Rorick AM et al. (2007), PP2A:B56epsilon is required for eye induction a...

XB-ART-34825

Dev Biol 2007 Feb 15;3022:477-93. doi: 10.1016/j.ydbio.2006.10.011.

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PP2A:B56epsilon is required for eye induction and eye field separation.

Rorick AM , Mei W , Liette NL , Phiel C , El-Hodiri HM , Yang J .

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Eye induction and eye field separation are the earliest events during vertebrate eye development. Both of these processes occur much earlier than the formation of optic vesicles. The insulin-like growth factor (IGF) pathway appears to be essential for eye induction, yet it remains unclear how IGF downstream pathways are involved in eye induction. As a consequence of eye induction, a single eye anlage is specified in the anterior neural plate. Subsequently, this single eye anlage is divided into two symmetric eye fields in response to Sonic Hedgehog (Shh) secreted from the prechordal mesoderm. Here, we report that B56epsilon regulatory subunit of protein phosphatase 2A (PP2A) is involved in Xenopus eye induction and subsequent eye field separation. We provide evidence that B56epsilon is required for the IGF/PI3K/Akt pathway and that interfering with the PI3K/Akt pathway inhibits eye induction. In addition, we show that B56epsilon regulates the Hedgehog (Hh) pathway during eye field separation. Thus, B56epsilon is involved in multiple signaling pathways and plays critical roles during early development.

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Species referenced: Xenopus laevis
Genes referenced: arx cer1 chdh chrd eomes foxa2 gal.2 gli1 gli3 gsc igf1 ins kif11 lhx2 mapk1 myc myod1 nodal odc1 otx2 pax2 pax6 pik3ca pik3cb pik3cg ptch1 ptch2 ptpa rax shh six3 six6 smo sox3 tbx2

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	Fig. 1. Dose-dependent effects of B56ε depletion on eye development. (A) Whole mount in situ hybridization showing the expression of rx (upper panels) and six3 (lower panels) in stage 20 control embryos, 2.5 ng εmor injected embryos, 5 ng εmor injected embryos, and 7.5 ng εmor injected embryos. εmor was injected into two dorsal animal blastomeres at the 8-cell stage. (B) Western blot showing the effects of εmor and 5mis on the translation of B56ε-FLAG. Myc-EGFP was used as a control for injection and loading. Morpholinos (5 ng) were injected into one of the dorsal animal blastomere at the 8-cell stage. Subsequently, a mixture of B56ε (200 pg) and Myc-EGFP (50 pg) was injected into the same blastomere. Injected embryos were harvested at the late gastrula stage. (C) Whole embryo phenotypes at the tadpole stage showing the effect of morpholinos injection on early embryonic development. Embryos were either uninjected (top), or bilaterally injected with εmor (5 ng, middle left; 7.5 ng, lower left), or injected with 5mis (5 ng, middle right; 7.5 ng, lower right) into both dorsal animal blastomeres at the 8-cell stage. (D) Whole mount in situ hybridization showing the expression of rx (upper panels) and six3 (lower panels) in stage 18 control embryos, 5 ng of 5mis injected embryos, and 7.5 ng of 5mis injected embryos. 5mis was injected into two dorsal animal blastomeres at the 8-cell stage. Note that less than 30% of embryos injected with 7.5 ng of 5mis exhibited fused rx or six3 expression at this stage. The rest of embryos were normal (not shown).
	Fig. 2. B56ε plays a direct role in the regulation of rx expression. (A) RT-PCR showing that maternal depletion of B56ε (oocyte injection) reduced the expression of dorsal specific genes (dickkopf-1, goosecoid, cerberus, and Xenopus nodal related3 (xnr3)) and enhanced the expression of a ventral-specific gene (sizzled) at stage 11. Injection of 7.5 ng of εmor into two dorsal animal blastomeres at the 8-cell stage did not affect the expression of above genes. ODC was used as the loading control. (B) Whole mount in situ hybridization showing that the expression of chd in a control embryo and an embryo injected with 7.5 ng of εmor. (C) Whole mount in situ hybridization showing the expression of rx in a stage 18 uninjected embryo, an embryo injected with 0.5 ng of εmor, an embryo injected with 1 ng of εmor, and an embryo injected with 1.5 ng of εmor. εmor was injected into one of A1 blastomeres at the 32-cell stage. Lineage tracer, n-β-gal, was co-injected with εmor to indicate the side of injection. (D) Whole mount in situ hybridization showing the expression of rx (stage 12.5), sox3 (pan-neural, stage 12.5), otx2 (pan-forebrain, stage 12.5), and pax2 (midbrain, stage 14), arx (diencephalons, stage 14), and eomesodermin (telencephalon, stage 26/27) in control embryos (upper panels) and embryos injected with εmor (4.5 ng, lower panels). The right side was injected as indicated by the Red-gal staining. Embryos were injected unilaterally at the 8-cell stage.
	Fig. 3. B56ε is required for the induction of a subset of EFTFs. (A) Whole mount in situ hybridization showing the expression of rx, lhx2, optx2, six3, and pax6 in stage 14 uninjected embryos (upper panels) and embryos bilaterally injected with 7.5 ng of εmor (lower panels). (B) RT-PCR showing the expression of rx, lhx2, pax6, six3, and ET in control embryos, embryos bilaterally injected with 5 ng or 7.5 ng of εmor from stage 10 (the beginning of gastrulation) to stage 15 (mid-neurula stage). ODC was used as loading control. (C) Whole mount in situ hybridization showing the expression of rx (left column) and lhx2 (right column) in stage 14 uninjected embryos (upper panels), embryos injected with εmor (7.5 ng, middle panels), and embryos injected with εmor (7.5 ng) and ε-c (100 pg) (lower panels). Both dorsal animal blastomeres were injected at the 8-cell stage. Morpholino and ε-c were injected sequentially. Note that the expression of rx and lhx2 was partially rescued by ε-c.
	Fig. 4. B56ε is required for IGF1-induced EFTFs expression. (A) Whole mount in situ hybridization showing that εmor injection blocked IGF1-induced rx expression (upper panels), without affecting IGF1-induced otx2 (middle panels) and sox3 (lower panels) expression in whole embryos (stage 14). IGF1 (2 ng) was unilaterally injected at the 8-cell stage into either wild-type embryos, or embryos that were previously injected with 5 ng of εmor. The right side was injected. (B) RT-PCR showing εmor injection blocked IGF1-induced rx, pax6, six3, and lhx2 expression, while IGF1-induced otx2 and sox3 expressions remain unaffected. εmor (10 ng) was injected at the 1-cell stage. IGF1 (2 ng) was radially injected into the animal pole of each blastomere at the 4-cell stage. Animal caps were dissected at late blastula stage and harvested at stage 13.
	Fig. 5. The PI3K/Akt pathway is required for IGF1-induced EFTFs. (A) Western blot showing that εmor injection blocked Akt phosphorylation induced by IGF1, but had no effect on Akt phosphorylation induced by P110. In addition, εmor had no effect on IGF1-induced ERK phosphorylation. εmor (10 ng) and RNA encoding IGF1 (2 ng) or P110 (1 ng) were injected sequentially, with εmor being injected at the 1-cell stage and RNAs being injected at the 4-cell stage. Animal caps were dissected at stage 8/9 and harvested at stage 13. (B) RT-PCR results showing LY294002, a PI3K inhibitor, blocked IGF1-induced expression of rx, lhx2, pax6, and six3, without affecting IGF1-induced otx2 and sox3 expressions in animal cap assay. IGF1 was injected as described above. Caps were dissected at late blastula stage and harvested at stage 13. Some caps were exposed to LY294002 from stage 9. (C) δp85 (2 ng) and dnAkt (2 ng) blocked IGF1 (2 ng) induced rx (upper panels), but not IGF1-induced otx2 (middle panels) and sox3 (lower panels) in whole embryos. One of the dorsal animal blastomeres was injected at the 8-cell stage. The right side was injected.
	Fig. 6. The PI3K/Akt pathway is required for eye induction. (A) Injection of δp85 (2 ng) or dnAkt (2 ng) into dorsal animal blastomeres at the 8-cell stage blocked eye formation. (B) δp85 and dnAkt blocked the expression of rx (upper panels) and lhx2 (lower panels) at stage 14. (C) Injection of δp85 or dnAkt into A1 blastomere at the 32-cell stage impaired eye formation on the injected side (left) when embryos reached tadpole stage. Left side was injected. (D) Injection of δp85 (middle panels) or dnAkt (lower panels) into A1 blastomere at the 32-cell stage reduced the expression of rx, but not myoD, otx2, and sox3. n-β-gal was used as a lineage tracer.
	Fig. 7. B56ε is required for eye field separation. (A) Dorsal (left column) and lateral (right column) views of an uninjected embryo (upper panels), an embryo injected with εmor (3.5 ng) (middle panels), and an embryo injected with εmor and ε-c (100 pg) (lower panel). (B) The expression of rx (upper panels) and six3 (lower panels) in stage 18 control embryos (left column), embryos injected with εmor (middle column), and embryos injected with εmor and ε-c (right column). Embryos were bilaterally injected at the 8-cell stage.
	Fig. 8. B56ε is required for the expression of endogenous Hh target genes. (A) Whole mount in situ hybridization showing the expression of shh (upper panels) and ptc-1 (lower panels) was reduced by εmor (3.5 ng) injection at stage 14/15. The reduced shh and ptc-1 expression was partially rescued in embryos injected with εmor and ε-c (100 pg). (B) RT-PCR showing εmor (3.5 ng) injection reduced ptc-1, foxA2, and shh expression from late gastrula stage. ODC was the loading control. (C) The expression of ptc-1, ptc-2, foxA2, and shh in tadpole stage control embryos (left column) and embryos injected with 3.5 ng of εmor (right column). Embryos were bilaterally injected at the 8-cell stage.
	Fig. 9. B56ε is required for the Hh pathway. (A) Whole mount in situ hybridization showing εmor (3.5 ng) injection blocked Shh (1 ng)-induced rx reduction (upper panels) and ptc-1 expression (lower panels) in whole embryos. Embryos were bilaterally injected at the 8-cell stage. (B) RT-PCR showing εmor (10 ng) injection blocked ptc-1 and foxA2 induced by shh (1 ng), smo-M2 (2.5 ng), and gli1 (0.5 ng) in neuralized animal caps. εmor and RNAs were injected sequentially at the 1-cell stage. Animal caps were dissected at stage 8/9 and harvested at stage 14. (C) RT-PCR showing εmor injection (10 ng) blocked ptc-1 expression induced by wild-type Gli1 (500 pg), but not by Gli-VP (50 pg and 100 pg). Embryos were injected and manipulated as described above. (D) Western blot showing overexpression of Shh inhibited the processing of overexpressed Myc-Gli3 (0.5 ng) in animal caps. The effect of Shh (1 ng) on Myc-Gli3 processing was not affected by εmor (10 ng) injection. Embryos were injected and manipulated as described above, except that caps were harvested at stage 12. (E) Co-IP results showing overexpression of Shh (1 ng) dissociated complex formation between FLAG-Cos2 (1 ng) and Myc-Gli1 (1 ng) in animal caps. Injection of εmor (10 ng) did not affect this complex dissociation induced by Shh. Embryos were injected and manipulated as described above. (F) Whole mount in situ hybridization showing the expression of rx in (from left to right) a control embryo, an embryo injected with εmor (3.5 ng), an embryo injected with Gli-VP (100 pg), and an embryo injected with Gli-VP and εmor. Embryos were bilaterally injected at the 8-cell stage.