3 and siamois
expression (Fig. 5B)
(Sokol and Melton, 1991
) and
Nodal and Activin (Smith et al.,
1990
; Sokol and Melton,
1991
) signaling, as detected by the inhibition of the expression
of Chordin, Brachyury and Wnt8 induced by Xnr1 and Activin
(Fig. 5C,D).
|
Based on the expression and biological activities of Coco, we propose that
an endogenous role of Coco might be to regulate fate determination in the
ectoderm through an inhibition of TGFß signals. In order to test whether
Coco can interact with BMP/TGFßs proteins, we co-injected synthetic RNAs
encoding tagged Coco protein together with tagged BMP4 or Xnr1 constructs into
animal caps and tested for direct binding in immunoprecipitation experiments
(Fig. 5E,F). Indeed, we found
that we can detect biochemical binding and immunoprecipitate Coco protein with
BMP4 and Xnr1 in this assay. We postulate that this interaction is likely to
inhibit the signaling input of the two TGFß ligands. In addition, and as
an independent way to assess whether Coco's bioactivities are due to direct
interference with the BMP and Wnt signaling pathways, we tested whether Coco
could prevent the transcriptional activation of Wnt and BMP-responsive
promoters (Fig. 5G,H). Embryos
were injected the Wnt-responsive promoter TOP-FLASH
(Hoppler et al., 1996)
together with Wnt8 RNA or co-injected with Wnt8 and
Coco RNAs (Fig. 5H).
Indeed, we found that there was a significant repression of this promoter by
Coco. Similarly, Coco was able to completely inhibit the activation of the
Bmp4 responsive promoter [Bmp Response Element, BRE4
(Hata et al., 2000
)] by BMP4
(Fig. 5G).
In addition we tested whether Coco could inhibit the activity of another signaling molecule, FGF. We cultured animal caps with or without Coco in the presence of FGF and analyzed them at neurula stages for mesoderm formation. We found that in this assay Coco could not prevent mesoderm formation, suggesting the activity of Coco is specific for selected signaling molecules (data not shown) and is not promiscuous.
Involvement of Coco in ectodermal competence
The maternal expression of Coco makes it a unique gene among the
large family of BMP inhibitors. Several BMPs, Wnts and Nodal-related factors
(Cui et al., 1995;
Hemmati-Brivanlou and Thomsen,
1995
; Onuma et al.,
2002
) are inherited maternally. However, no inhibitors are
reported to be expressed during these early stages. Therefore, a potential
function of Coco might be to block maternal signaling by these
molecules in the prospective ectodermal space. Coco is widely
expressed in the ectoderm until the end of gastrulation in Xenopus at
stage 12. The timing of the decline of the mRNA coincides with the loss of
competence of ectodermal cells to respond to mesoderm-inducing signals
(Green et al., 1990
;
Domingo and Keller, 2000
).
Therefore, we investigated whether the presence of Coco in the
ectoderm at specific stages could inhibit mesoderm formation or affect the
competence of the ectoderm to respond to mesoderm-inducing signals. Animal cap
explants can respond to activin and become mesoderm, although the
responsiveness of the explants declines over time until stage 12, at which
point they are no longer able to respond
(Green et al., 1990). We
therefore tested whether Coco RNA would alter this responsiveness
temporally or qualitatively, by exposing dissected caps to activin protein at
different time points (Fig.
5I). Indeed, animal caps expressing Coco responded
differently to Activin over time (Fig.
5I). In contrast to control explants, Coco-injected caps
failed to express mesodermal markers following Activin exposure at earlier
stages, suggesting that Coco can indeed change the timing of the
responsiveness of ectodermal cells to Activin. It is noteworthy that the
levels of Coco RNA used in this experiment are not sufficient to
block mesoderm induction following exposure to activin protein from the
blastula stages (Fig. 5I).
However, if Activin was added to Coco-injected caps at or after stage
10 (beginning of gastrulation), no mesoderm induction was observed compared to
control explants. These results strongly suggest that Coco changes the
responsiveness of the ectoderm to mesoderm inducing signals, and that the
effect might not necessarily be due to direct binding exclusively. Although it
is possible that Coco induces a prior fate change in the ectoderm, which would
lead to an altered responsiveness of the explants to Activin, this result is
consistent with the in vivo inhibition of mesodermal gene expression by Coco.
Furthermore, the secondary structures induced by Coco expression lack
axial tissues (Fig. 3N), and
the primary axis shows a loss of axial muscle tissue, further suggesting that
Coco inhibits mesoderm formation in vivo.
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DISCUSSION |
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The induction of mesoderm in vivo is thought to occur as a consequence of
TGFß signaling (Smith et al.,
1989; Harland and Gerhart,
1997
; Schier and Shen,
2000
). In particular, the role of Nodal signaling in mesoderm
specification is strongly supported by biochemical and genetic evidence
(Schier and Shen, 2000
).
Therefore, it is predicted that Nodal signals must be blocked to allow
ectoderm to be appropriately patterned
(Thisse et al., 2000
) during
gastrulation. This suggests that a function of Coco might be to inhibit
mesoderm-inducing signals operating in the ectoderm. We have shown that Coco
can bind and inhibit Xnr1, as well as BMP4, Activin and Wnt8. These results,
combined with the overall inhibitory effects of Coco on mesodermal gene
expression in vivo and in explants, suggests that Coco's bioactivities are
largely due to its inhibitory effects on nodal and activin signaling. Amongst
the known TGFß inhibitors, Coco is the only member whose expression is
consistent with a role in inhibiting mesodermal signals in the ectoderm. By
contrast, the other two known Nodal inhibitors, Antivin
(Tanegashima et al., 2000
) and
Cerberus (Piccolo et al.,
1999
), and the Wnt inhibitor Dkk
(Glinka et al., 1998
) are not
expressed during those stages in the ectoderm. Further experiments are
required such as loss of function of Coco to confirm this role of Coco in
embryonic patterning.
The TGFß inhibitory activities of Coco might also act to restrict the mesodermal domain to the characteristic ring of cells prior to involution. The animal-to-vegetal gradient of Coco RNA in the egg and early embryo suggest that two opposing gradients of TGFß activity might act to shape the future mesodermal domains in the embryo. It has been well established that the vegetally localized gradients of VegT and Vg1 expression act to promote mesoderm formation. Therefore, Coco activity might act to restrict the activity of Vg1 and potentially other TGFß ligands to the vegetal and equatorial regions of the embryo, and ensure a tight domain of mesodermal gene expression.
Altogether, we have identified a maternal BMP, TGFß and Wnt inhibitor,
whose expression and biological activities are consistent with a role in the
regulation of ectodermal competence, to ensure proper ectodermal patterning
during gastrulation. Coco expression in the ectoderm might also act
to lower overall levels of BMP signals, so that additional BMP inhibitors
expressed in the organizer can induce the formation of the nervous system.
Therefore, expression of Coco in the entire ectodermal region prior
to gastrulation might act to prevent fate specification in the ectoderm and
ensure the maintenance of the stem-cell-like properties exhibited by
ectodermal cells (Tiedemann et al.,
2001). Interestingly, the mouse and human homologs of Coco are
also expressed in undifferentiated, multipotent stem cells suggesting that
this potent new inhibitor might fulfil similar functions during mammalian
embryogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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