Peter Nemes (Presenter)
George Washington University
Bio: Peter Nemes, Ph.D., is an Assist. Prof. of Chemistry (2013–Present) at the George Washington University, whence he obtained his Ph.D. in Chemistry (Advisor: Prof. Akos Vertes). He completed his postdoctoral research in Bioanalytical Chemistry for Neurobiology in Prof. Jonathan Sweedler’s laboratory at the University of Illinois—Urbana-Champaign. Dr. Nemes’ research is focused on advancing MS to assess the metabolome and proteome of volume-limited samples, specifically single cells, during early development of the embryo and the central nervous system. He has (co)authored 4 book chapters, 28 peer-reviewed publications, 60+ presentations at (inter)national conferences. He is the co-inventor of the LAESI-MS technology. In 2015, Dr. Nemes was appointed a Beckman Young Investigator by the Arnold and Mabel Beckman Foundation.
Authorship: Peter Nemes (1), Rosemary M. Onjiko (1), Erika P. Portero (1), and Sally A. Moody (2)
(1) Department of Chemistry and (2) Department of Anatomy & Regenerative Biology, The George Washington University, Washington DC, 20052
| Short Abstract Knowledge of all molecules in embryonic cells raises a potential to holistically understand basic processes that orchestrate the development of the normal embryo. However, the complex three-dimensional and spatiotemporally evolving structure of the embryo poses significant analytical challenges in capturing molecular differences between cells using mass spectrometry, particularly at the level of metabolites that are complex and also dynamic. We describe here a microsampling approach to find metabolic differences between identified single cells in the 16-cell frog (Xenopus laevis) embryo. In combination with functional studies, we discover metabolites that are able to alter the developmental fate of embryonic cells. |
Long Abstract
Introduction. Characterization of biomolecular pathways underlying the patterning of the body axis is essential to understanding normal embryonic development and finding therapeutics for congenital anomalies. How small molecules, particularly metabolites, are implicated in the establishment of developmental axes and tissue fates is little known because it has not been technologically feasible to measure a broad spectrum of small molecules in single embryonic cells (blastomeres). Recent whole-embryo analyses on Xenopus [1] and zebrafish [2] found that gross transcriptomic, proteomic, and metabolic changes delineate the different embryonic developmental stages, leading to our hypothesis that metabolomic regulation is heterogeneous also on the level of the single blastomere during early embryogenesis. To test this hypothesis, we utilized a custom-built single-cell mass spectrometry (MS) technology [3] to compare metabolite production between single blastomeres that reproducibly demarcate the three developmental axes and give rise to different tissue types in the normal embryo [4] using the South African clawed frog (Xenopus laevis), a popular model in cell and developmental biology.
Methods. Adult frogs (Xenopus laevis) were maintained in a breeding colony following protocols approved by the GW Institutional Animal Care and Use Committee (approval number A311). Single-cell measurements utilized a custom-built capillary electrophoresis electrospray ionization (CE-ESI) MS system that we built based on our earlier prototype [3]. In this work, we extended the performance of this instrument to a lower limit of detection below 10 nM, or 60 amol, for various metabolites with a measurement reproducibility of ∼5% relative standard deviation (RSD) for separation time and <25% RSD for quantitation. Metabolites were electrophoretically separated in 1% formic acid at 20 kV potential, ionized by a custom-built CE electrospray ionization source (sheath-flow design), and detected by a Qq-TOF high-resolution mass spectrometer capable of single- and tandem-stage operational modes. Metabolite identifications were facilitated by collision-induced dissociation at 17 eV energy in nitrogen collision gas. The mass spectrometric data were analyzed using multivariate tools in an unsupervised (hierarchical cluster analysis, HCA; principal component analysis, PCA) and supervised (partial least squares discriminant analysis, PLSDA) manner. Significant differences were marked by a p value < 0.05 (Student’s t-test) and fold change > 2.0.
Results. To address our hypothesis, we developed a microanalytical workflow to determine the metabolic composition of single blastomeres that occupy the dorsal-ventral, animal-vegetal, and left-right axes of the Xenopus embryo at different developmental stages. We isolated left and right blastomeres in the animal-dorsal quadrant (D1 cell) of the embryo and three distinct blastomeres that reproducibly give rise to different tissue types in the 16-cell embryo: the animal-dorsal cell (D11, precursor to the brain, retina), the animal-ventral (V11, precursor to the epidermis), and the vegetal-ventral cell (V21, precursor to the hindgut). Immediately after microdissection, these blastomeres were transferred to cold methanol to quench enzymatic activity, the samples were lyophilized, and their metabolomes extracted in 5 µL of 50% methanol (0.5% acetic acid). The extracts were sonicated and centrifuged before measuring a 10–20 nL fraction using the CE-MS platform. This microanalytical workflow enabled the qualitative and quantitative characterization of small molecules in the single blastomeres with trace-level detection. Of the more than 130 different metabolite signals were detected in the single blastomeres, 80 were consistently measured between the D11, V11, and V21 blastomeres between multiple technical and biological replicates; further analysis was limited to these features. Forty of these signals were identified based on accurate mass measurements, isotope distribution analysis, and comparison to separation (migration) time and tandem mass spectrometry data recorded on related chemical standards. Approximately 90% of these metabolites were mapped to major metabolic pathways that range from the synthesis of basic amino acids and classical neurotransmitters to energy carriers and photoprotectants.
To compare the small-molecular composition along the body axes, we performed unsupervised PCA and HCA on the CE-ESI-MS data for the D11, V11, and V21 blastomeres. A total of n = 5 biological replicates (different cells from different embryos) were measured in this portion of the study to ensure sufficient statistical power. In the resulting scores plot, the D11, V11, and V21 sample data formed distinct data clusters, revealing unique metabolite profiles between these cell types. These characteristic differences were also evidenced by differential clustering of the extracts in HCA plot. Furthermore, certain metabolites were present in statistically significantly different ion abundances between the cell types (p < 0.05). For instance, serine accumulated in the D11 blastomeres but not in the others. In agreement, serine is a known neurotropic factor for the development of embryonic nervous system, and D11 blastomeres are precursors to the brain, retina, and spinal cord. Next, we tested whether metabolic differences existed also along the left-right axis. To probe deeper into the metabolome with higher sensitivity, we developed a triplex strategy to extract polar and polar metabolites from left and right D1 blastomeres in the 8-cell embryo. PLSDA supplemented revealed 12 distinct metabolites that were differentially enriched between the left and right D1 blastomeres (p < 0.05 and fold change > 2), which were further confirmed via statistical tools. That single blastomeres foster metabolically heterogeneous composition along the animal-vegetal, dorsal-ventral, and left-right axes in the 8- and 16-cell embryo was previously unknown and is surprising considering that the transcriptome is indicative of cell asymmetry only along the animal-vegetal axis.
Last, we tested the developmental significance of some of the identified metabolites that had differential accumulation across the body axes. Using external concentration calibration, we determined their absolute concentration in the D11 and V11 blastomeres. Afterward, we microinjected D11-rich metabolites as standards into V11 blastomeres at their nature concentration, and vice versa, while using the green fluorescent protein to track cell fates. Surprisingly, as a result of microinjection, the developmental fate of these blastomeres was altered, demonstrating small molecules that interfere with cellular differentiation.
Conclusions. These results demonstrate metabolic cell heterogeneity at a relatively early stage of embryonic development (8- and 16-cell embryo), long before translation of the embryo’s own genome is known to begin. This work underscores the central role that bioanalytical chemistry and instrument development holds in translational research. We anticipate that the single-cell MS, such as the technology presented here, will serve as a new research tool for cell and developmental biology to allow for asking new types of questions to better our understanding and treatment of human health.
References & Acknowledgements:
References.
[1] L. Vastag, P. Jorgensen, L. Peshkin, R. Wei, J. D. Rabinowitz, M. W. Kirschner, Remodeling of the metabolome during early frog development, PlosOne 2011, 6, e16881.
[2] S. M. Huang, F. G. Xu, S. H. Lam, Z. Y. Gong, C. N. Ong, Metabolomics of developing zebrafish embryos using gas chromatography- and liquid chromatography-mass spectrometry. Molecular Biosystems. 2013, 9, 1372-1380.
[3] P. Nemes, S. S. Rubakhin, J. Aerts, and J. V. Sweedler, Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry, Nat. Protoc. 2013, 8, 783–799.
[4] R. M. Onjiko, S. A. Moody, and P. Nemes, Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo, Proc. Nat. Acad. Sci. USA 2015, 112, 6545–6550.
Acknowledgment. This work was supported by the National Institutes of Health Grant GM114854 (to P.N.) as well as the GW Dept. Chem. Start-Up Funds (to P.N.) and the Columbian College Facilitating Fund (to P.N. and S.A.M.).
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