Extended data Figure 1: Analysis of the intracellular pH (pHi) in the chicken embryo in vivo.

(a) Ratiometric live expression of pHluorin (488/405 nm) detected in the posterior domain of electroporated embryos exposed to different pH buffers and Nigericin and Valinomycin (n=7). Fluorescence intensity is shown by pseudocolor image (Fire color) using image J. Yellow signal indicates lower pH. Ventral view, Anterior to the left. Scale bar :100 μm.

(b) Each dot represents the average 488/405 nm signal ratio of ~300 single cells segmented in one embryo (n=2 for pH 5.5, n=3 for pH 6.5, n=2 for pH 7.5). Embryos were treated for 20 min in different pH buffers with the protonophores Nigericin and Valinomycin, before live imaging.

(c) Micro-dissected posterior PSM incubated with 20 μM BCECF (n=7). Left panel: fluorescent intensities for excitation at 405 nm (blue) and 488 nm (green). Right panel: 488/405 nm ratio. Scale: 100 μm.

(d) Fluorescence 488/405 nm ratios along the PSM. Each colored line corresponds to an explant (n=7 from two independent experiments). med: medial PSM; pos: posterior region. Two-sided paired t-test. **P=0.009

(e-f) Whole-mount in situ hybridization of 2-day chicken embryos cultured at different pH and hybridized with MSGN1 (pH5.3: n=4, pH7.2: n=3, pH7.6: n=3) (e) and SAX1 (pH5.3: n=6, pH7.2: n=4, pH7.6: n=5) (f). Ventral view, anterior to the top. Scale bar: 100 μm.