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Figure 1. Synopsis of the 13-catenin mutants used in this work.
Prefix 13, untagged; prefix HT, HA-tagged; and prefix MT, myctagged.
13-Catenin sequence contains a gap (white case) between
repeats 10 and 11. See Materials and Methods for the~precise coding
sequence of each construct.
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Figure 2. Assay for 13-catenin signaling: axis duplication in Xenopus
embryos. (A) Example of tadpole with two complete body
axes, including two heads, obtained by injection of 0.75 ng of a
truncated 13-catenin mutant mRNA, MT10, in the ventral side of
a cleaving embryo. (B) Normal tadpole developed from an uninjected
embryo.
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Figure 3. Association of mutants
HT6-HT9 with endogenous
C-cadherin and APC.
(a) Expression of full-length
13-catenin (HT1) and mutants
HT6-HT9 in Xenopus embryos
injected with 3 ng
mRNA of each construct.
The mutants were detected
with a mixture of two pAb
raised against the NH2 and
COOH terminus of Xenopus
13-catenin, respectively. (b)
Detection of C-cadherin in
[3-catenin mutant immunoprecipitates
under mild conditions
(nonionic detergent).
Mutants were immunoprecipitated
with anti-HA mAb
12CA5 in the presence of 1%
NP-40. C-cadherin is associated
with HT1, HT8 and HT9, but not HT6 and HT7. (c) Detection
of C-cadherin in [3-catenin mutant immunoprecipitates under
harsher conditions (anionic detergent). Mutants were
immunoprecipitated with anti-HA mAb 12CA5 in the presence
of RIPA buffer, containing 0.2% SDS, 1% Triton X-100 and
0.5% deoxycholate. C-cadherin is associated with HT1 and HT9,
but not HT6, HT7, nor HT8. (d) Detection of APC in 13-catenin
mutant immunoprecipitates. Mutants were immunoprecipitated
with the anti-HA mAb 12CA5 in the presence of NP-40. C-cadherin
is associated with HT1, HT8 and HT9, but not HT6 and HT7.
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Figure 4. Lack of association between MT10 and C-cadherin. (a)
Expression of full-length 13-catenin (MT1) and MT10, containing
only the first nine armadillo repeats, in Xenopus embryos injected
in the two ventral blastomeres of a 4-cell stage embryo
with 2 x 3 ng mRNA. MT1 and MT10 were detected with the
anti-myc tag mAb 9E10.2. (b) Detection of MT1 and MT10 after
immunoprecipitation with anti-myc mAb 9E10.2 in the presence
of 1% NP-40. (c) Detection of C-cadherin in MT1 and MT10 immunoprecipitates
(1% NP-40). The first lane shows a control immunoprecipitation
from uninjected embryos. C-cadherin (arrow)
is associated only with full-length 13-catenin, but not MT10.
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Figure 5. Ventralization of Xenopus embryos by overexpression
of C-cadherin in the dorsal inducing region. (A) When two dorsal
blastomeres of 4-cell stage embryos were injected at the vegetal
pole with 4--5 ng C-cadherin mRNA, the embryos developed into
ventralized tadpoles (no head, diminished dorsal structures, hypertrophy
of ventral tissues). (B) After injections of the same
RNA in the ventral side, the embryos developed normally.
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Figure 6. Inhibition of 13-catenin-indueed axis duplication by coexpression
of C-cadherin. (A and C) Embryos injected with 0.3
ng full-length I~-catenin (HT1) mRNA developed duplicated axis,
visible at neurula stage by the two pigmented neural tubes (,4)
and at tadpole stage as two complete body axis, including two
heads. (B and D) Embryos coinjected with 0.3 ng HT1 and 5 ng
C-cadherin mRNA have only one axis (B) and develop into normal
tadpoles (D).
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Figure 7. Redistribution of fulllength
~3-catenin (MT1) but not
MT10 by coexpression of C-cadherin.
Full-length 13-catenin
(MT1) or MT10 (both 1.5 ng
mRNA), expressed alone (A
and C) or coexpressed with excess
C-cadherin (5 ng mRNA)
(B and D) were immunolocalized
at early gastrula stages by
indirect immunofluorescence on
frozen sections with anti-myc
mAb 9E10.2. (A '-D') HOECHST
staining to visualize nuclei on
the same sections. Cell-cell
boundaries are outlined with
dotted lines in panels C' and D '.
(A) Full-length MT1 expressed
alone is detected in the cytoplasm
(i.e., between the yolk
platelets, which appear as large
dark granules), and accumulates
at the plasma membrane
and in the nuclei (arrowheads).
(B) In the presence of excess
C-cadherin, MT1 is completely
redistributed to the plasma membrane
and intracellular vesicles
(arrows). Cytoplasm and nuclei
(arrowheads) are depleted. (C)
The mutant MT10 expressed
alone is localized in the cytoplasm
and the nuclei (arrowheads),
but not at the membrane.
The cell periphery often
appears brighter because it contains
less yolk. The peripheral
cytoplasmic staining closely
matches the irregular outline of
the yolk platelets (small arrows)
and can be unambiguously distinguished
from the sharper,
straight line of membrane staining
observed for full-length
13-catenin (A and B), endogenous
13-catenin and C-cadherin
(not shown, see Fagotto and
Gumbiner, 1994). (D) MT10 coexpressed
with C-cadherin is distributed
throughout the cytoplasm,
with no enrichment at the
cell membrane. Some punctate
intracellular staining is observed
(arrow). Arrowheads point to
nuclei. Bar, 20 t~m.
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Figure 8. Redistribution of full-length 13-catenin (MT1) but not
MT10 by coexpression of C-cadherin: cell fractionation. Late
blastula embryos expressing MT1 alone (a), MT1 with excess
C-cadherin (b), MT10 alone (c), and MT10 with excess C-cadherin
(d) were fractionated as described in Materials and Methods
into a membrane glycoprotein (cadherins) fraction (G); a
particulate noncadherin-bound fraction (P), and a soluble fraction
(S). MT1 and MT10 were detected on Western blots with
anti-myc mAb 9E10.2.
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