Single-cell mass spectrometry with multi-solvent extraction identifies metabolic differences between left and right blastomeres in the 8-cell frog (Xenopus) embryo

Abstract

Single-cell metabolic mass spectrometry enables the discovery (untargeted) analysis of small molecules in individual cells. Using single-cell capillary electrophoresis high-resolution mass spectrometry (CE-HRMS), we recently uncovered small-molecule differences between embryonic cells located along the animal–vegetal and dorsal–ventral axes of the 16-cell frog (Xenopus laevis) embryo, raising the question whether metabolic cell heterogeneity also exists along the left–right body axis. To address this question, we here advance single-cell CE-HRMS for identifying and quantifying metabolites in higher analytical sensitivity, and then use the methodology to compare metabolite production between left and right cells. Our strategy utilizes multiple solvents with complementary physicochemical properties to extract small molecules from single cells and improve electrophoretic separation, increasing metabolite ion signals for quantification and tandem HRMS. As a result, we were able to identify 55 different small molecules in D1 cells that were isolated from 8-cell embryos. To quantify metabolite production between left and right cells, we analyzed n = 24 different D1 cells in technical duplicate–triplicate measurements. Statistical and multivariate analysis based on 80 of the most repeatedly quantified compounds revealed 10 distinct metabolites that were significantly differentially accumulated in the left or right cells (p < 0.05 and fold change ≥ 1.5). These metabolites were enriched in the arginine–proline metabolic pathway in the right, but not the left D1 cells. Besides providing analytical benefits for single-cell HRMS, this work provides new metabolic data on the establishment of normal body asymmetry in the early developing embryo.

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)

“…These developments enabled femtogram (zeptomole) limit of detection (38) for protein digests from cell populations. For example, we recently developed metabolomic CE microflow electrospray ionization (ESI) HRMS platforms (26) for measuring metabolites (27,39) and proteins (36) in single Xenopus blastomeres. Using microdissection to isolate single blastomeres, tandem mass tags to enable multiplexing quantification, and bottom-up proteomics, CE-ESI-HRMS was able to quantify 130 -150 different protein groups (isoforms) in common between multiple single blastomeres in the 16-cell Xenopus embryo.…”

From cytoskeletal dynamics to organ asymmetry: a nonlinear, regulative pathway underlies left–right patterning

“…This process is best understood in frog embryos, but has also been observed and functionally implicated in amphioxus [149], sea urchin [146,150,151], C. elegans [152 -154], zebrafish [96,155,156] and recently humans [157,158]. Indeed, a recent analysis of the differences between blastomeres at the very earliest stages of embryo development identified distinct metabolites in L versus R cells in the eight-cell embryo using mass spectrometry [88]. The majority of these metabolites themselves have roles in functional regulation of ion transport [159 -167], suggesting possible feedback loops in electrophysiology that could be important amplifying mechanisms for initially subtle LR asymmetry.…”

“…From data in a range of model species, it is clear that numerous aspects of development, including maternal protein localization [84,[95][96][97], Wnt signalling [98] and small signalling molecules [88], are already consistently asymmetric long before cilia appear: most animal embryos can tell their left from their right at very early stages. Thus, the search for the origin of asymmetry has been extended far upstream of neurulation [22,99].…”

“…Recent functional data revealed conserved mechanisms in a range of organisms from plants to mammals, which establish asymmetry without a ciliated structure, or long before it forms; indeed, many phyla including some vertebrates determine their LR axis very early after fertilization [80][81][82][83][84][85][86][87][88][89][90]. Even mouse embryos are known to exhibit molecular and functional asymmetries (e.g.…”

Introduction to provocative questions in left–right asymmetry

“…mechanosensitive cilia, morphogen gradients or vesicular particles. More recent studies have shown that primary cilia are not in fact calcium-responsive mechanosensors [8], and that asymmetries in cleavage-stage frog embryos have not only consistently asymmetric metabolic profiles [9], but also protein components with functional roles in asymmetry development [10]. Consistent with the existence of L/R asymmetry at such an early stage of development, Dasgupta and Amack [6] used mosaic cell labelling and time-lapse imaging in the zebrafish embryo to demonstrate that ciliated cells within Kupffer's vesicle-a structure that is functionally analogous to the embryonic node and alternatively termed the 'L/R organizer'-are non-stochastically arranged, suggesting that they may be propagating L/R positional information that exists prior to L/R Organizer morphogenesis and hence, generation of directional fluid flow.…”