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Adv Sci (Weinh)
2024 Mar 01;119:e2305401. doi: 10.1002/advs.202305401.
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Chemo-Selective Single-Cell Metabolomics Reveals the Spatiotemporal Behavior of Exogenous Pollutants During Xenopus Laevis Embryogenesis.
Li P
,
Gao S
,
Qu W
,
Li Y
,
Liu Z
.
Abstract
In-depth profiling of embryogenesis-associated endogenous and exogenous metabolic changes can reveal potential bio-effects resulting from human-made chemicals and underlying mechanisms. Due to the lack of potent tools for monitoring spatiotemporal distribution and bio-transformation behavior of dynamic metabolites at single-cell resolution, however, how and to what extent environmental chemicals may influence or interfere embryogenesis largely remain unclear. Herein, a zero-sample-loss micro-biopsy-based mass spectrometric platform is presented for quantitative, chemo-selective, high-coverage, and minimal-destructive profiling of development-associated cis-diol metabolites, which are critical for signal transduction and epigenome regulation, at both cellular level and tissue level of Xenopus laevis. Using this platform, three extraordinary findings that are otherwise hard to achieve are revealed: 1) there are characteristically different cis-diol metabolic signatures among oocytes, anterior and posterior part of tailbud-stage embryos; 2) halogenated cis-diols heavily accumulate at the posterior part of tailbud-stage embryos of Xenopus laevis; 3) dimethachlon, a kind of exogenous fungicide that is widely used as pesticide, may be bio-transformed and accumulated in vertebrate animals in environment. Thus, this study opens a new avenue to simultaneously monitoring intercellular and intraembryonic heterogeneity of endogenous and exogenous metabolites, providing new insights into metabolic remolding during embryogenesis and putting a warning on potential environmental risk.
Figure 1
Schematic illustration of the workflow for single oocyte cells and tailbud-stage embryo analysis. The multiscale analysis on embryogenesis begins with 1) sampling a single oocyte or single embryo and performing extraction, washing, desorption, and nano-ESI MS-based fingerprinting. 2) Morphology analysis for detecting eye size at Nieuwkoop-Faber stage ≈27 and the body length of tadpoles at Nieuwkoop-Faber stage ≈45. 3) Behavioral analysis for detecting the residence times within 1 min of each tadpole over the white and black backgrounds.
Figure 2
Development of a micro-biopsy-based profiling platform for oocytes and embryos. A, B) SEM characterization of sampling micropipettes. C) MS spectrum of direct analysis of a mixture containing an equal concentration of standard adenosine, 3-methyluridine, 2′-deoxyadenosine, 2′-deoxyuridine, and thymidine and MS spectrum of compounds extracted from boronate affinity micropipette from the above solution. D) Optimization of extraction time (n = 3 independent repeats; mean ± s.d.). E) Optimization of cyclic number for the solvent evaporation step (n = 3 independent repeats; mean ± s.d.). F) Home-built sampling platform and real-time monitoring platform. G) Sampling process for single oocyte cells and different parts of tailbud-stage embryos.
Figure 3
Morphology and behavioral biology analysis. A) Photos of eyes of sampled and unsampled embryos at the Nieuwkoop–Faber stage around 27 (N = 5 for each group). B) Photos of tadpoles developed from sampled and unsampled embryos at the Nieuwkoop–Faber stage around 45 (N = 4 for each group). Comparison of C) area of eyes at Nieuwkoop-Faber stage around 27 (N = 5 for each group), D) tadpole size at Nieuwkoop-Faber stage around 45 (N = 4 for each group), and E) time to stay at the lighter background (N = 4 for each group) between sampled and unsampled embryos (n.s. means non-significant).
Figure 4
Mapping of cis-diol metabolites of the oocyte and organogenesis-stage embryo. Raw typical MS spectra of A) oocyte cell, anterior and posterior part of tailbud-stage embryo. B) PCA analysis of three kinds of samples. C) Venn diagram of three kinds of samples. D) Pie chart of ClassyFire categories to classify the metabolite diversity of all annotated cis-diols. E) Mulberry chart of annotated cis-diols to understand the relationship of them with three kinds of samples.
Figure 5
Spatiotemporal cis-diol metabolome atlas from developing embryos. Difference in time dimension: comparison of cis-diols between single oocyte cells and anterior A) or posterior B) part of tailbud-stage embryos with OPLS-DA and Volcano plot. C) Difference in spatial dimension: comparison of cis-diols between anterior and posterior part of tailbud-stage embryos with OPLS-DA and Volcano plot. D) False-color heatmap and hierarchical clustering of small molecules detected in oocytes, anterior and posterior part of tailbud-stage embryos (N = 7 for each group). Metabolite abundances are normalized and transformed by log10 and shown in false color for the 25 statistically most significant features. E) Multiscale heterogeneity of 3′,4′-dihydroxychalcone, 4-tert-butylcatechol, pentaerythritol mononitrate, 1-(3,5-dichlorophenyl)pyrrole-2,3,5-triol, niazicin and 2,4-dihydroxy-1,4-benzoxazin-3-one glucuronide among oocytes, anterior and posterior part of tailbud-stage embryos.
Figure 6
Expression Landscape of halogenated cis-diols and discovery of exogenous pesticide. A) Comparison of halogenated cis-diols in three kinds of samples. B) Enzyme enrichment analysis of identified cis diols. C) Illustration of how dimethachlon was converted into 1-(3,5-dichlorophenyl)pyrrole-2,3,5-triol and extracted by boronate affinity probes. D) Illustration of interaction between dimethachlon and androgen receptor.
Figure S1. Comparison of the micro-sampling tools-based MS between reported papers and this
work. (A) In previous work, operation process of liquid-phase extraction-based single-cell
metabolomics. (B) In the present work, a solid-phase extraction-based single-cell metabolomics
was developed.
Figure S2. Comparison of the amounts of adenosine and deoxyadenosine captured by a boronic
acid-functionalized extraction micropipette.
Figure S3. Assessment of reproducibility of developed method. (A) Coefficient of variation for
testing adenosine and guanosine with and without of IS to calibrate. (B) Corresponding raw MS
spectra for testing adenosine and guanosine.
Figure S4. Survival analysis at key developmental stages (NF, Nieuwkoop-Faber) for N=41
embryos in the without sampling group, N=16 embryos in the posterior sampling group, N=25
embryos in the anterior sampling group.
Figure S5. PCA analysis of cis-diols in time dimension. Comparison of (A) oocyte cells and anterior
part of tailbud-stage embryos, and (B) oocyte cells and posterior part of tailbud-stage embryos.
Figure S6. Significant features identified by OPLS-DA of (A) oocyte cells and anterior part of
tailbud-stage embryos, and (B) oocyte cells and posterior part of tailbud-stage embryos. The
features are ranked by variable importance on projection value.
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