Fig. 5: Regulatory activity of naive versus formative pluripotency enhancers in vivo. | Nature Communications

Fig. 5: Regulatory activity of naive versus formative pluripotency enhancers in vivo.

From: Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression

Fig. 5

a Representation of enhancer-2 and enhancer-7-3 deletions in Myc GFP/GFP fertilized eggs by CRISPR-Cas9. Crosses were then set up to generate MycGFP/GFP with enhancer-2 (∆2) or enhancer-7-3 (∆7-3) deletions in the littermate embryos. Confocal images show immunostaining against GFP and DAPI in E3.5 embryos of genotypes MycGFP/GFP (b), KO for enhancer-2 (c), and KO for enhancer-7-3 (d). Scale bar = 30 microns. N = 8 MycGFP/GFP, 3 MycGFP/GFP∆2/+, 3 MycGFP/GFP∆2/∆2, 10 MycGFP/GFP∆7-3/+; 7 MycGFP/GFP∆7-3/∆7-3. Dot plots showing GFP-MYC expression levels in trophectoderm (TE) and epiblast cells (EPI) of MycGFP/GFP ∆2-heterozygous and ∆2-homozygous embryos (e) and in WT, MycGFP/GFP and MycGFP/GFP ∆7-3-heterozygous and ∆7-3-homozygous embryos (f). Each dot represents the mean intensity per cell measured in individual embryos. The number of cells analyzed by embryo/tissue is provided in Supplementary Fig. 8. For e and f, ordinary one-way ANOVA with Šídák’s multiple comparisons test and two-tailed P-values; ns: P-value > 0,05; *P-value = 0.0363; ***P-value = 0.0003; ****P-value < 0,0001. Confocal images show immunostaining against GFP and DAPI in post-implantation embryos of control MycGFP/GFP (g), complete KO for enhancer-2 (∆2) (i, j) or for enhancer-7-3 (∆7-3) (k, l). Scale bar = 30 microns. g´–l´, Heatmap representation of the anti-GFP channel of each embryo shown in g–l. Scale bar = 30 microns. N = 3 MycGFP/GFP, 7 MycGFP/GFP∆2/+, 3 MycGFP/GFP∆2/∆2, 6 MycGFP/GFP∆7-3/+ and 5 MycGFP/GFP∆7-3/∆7-3 embryos. Dot plot shows GFP-MYC expression levels in extraembryonic ectoderm (EE) and epiblast cells (EPI) in Δ2 (m) and in WT, MycGFP/GFP, and Δ7-3 (n) embryos. Each dot represents the mean intensity per cell measured in individual embryos. The number of cells analyzed by embryo/tissue is provided in Supplementary Fig. 8. Ordinary one-way ANOVA with Šídák’s multiple comparisons test; ns: P-value > 0,05; **P-value = 0.0024 for EE and 0.0012 for EPI; ***P-value < 0.0006. Source data for all graphs are available from the Source Data file and raw data from Figshare (see “Data availability” section).

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