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Fig. 3. Depletion of kermit 2/XGIPC specifically inhibits Xenopus eye development. (A-D) Depletion of kermit 2 does not affect the expression of dorsal mesoderm markers chordin and goosecoid. Embryos are viewed from the vegetal side and dorsal is towards the top. Control morpholino or kermit 2 morpholino (40 ng) was injected into two dorsal animal blastomeres of four-cell embryos. Embryos were fixed at the gastrula stage (stage 10) and whole-mount in situ hybridization was performed for chordin (A,B) and goosecoid (C,D). (E-L) Depletion of kermit 2 specifically reduces marker gene expression within the presumptive eye field in stage 20 embryos. Embryos are viewed from the anterior side with dorsal towards the top. Control or kermit 2 morpholino was injected into one dorsal animal blastomere of four-cell/eight-cell embryos with 500 pg of mRNA for nuclear β-galactosidase. Embryos were fixed at stage 20 andβ -galactosidase activity was measured in situ (red) followed by whole-mount in situ hybridization for Bf1 (E,F), Otx2 (G,H), Pax6 (I,J) and Xrx (K,L). Bf1 expression was not affected by depletion of kermit 2 (F). Expression of Otx2 (H) and Pax6 (J) were reduced only within the presumptive eyeforming region (red arrowheads) in embryos injected with kermit 2 morpholino. The expression of the eye marker Xrx was also inhibited (L red arrowhead). (M-P) Depletion of kermit 2 does not reduce Xrx (N) or Pax6 (P) expression in early neurula stage embryos (stage 14). (Q-S) Kermit 2 mRNA restored Xrx expression in kermit 2-depleted embryos. Red arrowhead in R indicates strongly reduced Xrx expression within presumptive eye domain in kermit 2-depleted embryo; red arrow in S indicates recovered Xrx expression in embryo co-injected with kermit 2 morpholino and kermit 2 mRNA. |
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Fig. 7. Activation of PI3K partially rescues eye development in kermit 2/XGIPC-depleted embryos. (A) p110*, a constitutively active subunit of PI3 kinase, induces AKT phosphorylation/activation in stage 10 animal cap explants. mRNA encoding p110* was injected into fertilized eggs and animal caps were explanted at the gastrula stage and analyzed by western blot with antibodies recognizing AKT phosphorylated at serine-473 (upper panel) or general anti-AKT antibodies (lower panel). (B) p110* partially rescues eye development in kermit 2-depleted embryos. Kermit 2 morpholino (40 ng), 3 ng of p110* mRNA, or both, were injected into two dorsal animal blastomeres of four-cell/eight-cell embryos. No apparent eyes are present in embryos injected with the kermit 2 morpholino, while small eyes are present in over 70% of embryos co-injected with p110* mRNA and morpholino. p110* injection alone does not affect embryo development. (C) The percentage of embryos with normal, small or absent/miniscule eyes is summarized. (D) p110* partially recovers the expression of Xrx in kermit 2-depleted embryos. One dorsal-animal blastomere of four-cell/eight-cell embryos was injected with kermit 2 morpholino with or without p110* mRNA (right side, indicated by GFP lineage tracer) and harvested at stage 20. Whole-mount in situ hybridization was performed for Xrx. Embryos are viewed from the anterior side with dorsal towards the top. |
