December 1, 2003;
PP2A:B56epsilon is required for Wnt/beta-catenin signaling during embryonic development.
The Wnt/beta-catenin pathway plays important roles during embryonic development and growth control. The B56 regulatory subunit of protein phosphatase 2A (PP2A
) has been implicated as a regulator of this pathway. However, this has not been investigated by loss-of-function analyses. Here we report loss-of-function analysis of PP2A
:B56epsilon during early Xenopus embryogenesis. We provide direct evidence that PP2A
:B56epsilon is required for Wnt/beta-catenin signaling upstream of Dishevelled
and downstream of the Wnt ligand. We show that maternal PP2A
:B56epsilon function is required for dorsal development, and PP2A
:B56epsilon function is required later for the expression of the Wnt target gene engrailed, for subsequent midbrain
boundary formation, and for closure of the neural tube. These data demonstrate a positive role for PP2A
:B56epsilon in the Wnt pathway.
[+] show captions
Fig. 1. Expression pattern of PP2A:B56ϵ during early embryonic development of Xenopus. (A,B) Expression of PP2A:B56ϵ in the early blastula. (A) PP2A:B56ϵ 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:B56ϵ was analyzed by RT-PCR. EF1α was used as loading control. Xnr6 (dorsally restricted expression) was used as a control for accurate dissection. (B) PP2A:B56ϵ 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:B56ϵ was analyzed by RT-PCR. VegT (vegetal pole restricted expression) was used as a control for accurate dissection. (C-F) PP2A:B56ϵ expression as determined by in situ hybridization of hemisections. (C) PP2A:B56ϵ 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:B56ϵ expression in the anterior neural ectoderm. (G) PP2A:B56ϵ expression in a stage 33 tadpole. Arrows in E and F indicate the anterior edge of PP2A:B56ϵ expression domain in the neural ectoderm.
Fig. 4. PP2A:B56ϵ 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:B56ϵ 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:B56ϵ morpholino (middle column), or PP2A:B56ϵ morpholino together with PP2A:B56ϵ-c mRNA (right column). All embryos were co-injected with nuclear β-galactosidase mRNA as a lineage tracer. Control (left column) is n-β-gal alone.
Fig. 5. Regulation of en2 expression and β-catenin stability by PP2A:B56ϵ in the neural ectoderm at stage 14. (A) RT-PCR showing that en2 is reduced by PP2A:B56ϵ 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:B56ϵ morpholino injection at stage 14. (B,D) Control embryos; (C,E) embryos injected with PP2A:B56ϵ morpholino unilaterally. (F) Western blot showing injected β-catenin was less stable in the neural ectoderm of PP2A:B56ϵ knockdown embryos. EFGP was used as a control for both injection and loading.
Fig. 2. Maternal PP2A:B56ϵ is required for dorsal development. (A) Morpholino antisense oligonucleotide directed against PP2A:B56ϵ blocks the translation of flag-tagged PP2A:B56ϵ RNA, but not flag-tagged PP2A:B56ϵ-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:B56ϵ-depleted embryo also at stage 10+, showing absence of dorsal lip. (D) RT-PCR showing the gene expression profile in maternal PP2A:B56ϵ and β-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:B56ϵ-depleted embryos could be rescued by PP2A:B56ϵ-c mRNA.
Fig. 6. PP2A:B56ϵ 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:B56ϵ morpholino bilaterally; (C) embryo injected with 2.5 ng of PP2A:B56ϵ morpholino and 20 pg of PP2A:B56ϵ-c mRNA bilaterally. (D-F) Anterior view of stage 16 embryos. (D) Control embryo; (E) embryo injected with 2.5 ng of PP2A:B56ϵ morpholino bilaterally; (F) embryo injected with 2.5 ng of PP2A:B56ϵ morpholino and 20 pg of PP2A:B56ϵ-c mRNA bilaterally. (G-I) Tadpole stage. (G) Control; (H) embryo was injected with 2.5 ng of PP2A:B56ϵ morpholino; (C) embryo was injected with 2.5 ng of PP2A:B56ϵ morpholino and 20 pg of PP2A:B56ϵ-c mRNA.
Fig. S1. Overexpression of PP2A:B56e leads to anterior truncation without inhibiting dorsal specific gene expression during gastrulation. (A) Control tadpoles from uninjected embryos. (B) Tadpoles after injection of 100pg PP2A:B56e mRNA into two dorsal blastomeres at the four-cell stage. Nineteen out of a total 37 injected embryos showed anterior truncation. Embryos injected with 100pg of PP2A:B56e mRNA into both ventral blastomeres at the four-cell stage develop normally, while injection of higher dose of PP2A:B56e RNA inhibits cell division (data not shown). (C) Chordin expression in a control stage 10 embryo. (D) Chordin expression at stage 10 is unaffected by injection of PP2A:B56e mRNA. (E) The expression of siamois and Xnr3, as determined by RT-PCR, is normal in PP2A:B56e-injected embryos at stage 10. (F) expression of otx2, but not of en2 or wnt1, is reduced on the injected side in embryos injected with 100pg of PP2A:B56e mRNA into one dorsal blastomere at the four-cell stage (embryos were coinjected with lacZ mRNA as a lineage tracer).