ZFIN ID: ZDB-FIG-200817-13
He et al., 2020 - Nanog safeguards early embryogenesis against global activation of maternal β-catenin activity by interfering with TCF factors. PLoS Biology   18:e3000561 Full text @ PLoS Biol.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagents:
Anatomical Terms:
Stage Range: 1-cell to Adult
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: 64-cell to 75%-epiboly

Fig 4 Maternal β-catenin activity is hyperactivated in <italic>nanog</italic> mutant.

(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.

Gene Expression Details
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay
chrd WT standard conditions Sphere whole organism RTPCR
30%-epiboly whole organism ISH
nanogihb97/ihb97 standard conditions Sphere whole organism RTPCR
30%-epiboly whole organism ISH
nanogihb97/ihb97 + MO1-ctnnb1 standard conditions Sphere whole organism RTPCR
30%-epiboly whole organism ISH
nanogihb97/ihb97 + MO1-ctnnb2 standard conditions Sphere whole organism RTPCR
30%-epiboly whole organism ISH
nanogihb98/ihb98 standard conditions 30%-epiboly whole organism ISH
Sphere whole organism RTPCR
nanogihb98/ihb98 + MO1-ctnnb1 standard conditions 30%-epiboly whole organism ISH
Sphere whole organism RTPCR
nanogihb98/ihb98 + MO1-ctnnb2 standard conditions Sphere whole organism RTPCR
30%-epiboly whole organism ISH
dharma WT standard conditions Sphere whole organism ISH
Sphere whole organism RTPCR
nanogihb97/ihb97 standard conditions Sphere whole organism ISH
Sphere whole organism RTPCR
nanogihb97/ihb97 + MO1-ctnnb1 standard conditions Sphere whole organism ISH
Sphere whole organism RTPCR
nanogihb97/ihb97 + MO1-ctnnb2 standard conditions Sphere whole organism RTPCR
Sphere whole organism ISH
nanogihb98/ihb98 standard conditions Sphere whole organism RTPCR
Sphere whole organism ISH
nanogihb98/ihb98 + MO1-ctnnb1 standard conditions Sphere whole organism RTPCR
Sphere whole organism ISH
nanogihb98/ihb98 + MO1-ctnnb2 standard conditions Sphere whole organism ISH
Sphere whole organism RTPCR
nanog WT standard conditions 1-cell Not Detected whole organism WB
64-cell whole organism WB
1k-cell whole organism WB
Sphere whole organism nucleus WB
Shield whole organism WB
75%-epiboly whole organism WB
Bud Not Detected whole organism WB
Adult Not Detected oocyte WB
nanogihb97/ihb97 standard conditions 1-cell Not Detected whole organism WB
64-cell Not Detected whole organism WB
1k-cell Not Detected whole organism WB
Sphere Not Detected whole organism WB
Shield Not Detected whole organism WB
75%-epiboly Not Detected whole organism WB
Bud Not Detected whole organism WB
Adult Not Detected oocyte WB
nanogihb98/ihb98 standard conditions 1-cell Not Detected whole organism WB
64-cell Not Detected whole organism WB
1k-cell Not Detected whole organism WB
Sphere Not Detected whole organism WB
Shield Not Detected whole organism WB
75%-epiboly Not Detected whole organism WB
Bud Not Detected whole organism WB
Adult Not Detected oocyte WB
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
nanogihb97/ihb97 standard conditions 64-cell whole organism nanog expression absent, abnormal
1k-cell whole organism nanog expression absent, abnormal
Sphere canonical Wnt signaling pathway increased efficacy, abnormal
Sphere negative regulation of canonical Wnt signaling pathway decreased occurrence, abnormal
Sphere whole organism chrd expression increased amount, abnormal
30%-epiboly whole organism chrd expression increased distribution, abnormal
Sphere whole organism dharma expression increased amount, abnormal
Sphere whole organism dharma expression increased amount, abnormal
Sphere whole organism dharma expression increased distribution, abnormal
Sphere whole organism nanog expression absent, abnormal
Shield whole organism nanog expression absent, abnormal
Shield whole organism dorsalized, abnormal
75%-epiboly whole organism nanog expression absent, abnormal
nanogihb97/ihb97 + MO1-ctnnb1 standard conditions Sphere canonical Wnt signaling pathway increased efficacy, abnormal
Sphere negative regulation of canonical Wnt signaling pathway decreased occurrence, abnormal
Sphere whole organism chrd expression increased amount, abnormal
30%-epiboly whole organism chrd expression increased distribution, abnormal
Sphere whole organism dharma expression increased amount, abnormal
Sphere whole organism dharma expression increased distribution, abnormal
Shield whole organism dorsalized, abnormal
nanogihb97/ihb97 + MO1-ctnnb2 standard conditions Sphere canonical Wnt signaling pathway process efficacy, normal
Sphere regulation of canonical Wnt signaling pathway normal occurrence, normal
Sphere whole organism chrd expression amount, ameliorated
30%-epiboly whole organism chrd expression position, ameliorated
Sphere whole organism dharma expression amount, ameliorated
Sphere whole organism dharma expression position, ameliorated
Shield whole organism dorsalized, ameliorated
nanogihb98/ihb98 standard conditions 64-cell whole organism nanog expression absent, abnormal
1k-cell whole organism nanog expression absent, abnormal
Sphere canonical Wnt signaling pathway increased efficacy, abnormal
Sphere negative regulation of canonical Wnt signaling pathway decreased occurrence, abnormal
Sphere whole organism chrd expression increased amount, abnormal
30%-epiboly whole organism chrd expression increased distribution, abnormal
Sphere whole organism dharma expression increased amount, abnormal
Sphere whole organism dharma expression increased distribution, abnormal
Sphere whole organism nanog expression absent, abnormal
Shield whole organism nanog expression absent, abnormal
Shield whole organism dorsalized, abnormal
75%-epiboly whole organism nanog expression absent, abnormal
nanogihb98/ihb98 + MO1-ctnnb1 standard conditions Sphere canonical Wnt signaling pathway increased efficacy, abnormal
Sphere negative regulation of canonical Wnt signaling pathway decreased occurrence, abnormal
Sphere whole organism chrd expression increased amount, abnormal
30%-epiboly whole organism chrd expression increased distribution, abnormal
Sphere whole organism dharma expression increased amount, abnormal
Sphere whole organism dharma expression increased distribution, abnormal
Shield whole organism dorsalized, abnormal
nanogihb98/ihb98 + MO1-ctnnb2 standard conditions Sphere canonical Wnt signaling pathway process efficacy, normal
Sphere regulation of canonical Wnt signaling pathway normal occurrence, normal
Sphere whole organism chrd expression amount, ameliorated
30%-epiboly whole organism chrd expression position, ameliorated
Sphere whole organism dharma expression amount, ameliorated
Sphere whole organism dharma expression position, ameliorated
Shield whole organism dorsalized, ameliorated
Acknowledgments:
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