Comparative Metabolomics of Small Molecules Specifically Expressed in the Dorsal or Ventral Marginal Zones in Vertebrate Gastrula

Abstract

Many previous studies have reported the various proteins specifically secreted as inducers in the dorsal or ventral regions in vertebrate gastrula. However, little is known about the effect on cell fate of small molecules below 1000 Da. We therefore tried to identify small molecules specifically expressed in the dorsal marginal zone (DMZ) or ventral marginal zone (VMZ) in vertebrate gastrula. Small intracellular and secreted molecules were detected using explants and supernatant samples. Hydrophilic metabolites were analyzed by capillary ion chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and lipids were analyzed by supercritical fluid chromatography-tandem mass spectrometry. In total, 190 hydrophilic metabolites and 396 lipids were identified. The DMZ was found to have high amounts of glycolysis- and glutathione metabolism-related metabolites in explants, and the VMZ was richer in purine metabolism-related metabolites. We also discovered some hydrophilic metabolites and lipids differentially contained in the DMZ or VMZ. Our research would contribute to a deeper understanding of the cellular physiology that regulates early embryogenesis.

Keywords: Spemann organizer; Xenopus laevis; dorsal–ventral patterning; early embryogenesis; metabolomic analysis.

Figure 1

Correlations of the hydrophilic metabolites in explants and supernatants.

Figure 2

Identification of hydrophilic metabolites differentially expressed between the DMZ and VMZ in explants (DMZ and VMZ explants). (A) Principal component analysis (PCA) score visualizing the relationship between the DMZ (red circle) and VMZ (blue triangle) explants using hydrophilic metabolites. The contribution ratios were 14.9% and 13% for PC2 and PC3, respectively. (B) Partial least squares–discriminant analysis (PLS-DA) score plots for the DMZ (red circle) and VMZ (blue triangle) explants. (C) Hierarchical clustering analysis (HCA) for the top 15 metabolites by variable importance in projection (VIP) scores in explants. Rows display the metabolite, and columns represent the sample. Metabolites with relatively low contents are displayed in blue, whereas metabolites with relatively high contents are displayed in red. The brightness of each color corresponds to the magnitude of the difference compared with the mean value. The number at the end of each sample name corresponds to samples made from the same embryos. (D–F) Comparison of the concentrations of metabolites that differed notably between the DMZ and VMZ explants. Symbols represent each sample, and the bar graph indicates the mean ± SD (n = 6, each sample from 20 explants). * FDR adjusted p < 0.05 (Welch’s t-test, Benjamini–Hochberg procedure). (D) Bar graphs of the metabolites involved in glycolysis in the top 15 metabolites by VIP scores. (E) Bar graphs of the metabolites involved in glutathione metabolism in the top 15 metabolites by VIP scores. (F) Bar graphs of the metabolites involved in purine metabolism in the top 15 metabolites by VIP scores.

Figure 3

Comparative analysis of the characteristic hydrophilic metabolites identified in supernatants. (A) PLS-DA score plots for the supernatants of the DMZ (red circle) and VMZ (blue triangle) samples (DMZ and VMZ supernatants) based on hydrophilic metabolite data. (B) Comparison of hypoxanthine, guanine, and glucuronic acid concentrations in the DMZ and VMZ supernatants. Symbols represent each sample and the bar graph indicates the mean ± SD (n = 6, each sample from 20 explants).

Figure 4

Lipid characteristics between the DMZ and VMZ in explants and supernatants. (A) Correlations of the lipids detected between explants and supernatants. (B) Bar graphs showing the proportion of each lipid class in the total lipid content. (C) Enrichment of lipid classes in explants and supernatants. The x-values are the ratios obtained by dividing the proportion in supernatants by that in explants. Any lipid class whose value was zero was excluded. TAG, triacylglycerol; DAG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PE (p), alkenyl-acyl phosphatidylethanolamine; SM, sphingomyelin; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; Cer, ceramide; HexCer, hexosylceramides.