Figure 1: A glycolysis-dependent pHi gradient in the tail bud.

(a) (Left) Tail bud (TB) and presomitic mesoderm (PSM) region in 2-day chicken embryo; pHi gradient (pink). (Right) Corresponding region electroporated with pHluorin (n=13). Yellow: acidic regions. A: anterior. P: posterior.

(b) 488/405 nm ratio in cells in a representative embryo (n=3).

(c) Average 488/405 nm ratio of PSM region in control (Ctl) (n=8) and 2DG-treated (n=8) embryos. Two-sided unpaired t-test, *p=0.01.

(d) Normalized Average 488/405 nm ratio in control (n=8) and 2DG-treated (n=8) embryos. Red lines: Mean (±SD) of different embryos. Two-sided paired t-tests, * p=0.02; n.s. p=0.69.

(e) Axis elongation in 2-day embryos in vitro: (Top) pH7.2, 8.3mM (n=6), 0.83mM (n=3) and 0 mM Glucose (n=6). (Bottom) pH7.2 (n=6), pH6.0 (n=5) and pH5.3 (n=8). Two-way Anova followed by Tukey’s multiple comparison test. ***p<0.0005.

(f) Average 488/405 nm ratio of PSM region of 2-day embryos at pH7.2 with 8.3 mM (n=6), 0.83 mM (n=5) and 0 mM Glucose. Two-sided unpaired t-test. *p=0.01; **p=0.001; n.s. p>0.05.

(g) Normalized Average 488/405 nm ratio. Embryos cultured at pH7.2: 8.3 mM (n=6), 0.83 mM (n=5) and 0 mM Glucose (n=6). Compared average ratios +/− SD (ends of the red line) of pooled samples. Two-sided paired t-test. *p=0.002. n.s. p>0.05.

(h) Average 488/405 nm fluorescence ratio of 2-day chicken embryos at pH5.3: n=4, pH6.0: n=4, pH7.2: n=4, pH7.6: n=4; (two independent experiments). Two-sided unpaired t-test. *p<0.01; **p<0.001; n.s. p>0.05.

(i) MSGN1, SAX1, SOX2 and AXIN2 expression in the posterior region of 2-day chicken embryos at different pH (n=4 for each gene). Data normalized with pH7.2 samples. Tukey’s multiple comparison test. * p<0.05, ***p<0.001.

(j) 2-day chicken embryos hybridized with AXIN2 (pH5.3: n=6, pH7.2: n=6, pH7.6: n=4) and SOX2 (pH5.3: n=6, pH7.2: n=4, pH7.6: n=5) probes.

(b-h) Mean +/− SD. (a, b, j) Ventral views, anterior to the top. Bar, 100 μm. Exact p values can be found in the corresponding Source Data files.