Fig. 3: Glucose metabolism modulates ERK signalling in the epiblast.
From: Selective utilization of glucose metabolism guides mammalian gastrulation
a, Western blot showing total ERK1/2 and phosphorylated (p-)ERK(T202/Y204) in EpiSCs after 12 h of the indicated treatment. n = 2 experimental replicates. For gel source data, see Supplementary Fig. 1. b, Left, schematic of tetraploid complementation assay, which generates embryos in which all cells in the embryo proper are derived from ERK-KTRmClover 2N mouse embryonic stem cells. Right, live imaging of ERK-KTRmClover signal in a representative embryo generated from a tetraploid complementation assay. Scale bars, 40 µm. c, Representative gastrulas are shown for ERK-KTRmClover signal under control (n = 10), 2-DG and BrPA (n = 8), NaClO3 (n = 4), azaserine (n = 8) and azaserine with GlcNAc rescue (n = 8) treatments. n = 3 independent experiments. Scale bars, 20 µm. Also see Extended Data Fig. 8d,e. d, Nuclear:cytoplasmic ratio of ERK-KTRmClover in EpiSCs treated as indicated. Data are mean ± s.e.m. Control (n = 310 cells quantified) was significantly different from all other conditions (PD0325901 (PD), n = 188 cells; 2-DG, n = 166 cells; azaserine, n = 193 cells; NaClO3, n = 120 cells; PD0325901 + FGF2, FGF4 and FGF8 (FGFs), n = 201 cells; 2-DG + FGFs, n = 178 cells; azaserine + FGFs, n = 186 cells; NaClO3 + FGFs, n = 132 cells). Dunnett’s multiple comparison test following ordinary one-way ANOVA; P values are shown. e,f, Representative images (e) and nuclear:cytoplasmic ratio of mClover signal (f) of ERK-KTRmClover embryonic stem cells differentiated to mesoderm under control (n = 75 cells), azaserine, 2-DG or PD0325901 (n = 50 cells each) conditions. Black horizontal line represents the mean. Dunnett’s multiple comparison test following ordinary one-way ANOVA; P values are shown. All comparisons shown are to control. Scale bars, 40 µm. g, Directed-differentiated mesoderm cells in nutrition-sparse medium (free of glucose, pyruvate and glutamine) and rescue conditions (selective reintroduction of the excluded nutrients) for 6 h. Black horizontal line represents the mean. Dunnett’s multiple comparison test following ordinary one-way ANOVA; P values are shown. Control, n = 83 cells; nutrient depleted, n = 41 cells; glucose only, n = 70 cells; glutamine only, n = 66 cells; pyruvate only, n = 47 cells.
