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 ß-galactosidase mRNA was co-injected as a lineage tracer. Embryos were fixed at stage 20 and ß-galactosidase activity was measured in situ (red) followed by whole-mount in situ hybridization for Pax6.