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Fig. 3
Nuclear Net1 enhances Nodal signaling and mesendoderm formation in a GEF-independent manner. (A,B) Endogenous Net1 primarily localizes in the zebrafish embryonic cell nucleus. Nuclear and cytosolic fractions were extracted from wild-type embryos at indicated stages (A) or from sqt mRNA-injected embryos at the shield stage (B). Then these resulting samples were assessed by western blotting with the indicated antibodies. (C) Net1 promotes Nodal signaling in the nucleus. HeLa cells were transfected with the ARE-luciferase reporter together with Net1, Net1-NLS or Net1-ΔN-NES. After 36 h of transfection, the cells were treated with or without TGF-β1 overnight, then harvested for luciferase assays. **P<0.01; ***P<0.001; NS, not significant (Student's t-test). (D) Nuclear Net1 facilitates mesendoderm induction. At the sphere stage, zebrafish embryos injected with net1 2Mix or net1-NLS 2Mix or net1-ΔN NES 2Mix were exposed to UV light, and then harvested for whole-mount in situ hybridization to detect the expression of gsc and sox17 at the shield and 75% epiboly stages. (E,F) Overexpression of the nuclear, but not cytosolic, Net1 rescues the mesendoderm defects in net1 morphants. Embryos were injected with cMO, net1 MO1, net1-NLS 3Mix or net1-ΔN NES 3Mix. Embryos injected with net1-NLS 3Mix or net1-ΔN NES 3Mix were exposed to UV light at the sphere stage. The expression of gsc (E) and sox17 (F) was examined by in situ hybridization at the shield and 75% epiboly stages, respectively. (G,H) Net1 promotes Nodal signaling independently of its GEF activity. HeLa cells were co-transfected with the ARE-luciferase reporter and wild-type Net1 and its GEF-deficient mutants (G) or nuclear Net1 and its GEF-deficient mutants (H). After 36 h of transfection, the cells were treated with or without TGF-β1 overnight, then harvested for luciferase assays. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). (I) The influence of nuclear Net1 and its GEF-deficient mutants in mesendoderm formation. Zebrafish embryos were injected with net1-NLS 2Mix, NLS-L266E 2Mix and NLS-W437L 2Mix, respectively. These embryos were exposed to UV at the sphere stage, then harvested for whole-mount in situ hybridization. In D,E,F and I, the percentages (mean±s.d.) of the affected embryos are shown as calculated from three independent biological repeats with ∼20–30 embryos in each group. Results in C,G and H are mean±s.d. (n=3). UIC, uninjected control.