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First published online 1 October 2003
doi: 10.1242/dev.00762

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PP2A:B56{epsilon} is required for Wnt/ß-catenin signaling during embryonic development

Jing Yang, Jinling Wu, Change Tan and Peter S. Klein*

Department of Medicine (Hematology-Oncology) and Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, 364 Clinical Research Building, 415 Curie Blvd, Philadelphia, PA 19104, USA



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  		Fig. 1. Expression pattern of PP2A:B56{epsilon} during early embryonic development of Xenopus. (A,B) Expression of PP2A:B56{epsilon} in the early blastula. (A) PP2A:B56{epsilon} expression along the dorsal-ventral axis: 500-cell stage embryos were dissected into dorsal and ventral halves. RNA was prepared from each half. Expression of PP2A:B56{epsilon} was analyzed by RT-PCR. EF1{alpha} was used as loading control. Xnr6 (dorsally restricted expression) was used as a control for accurate dissection. (B) PP2A:B56{epsilon} expression along the animal-vegetal axis: 500-cell stage embryos were dissected into animal, marginal zone, and vegetal pieces. RNA was prepared from each and expression of PP2A:B56{epsilon} was analyzed by RT-PCR. VegT (vegetal pole restricted expression) was used as a control for accurate dissection. (C-F) PP2A:B56{epsilon} expression as determined by in situ hybridization of hemisections. (C) PP2A:B56{epsilon} expression in the dorsal marginal zone of an early gastrula stage (stage 10) embryo. (D. Expression in marginal zone of a midgastrula (stage 11) embryo; (E) expression in neural ectoderm of a midneurula stage (stage 15) embryo. (F) Higher magnification of E showing PP2A:B56{epsilon} expression in the anterior neural ectoderm. (G) PP2A:B56{epsilon} expression in a stage 33 tadpole. Arrows in E and F indicate the anterior edge of PP2A:B56{epsilon} expression domain in the neural ectoderm.

 


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  		Fig. 2. Maternal PP2A:B56{epsilon} is required for dorsal development. (A) Morpholino antisense oligonucleotide directed against PP2A:B56{epsilon} blocks the translation of flag-tagged PP2A:B56{epsilon} RNA, but not flag-tagged PP2A:B56{epsilon}-c, in Xenopus oocytes, as determined by western blotting. EGFP mRNA was coinjected and western blotted with anti-GFP as a control. (B) Vegetal view of an uninjected host-transfer embryo at the onset of gastrulation (stage 10+), showing formation of the dorsal lip of the blastopore. (C) Vegetal view of a maternal PP2A:B56{epsilon}-depleted embryo also at stage 10+, showing absence of dorsal lip. (D) RT-PCR showing the gene expression profile in maternal PP2A:B56{epsilon} and ß-catenin-depleted embryos at midgastrula stage (stage 11). (E) RT-PCR showing reduced expression of Xnr5 and Xnr6 at the 1,000-cell stage and reduced expression of siamois and Xnr3 at gastrula stage (stage 10). (F) RT-PCR showing reduction of dorsal gene expression in maternal PP2A:B56{epsilon}-depleted embryos could be rescued by PP2A:B56{epsilon}-c mRNA.

 


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  		Fig. 3. PP2A:B56{epsilon} is required for Wnt/ß-catenin signaling. (A) Western blot showing endogenous cytoplasmic ß-catenin in maternal PP2A:B56{epsilon}-depleted embryos at stage 9. hn-RNP was used as a loading control. (Shown here is a representative result from three different experiments). (B) RT-PCR showing that injection of mRNA encoding VP16-Xtcf3 (2 pg), ß-catenin (500 pg), GID (1 ng), and Xdsh (2 ng) rescue siamois and Xnr3 expression in maternal PP2A:B56{epsilon}-depleted embryos at stage 10, while Wnt8b (20 pg) does not. ODC was used as loading control. Wnt8b (20 pg), Xdsh (2 ng), GID (1 ng), ß-catenin (500 pg), and VP16-Xtcf3 (2 pg) induce similar percentages of secondary axis when injected ventrally into normal embryos at the 4-cell stage (not shown).

 


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  		Fig. 4. PP2A:B56{epsilon} is required for MHB and anterior hindbrain gene expression in stage 16 neurulae. (A) The expression of otx2, en2, wnt1, krox20 and hoxB9 at the mid-neurula stage (stage 16) in control embryos (upper panels) and embryos injected with 2.5 ng PP2A:B56{epsilon} morpholino (lower panels) into both dorsal animal blastomeres at the 8-cell stage. For otx2, en2 and wnt1: anterior view; krox20 and hoxB9: dorsal view, with anterior upper most). (B) The expression of en2 and krox20 in embryos injected unilaterally with PP2A:B56{epsilon} morpholino (middle column), or PP2A:B56{epsilon} morpholino together with PP2A:B56{epsilon}-c mRNA (right column). All embryos were co-injected with nuclear ß-galactosidase mRNA as a lineage tracer. Control (left column) is n-ß-gal alone.

 


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  		Fig. 5. Regulation of en2 expression and ß-catenin stability by PP2A:B56{epsilon} in the neural ectoderm at stage 14. (A) RT-PCR showing that en2 is reduced by PP2A:B56{epsilon} morpholino at stage 14. (B-E) Whole-mount in situ hybridization showing pax2.1 (B,C) and XHR1 (D,E) are not reduced by PP2A:B56{epsilon} morpholino injection at stage 14. (B,D) Control embryos; (C,E) embryos injected with PP2A:B56{epsilon} morpholino unilaterally. (F) Western blot showing injected ß-catenin was less stable in the neural ectoderm of PP2A:B56{epsilon} knockdown embryos. EFGP was used as a control for both injection and loading.

 


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  		Fig. 6. PP2A:B56{epsilon} is required for neural tube closure and formation of head structures. (A-C) Dorsal views of stage 16 embryos. (A) Control; (B) embryo injected with 2.5 ng of PP2A:B56{epsilon} morpholino bilaterally; (C) embryo injected with 2.5 ng of PP2A:B56{epsilon} morpholino and 20 pg of PP2A:B56{epsilon}-c mRNA bilaterally. (D-F) Anterior view of stage 16 embryos. (D) Control embryo; (E) embryo injected with 2.5 ng of PP2A:B56{epsilon} morpholino bilaterally; (F) embryo injected with 2.5 ng of PP2A:B56{epsilon} morpholino and 20 pg of PP2A:B56{epsilon}-c mRNA bilaterally. (G-I) Tadpole stage. (G) Control; (H) embryo was injected with 2.5 ng of PP2A:B56{epsilon} morpholino; (C) embryo was injected with 2.5 ng of PP2A:B56{epsilon} morpholino and 20 pg of PP2A:B56{epsilon}-c mRNA.

 

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© The Company of Biologists Ltd 2003