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First published online 3 January 2007
doi: 10.1242/dev.02739

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Wnt11/ß-catenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin

¹ Division of Developmental Biology, Cincinnati Children's Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
² Department of Biological Sciences, The University of Iowa, 257 Biology Building, Iowa City, IA 52242-1324, USA.

View larger version (74K): [in this window] [in a new window]   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 Xsox17 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.).

View larger version (18K): [in this window] [in a new window]   Fig. 2. LRP6 and Wnt11 depletion cause similar effects on dorsal gene expression. The expression of Wnt target genes (siamois, Xnr3) assayed by real-time RT-PCR at stage E9.5-E10.25 in sibling embryos depleted of Wnt11 or LRP6. Expression of Wnt target genes was reduced in both cases, whereas that of Xvent1, Xvent2 and BMP4 were not.

View larger version (67K): [in this window] [in a new window]   Fig. 3. LRP6 is downstream of Wnt11 and upstream of ß-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 ß-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. ß-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.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Wnt11-LRP6 signaling regulates axin levels in the early embryo. (A) Western blot of embryos derived from wild-type and LRP6-depleted oocytes (LRP6-) that were frozen at the 8-cell (stage 4) and early blastula (stage 7) stages and probed with an affinity purified axin polyclonal antibody. Blots were stripped and re-probed for ß-catenin, and -tubulin antibody was used as a loading control. Axin levels were increased in LRP6-depleted embryos (arrow; increased by 47% compared with control). ß-catenin levels were decreased by LRP6 depletion (arrowhead; decreased by 17% compared with controls). (B) Western blot of embryos derived from wild-type and Wnt11-depleted oocytes (Wnt11-) that were frozen at stage 4 and probed with an affinity purified axin polyclonal antibody. Blots were stripped and re-probed for ß-catenin. Axin levels were increased (arrow; 46% compared to control) in Wnt11-depleted embryos and ß-catenin levels were decreased (arrowhead; 23% compared with control). (C) A model of LRP6 function in the axis-forming pathway. (Ci) Sperm activates the cortical rotation movements of the first cell cycle, leading to a dorsal asymmetry of Wnt11 mRNA and protein, which causes increased Wnt11 signaling dorsally (purple arrows). (Cii) By the 8-cell stage, Wnt11 protein is secreted, mostly by dorsal vegetal cells, and binds to LRP6. Signaling is shown between a dorsal vegetal and animal cell, although LRP6 is likely to be present in both cells, and autocrine signaling may occur. In LRP6-depleted embryos, Wnt11 molecules are secreted dorsally but are unable to activate the pathway. (Ciii) An enlargement of the animal cells shown in Fig. 3Cii at the 8-cell stage. Association of Wnt11 with the extracellular domain of LRP6 causes the degradation of axin. ß-catenin enters nuclei and binds to Xenopus Tcf3 on the dorsal side. In LRP6-depleted embryos, Wnt11 molecules do not bind LRP6, so axin increases in concentration and ß-catenin is degraded to the same extent on dorsal and ventral sides, and does not enter nuclei.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Testing the predictions of the axin-degradation model of axis formation. (A) LRP6 is not a dorsally localized mRNA. Real-time RT-PCR analysis of groups of two wild-type embryos, and four dorsal- or ventral-half embryos at the 32-cell stage shows that endogenous LRP6 mRNA was not enriched dorsally, in comparison with Wnt11 mRNA. Data from two separate sets of embryos is shown. (B,C) Axin protein is less abundant dorsally than ventrally in wild-type 8-cell-stage embryos. (B) Total axin protein in western blots of ten dorsal- and ten ventral-half embryos at stage 4. Axin levels were reduced dorsally compared with ventrally. Blots were stripped and re-probed for ß-catenin levels. (C) Total axin protein levels in a series of sibling embryos from the 8- to the 64-cell stage. Only at the 8-cell stage was axin at a low level dorsally compared with ventrally. ß-catenin was enriched dorsally compared with ventrally throughout the 16- to 64-cell stage. (D) The dorsal enrichment of ß-catenin is lost in LRP6-depleted embryos. Two experiments showing western blots of ten dorsal or ten ventral halves of wild-type and LRP6-depleted embryos hemisected and frozen at the 32-cell stage, and probed with a ß-catenin polyclonal antibody. ß-catenin levels were enriched dorsally in control dorsal halves compared to ventral halves (32% more in experiment 2), and this difference was lost in LRP6-depleted embryos (dorsal halves have 8% less ß-catenin in experiment 2). (E) Embryos depleted of LRP6 and axin are less dorsalized than axin-depleted embryos. Real-time RT PCR analysis of the expression of Wnt target genes at the early gastrula stages in wild-type control embryos and embryos depleted of LRP6, axin or both LRP6 and axin. Axin depletion causes the upregulation, which is partially rescued by the depletion of LRP6, of Xnr3 and siamois.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Wnt11-LRP6 signaling regulates axin levels in the stage-6 oocyte. (A) Western blots of wild-type control and LRP6-depleted (LRP6-) stage-6 oocytes frozen after 3 days in culture and probed with an affinity purified axin polyclonal antibody. Axin levels were increased in LRP6-depleted oocytes compared with controls (*36% and 46% increases in experiment 1 and 2, respectively). (B) Western blots of wild-type, manually defolliculated oocytes (Control) and collagenase-treated oocytes frozen after 3 days in culture and probed with an axin antibody. Axin levels were unchanged as a result of culture without the follicle cell layer. Two repeats of the experiment are shown. (C) Western blots of wild-type and Wnt11-depleted (Wnt11-) stage-6 oocytes frozen after 3 days in culture and probed with an axin antibody. Axin levels were increased in Wnt11-depleted oocytes compared with controls (increased by 128%; range 10-128% in three experiments). (D) Western blots of wild-type control and LRP6-depleted (LRP6-) stage-6 oocytes that were frozen after 3 days in culture and probed with a ß-catenin antibody. Oocytes were non-matured or stimulated with progesterone and cultured for 8 hours before freezing. ß-catenin was expressed at low levels in oocytes before maturation, compared with the levels after maturation. LRP6 depletion reduced the amount of ß-catenin protein in matured oocytes (23% less ß-catenin compared with controls). (E) Western blots of wild-type and axin-depleted (Axin-) stage-6 oocytes that were frozen after 3 days in culture and probed with a ß-catenin antibody. ß-catenin levels were increased in axin-depleted oocytes compared with controls (99% increase compared with controls). (F) A model for steady-state canonical Wnt signaling. Axin is continually synthesized in full-grown, non-matured oocytes and degrades cytoplasmic ß-catenin. Wnt11 is also continually synthesized and secreted, binds to LRP6, and degrades some axin protein. Thus, a steady-state level of axin is maintained by Wnt signaling, which, in turn, keeps ß-catenin at a low level in the cytoplasm. In LRP6-depleted oocytes, Wnt11 is secreted, but signals cannot be transduced. Thus, axin levels increase, and ß-catenin is further reduced.

View larger version (57K): [in this window] [in a new window]   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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