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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. 4. Interaction between kermit2/XGIPC and XIGF1R in eye development. (A) Coimmunoprecipitation of kermit 2 and full-length XIGF1R. Embryos were injected at the one-cell stage with mRNAs encoding XIGF1R (2 ng) and GFP-tagged kermit 2 (1 ng), and cultured until the gastrula stage. XIGF1R/kermit 2 complexes were immunoprecipitated from embryo lysates with anti-GFP antibody and XIGF1R was visualized by western blotting. Mouse IgG was used as a negative control. (B) Kermit 2 morpholino and DN-IGFR synergistically inhibit eye formation in embryos. Kermit 2 morpholino (20 ng), DN-IGFR mRNA (500 pg), or both, were injected into two dorsal animal blastomeres of four-cell/eight-cell embryos. Embryos were cultured until tadpole stages to score phenotypes. The percentage of embryos with either strong or mild reduction in eyes is tabulated in the panel on the right side. (C) Kermit 2 morpholino and DN-IGFR synergistically reduce expression of Pax6 in presumptive eye domain (arrowhead). Microinjections were performed as in Fig. 2B. Nuclear β-galactosidase mRNA was co-injected as a lineage tracer. Embryos were fixed at stage 20 and β-galactosidase activity was measured in situ (red) followed by whole-mount in situ hybridization for Pax6. |
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Fig. 8. Kermit 2/XGIPC is required for cell survival, but not for cell proliferation in Xenopus. (A-F) Control morpholino (A), kermit 2 morpholino alone (B,E,F), kermit 2 morpholino with 3 ng of kermit 2 mRNA (C) or 1 ng of DN-IGFR (D) was injected into the right side dorsal animal blastomere of four-cell/eight-cell embryos. The left side serves as a control. TUNEL staining and phosphorylated histone H3 staining were performed on neurula stage embryos. Embryos are viewed from the dorsal side, anterior towards the top. Compared with the control side (left side), the side injected with kermit 2 morpholino (right side) shows a substantial increase in the number of TUNEL-positive nuclei (B), without apparent change in the number of mitotic cells (E,F). The increased apoptosis in kermit 2-depleted embryos can be rescued by co-expression of kermit 2 mRNA lacking the 5′UTR (C). DN-IGFR also leads to elevated cell death on the injected side, as shown in D. (G-I) Co-injection of Bcl2 mRNA recovers Pax6 eye expression in kermit 2-depleted embryos. Microinjections were performed as above and nβ-gal was used as the lineage tracer. Embryos were fixed at stage 20 andβ -galactosidase activity was measured in situ (red) followed by whole-mount in situ hybridization for Pax6. Red arrowhead in H indicates strongly reduced eye region in kermit 2 morpholino-injected embryo and red arrow in I indicates recovered eye expression domain by Bcl2 mRNA. |
