Transitions in the proteome and phospho-proteome during Xenopus laevis development
Elizabeth Van Itallie, Matthew Sonnett, Marian Kalocsay, Martin Wühr, Leonid Peshkin, Marc W Kirschner
Dev Biol. 2025 Sep:525:155-171. doi: 10.1016/j.ydbio.2025.05.022. Epub 2025 Jun 2.
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Highlights
• Before the early tadpole period, gene expression proteins change in level.
• Organogenesis proteins increase dramatically later in the embryonic period.
• Tissue specific proteins drive the increase in protein amount in the tadpole.
• Cell cycle and gene expression phosphorylation dominate the phospho-proteome.
• Novel cross-species pipeline links phospho-sites in Xenopus and human.
Abstract
Vertebrate development from an egg to a complex multi-cell organism is accompanied by multiple phases of genome-scale changes in the repertoire of proteins and their post-translational modifications. While much has been learned at the RNA level, we know less about changes at the protein level. In this paper, we present a deep analysis of changes of ∼15,000 proteins and ∼11,500 phospho-sites at 11 developmental time points in Xenopus laevis embryos ranging from the stage VI oocyte to the juvenile tadpole. We find that the most dramatic changes to the proteome occur during the transition to functional organ systems, which occurs as the embryo becomes a tadpole. At that time, the absolute amount of non-yolk protein increases two-fold, and there is a shift in the balance of expression from proteins regulating gene expression to receptors, ligands, and proteins involved in cell-cell and cell-environment interactions. Between the early and late tadpole, the median increase for membrane and secreted proteins is substantially higher than that of nuclear proteins. To begin to appreciate changes at the post-translational level, we have measured quantitative phospho-proteomic data across the same developmental stages. In contrast to the significant protein changes that are concentrated at the end of the time series, the most significant phosphorylation changes are concentrated in the very early stages of development. A clear exception are phosphorylations of proteins involved in gene expression: these increase just after fertilization, with patterns that are highly correlated with the underlying protein changes. To facilitate the interpretation of this unique phospho-proteome data set, we created a pipeline for identifying homologous human phosphorylations from the measured Xenopus phospho-proteome. Collectively, our data reveal multiple coordinated transitions in protein and phosphorylation profiles, reflecting distinct developmental strategies and providing an extensive resource to further explore developmental biology at the proteomic and phospho-proteomic levels.
Fig. 1. Temporal Profiling of the Xenopus laevis Proteome and Phospho-proteome Across Development. A. The eleven Xenopus laevis developmental stages collected for proteomic and phospho-proteomic measurement spaced according to Nieuwkoop and Faber times for temperatures between 22 and 24 °C and mapped to broad developmental classifications. (The Stage 41 illustration is actually Stage 40 for convenience. See Methods for Illustration information.) B. (i) 14,892 proteins were quantified; 61.4 % measured in all three replicates. (ii) 11,422 phospho-forms were quantified; 14.3 % were measured in all three replicates. C. Representative Examples: (i) SF3B1 protein and (ii) SF3B1 phospho-form T-429 are measured in all three replicates. SF3B1 replicate Pearson correlation coefficients (0.85 for protein and 0.87 for phospho-form) align with the median values for all measurements (Median Pearson of 0.90 for 14.9k proteins and 0.87 for 11.4k phospho-forms respectively; Fig. 2C).
Fig. 2. Distinct Protein Expression Clusters Correspond to Major Developmental Transitions. A. Twelve k-means clusters of relative protein trends were merged into eight summary trends ordered by the time that the first dynamic event in the trend occurs. The median trend of the cluster is the colored line; the ten to ninety percentile region is shown with shading. The pie chart shows the fractions of total proteins in each cluster. B. Over-representation analysis of biological process GO gene sets shows that, as development progresses, dynamic clusters are associated with proliferation, patterning and differentiation, organogenesis, and tissue function. For each GO protein list shown in black, the p-value for the over-representation of those proteins in the gene set relative to all proteins is shown with a circle, whose area is proportional to the -log10 p-value (hypergeometric) and has the appropriate cluster color. All GO protein lists are overrepresented in at least one cluster with FDR≤5 %. (See Spreadsheet S4 for the entire set of over-represented sets). C. (i) Median trend relative protein levels consistent with the transitions in the mitotic cell cycle across development. Securin/PTTG1 is highest in the metaphase arrested egg and decreases following fertilization. CDK4 levels increase following Stage 9 as the embryo enters the period where entry into S phase is under regulation. (See Fig. S4 for individual replicate trends). (ii,iii) Expression of proteins connected to tissue differentiation and organogenesis: for oligodendrocytes (ii) and gut (iii) (See Fig. S4 for individual replicate trends). The plots illustrate the temporal progression through these phases, as supported by the GO overrepresentation analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Two-Fold Proteome Mass Increase from Late Tailbud to Tadpole is Predominantly Driven by Tissue-Specific Proteins. A. Amount of non-yolk protein per egg/embryo (average of ten) at ten developmental stages. The mean of three technical replicates is shown with 95 % confidence intervals. B. The total amount of protein, in picomoles, at the measured developmental stages. (See Methods and Fig. S10 for more details about estimation of moles per protein per timepoint). The dots are the mean of three replicates and the error bars are the standard deviation. Red lines show the developmental stages of embryo drawings. C. Median trend (bars) absolute change of proteins by annotated localization for Stage 30–41 (lighter shade) and Stages 41–48 (darker shade). Open circles are the values for each replicate for each subset. Statistical significance was determined using the Kruskal Wallis test with Bonferroni correction for 20 comparisons (α = 0.0025). D. Absolute protein amount median trends for three different extracellular matrix proteins. Individual replicates are shown in Supplemental Fig. 11A. E. Median trend (bars) absolute change of proteins with the localization classifications additionally stratified by tissue specificity (TS) (not tissue specific: no black lines, tissue specific: black lines; left and right, respectively) for Stages 30–41 (lighter shade) and Stages 41–48 (darker shade). Open circles are the values for each replicate for each subset. Asterisks show the comparisons where the median trend and all three replicates are statistically significantly different, where statistical significance for a comparison is assessed with the Kruskal Wallis test, Bonferroni correction for eighty comparisons (α = 0.000625). F. The absolute amount of protein across development for three tissue specific membrane proteins and tissue specific secreted proteins. Individual replicates are shown in Supplemental Fig. 11B,C. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Adapted with permission from Elsevier on behalf of Developmental Biology: Van Itallie et al. (2025). Transitions in the proteome and phospho-proteome during Xenopus laevis development. Dev Biol. 2025 Sep:525:155-171. doi: 10.1016/j.ydbio.2025.05.022. Epub 2025 Jun 2.