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Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been mostly limited to transcripts and a few proteins, the latter due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been known to be the origin of signals that pattern the mesoderm and central nervous system. Molecular screens of the SMO have identified several genes responsible for the ability of the SMO to establish the body axis. Nonetheless, a comprehensive study of proteins and metabolites produced specifically in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the remainder of the embryo using high-resolution mass spectrometry (HRMS). Quantification of ~4,600 proteins and a panel of targeted metabolites documented differential expression for 460 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the SMO. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO. Chemical perturbation of the redox gradient perturbed mesoderm involution during early gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.
Fig. 1.
Workflow for lineage tracing and dual proteomic-metabolomic screening of the SMO in X. laevis and HRMS. (A) The dorsal midline equatorial cells D112 and D212 of the 32-cell embryo (Left panel) were lineage traced to encompass the majority of SMOtissue, shown in the hemisected gastrula at stage 10 (Right panel). (B) The lineage-traced tissues were dissected to pool five SMO tissues and the remainder of the embryo (RE) as one biological replicate. (C) Three different pools of tissues were processed for tandem/multistage HRMS quantification of the proteome via isobaric barcoding bottom-up proteomics (MS3) and metabolite intermediates via targeted tandem HRMS (MS2) metabolomics. The resulting proteo-metabolome dataset was statistically analyzed and interpreted using gene ontology (GO) and Ingenuity Pathway Analysis (IPA). (Scale bars, 250 µm.) Key to embryonic axes: AN, animal; D, dorsal; V, ventral; and VG, vegetal.
Fig. 2.
Proteomic profiling of the SMO vs. the remainder of the embryo (RE). (A) Of ~4,600 proteins quantified, 460 showed differential enrichment in either the SMO (green) or the RE (black). (B) Functional annotation of the 15 canonical pathways with most significant z-scores via Ingenuity Pathway Analysis. (C) Highly expressed proteins in the SMO based on HRMS (orange circle) compared to elevated gene expression in the SMO from ref. 14 (gray circle) and (D) single-cell RNA-sequencing from ref. 44. Analysis of biological pathway overrepresentation for gene products enriched both based on HRMS and single-cell RNA-seq using PantherDB with Bonferroni correction for multiple testing.
Fig. 3.
Proteome and targeted metabolite profiling of energy production between the SMO and remainder of the embryo using HRMS. Protein downregulation in glycolysis and upregulation in oxidative phosphorylation and TCA intermediates led to the enrichment of several metabolite intermediates in the SMO. Key to labels: CitA, citric acid; En., energy; F1,6BP, fructose-1,6-biphospate; G3P, glyceraldehyde-3-phospate; MalA, malic acid; α-KG, α-ketoglutaric acid.
Fig. 4.
Our biochemical model of elevated oxidative stress in the SMO region. H2O2-sensitive fluorescent imaging of the (A) vegetal, (B) dorsal, and (C) animal poles of stage 10 X. laevis embryos. (D) Background-normalized fluorescence intensity for BioTracker green H2O2 live cell dye measured in the SMO, dorsal, and vegetal regions of the embryo. Key: *P < 0.05 (paired Wilcoxon signed ranks test). (E) Our working molecular model proposes active generation of H2O2 based on enrichment of the redox proteins Sod and Prdx as well as downregulation of Gpx, which converts glutathione (GSH) to oxidized glutathione (GSSG) to buffer the redox system. (F) HRMS testing of the hypothesis through quantification of the redox system proteome. Key: *P < 0.05 (Student’s t test). (G) Experimental profiling of GSH/GSSG using a luminescence-based assay (Left panel) and targeted LC-MS metabolomics (Right panel) validated elevated redox stress. Key: *P < 0.05 (Student’s t test). (Scale bars, 250 µm.)
Fig. 5.
ROS enrichment in the SMO impairs mesoderm involution during gastrulation. (A) In our experimental approach, the SMO region of stage 10 embryos was injected with the antioxidant N-acetylcysteine (NAC) or control 20% Steinberg’s solution (SS) and cultured to either stage 10.5 or 12.5 for immediate assessment of ROS enrichment using an H2O2-sensitive Green Live Cell Dye (BioTracker) or (B) in situ hybridization analysis of the indicated genes. (C) At stage 10.5 to 11, Tbxt expression (blue) encircles the yolk plug in both control (Ctrl) and NAC-treated embryos. However, in NAC-treated embryos, the intensity of the staining is weaker, and the diameter of the yolk plug (red line in Ctrl) is significantly larger (*P < 0.0001). Vegetal view with dorsal to the top. (D) Tbxt-stained embryos were bisected in the midsagittal plane, and the length of the archenteron, indicating mesoderm involution, was measured (red lines). The archenteron was significantly smaller in NAC-treated embryos (*P < 0.0001). Note that Tbxt-positive mesoderm had reached the blastopore lip in controls (red arrow) but not in NAC-treated embryos. Dorsal to the top. (E) In stage 12.5 control embryos, Chrd-expressing mesoderm has involuted inside and the diameter of the blastopore is small (red line). In NAC-treated sibling embryos, the blastopore is significantly larger (*P < 0.0001), and Chrd-expression is absent from the dorsal midline. Dorsal-vegetal views. (F) Similarly, neural plate expression of Sox2 is missing from the dorsal midline. Dorsal views. (G) Chrd-stained embryos were bisected in the midsagittal plane, and the length of the archenteron, indicating extent of mesoderm involution, was measured (dotted red lines). The archenteron was significantly smaller in NAC-treated embryos (*P < 0.0001). Dorsal to the top. (Scale bars, 125 µm.)
Figure S1. Quantitative comparison of glycolysis and mitochondrial proteins
between the Spemann-Mangold Organizer (SMO) and the remainder of the
embryo (RE). Key to statistics: Horizontal dashed line, statistical threshold at p <
0.05 (Student’s t-test); Vertical dashed line, fold change = 1.
Figure S2. Metabolite concentration calibration curves for a panel of
intermediates from (A) glycolysis, (B) phosphate energy pool (ATP, ADP, AMP),
and (C) mitochondrial activity and including the TCA cycle. The lower limit of
quantification (LLoQ) is exemplified. Key: R2, coefficient of linear regression.
Figure S3. Metabolite profiling of the SMO and RE for targeted intermediates of
(A) glycolysis, (B) the adenylate energy pool, and (C) mitochondrial activity
including the TCA cycle. In each figure, the relative intensity (Rel. Int.) compares
the absolute concentration that was determined based on external concentrationcalibration curves for each different metabolite. Key to metabolites: CitA, citric
acid; F1,6BP, fructose-1,6-biphospate; G3P, glyceraldehyde-3-phospate; GluA,
glutamic acid; MalA, malic acid; α-KG, α-ketoglutaric acid. Key to statistics: *, p <
0.05; **, p < 0.005; ns., not significant (Student’s t-test).
Figure S4. Validation of ROS reduction via injection of N-acetyl cystine (NAC).
Embryos injected with 20% Steinberg’s Solution and without injection served as
controls. A Key: *, p < 0.05 (Student’s t-test).
