Metabolic Comparison of Dorsal versus Ventral Cells Directly in the Live 8-cell Frog Embryo by Microprobe Single-cell CE-ESI-MS.

Single-cell mass spectrometry (MS) empowers the characterization of metabolomic changes as cells differentiate to different tissues during early embryogenesis. Using whole-cell dissection and capillary electrophoresis electrospray ionization (CE-ESI) MS, we recently uncovered metabolic cell-to-cell differences in the 8- and 16-cell embryo of the South African clawed frog (Xenopus laevis), raising the question whether metabolic cell heterogeneity is also detectable across the dorsal-ventral axis of the 8-cell embryo. Here, we tested this hypothesis directly in the live embryo by quantifying single-cell metabolism between the left dorsal-animal (D1L) and left ventral-animal (V1L) cell pairs in the same embryo using microprobe single-cell CE-ESI-MS in the positive ion mode. After quantifying ~70 molecular features, including 52 identified metabolites, that were reproducibly detected in both cells among n = 5 different embryos, we employed supervised multivariate data analysis based on partial least squares discriminant analysis (PLSDA) to compare metabolism between the cell types. Statistical analysis revealed that asparagine, glycine betaine, and a yet-unidentified molecule were statistically significantly enriched in the D1L cell compared to V1L (p < 0.05 and fold change ≥ 1.5). These results demonstrate that cells derived from the same hemisphere (animal pole) harbor different metabolic activity along the dorsal-ventral axis as early as the 8-cell stage. Apart from providing new evidence of metabolic cell heterogeneity during early embryogenesis, this study demonstrates that microprobe single-cell CE-ESI-MS enables the analysis of multiple single cells in the same live vertebrate embryo.