Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo

Abstract

Spatial and temporal changes in molecular expression are essential to embryonic development, and their characterization is critical to understand mechanisms by which cells acquire different phenotypes. Although technological advances have made it possible to quantify expression of large molecules during embryogenesis, little information is available on metabolites, the ultimate indicator of physiological activity of the cell. Here, we demonstrate that single-cell capillary electrophoresis-electrospray ionization mass spectrometry is able to test whether differential expression of the genome translates to the domain of metabolites between single embryonic cells. Dissection of three different cell types with distinct tissue fates from 16-cell embryos of the South African clawed frog (Xenopus laevis) and microextraction of their metabolomes enabled the identification of 40 metabolites that anchored interconnected central metabolic networks. Relative quantitation revealed that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos. Altering postfertilization cytoplasmic movements that perturb dorsal development confirmed that these three cells have characteristic small-molecular activity already at cleavage stages as a result of cell type and not differences in pigmentation, yolk content, cell size, or position in the embryo. Changing the metabolite concentration caused changes in cell movements at gastrulation that also altered the tissue fates of these cells, demonstrating that the metabolome affects cell phenotypes in the embryo.

Keywords: Xenopus; embryo development; mass spectrometry; metabolomics; single cell.

Fig. 1.

Our experimental workflow to uncover small-molecular activity during early-stage embryo development. Single blastomeres were identified and dissected from 16-cell frog (X. laevis) embryos and their metabolomes extracted and measured by a custom-designed single-cell CE-µESI-MS. (Scale bars, 250 µm.)

Fig. 2.

False-color heat map and hierarchical clustering of small molecules detected in single D11, V11, and V21 blastomeres of 16-cell Xenopus embryos. Metabolite abundances are normalized relative to the mean and shown in false color for the 40 statistically most significant features (P < 0.05). Each blastomere has a unique identifier (bottom axis) with solid lines connecting the technical replicates for four of the n = 5 biological replicates that were measured for each cell type. The data reveal that D11, V11, and V21 cells have distinct small-molecular activities during early embryogenesis. Abbreviations of small molecules are given in SI Appendix, Table S2.

Fig. 3.

Significant differences in the metabolic activity of blastomeres in the animal–vegetal–dorsal–ventral domains of the embryo. (Left) Each data point plots the metabolite signal abundance ratio vs. statistical significance for different small molecules between the D11/V11 (black filled circle), D11/V21 (gray filled circle), and V11/V21 (open circle) cell types. (Right) Box-whisker plots compare cases of select metabolites that have similar and biologically statistically significantly different concentrations in the D11, V11, and V21 blastomeres (square is mean, box is 1 × SE, and whisker is 1.5 × SE.) Statistical significance is marked at *P < 0.05 and **P < 0.005. Small molecules are listed in SI Appendix, Table S2.

Fig. 4.

Functional evaluation of small-molecular differences between D11, V11, and V21 blastomeres. Differential metabolic activity between the D11/V11 and D11/V21 blastomere types that is characteristic of the untreated (UT, black) embryo was lost for most metabolites in UV light-ventralized (UV, red) embryos. These results establish that the small-molecular cell heterogeneity in the 16-cell embryo is driven by blastomere type rather than the location, size, or pigmentation of the cells. Small molecules are listed in SI Appendix, Table S2. Statistical and biological significances are tabulated in SI Appendix, Table S3.

Fig. 5.

Assessment of developmental significance for blastomere type-characteristic metabolites by microinjection and cell lineage tracking. Larval stages (side views, dorsal to the top) injected with gfp mRNA alone (D11 ctrl; V11 ctrl) or metabolites plus gfp mRNA (m_V11D11; m_D11V11). Green fluorescent cells are descended from the injected blastomere. (Left) In D11 ctrl embryos, there are large numbers of cells in the brain (b), retina (r), olfactory pit (olf), and central somite (cs). In V11-metabolite-injected D11 embryos (m_V11D11), the numbers of descendants in brain and central somite are significantly reduced, and those in the ventral epidermis (epi) are increased. (Right) In V11 ctrl embryos, there are only small numbers of cells in the olfactory pit, lens (l), and otocyst (oto). In D11-metabolite-injected V11 embryos (m_D11V11), the numbers of descendants in these head structures are increased. Quantitation of the number of cells in selected tissues show significant differences between control and metabolite-injected clones. Statistical significance is marked at *P < 0.05 and **P < 0.005. (Scale bars, 275 μm for all images.)