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Fig. 7. Exogenous ß-catenin is degraded in oocytes. (A)
Western blot of wild-type unmatured oocytes injected with 100 pg myc-tagged
ß-catenin mRNA (Con) and oocytes injected with 100 pg myc-tagged
ß-catenin mRNA and treated with the proteasome inhibitor MG132
(+MG132), frozen after 3 days in culture and probed with an anti-myc antibody.
Myc-tagged ß-catenin protein expression was enhanced in the presence of
the proteasome inhibitor (44% increase compared with control levels),
suggesting that ß-catenin is degraded by a proteasomal pathway.
(B) Western blot of wild-type oocytes injected with 100 pg myc-tagged
ß-catenin mRNA (ßcat-myc) compared to oocytes co-injected
with 100 pg myc-tagged ß-catenin mRNA together with 400 pg
LRP6 mRNA (ßcat-myc +LRP6) frozen after 3 days in culture and
probed with an anti-myc antibody. One group of oocytes was matured for 8 hours
after progesterone stimulation (Matured oocyte) before freezing. Myc-tagged
ß-catenin protein expression was enhanced in the presence of LRP6, both
before (increased by 32%) and after (increased by 62%) maturation. (C)
The ventralized phenotype of LRP6-depleted embryos (bottom left; 6/7
ventralized) was rescued by 50 pg ß-catenin mRNA when injected
at the 4-cell stage (top right; 0/15 ventralized), but not by injection of 50
pg ß-catenin mRNA in the stage-6 oocyte before maturation
(bottom right; 7/7 ventralized; 16 died at cleavage stage). (D) The
expression of the Wnt target genes siamois and Xnr3 assayed
by real-time RT-PCR at the beginning and early gastrula stages (stage 10 and
10.5) in embryos that were siblings to those shown in C. Expression of Wnt
target genes was severely reduced in LRP6-depleted embryos, and were partially
rescued by the injection of ß-catenin mRNA after, but not
before, fertilization. (E) Western blot of sibling embryos to those
shown in C, frozen at the 64-cell stage and analyzed for total ß-catenin
protein. Injection of LRP6 oligo reduced ß-catenin levels, which was
rescued by the injection of ß-catenin mRNA at the 4-cell stage,
but not by injection of ß-catenin mRNA in the oocyte. (F)
Stabilized ß-catenin mRNA (ptßcat-myc, 20 pg) rescued LRP6
depletion when injected into oocytes (8/12 cases dorsalized), in comparison to
20 pg wild-type ß-catenin mRNA (8/8 ventralized). Embryos at
tailbud stage. (G) Western blot of lysates of LRP6-depleted oocytes
that were siblings to those used in F. Protected ß-catenin protein
accumulates after 48 hours in culture, more so than wild type ß-catenin.
(H) The expression of the Wnt target genes siamois and
Xnr3, assayed by real-time RT-PCR at stage 9.5 in sibling embryos to
those in F. Wnt target genes were severely reduced in expression in
LRP6-depleted embryos, and were partially rescued by the injection of
ptß-catenin mRNA before fertilization. Xfz7 mRNA did
not rescue siamois and Xnr3 expression.
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