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The vertebrate body plan is specified in the early embryo through the inductive influence of the organizer, a special region that forms on the dorsalmost side of the embryo at the beginning of gastrulation. In Xenopus, the homeobox gene Siamois is activated prior to gastrulation in the area of organizer activity and is capable of inducing a secondary body axis when ectopically expressed. To elucidate the function of endogeneous Siamois in dorsoventral axis formation, we made a dominant repressor construct (SE) in which the Siamois homeodomain was fused to an active repression domain of Drosophila engrailed. Overexpression of 1-5 pg of this chimeric mRNA in the early embryo blocks axis development and inhibits activation of dorsal, but not ventrolateral, marginal zone markers. At similar expression levels, SE proteins with altered DNA-binding specificity do not have the same effect. Coexpression of mRNA encoding wild-type Siamois, but not a mutated Siamois, restores dorsal development to SE embryos. Furthermore, SE strongly blocks axis formation triggered by beta-catenin but not by the organizer product noggin. These results suggest that Siamois function is essential for beta-catenin-mediated formation of the Spemann organizer, and that Siamois acts prior to noggin in specifying dorsal development.
Fig. 1. Constructs used in this study. An engrailed repression domain contained within the first 296 amino
acids was fused with the Siamois homeodomain to generate SE. The control construct (ÆEn) contains only the
engrailed part of the fusion. The constructs SEQ>K and SEQ>E contain a glutamine-to-lysine (Q>K) or
glutamine to glutamic acid (Q>E) mutation at position 50 of the homeodomain. Sia Q>K is identical to wildtype
Siamois except for a glutamine-to-lysine substitution at position 50 of the homeodomain.
Fig. 2. Siamois function is required
for dorsal development. Embryos
were injected with 5 pg of SE
mRNA (A,B,D,F), 5 pg of SEQ>E
mRNA (E) or 5 pg of ÆEn mRNA
(G) into both dorsal blastomeres at
the 4-cell stage. (A) Early gastrulae
(stage 10+). Uninjected control
embryo is at top right. (B) Lack of
neural tube and head structures in
SE embryos at stage 21-22.
(C) Uninjected stage 21-22
neurulae. Arrowhead points to the
cement gland. (D) Ventralized
phenotype of SE embryos at the
equivalent of stage 36. (E)
Embryos injected with SEQ>E
mRNA are indistinguishable from
uninjected and ÆEn mRNAinjected
controls. (F,G) Crosssections
of injected embryos
equivalent to normal embryos at
stage 36. Bar in F, also refers to G,
is 250 mm. Abbreviations: dl,
dorsal lip; nt, neural tube; nc,
notochord; mu, muscle
Fig. 3. SE overexpression leads to a loss of dorsal marginal zone
markers. Embryos at the 4- to 8-cell stage were injected with a total
of 20-30 pg of mRNA as indicated and cultured until stage 10.5 for
northern analysis. Total RNA from 2.5 embryos was loaded per lane.
Fibronectin is a loading control.
Fig. 4. SE constructs are well-expressed in injected embryos.
Embryos were injected equatorially in each blastomere at the 4-cell
stage with (A) 5 pg of SE or 5 pg of SEQ>K mRNA, and (B) 10 pg
of SE or 5 pg of SEQ>E. Lysates were prepared at stage 11 for
western analysis with 9E10 myc-specific monoclonal antibodies.
Fig. 5. Wild-type Siamois mRNA corrects dorsal deficiences of SE
embryos. Embryos were injected in both dorsal blastomeres at the 4-
cell stage with either (center) 5 pg of SE mRNA alone or with a
mixture of (left) 5 pg of SE and 10 pg Siamois mRNAs, or (right) 5
pg of SE and 10 pg of Sia Q>K mRNAs. Injections of SE mRNA
alone resulted in ventralized embryos indistinguishable from those in
Fig. 2D.
Fig. 6. SE inhibits Siamois-mediated activation of a goosecoid
promoter element. A representative experiment is shown. Embryos
were microinjected in both ventral blastomeres at the 4-cell stage
with -155gsc/Luc plasmid DNA (Watabe et al., 1995), either alone
or in combination with indicated mRNAs. For luciferase activity
measurements, lysates were prepared from embryos at stage 10+.
Data are expressed as the means from triplicate samples ± the
standard deviations.
Fig. 7. SE blocks the axis rescuing activity of b-
catenin, but not noggin. UV-treated embryos
were microinjected subequatorially at the 4- to
8-cell stage with 30 pg of b-catenin mRNA
(B,C) or 5 pg of noggin mRNA (D,E), alone or
in combination with 5 pg of SE mRNA (C,E).
(A) Uninjected UV-treated embryos.
