Fig 4

(A) western blot showing translation of Nanog protein totally disappeared in MZnanog. Nanog protein can be detected as early as the 64-cell stage and vanished at 75% epiboly stage in WT embryos, whereas no Nanog protein was detected in MZnanog mutant embryos. (B) Immunolocalization of Nanog on cryosections of WT and MZnanog embryos at 4 hpf. Nanog is localized in the cell nuclei of WT embryos and disappeared in MZnanog embryos. Nuclei were co-stained with DAPI. Scale bar, 50 μm. (C) WISH analysis showing the injection of low dose of ctnnb2 mRNA (200 pg) induced slight up-regulation of boz and chd in WT embryos and induced massive expression of boz and chd in MZnanog embryos. boz was detected at 4 hpf, and chd was detected at 4.5 hpf. Scale bar, 100 μm. (D) Statistical analysis of the embryos in panel C. N represents analyzed embryo number. (E) Knockdown of ctnnb2 but not ctnnb1 rescued the developmental defects of MZnanog at gastrula stage. The numbers below the morphology pictures are the number of embryos showing representative phenotype/total number of embryos. Scale bar, 500 μm. (F) WISH analysis showing expression of chd and boz were expanded in MZnanog embryos, and knockdown of ctnnb2 (β2 MO) but not ctnnb1 (β1 MO) rescued these defects. boz was detected at 4 hpf, and chd was detected at 5 hpf. Scale bar, 100 μm. (G) Statistical analysis of the embryos in panel F. N represents analyzed embryo number. (H) Relative mRNA level of boz and chd in the embryos of WT, MZnanog, MZnanog co-injected with ctnnb1 MO (+ β1 MO) and MZnanog co-injected with ctnnb2 MO (+ β2 MO). Error bars, mean ± SD, **P < 0.01; NS means no significant difference. (I) TOPflash assay showing β-catenin transcriptional activity was significantly up-regulated in the embryos of MZnanog compared with WT, and co-injection of ctnnb2 MO (+ β2 MO) but not ctnnb1 MO (+ β1 MO) into MZnanog embryos significantly decreased the transcriptional activity. Error bars, mean ± SD, **P < 0.01, ***P < 0.001. The P values in this figure were calculated by Student t test. The underlying data in this figure can be found in S1 Data. hpf, hours post fertilization; MO, morpholino; MZnanog, maternal zygotic mutant of nanog; RT-qPCR, reverse-transcription quantitative PCR; WISH, whole-mount in situ hybridization; WT, wild type.