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Dev Biol
2003 Jan 01;2531:125-38. doi: 10.1006/dbio.2002.0867.
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Nodal signaling in Xenopus gastrulae is cell-autonomous and patterned by beta-catenin.
Hashimoto-Partyka MK
,
Yuge M
,
Cho KW
.
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The classical three-signal model of amphibian mesoderm induction and more recent modifications together propose that an activin-like signaling activity is uniformly distributed across the vegetal half of the Xenopus blastula and that this activity contributes to mesoderm induction. In support of this, we have previously shown that the activin-response element (DE) of the goosecoid promoter is uniformly activated across the vegetal half of midgastrula-stage embryos. Here, we further examine the nature of this activity by measuring DE activation by endogenous signals over time. We find that the spatiotemporal pattern of DE activation is much more dynamic than was previously appreciated and also conclude that DE(6X)Luc activity reflects endogenous nodal signaling in the embryo. Using both the DE(6X)Luc construct and endogenous Xbra and Xgsc expression as read-outs for nodal activity, and the cleavage-mutant version of Xnr2 (CmXnr2) to regionally suppress endogenous nodal activity, we demonstrate that nodal signals act cell-autonomously in Xenopus gastrulae. Nodal-expressing cells are unable to rescue either reporter gene activation or target gene expression in distant nodal-deficient cells, suggesting that nodals function at short range in this context. Finally, we show that DE activation by endogenous signals occurs in the absence of dorsal beta-catenin-mediated signaling, but that the timing of dorsal initiation is altered. We conclude that nodal signals in Xenopus gastrulae function cell autonomously at short ranges and that the spatiotemporal pattern of this signaling along the dorsoventral axis is regulated by maternal Wnt-like signaling.
FIG. 1. DE activation reflects a wave of activity that initiates
dorsally. Embryos were injected with the reporter construct 6X(DE)-
Luc into the C1 or C4 blastomere at the 32-cell stage and assayed
for reporter activity at indicated stages. No activity was detected
before stage 10. Data from a single representative experiment are
shown.
FIG. 2. Nodal signaling is required for endogenous DE activation.
CmXnr2 was injected subequatorially into the two dorsal blastomeres
at the 4-cell stage. Then, 6X(DE)Luc was injected into the
C1 blastomere at the 32-cell stage. Data from a single representative
experiment are shown.
FIG. 3. Ectopic Xnr expression activates 6X(DE)Luc in animal
caps. Xnr mRNAs and 6X(DE)Luc reporter were injected into the
animal pole of all cells at the four-cell stage (total amount of
mRNA per embryo is indicated). Caps were isolated at stage 8 and
allowed to develop until intact sibling stage 10.5, when they were
harvested and assayed for luciferase activity. Relative activities
cannot be compared as the different Xnrs are in different parent
plasmids.
FIG. 4. Possible mechanisms of 6X(DE)Luc activation.
FIG. 5. Blockage of endogenous nodal signaling has local effects.
CmXnr2 mRNA was injected subequatorially into the two dorsal
(A) or ventral (B) blastomeres at the 4-cell stage, and 6X(DE)Luc was
then injected into the C1 or C4 blastomeres at the 32-cell stage.
Embryos were harvested at stage 10.5 and luciferase activity was
assayed. In each case, data from a single representative experiment
are shown.
FIG. 6. DE activation occurs tissue-autonomously. (A) 6X(DE)Luc
was injected at the 32-cell stage into the C4 blastomere and VMZ
explants were harvested at late stage 7. Intact embryos or VMZ
explants were harvested at intact sibling stage 10 or 10.5, and
luciferase activity was assayed. Remains of the embryos from
which VMZ tissue was taken were assayed and were devoid of
reporter activity (data not shown). (B) Similar to (A), 6X(DE)Luc was
injected into the C1 blastomere and DMZ explants were assessed
for luciferase activity. In each case, data from a single representative
experiment are shown.
FIG. 7. Nodal signals are cell-autonomous in Xenopus gastrulae. CmXnr2 mRNA, together with rhodamine dextran, was injected into a
single cell in each of the B and C tiers at the 32-cell stage (300 pg per blastomere). Embryos were allowed to develop to gastrula stages, fixed,
and analyzed by whole-mount in situ hybridization for Xgsc (a–c andm–o) or Xbra (g–i, p–r) expression. Control embryos were injected with
rhodamine dextran only (d–f and j–l). All embryos injected with CmXnr2 mRNA disrupted either Xbra (n 100) or Xgsc (n 50) expression.
Injection into a single cell in the C tier at the 32-cell stage with CmXnr2 (300 pg per blastomere) and Xnr1 (300 pg per blastomere) mRNAs
(v–x) rescued Xgsc expression compared with embryos injected in the same experiment with only CmXnr2 mRNA (s–u). Rescue occurred
in 7 of 9 embryos in which the normal Xgsc expression domain overlapped with the rhodamine dextran-labeled clone of cells. Arrowhead
indicates disruption of dorsal lip formation, indicating that dorsal lip formation is perturbed by inheritance of CmXnr2 mRNA, although
Xgsc expression is restored. Endogenous Xnr3 expression persisted in CmXnr2-positive cells in 8 of 9 embryos in which the normal Xnr3
expression domain overlapped with the rhodamine dextran-labeled clone of cells (z–bb).
FIG. 8. NXtcf3 disturbs the initial dorsal–ventral pattern, but
not the final level of DE activation. A total of 1 ng of NXtcf3
mRNA was injected subequatorially into the two dorsal blastomeres
at the 4-cell stage, then the C1 or C4 blastomere was
injected at the 32-cell stage. Embryos were harvested at the
indicated stages and assayed for luciferase activity. To assess
NXtcf3 mRNA efficacy, activation of –357XtwnLuc (a known
target of -catenin-mediated Wnt signaling; Laurent et al., 1997)
was assayed and was suppressed to background levels in NXtcf3
injected relative to uninjected embryos (data not shown). The data
from a single representative experiment are shown.
FIG. 9. Transcripts of endogenous Xnr 1, 2, and 4 mRNA expression
persist in the absence of dorsal -catenin-mediated signaling.
A total of 1 ng of NXtcf3 mRNA was injected subequatorially into
the two dorsal blastomeres at the four-cell stage. Embryos were
harvested at the indicated stages and total RNA was collected for
RT-PCR analysis. NXtcf3-injected embryos that were allowed to
develop to uninjected sibling late tailbud stages were completely
ventralized according to the standard dorsoanterior index scale
(Kao and Elinson, 1988). DAI 0, 1, or 2 for 24/25 NXtcf3
mRNA-injected embryos.
