Fig. 3

Signalling pathways involved in segregation of lineages. a Experimental design of embryo treatments. b Bright field and IF staining for indicated markers; embryos were counterstained with DAPI (merge). Scatter dot plots of NANOG-, SOX17-positive cells and total cell numbers (black bar indicates mean) of control pig morula (M, n = 7), early blastocysts (EB, n = 8), mid-blastocysts (MB, n = 9) and late blastocysts (LB, n = 5). c Bar charts indicating percentage of ICM cells expressing indicated markers in control embryos. d Gene expression of LIF/IL6 and its cognate receptors in pig embryos. e Bar charts indicating percentage of ICM cells expressing indicated markers after different treatments. f Scatter plots show proportion of cells stained for the indicated markers in control embryos (EB n = 8, MB n = 9, LB n = 5) and embryos treated with JAKi: 10 µM AZD1480 (EB n = 11, MB n = 4, LB n = 8), PI3Ki: 10 µM LY294002 (EB n = 7, MB n = 5), TGFβi: 20 µM SB431542 (EB n = 3, MB n = 7, LB n = 7), MEKi: 10 µM PD0325901 (MB n = 7), WNTi: 3 µM IWP2 (MB n = 9), IL6: 10 ng ml⁻¹ (MB n = 8) and TGFβ: 10 ng ml⁻¹ (MB n = 8). g Scatter plots show the number of cells stained in Sham controls (n = 24), homozygous KO (IL6^−/−) (n = 9) and mosaic embryos (n = 6) after CRISPR/Cas9 gene editing. ICM cells were determined by counting SOX2 positive cells and TE cells were calculated by subtracting SOX2 cells from the total cell count. Ctr: control, PM: pre-morula. For c, e: *p ≤ 0.05, two-way ANOVA. For f, g: *p ≤ 0.05, Mann–Whitney test. Source data are provided as a Source Data file. Scale bars: 50 µm