ZFIN ID: ZDB-FIG-200817-15
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:
Anatomical Terms:
Stage Range: Shield to 1-4 somites
PHENOTYPE:
Fish:
Observed In:
Stage Range: Shield to 1-4 somites

Fig 6 N-terminal of Nanog is required for its Wnt/β-catenin repressive activity.

(A) WISH analysis showing the expression of mesendoderm marker, mxtx2, strictly zygotic gene, blf, and microRNA-430 precursor (mir-430), and miR-430 target, sod1 in embryos of WT, MZnanog, MZnanog injected with nanog_FL, nanog_ΔC, or vp16-nanog mRNA. Expression of mxtx2, blf, and mir-430 was reduced, even absent, whereas expression of sod1 was significantly increased in MZnanog embryos; overexpression of nanog_FL, nanog_ΔC, or vp16-nanog restored the expression of mxtx2, blf, and mir-430 and cleaned the expression of sod1 in MZnanog embryos. mxtx2, blf, and sod1 were detected at 6 hpf, and mir-430 was detected at 4 hpf. Scale bar, 100 μm. (B) Statistical analysis of embryos in panel A. N represents analyzed embryo number. (C) Relative mRNA level of mxtx2, blf, and sod1 in WT, Mznanog, and the rescued embryos at 6 hpf examined by RT-qPCR analysis. Error bars, mean ± SD, **P < 0.01. (D) Overexpression of nanog_FL, nanog_ΔC, and vp16-nanog rescued the developmental defects of MZnanog. Both of nanog_FL and nanog_ΔC rescued embryos showed WT-like phenotype, whereas vp16-nanog rescued embryos still showed a forebrain defective phenotype. Phenotype was observed at 36 hpf. Scale bar, 100 μm. The numbers below the morphology pictures indicate number of embryos showing representative phenotype/total number of embryos. (E) WISH analysis showing the expression of neuroectoderm marker otx2 and forebrain marker six3b in embryos of WT, MZnanog, MZnanog injected with nanog_FL, nanog_ΔC, or vp16-nanog mRNA at 90% epiboly (for otx2) and 2-somite stage (for six3b). Expression of otx2 and six3b was absent in MZnanog embryos and restored by overexpression of nanog_FL or nanog_ΔC but not vp16-nanog. Red arrows indicate the absent expression of otx2 or six3b. Scale bar, 100 μm. (F) Statistical analysis of the embryos in panel E. N represents analyzed embryo number. (G) TOPflash assay showing co-transfection of nanog_FL (2 μg) or nanog_ΔC (2 μg) but not vp16-nanog (2 μg) significantly inhibited the up-regulated β-catenin transcriptional activity induced by β-catenin (0.5 μg) in HEK293T cells. Error bars, mean ± SD, **P < 0.01; NS means no significant difference. 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; nanog_FL, full length of Nanog; nanog_ΔC, C-terminal truncated Nanog; RT-qPCR, reverse-transcription quantitative PCR; vp16-nanog, Nanog homeodomain fusion with Vp16; WISH, whole-mount in situ hybridization; WT, wild type.

Gene Expression Details
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay
blf WT standard conditions Shield whole organism ISH
nanogihb97/ihb97 standard conditions Shield whole organism ISH
nanogihb98/ihb98 standard conditions Shield whole organism ISH
mxtx2 WT standard conditions Shield presumptive mesoderm ISH
nanogihb97/ihb97 standard conditions Shield whole organism ISH
nanogihb98/ihb98 standard conditions Shield whole organism ISH
otx2b WT standard conditions 90%-epiboly neuroectoderm ISH
nanogihb97/ihb97 standard conditions 90%-epiboly Not Detected neuroectoderm ISH
nanogihb98/ihb98 standard conditions 90%-epiboly Not Detected neuroectoderm ISH
six3b WT standard conditions 1-4 somites forebrain neural keel ISH
nanogihb97/ihb97 standard conditions 1-4 somites Not Detected forebrain neural keel ISH
nanogihb98/ihb98 standard conditions 1-4 somites Not Detected forebrain neural keel ISH
sod1 WT standard conditions Shield whole organism ISH
nanogihb97/ihb97 standard conditions Shield whole organism ISH
nanogihb98/ihb98 standard conditions Shield whole organism ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
nanogihb97/ihb97 standard conditions Shield whole organism blf expression decreased amount, abnormal
Shield whole organism mxtx2 expression decreased amount, abnormal
Shield whole organism sod1 expression increased amount, abnormal
90%-epiboly neuroectoderm otx2b expression absent, abnormal
1-4 somites forebrain neural keel six3b expression absent, abnormal
nanogihb98/ihb98 standard conditions Shield whole organism blf expression decreased amount, abnormal
Shield whole organism mxtx2 expression decreased amount, abnormal
Shield whole organism sod1 expression increased amount, abnormal
90%-epiboly neuroectoderm otx2b expression absent, abnormal
1-4 somites forebrain neural keel six3b expression absent, abnormal
Acknowledgments:
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ PLoS Biol.