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Dev Biol
2001 Jun 01;2341:42-54. doi: 10.1006/dbio.2001.0253.
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Axis induction by wnt signaling: Target promoter responsiveness regulates competence.
Darken RS
,
Wilson PA
.
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The modulation of inductive competence is a major theme in embryonic development, but, in most cases, the underlying mechanisms are not well understood. In principle, the capacity of extracellular signals to elicit particular responses could be regulated by changes in cell surface receptors, in intracellular signaling pathways, or in the responsiveness of individual target gene promoters. As an example of regulated competence, we have examined dorsal axis induction in Xenopus embryos by Wnt signaling. Competence of Wnt proteins such as Xwnt-8 to induce an ectopic axis or the dorsal early response genes siamois and Xnr3 is lost by the onset of gastrulation, when these same ligands now produce a distinct set of "late" effects, including anterior truncation and induction of the midbrain/hindbrain marker engrailed-2. Although other Wnts apparently make use of alternative signaling mechanisms, we demonstrate that late-expressed Xwnt-8 continues to employ the canonical Wnt signaling pathway used earlier in dorsal axis induction, stabilizing cytosolic beta-catenin, and activating gene expression through Tcf/Lef transcription factors. Moreover, an activated, hormone-inducible version of XTcf-3 (TVGR) that can reproduce both early and late Wnt responses when activated at appropriate stages becomes unable to induce siamois and secondary axes at the same time as Wnt ligands themselves. Finally, we show that TVGR also loses the ability to induce expression of a reporter construct containing a small fragment of the siamois promoter, implying that this fragment contains sequences governing the loss of Wnt responsiveness before gastrulation. Together, these results argue that the competence of Wnts to induce a dorsal axis is lost in the nucleus, as a result of changes in the responsiveness of target promoters.
FIG. 1. Wnts cease to induce siamois or Xnr3 expression by the start of gastrulation. (A) RT-PCR analysis of animal cap explants shows
that Xwnt-8 RNA (20 pg) but not DNA (10–200 pg) induces the dorsal target genes siamois and Xnr3. Explants were isolated at early stage
9 and harvested at stage 10.25 in pools of 5. Xbra is a general mesodermal marker; EF1-a is an ubiquitously expressed message. RT-: mock
reverse-transcribed whole embryo sample. (B) Animal cap explants express siamois and Xnr3 after exposure to conditioned medium (CM)
from Wnt-3A-transfected L cells at stage 7 or 8, but not when exposed at stage 10. W, Wnt-3A conditioned medium; C, control conditioned
medium. Samples were harvested at stage 10.5 (2 h after stage 10 exposure). (C) In contrast, when this conditioned medium is applied to
noggin-injected caps at stage 10, it strongly induces the late Wnt response gene engrailed-2 (Eng-2). Caps were harvested at stage 18. Muscle
actin (MA) is a mesodermal marker. None of the markers tested were induced by control medium from untransfected L cells.
FIG. 2. Xwnt-8 plasmid activates the b-catenin/Tcf pathway. (A)
Post MBT Xwnt-8 expression causes accumulation of cytosolic
b-catenin. b-Catenin levels were examined by Western blot in
animal caps from embryos injected in the animal hemisphere (4/4
cells) with a total of 20 pg of Xwnt-8 RNA or 20 or 100 pg of Xwnt-8
DNA. Pools of 10 caps were harvested at stage 10 and incubated
with concanavalin A (Con A) beads to remove cadherin-bound
b-catenin (see Materials and Methods); the resulting supernatant
(SN) is enriched for free b-catenin. Total levels of b-catenin from
these samples before Con A treatment are also shown. (B) Xwnt-8
DNA induction of engrailed-2 (Eng-2) is blocked by DN XTcf-3.
Animal caps from embryos coinjected with 250 pg noggin RNA and
10 pg of Xwnt-8 DNA with or without DN XTcf-3 (500 pg) RNA
were harvested at stage 17/18 and analyzed by RT-PCR. Animal
caps from embryos coinjected with 250 pg noggin RNA and 20 pg
VP16-XTcf-3 (TV) DNA were harvested and analyzed in the same
manner.
FIG. 3. Activated, hormone-inducible XTcf-3 (TVGR) induces second axes and siamois expression during cleavage stages but not at the
start of gastrulation. (A) XTcf-3 constructs. Dominant-negative XTcf-3 (DN XTcf-3) lacks the b-catenin-binding domain (b-cat. BD). In the
activated form XTcf-3-VP16 (TV), this domain is replaced by the activation domain (AD) from VP16. An inducible version (TVGR) was
constructed by added the hormone binding domain of the glucocorticoid receptor (Gluc. Rec.) to the C terminus. See Materials and Methods
for details. (B, C) Ventral marginal zone injection of 2 pg of TVGR (2/4 cells) has no significant effect when the inducing hormone
dexamethasone (dex) is not added or added at stage 10. Embryos induced during cleavage stages (4 cell), however, develop second axes at
a high frequency. Results from a representative experiment are graphed in (C); typical individuals are shown in (B) at stage 45. Secondary
axes that contained at least one eye were scored as complete. Each treatment pool contained approximately 30 embryos. (D) Siamois and
Xnr3 are expressed in animal caps from embryos injected with 2 pg TVGR when the construct is induced during cleavage stages, but not
when hormone is either not provided or is added at stage 10. These genes are not expressed by animal caps from uninjected embryos (UI),
whether or not these are exposed to dexamethasone. Explants were cut at stage 9 and harvested for RT-PCR at stage 11–11.5.
FIG. 4. TVGR RNA and protein persist through gastrula stages.
(A) RT-PCR analysis shows that injected TVGR RNA is present
through gastrula stages. Embryos were injected in the animal
hemisphere with a total of 2 pg of TVGR RNA and harvested at
various stages in pools of 5. Uninjected embryos were harvested at
stage 10. ODC is an ubiquitously expressed message. (B) TVGR
protein is present at least through late gastrula stages. RNA
encoding myc-tagged TVGR (TVGR-MT) was injected as in (A), and
embryos were harvested in pools of 10. Western blot analysis using
a myc antibody shows that TVGR protein persists through stage
12.5. As a loading control, this blot was reprobed with an antibody
for b-catenin.
FIG. 5. TVGR is still active at gastrula stages and can reproduce late Xwnt-8 effects. (A) TVGR produces a late embryonic phenotype
similar to that seen with Xwnt-8 plasmid. TVGR RNA (8 pg) was injected into dorsal/animal blastomeres at the 8-cell stage; inducing
hormone was added at stage 10. Representative uninjected, uninduced (no dex), and stage-10-induced embryos are shown here at stage 44.
An embryo injected in the same location with 100 pg of Xwnt-8 DNA is shown for comparison. (B) In the presence of noggin, TVGR turns
on expression of engrailed-2 and krox-20 when activated at either cleavage or gastrula stages. Animal caps were cut from embryos injected
with 250 pg noggin RNA (Ng) or coinjected with noggin and 2 pg of TVGR RNA, harvested at stage 18, and analyzed by RT-PCR. (C) The
addition of noggin RNA does not affect TVGR’s ability to turn on expression of siamois and Xnr3 when induced early but not late (compare
with Fig. 3D). Embryos were treated as in (B) but harvested at stage 11.
FIG. 6. TVGR activates the siamois promoter when induced early
but not late. A luciferase reporter plasmid containing a 0.8-kb
fragment of the siamois promoter was injected into the ventral/
animal region of four cell embryos with or without 2 pg of TVGR
RNA. Embryos injected with both constructs were then induced
with dexamethasone at cleavage stages, at stage 10, or not at all.
Three sets of four embryos were harvested at stage 12.5 for each
condition. A representative experiment is shown.
