Journal: Nucleic Acids Research

Article Title: Nascent transcriptome of embryonic genome activation reveals a regulatory axis linking transcriptional priming to early lineage specification in mouse embryos

doi: 10.1093/nar/gkag173

Figure Lengend Snippet: KLF17 participates in the first lineage specification. ( A ) Quantification of the developmental rate at the indicated timepoints from four independent experiments. Sample sizes: control (Ctrl): n = 17, 20, 23, and 21; KO: n = 10, 28, 37, and 15; pKO: n = 17, 15, 48, and 23; mKO: n = 44, 28, 41, and 15. ( B ) Representative images of OCT4 and CDX2 signal in blastocysts, alongside three-dimensional (3D) reconstruction. Scale bar, 20 µm. ( C ) Quantitative analysis of total, TE, and ICM cell numbers (left) with corresponding proportions (right) in blastocysts across experimental groups in B. Sample sizes: Ctrl: n = 16, KO: n = 9, mKO: n = 21, pKO: n = 13. One-way ANOVA followed by Dunnett’s test. ** P < 0.01; *** P < 0.001; NS, not significant. ( D ) Scheme of exogenous KLF17 expression. Zygotes were injected with Klf17-Gfp mRNA and collected at the 2C stage for immunostaining. Scale bar, 20 µm. ( E ) Relative exogenous KLF17 expression level analysis in 2C embryos. Each dot represents one blastomere, and each column represents one embryo. Hoechst staining as an internal control for normalization. Sample sizes: n = 34. ( F ) Scheme of Klf17 knockdown (KD) and overexpression (OE). One blastomere of two-cell embryos was cytoplasmically injected with Klf17 siRNA or Klf17 mRNA, together with Myr-Gfp mRNA. The embryos were cultured to the blastocyst stage, and the lineage contribution (ICM/TE) of the progeny derived from the injected blastomere analyzed. Scale bar, 20 µm. ( G ) Analysis of the distribution of progeny of injected blastomeres at the blastocyst stage based on 3D reconstruction. Sample sizes: Ctrl: n = 23, Klf17 OE: n = 23, Klf17 KD: n = 16. Inter-group comparisons are present on the left by using one-way ANOVA followed by Dunnett’s test. Intra-embryo comparisons are present on the right by using paired t -tests. * P < 0.05; NS, not significant.

Article Snippet: After blocking, the embryos were incubated with primary antibody (Oct3/4 Antibody (C-10), Santa Cruz, sc-5279; CDX2 (D11D10) Rabbit mAb, Cell Signaling Technology, 12306; GFP Polyclonal Antibody, Invitrogen, PA1-9533) diluted with blocking solution overnight at 4°C.

Techniques: Control, Expressing, Injection, Immunostaining, Staining, Knockdown, Over Expression, Cell Culture, Derivative Assay