Fig. 2: Epiblast occurs in the absence of Smad4. | Nature Communications

Fig. 2: Epiblast occurs in the absence of Smad4.

From: A stepwise mode of TGFβ-SMAD signaling and DNA methylation regulates naïve-to-primed pluripotency and differentiation

Fig. 2

A The scheme showed the procedure of EpiLC transition from mESC (top). Briefly, mESCs turned into EpiLCs after mESCs were shifted from 2i+LIF medium to N2B27 medium with Activin A and bFGF for 6 days. This experiment was repeated 5 times. Representative images showing cell morphology changes during the transition in WT, S4KO, S2/3DKO, and S2/3DKO rescued with Smad2/3 (bottom) are presented here. Scale bar: 100 μm. B mRNA levels of pluripotent genes (Nanog, Oct4) and post-implantation epiblast genes (Fgf5, Dnmt3a/3b, T) during the induction of EpiLCs were analyzed by qRT-PCR. Data are presented as mean values +/- SD. n = 3 independent experiments. P values were calculated by two-way ANOVA test with Geisser-Greenhouse correction. C Venn diagram indicated the overlapping peaks among Smad4 ChIP-seq in WT, Smad2/3 ChIP-seq in WT and Smad2/3 ChIP-seq in S4KO. Genes adjacent to 7926 peaks were annotated, in which primed epiblast genes were marked below. D Heatmap of ChIP-seq tag densities for Smad2/3 and Smad4 were within −1.5/ + 1.5 kb genomic regions surrounding the centers of 11,080 high-confidence Smad2/3 binding sites in S4KO D3 EBs with AC treatment. SB, SB431542, a selective inhibitor of TGFβR; AC, Activin A, an available ligand for Nodal/Activin receptors. E Gene track view of the Fgf5, Dnmt3a, Dnmt3b and T loci. Smad2/3 and Smad4 ChIP-seq were performed in SB- or AC-treated D3 EBs and in D0 non-treated mESCs. Tag densities were normalized to reads per kilobase per million mapped reads (RPKM). The gene structures from RefSeq are schematically represented at the bottom.

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