Fig. 2: Glucose metabolism via HBP is necessary for mesoderm fate acquisition and maintenance.
From: Selective utilization of glucose metabolism guides mammalian gastrulation
a, Schematic of the three branches of glucose metabolism: HBP, glycolysis pathway and pentose phosphate pathway (PPP). GFAT, glutamine fructose-6-phosphate amidotransferase; PFK, phosphofructo-1-kinase; TCA, tricarboxylic acid. b, Representative z-sections of embryos in the indicated conditions following 12 h ex vivo culture. Inhibition of glucose metabolism (2-DG + BrPA) and HBP (Azaserine) impairs primitive streak elongation (dashed cyan lines) and reduces expression of T-box transcription factor T (also known as Brachyury) (cyan). Bottom, quantification of streak distal elongation lengths in embryos treated as follows: pre-culture control (n = 7), control (after 12 h of culture, n = 44), glucose metabolism inhibition (2-DG and BrPA; n = 16), 2-DG and BrPA with galactose rescue (n = 8), glycolysis inhibition (YZ9; n = 9), glycolysis inhibition (shikonin; n = 9), HBP inhibition (azaserine; n = 21), no glucose, no glutamine (nutrient sparse) plus galactose (n = 6), pentose phosphate pathway inhibition (6-AN; n = 4), ATP synthase inhibition (oligomycin; n = 8) and lactate dehydrogenase inhibition (galloflavin; n = 6). Data are mean ± s.e.m. Tukey’s multiple comparison test following ordinary one-way ANOVA; P values are shown. Scale bars, 40 µm. c, GLUT1 (heat map intensity colours via LUT in Fiji) expression in epiblast co-localizes to regions of intact basement membrane identified via laminin staining (n = 28 embryos). Scale bars, 40 µm. d, Quantification of basement membrane (BM) distal breakdown length (yellow arrows), identified from laminin expression. Pre-culture control (n = 7), control (after 12 h of culture; n = 42), glucose metabolism inhibition (2-DG and BrPA; n = 16), 2-DG and BrPA with GlcNAc rescue (n = 14), glycolysis inhibition (YZ9; n = 12), glycolysis inhibition (shikonin; n = 3), HBP inhibition (azaserine; n = 11), azaserine with GlcNAc rescue (n = 7), pentose phosphate pathway inhibition (6-AN; n = 4), ATP synthase inhibition (oligomycin; n = 6), and lactate dehydrogenase inhibition (galloflavin; n = 6). Data are mean ± s.e.m. Tukey’s multiple comparison test following ordinary one-way ANOVA; P values are shown. Scale bars, 40 µm. e, Representative outcome of in vitro EMT assay with DQ gelatin applied on epiblast-like stem cells (EpiSCs) at differentiation stage (day 2 (Extended Data Fig. 5a)). Bottom, the number of DQ+ clusters identified per imaging field (n = 25 fields quantified over 2 independent experimental replicates). Dunnett’s multiple comparison test following ordinary one-way ANOVA; P values are shown. Scale bars, 100 µm.
