Extended Data Fig. 3: Glucose metabolism via HBP controls mesoderm fate acquisition.
From: Selective utilization of glucose metabolism guides mammalian gastrulation
(a) Left: Schematic of glucose metabolism and its three branches: light-grey = Hexosamine Biosynthetic Pathway (HBP); pink = core/late-stage glycolysis; green = Pentose Phosphate Pathway (PPP). Red text indicates chemical inhibitors and their metabolic enzyme targets in blue. (b) Representative z-sections showing the expression pattern of SOX2, T/BRA, and LEF1 proteins in embryos treated with 2-DG+BrPA for 18 h ex vivo and (c) their Theiler developmental outcomes: Control (n = 17), 2-DG+BrPA (n = 24) across 6 experimental replicates; early-streak (ES), mid-streak (MS), late-streak (LS), no bud (OB), early bud (EB), late-bud (LB), early head fold (EHF). Scale bars represent 40 µm. Plots show mean ± SEM. On average, ~53% of control embryos develop to OB stage gastrulation, while ~73% of 2-DG+BrPA treated embryos only develop to MS stage gastrulation. (d) Dose response curves demonstrating efficacy of the inhibitors 2-DG (left; pre-culture, n = 7; control, n = 42; 0.5 mM, n = 5; 1 mM, n = 4; 2 mM, n = 7; 3 mM, n = 4; 5 mM, n = 5 embryos) and Azaserine (right; pre-culture, n = 7; control, n = 42; 1 μM, n = 3; 3 μM, n = 4; 5 μM, n = 12; 7 μM, n = 4; 10 μM, n = 3 embryos). Plots show mean and SD. Tukey’s multiple comparison tests following ordinary one-way ANOVAs. ****P < 0.0001 whenever indicated. Pre-culture vs. 1 mM 2-DG **P = 0.0066, pre-culture vs. 5 mM 2-DG **P = 0.0036, 1 mM 2-DG vs. 2 mM 2-DG **P = 0.0016, 1 mM 2-DG vs. 3 mM 2-DG *P = 0.0173, 2 mM 2-DG vs. 5 mM 2-DG *P = 0.0152, 3 mM 2-DG vs. 5 mM 2-DG *P = 0.0217. Pre-culture vs. 1 μM Azaserine *P = 0.0522, pre-culture vs. 3 μM Azaserine *P-0.0435, control vs. 5uM Azaserine ***P = 0.0006, 1 μM Azaserine vs. 10 μM Azaserine ***P = 0.0001, 5 μM Azaserine vs. 7 μM Azaserine ***P = 0.0001, 5 μM Azaserine vs. 10 μM Azaserine ***P = 0.0009. Please note that the “pre-culture” condition refers to the embryos at the beginning of the experiment, when they already exhibit some level of primitive streak formation. Drug concentrations that overall compromised embryo viability are considered toxic. Concentrations used in this study (boxed in red) altered primitive streak elongation without compromising embryo viability. (e) Representative images show mouse gastrula developmental outcomes following 12 hr metabolic inhibitor treatment. Also see Fig. 2b. A, Anterior; P, Posterior. Scale bars represent 40 µm. (f) Mouse gastrulas cultured in nutrition-sparse media (free of glucose, pyruvate, and glutamine), and rescue conditions (selective reintroduction of the excluded nutrients) to functionally validate the specific effects of chemical inhibitors. A, Anterior; P, Posterior. Scale bars represent 40 µm. Graph on the left shows mean ± SEM of SOX2 expression intensity (normalised to DAPI). Dunnett’s multiple comparison tests following ordinary one-way ANOVAs. Control (n = 4 embryos) compared to no nutrients (n = 3 embryos), glucose (n = 5 embryos), pyruvate (n = 5 embryos), glutamine (n = 4 embryos), and glucose + glutamine (n = 5 embryos for anterior epiblast, n = 3 embryos for PS + mesoderm). Graph on the right shows PS distal elongation % (Control, n = 7 embryos; no nutrient, n = 6 embryos; glucose, n = 8 embryos; pyruvate, n = 9 embryos; glutamine, n = 7 embryos). Plot shows mean + SD. (g) Representative z-sections showing similar PHOSPHO-HISTONE H3 localizations (cyan) in 2-DG-BrPA and Azaserine treated embryos compared to control embryos at the MS stage of gastrulation. Scale bars represent 40 µm. Plots show mean + SD. Ordinary one-way ANOVA. P-values shown.
