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The organizer in vertebrate embryos is responsible for the formation of the primary body axis. In amphibian embryos, the organizer forms in the dorsal marginal zone (prospective dorsal mesoderm) at a location determined by the point of sperm entry. Using inducible versions of axis-inducing proteins, it has been shown that, irrespective of the mode of secondary axis induction, organizer formation in the ventral marginal zone is temporally restricted from the midblastula transition to the onset of gastrulation. Here, we show that the competence of marginal zone cells to respond to organizer-inducing signals is under temporal control, one of the regulators being the homeobox transcription factor Xcad2. Overexpression of Xcad2 restricts the temporal competence for axis induction, whereas partial loss of function expands this competence, supporting our suggestion. We propose that Xcad2 competes with putative axis-inducing signals within the marginal zone to prevent expression of organizer-specific genes. Elimination of endogenous Xcad2 allows for the activation of organizer genes beyond the normal competence window during early/mid-gastrulation. We conclude that Xcad2, through its early expression in the ventrolateral marginal zone, terminates the competence of this embryonic region to respond to organizer-inducing signals by preventing the activation of organizer-specific genes.
FIG. 1. Chimeric proteins. Schematic representation of the different
Xcad2 and siamois constructs utilized. The Xcad2 and siamois
proteins were fused to the GR domain. The Xcad2 homeodomain
was fused to the engrailed repression domain or the VP16 activation
domain.
FIG. 2. Xcad2 modifies the competence of the mesoderm to
differentiate into Spemann’s organizer. (A–E) Embryos injected
ventrally with sia/GR mRNA and treated with dex at the stages
stated exhibit a high incidence of secondary axis induction (arrowheads)
as a result of activation at blastula stages. (F) Control
uninjected sibling embryo. (G–K). Embryos coinjected ventrally
with sia/GR and antisense Xcad2 RNA. The constructs were
activated at the stages as marked and exhibit secondary axis
induction also during gastrula activation. (L) Injection of antisense
Xcad2 RNA does not result in secondary axis induction. (M–Q)
Ventral coinjection of sia/GR with Xcad2/GR and activation at
different developmental stages. This combination results in very
weak secondary axis induction at all stages tested. (R) Ventral
injection of Xcad2/GR mRNA and activation at stage 8 result in a
weak ventralization.
FIG. 3. Xcad2 activity regulates organizer induction during gastrulation in the VMZ. (A) Embryos were injected ventrally with sia/GR
mRNA (4 pg) alone or in combination with antisense Xcad2 (1 ng), en/Xcad2 (160 pg), or Xcad2/GR (400 pg) RNA. The GR constructs were
activated at different developmental stages and the fraction of embryos with secondary axes determined. (B) Injection with a constant
amount of sia/GR RNA (8 pg) alone or with increasing amounts of Xcad2/GR mRNA (40, 200, and 400 pg). The fraction of secondary axes
induced was scored.
FIG. 4. Specificity of the Xcad2 antisense and antimorph constructs. (A–F) Phenotypic effects of overexpression of the different Xcad2
constructs. (A) Control embryo. (B) Xcad2 overexpression results in ventralized embryos. (C) Radial overexpression of the en/Xcad2 protein
results in an antimorphic phenotype opposite to that of Xcad2 overexpression. (D) Radial injection of antisense Xcad2 RNA results in dorsalized
embryos. (E) Coinjection of Xcad2 RNA and en/Xcad2 mRNA results in the phenotypic rescue of the effects of both constructs. (F) Injection of
Xcad2 mRNA together with antisense Xcad2 RNA rescues the phenotypes of the embryos. (G–J) The regulatory influence of the Xcad2
constructs on the ventrolateral downstream gene, Xpo. (G) Control Xpo expression pattern. (H) Xcad2 overexpression causes the expansion of Xpo
expression. Radial injection of either en/Xcad2 mRNA (I) or antisense Xcad2 RNA (J) downregulate Xpo expression similarly.
FIG. 5. Early dorsal Xcad2 expression represses the formation of the endogenous organizer. Embryos were injected dorsally with 480 pg
of Xcad2/GR mRNA and treated with dex at different developmental stages. At each time point, the embryos were divided into very mild
axial defects (DAI 4â5), moderate anterior effects (DAI 2â3), and strong inhibition of organizer activity (DAI 0â1). The graph represents the
compilation of a number of experiments where the percentage of each type of embryos is represented.
FIG. 6. Expression of organizer-specific genes during gastrulation depends on the competition between siamois and Xcad2. (A) Activation of
organizer-specific genes by siamois is also restricted to blastula stages. RT-PCR of RNA samples prepared at stage 11 from embryos injected with
sia/GR and treated with dex between stages 8 and 10.5. The genes tested were gsc, chordin, ADMP, BMP-4, and histone H4 as a loading control.
(B) The activity of siamois is restricted in explanted VMZ. Embryos were injected with sia/GR, en/Xcad2/GR, or a combination of both RNAs.
VMZ explants were prepared and treated with dex at the specified stages. RNA extraction was performed at about stage 12. (C) Xcad2 normally
downregulates the expression of organizer-specific genes during gastrulation. Antisense Xcad2 RNA was injected radially into four-cell embryos.
At the specified stages, injected and control embryos were processed for RNA extraction and RT-PCR analysis under identical conditions. (D)
Reduction of Xcad2 function allows siamois to activate organizer-specific genes during gastrulation. Sia/GR and Xcad2 antisense RNAs were
ventrally coinjected. Activation by dex was performed at the stages specified, and RNA extraction was performed at about stage 12 to all samples
together. In all experiments, the control samples are from uninjected embryos.
FIG. 6. Expression of organizer-specific genes during gastrulation depends on the competition between siamois and Xcad2. (A) Activation of
organizer-specific genes by siamois is also restricted to blastula stages. RT-PCR of RNA samples prepared at stage 11 from embryos injected with
sia/GR and treated with dex between stages 8 and 10.5. The genes tested were gsc, chordin, ADMP, BMP-4, and histone H4 as a loading control.
(B) The activity of siamois is restricted in explanted VMZ. Embryos were injected with sia/GR, en/Xcad2/GR, or a combination of both RNAs.
VMZ explants were prepared and treated with dex at the specified stages. RNA extraction was performed at about stage 12. (C) Xcad2 normally
downregulates the expression of organizer-specific genes during gastrulation. Antisense Xcad2 RNA was injected radially into four-cell embryos.
At the specified stages, injected and control embryos were processed for RNA extraction and RT-PCR analysis under identical conditions. (D)
Reduction of Xcad2 function allows siamois to activate organizer-specific genes during gastrulation. Sia/GR and Xcad2 antisense RNAs were
ventrally coinjected. Activation by dex was performed at the stages specified, and RNA extraction was performed at about stage 12 to all samples
together. In all experiments, the control samples are from uninjected embryos.
FIG. 7. Inhibition of secondary axis induction by Xcad2 can take place in the absence of protein synthesis. Embryos were injected with
sia/GR and Xcad2/GR mRNAs singly (A–H) or in combination (I–L) and treated with dexamethasone (dex) and cycloheximide (chx). At the
onset of gastrulation, the embryos were analyzed for changes in the expression of the organizer-specific gene, gsc. The blue arrows mark
the endogenous gsc expression; the red arrows point to the ectopic gsc expression.
