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Abstract Siamois, a Xenopus zygotic homeobox gene with strong dorsalising activity, is expressed in the dorsal-vegetal organiser known as the Nieuwkoop centre. We show that, in contrast to Spemann organiser genes such as goosecoid, chordin and noggin, Siamois gene expression is not induced following overexpression of mesoderm inducers in ectodermal (animal cap) cells. However, Siamois is induced by overexpressing a dorsalising Wnt molecule. Furthermore, like Wnt, Siamois can dorsalise ventral mesoderm and cooperate with Xbrachyury to generate dorsal mesoderm. These results suggest that Siamois is a mediator of the Wnt-signalling pathway and that the synergy between the Wnt and mesoderm induction pathways occurs downstream of the early target genes of these two pathways. Overexpression of Siamois in animal cap cells reveals that this gene can act in a non vegetal or mesodermal context. We show the following. (1) Animal cap cells overexpressing Siamois secrete a factor able to dorsalise ventralgastrulamesoderm in tissue combination experiments. (2) The Spemann organiser-specific genes goosecoid, Xnr-3 and chordin, but not Xlim.1, are activated in these caps while the ventralising gene Bmp-4 is repressed. However, the dorsalising activity of Siamois-expressing animal caps is significantly different from that of noggin- or chordin-expressing animal caps, suggesting the existence of other dorsalising signals in the embryo. (3) Ectodermal cells overexpressing Siamois secrete a neuralising signal and can differentiate into cement gland and, to a lesser extent, into neural tissue. Hence, in the absence of mesoderm induction, overexpression of Siamois is sufficient to confer organiser properties on embryonic cells.
Fig. 1. Activation of Siamois by the Wnt-signalling pathway but not
by mesoderm inducers in animal caps. mRNA of activin bB, bVg1,
Xnr2, Bmp-4 and bFGF was injected in the animal pole of 2-cell
embryos. The animal caps were excised at stage 9 and gene
expression was analysed by RNase protection assay at stage 10.
(A,B) Injection of mRNA coding for mesoderm inducers in animal
pole blastomere leads to the activation of Xbra and goosecoid (gsc)
but not of Siamois (Sia). (A) Both a short and a long exposure of the
autoradiograph are presented to demonstrate the lack of activation of
Siamois. (B) The Xbra and Siamois signals have been normalised
using the signal for the constitutively active FGFR. (C) Siamois but
not gsc or Xbra is activated by the Wnt-signalling pathway.
Exposure times were 5 days (top panel) and 24 hours (bottom panel).
The apparent decrease in the activation of Siamois at high
concentrations of Xwnt-8 mRNA is due to a weaker loading of the
corresponding lanes (see the FGFR signal). WE, whole embryo; C,
control uninjected animal caps.
Fig. 2. Siamois cooperates with Xbra to induce dorsal mesoderm in
animal caps. (A) Diagrammatic representation of the experiment:
embryos were injected into their animal poles at the two cell stage
with Xbra mRNA (2.5 ng), Siamois mRNA (50 pg), or with a
mixture of both. Animal caps were explanted at stage 9 and cultured
as doublets. (B-E) Stage 17 animal caps: (B) uninjected, (C) injected
with Xbra mRNA, (D) with Siamois mRNA or (E) with a mixture of
both. (F,G) Representative photographs of sections through animal
caps overexpressing Xbra (F) or Xbra and Siamois (G), cultured until
stage 32 and stained with the 12/101 muscle-specific mAb (black).
Strongly vacuolated notochord cells can be seen in the animal caps
overexpressing both Siamois and Xbra (arrows). Animal caps from
embryos injected with Siamois mRNA alone showed neither
morphological signs of mesodermal differentiation nor staining with
the 12/101 antibody (not shown).
Fig. 3. Effects of the overexpression of Siamois in embryonic ventral
mesoderm tissue. (A) Diagrammatic representation of the
experiment. Pieces of ventral marginal zone (composed mostly of
ventral mesoderm cells) were explanted at stage 10.25 from embryos
injected in their two ventral vegetal blastomeres at the 4-cell stage
with different amounts of Siamois mRNA. The marginal explants
were fixed when sibling embryos reached stage 26-28, then doublestained
with the muscle-specific antibody 12/101 and the notochord
specific antibody MZ15. (B) Double-stained ventral marginal zone
explants from embryos injected with dilutions (0.1-100 pg) of
Siamois mRNA. Cells labelled with the MZ15 and 12/101 antibodies
are stained red and brown, respectively.
Fig. 4. Siamois-, noggin-, and Xwnt-8 but not goosecoid expressing
animal caps secrete a dorsalising signal.
(A) Diagramatic representation of the experiment. Pieces
of ventral equatorial tissue (composed mostly of ventral
mesoderm cells) were dissected from embryos (stage
10.25) previously injected with the lineage tracer RLDx
(red). The explants were immediately combined with
blastula animal caps (stage 9) derived from embryos
previously injected with different amounts of Siamois (25
pg, 50 pg, 100 pg), noggin (25 pg, 50 pg, 100 pg), or gsc
(100 pg, 500 pg) mRNA or with 10 pg ofXwnt-8 mRNA.
The ‘sandwiches’ were cultured until their mesoderm
component reached the equivalent of stage 18 for analysis
of the presence of the muscle marker MyoD (B) or stage
26-28 for double immunostaining with the muscle and
notochord antibodies 12/101 and MZ15 (B,C, and not
shown). (B,C) Photographs of sections through
sandwiches composed of a piece of ventral mesoderm
tissue labelled with fluorescent RLDx combined with an
animal cap derived either from control uninjected embryos
(cont) or from embryos injected with 50 pg of noggin
mRNA (nog), 50 pg of Siamois mRNA (Sia), 500 pg of
gsc mRNA (gsc) or 10 pg of Xwnt-8. B) Left panels,
RLDx-labelled cells at stage 18; middle panels, MyoDpositive
cells in the same sections as the left panels; right
panels, 12/101-positive cells at stage 26. MyoD staining
was shown here in addition to 12/101 staining as it allows
a better visualisation of the relative localisations of
antibody and RLDx stainings, and demonstrates that
muscle-specific staining was seen only in the progeny of
the RLDx-labelled marginal zone cells and not in those of
the animal cap. (C) Left panels, RLDx-labelled cells at
stage 26. Right panels, 12/101-positive cells at stage 26.
No MZ15 staining was ever observed (not shown and
Fig. 5. Siamois triggers an organiser gene expression programme in
animal cap explants. RNase protection assays using total RNA
extracted from early gastrula animal cap sandwiches either
uninjected (C) or injected with Siamois, noggin, gsc or chordin
mRNAs. WE, whole embryo. (A) Effect of the injection of Siamois
mRNA on the activation of noggin, Siamois, chordin, gsc, Xlim.1,
Xnr-3 and Xbra. (B) Effect of the overexpression of Siamois, noggin,
gsc and chordin in animal caps on the steady state level of Bmp-4
mRNA. To determine the level of Siamois mRNA, animal cap
sandwiches were stopped at stage 10-10.25. Otherwise, the assays
were performed on stage 10.5-11 sandwiches. The arbitrary units
correspond to the ratio of the radioactive signal for the tested gene
over that for the FGF receptor, used here as a loading control (10-15
animal caps per experimental point). ND, not done.
Fig. 6. The windows of competence for dorsalisation by Siamois,
noggin and chordin are different. (A) Diagram of heterochronic
experiments. Pieces of ventral mesoderm tissue were dissected from
embryos previously injected with the lineage tracer RLDx (red).
They were conjugated with stage 9 animal caps from embryos
injected with the indicated amounts of Siamois, noggin or chordin
mRNAs either immediately after dissection or after culture in MBS
until the equivalent of stage 12 or 13. The ‘sandwiches’ were
cultured until their mesoderm component reached the equivalent of
stage 26-28, fixed, sectioned and analysed for muscle by
immunostaining with the muscle specific antibody 12/101.
(B) Quantification of the percentage of muscle cells in heterochronic
mesoderm-ectoderm conjugates. The percentage of muscle cells per
sandwich was estimated by calculating the ratio of the nuclei of
12/101-positive cells over the total number of nuclei of RLDxpositive
cells. The stage 9 animal caps used in the sandwiches were
derived from embryos injected with 25 pg (triangles) or 100 pg
(squares) of mRNAs for Siamois or noggin or with 600 pg (circles)
of chordin mRNA. Each point on the graph represents the average
muscle content in 10 analysed conjugates. VMZ, ventral marginal
Fig. 7. Neuralisation of Siamois-expressing animal caps. (A) Animal
caps were injected with 10 pg of Siamois mRNA, explanted at stage
9 and cultured in MBS as doublets until stage 30. Large cement
glands formed in over 90% of the injected caps (arrowheads).
(B) Section through a stage 30 animal cap injected with 100 pg of
Siamois mRNA and stained with the antibody 4d, specific for the
pan-neural marker N-CAM. (C) Diagrammatic representation of the
experiment showing the cement gland inducing ability of Siamois.
Animal caps injected with either Siamois mRNA (10, 33 or 100 pg)
or the lineage tracer RLDx were excised and combined at stage 9 and
cultured until stage 30. (D,E): Phase contrast (D) and fluorescent (E)
view of a recombinant between an animal cap injected with 10 pg of
Siamois mRNA and an RLDx-injected animal cap. Arrowheads mark
the limits of the induced cement gland. Note the presence of RLDxpositive
cells with the cement gland typical columnar shape.