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Figure 1. Truncated Wnt protein sequences. Alignment of the amino acid sequences for Dwnt-1 (wingless), mouse Wnt-1, and
Xwnt-8. Periods represent gaps in the sequence alignment; the position of conserved cysteine residues are indicated by the letter C
below the aligned sequences. The underlined letters represent the sequence of the truncated protein encoded by the Dwnt-1 mutant
alleles $84 and CE7 and the amino acid sequence of dnWnt-1 and dnXwnt-8, truncated Wnt-1 and Xwnt-8 proteins, respectively, with
dominant-negative activity. A truncation of Xwnt-8 at position 230, which results in a 30-amino-acid-shorter protein than dnXwnt-8,
had only weak inhibitory activity.
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Figure 2. dnXwnt-8 inhibits gsc induction and axis duplication
by Wnts. Development of tadpoles (A, C,E, G, anterior to the
left) and gsc in situ hybridization of gastrula-stage embryos
(B,D,F,H). Embryos were injected at the four-cell stage with
-300 pg of control RNA (A-D) or dnXwnt-8 RNA (E-H) into
each ventral blastomere and again at the 8- to 16-cell stage into
the ventral marginal zone with -25 pg of RNA encoding
Xwnt-8 (C-F) or -60 pg of A[3-catenin RNA (G,H). The RNA
amounts were based on first establishing the lowest amount of
each required to elicit a duplication of the axis in most embryos.
Embryos injected with control RNA develop with a single head
{A) and a single domain of gsc expression (B, indicated by arrowhead).
When injection of control RNA is followed by a second
injection of RNA encoding wild-type Xwnt-8, embryos develop
with a duplication of the embryonic axis (C, two heads
indicated by arrowheads) and with an ectopic domain of gsc
expression (D, two gsc expression domains indicated by arrowheads).
When dnXwnt-8 RNA injection precedes that of RNA
encoding wild-type Xwnt-8, axis duplication is repressed; embryos
develop with one head (E) and with a single domain of gsc
expression (F, indicated by arrowhead). Injection of dnXwnt-8
RNA does not affect the axis duplicating activity of subsequently
injected A[3-catenin RNA, and tadpoles develop with
two heads (G, indicated by arrowheads) and with an ectopic
domain of gsc expression (H, two gsc expression domains indicated
by arrowhead). Note that dnXwnt-8 RNA injections specifically
inhibit Wnt-induced axis duplication (see also Table 2).
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Figure 3. dnXwnt-8 blocks embryonic responses
to Xwnt-8 in a cell nonautonomous
manner. (A,B) Embryos were first injected at the
four-cell stage with either -300 pg of control
RNA (A) or dnXwnt-8 RNA (B) into the marginal
zone of both ventral blastomeres and
again at the 16-cell stage (A,B) into a single ventral
marginal blastomere with -50 pg of Xwnt-
8myc RNA. At gastrula stage 10, embryos were
fixed and stained for expression of the c-myc
epitope (dark brown staining), which revealed
that dnXwnt-8 does not affect the expression of
Xwnt-8myc [cf. B with A, control), although it
blocks its axis-duplicating activity (not shown,
but identical to Fig. 2E, F). (C) Drawing (Nieuwkoop
and Faber 1967) explaining the design of
the nonautonomy experiment in D-F, with five
cells (red hatched) being injected at the early
16-cell stage (indicated by arrows) with lineage
tracer (phenol red and 13-galactosidase RNA) and
~250 pg of RNAs [either dnXwnt-8 RNA or a
control RNA). Then, at the late 16-cell stage the
central cell [white cell within the red hatched
area indicated by an asterisk (*)] was injected
with -25 pg of Xwnt-8myc RNA. (D) Blastulastage
embryos injected as in C were processed
for [3-galactosidase activity (blue) and c-myc
epitope localization (light brown). Note that the
blue [3-galactosidase RNA-injected cells [indicated
by arrowhead) do not overlap with the
brown Xwnt-8myc RNA-injected cells [indicated
by an asterisk (*)]. (E) An endogenous and
an ectopic domain of gsc expression [solid arrowheads) are evident when control RNAs are injected into the five red cells as in C,
followed by Xwnt-8myc in the central cell. (F) In contrast, injection of dnXwnt-8 into the five red cells as in C, followed by Xwnt-8myc
into the central cell, had greatly reduced or no ectopic gsc expression (the shaded arrowhead indicates an ectopic domain of reduced
gsc expression in the embryo at right). Note that dnXwnt-8 is able to block the induction of gsc (cf. F with E, control), even if the
proteins are expressed in exclusive populations of cells, as shown with the complementary staining in D.
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Figure 4. dnXwnt-8 inhibits Xwnt-8 expressed after the blastula
stage. (A) RT-PCR analysis of gene expression in control
stage 12 animal explants for a control marker (histone H4),
a dorsal marginal zone marker (gsc), and a ventral marginal
zone marker (Xpo). Embryos were injected at the two-cell stage
into the animal pole of both blastomeres with -800 pg of
control RNA or dnXwnt-8 RNA (as indicated at top) and -80 pg
of CSKA Xwnt-8 plasmid DNA (where indicated at top), and
animal caps were explanted and treated with activin (where
indicated by + ) and subjected to analysis with RT-PCR. (B-D)
Histological sections through animal pole explants. Embryos
were injected at the two-cell stage into the animal pole of
both blastomeres with -200 pg of control RNA (B), with -200
pg of control RNA, and -30 pg of CSKA Xwnt-8 plasmid (C), or
-200 pg of dnXwnt-8 RNA and -30 pg of CSKA Xwnt-8 plasmid
(D). Animal pole explants were prepared at stage 8, then
cultured until sibling embryos reached stages 38-40. Note that
dnXwnt-8 RNA represses the formation of vesicular structures
induced by CSKA Xwnt-8 (see Table 3A). (E-G) Stage 40 embryos
(anterior to the left and dorsal on the top) that developed
from embryos injected at the four-cell stage into the marginal
zone of both dorsal blastomeres with -200 pg of control RNA
(E), -200 pg of control RNA mixed with -30 pg of CSKA
Xwnt-8 plasmid (F), or -200 pg of dnXwnt-8 RNA mixed with
-30 pg of CSKA Xwnt-8 plasmid (G). Note that dnXwnt-8 interferes
with the Xwnt-8-mediated repression of dorsoanterior
structures such as cement gland (indicated by small arrowheads}
and eyes (see also Table 3B).
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Figure 5. dnXwnt-8-injected embryos develop
dorsal mesoderm but form defective somites.
(A,C) Injection of -3 ng of control RNA into
cleavage-stage embryos leads to development
of normal stage 36--38 embryos, both in terms
of external morphology {A, anterior to left, dorsal
on top) and internal anatomy, as examined
in histological cross section through the trunk
region (C, dorsal on topJ. (B,D) When 1.5-3 ng
of dnXwnt-8 RNA is injected, development is
highly aberrant, embryos develop with a shortened
axis as well as a relatively enlarged head
region (B), and histological cross section
through such embryos (D) reveals that the notochord
is enlarged and that somitic muscle tissue
is reduced and disorganized. (E) Injection
into the marginal zone of all four blastomeres
of four-cell embryos with 1.5-3 ng of RNAs
encoding either control protein (bottom) or
dnXwnt-8 {top) results in normal expression of
gsc. (F) Similarly, 1.5-3 ng of dnXwnt-8 (top)
does not significantly reduce the level of expression
of Xnr3 compared with control embryos
(bottom). (en)Endoderm; (no)notochord;
(nt) neural tube; {sin) somitic muscle.
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Figure 6. dnXwnt-8 blocks induction of
XMyoDa and Xpo in the ventral and lateral
marginal zone, whereas ectopic Xwnt-8 induces
ectopic expression of these markers in
the dorsal marginal zone. In situ hybridization
(purple staining) of stage 101/4-101/2 embryos
with probes for XMyoDa (A,C,E,G,I) and Xpo
(B,D,F,H,J). (A,B) Control RNA (-3 ng) injections
into the marginal zone of all four blastomeres
of four-cell embryos lead to normal expression
of both the lateral mesodermal marker
XMyoDa (A) and the ventrolateral mesodermal
marker Xpo (B). (C,D) When 0.8-3 pg of
dnXwnt-8 RNA is injected, expression of
XMyoDa is abolished (C) and localized Xpo
RNA staining is greatly reduced (D) (see Table
4A). (E,F) Overexpression of wild-type Xwnt-8
(CSKA Xwnt-8, -100 pg) in one side of the ventrolateral
mesoderm of dnXwnt-8 RNA (-800
pg)-injected embryos rescues the expression of
the mesodermal markers XMyoDa (E, indicated
by arrowheads) and Xpo (F, indicated by arrowheads)
predominantly on the side of injection
(see also Table 4B). (G-J) In a gain-of-function
analysis, CSKA Xwnt-8 DNA (-100 pg) was injected
into the marginal zone of both dorsal
blastomeres at the four-cell stage (L J) to ectopically
express Xwnt-8 in the dorsal prospective
mesoderm. Whereas ventral injected embryos
have the normal dorsal gap of XMyoDa expression
[G, indicated by an open arrowhead; vegetal
view (left) and dorsal view (right) of the
same embryo; see Table 4C], ectopic Xwnt-8
expression results in the ectopic expression of
XMyoDa in most dorsal prospective mesoderm
[L indicated by a solid arrowhead; vegetal view
(left) and dorsal view (right) of the same embryo].
Similarly, whereas uninjected embryos
show only weak Xpo staining on the dorsal side
[H, indicated by an open arrowhead; vegetal
view (left) and dorsal view (right) of the same
embryo; see also Table 4C], ectopic Xwnt-8 expression
also causes strong expression of Xpo in
the dorsal prospective mesoderm [J, indicated
by a solid arrowhead, vegetal view (left) and
dorsal view (right) of the same embryo].
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