February 1, 2007;
Wnt11/beta-catenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin.
Current models of canonical Wnt signaling assume that a pathway is active if beta-catenin becomes nuclearly localized and Wnt target genes are transcribed. We show that, in Xenopus, maternal LRP6
is essential in such a pathway, playing a pivotal role in causing expression of the organizer
, and in establishing the dorsal axis. We provide evidence that LRP6
acts by degrading axin protein during the early cleavage
stage of development. In the full-grown oocyte
, before maturation, we find that axin levels are also regulated by Wnt11
. In the oocyte
regulates axin to maintain beta-catenin at a low level, while in the embryo
, asymmetrical Wnt11
signaling stabilizes beta-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality.
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Fig. 1. LRP6 is necessary for dorsal axis formation. (A) Two wild-type whole oocytes, and four animal and vegetal halves, or six animal, equatorial and vegetal thirds, were frozen and assayed by real-time RT-PCR for the expression of LRP6 and Wnt11 mRNAs. LRP6 mRNA was expressed throughout the oocytes. (B) Groups of two control and LRP6 antisense oligo-injected oocytes (2.5, 5 and 10 ng oligo) were incubated for 24 hours, and assayed for the expression of LRP6 and the related LRP5 mRNA. LRP6, but not LRP5, mRNA levels were reduced by the antisense oligo. (C) The phenotype of tailbud-stage embryos derived from oocytes injected with 500 pg mouse LRP6 mRNA (dorsalized) or 5 ng LRP6 antisense oligo (LRP6-; ventralized). (D) The LRP6-depleted ventralized phenotype at tailbud stage caused by the injection of LRP6 antisense oligo (middle row; 3 ng antisense oligo injected) was rescued by the injection of 100 pg mouse LRP6 mRNA (bottom row). LRP6 mRNA was injected 48 hours after oligo injection and 24 hours before oocyte maturation. (E) The expression of Wnt target genes (siamois, Xnr3, chordin and goosecoid) assayed by real-time RT-PCR at the early gastrula stage in sibling embryos to those in D. (F) The expression of endoderm marker Xsox17α and ventral mesodermal marker Xwnt8 was delayed in LRP6-depleted embryos at the late blastula stage, but reached wild-type levels of expression by the early gastrula stage in LRP6-depleted embryos. By comparison, Xnr3 expression remained off in LRP6-depleted embryos. (G) Transverse sections of tailbud-stage embryos derived from a control, LRP6-depleted and LRP6- + LRP6-mRNA-injected oocytes. LRP6 depletion resulted in a lack of dorsal structures, which was rescued by LRP6-mRNA injection. (H) TOPflash reporter activation in control and LRP6-depleted late blastulae after injection into two dorsal cells at the 4-cell stage. Error bars indicate the standard deviation from the mean (s.e.m.).
Fig. 3. LRP6 is downstream of Wnt11 and upstream of β-catenin in the axis-forming pathway. (A) The ventralized Wnt11-depletion phenotype at late neurula stage (top row; 9/9 cases ventralized), was rescued by 75 pg Wnt11 mRNA (second row; 0/8 cases ventralized), and by 75 pg LRP6 mRNA (third row; 0/12 cases ventralized), injected 24 hours before oocyte maturation. Additionally, 300 pg of LRP6 mRNA also rescued Wnt11-depleted embryos, but caused a dorsalized phenotype (fourth row; 0/8 cases ventralized). (B) The expression of Wnt target genes at the early gastrula stage (stage 10) in sibling embryos to those shown in A, assayed by real-time RT-PCR. LRP6 mRNA rescued the expression of siamois and Xnr3 in Wnt11-depleted embryos. (C) The ventralized LRP6-depletion phenotype at tailbud stage (red embryos; 9/9 cases ventralized) was not rescued by the injection of 100 pg Wnt11 mRNA 24 hours prior to maturation (bottom row; 6/6 cases ventralized). (D) The expression of Wnt target genes at the late blastula stage (stage 9.5) and early gastrula stages (stage 10) in embryos that were siblings to those shown in C, assayed by real-time RT-PCR. Wnt11 was not able to rescue the expression of Wnt target genes in LRP6-depleted embryos. (E) The ventralized LRP6-depletion phenotype (7/7 cases ventralized) shown at tailbud stage was rescued by the injection of 50 pg β-catenin mRNA into one dorsal cell at the 4-cell stage (6/20 cases ventralized). (F) The expression of Wnt target genes at the early gastrula stages (stage 10) in embryos that were siblings to those shown in C, assayed by real-time RT-PCR. β-catenin rescued the expression of Wnt target genes in LRP6-depleted embryos. (G) Lrp6-N-Myc interacts with Wnt11-HA in co-immunoprecipitation assays. Lrp6-N-Myc (1.5 ng) and Wnt11-HA (500 pg) were injected into two different blastomeres at the 4- to 8-cell stage. Embryos were harvested at stage 10.5. Panel on the right is a western blot of the embryo lysates, blotted with both anti-myc and anti-HA antibody. Panel on the left shows the result of the lysates immunoprecipitated with anti-HA antibody and blotted with anti-myc antibody. IgG band is indicated on the bottom of the left panel.
Fig. 7. Exogenous β-catenin is degraded in oocytes. (A) Western blot of wild-type unmatured oocytes injected with 100 pg myc-taggedβ -catenin mRNA (Con) and oocytes injected with 100 pg myc-taggedβ -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 β-catenin protein expression was enhanced in the presence of the proteasome inhibitor (44% increase compared with control levels), suggesting that β-catenin is degraded by a proteasomal pathway. (B) Western blot of wild-type oocytes injected with 100 pg myc-taggedβ -catenin mRNA (βcat-myc) compared to oocytes co-injected with 100 pg myc-tagged β-catenin mRNA together with 400 pg LRP6 mRNA (βcat-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-taggedβ -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 β-catenin mRNA when injected at the 4-cell stage (top right; 0/15 ventralized), but not by injection of 50 pg β-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 β-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 β-catenin protein. Injection of LRP6 oligo reduced β-catenin levels, which was rescued by the injection of β-catenin mRNA at the 4-cell stage, but not by injection of β-catenin mRNA in the oocyte. (F) Stabilized β-catenin mRNA (ptβcat-myc, 20 pg) rescued LRP6 depletion when injected into oocytes (8/12 cases dorsalized), in comparison to 20 pg wild-type β-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 β-catenin protein accumulates after 48 hours in culture, more so than wild type β-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 ptβ-catenin mRNA before fertilization. Xfz7 mRNA did not rescue siamois and Xnr3 expression.