Transcriptional level of Wnt/β-catenin components and nuclear accumulation of β-catenin are not increased in MZnanog.
(A) WISH and (B, C) RT-qPCR showed the maternal transcription of wnt8a1, wnt8a2, and ctnnb2 were not affected in MZnanog eggs when compared with WT eggs. Scale bar, 100 μm. Error bars, mean ± SD, *P < 0.05; NS means no significant difference. (D) Western blot analysis of total β-catenin and nuclear β-catenin (active β-catenin) in WT, Mznanog, and wnt8a overexpressed embryos. Anti-total β-catenin was used as the β-catenin expression control and anti-β-actin was used as the internal control. A total of 2 pg of wnt8a mRNA was injected at the 1-cell stage in WT and used as a positive control. Embryos were collected at 4 hpf. Experiments were carried out for triplicates. (E) Statistical analysis of active β-catenin/total β-catenin level in panel D. Error bars, mean ± SD, ***P < 0.001. (F) Immunolocalization of β-catenin on whole-mount embryos at the 512-cell stage shows that nuclear β-catenin in both the WT and MZnanog was localized in dorsal margin cells and nondorsal cells, and nuclear β-catenin localization was not stimulated in MZnanog embryos. Signals were observed at animal view. Nuclei were co-stained with DAPI. Scale bar, 50 μm. 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; MZnanog, maternal zygotic mutant of nanog; RT-qPCR, reverse-transcription quantitative PCR; WISH, whole-mount in situ hybridization; WT, wild type.