Quantitative proteomics reveals the dynamic proteome landscape of zebrafish embryos during the maternal-to-zygotic transition

Abstract

Maternal-to-zygotic transition (MZT) is central to early embryogenesis. However, its underlying molecular mechanisms are still not well described. Here, we revealed the expression dynamics of 5,000 proteins across four stages of zebrafish embryos during MZT, representing one of the most systematic surveys of proteome landscape of the zebrafish embryos during MZT. Nearly 700 proteins were differentially expressed and were divided into six clusters according to their expression patterns. The proteome expression profiles accurately reflect the main events that happen during the MZT, i.e., zygotic genome activation (ZGA), clearance of maternal mRNAs, and initiation of cellular differentiation and organogenesis. MZT is modulated by many proteins at multiple levels in a collaborative fashion, i.e., transcription factors, histones, histone-modifying enzymes, RNA helicases, and P-body proteins. Significant discrepancies were discovered between zebrafish proteome and transcriptome profiles during the MZT. The proteome dynamics database will be a valuable resource for bettering our understanding of MZT.

Keywords: Developmental biology; Proteomics.

Graphical abstract

Figure 1

Summary of the quantified proteins from zebrafish embryos by CZE-MS/MS and RPLC-MS/MS (A) Workflow of the iTRAQ-based quantitative experiment. Proteins were extracted from the embryos at four different stages in biological duplicate and digested. The peptides labeled with 8 channels of iTRAQ reagent were mixed and fractionated using high-pH RPLC, followed by low-pH RPLC-MS/MS and CZE-MS/MS analysis. (B) Overlaps of quantified proteins and peptides by RPLC-MS/MS and CZE-MS/MS. (C) Examples of reporter ion intensity correlations between biological replicates at the 64-cell stage from CZE-MS/MS and RPLC-MS/MS analyses. (D) Normalized reporter ion intensity ratio (RPLC/CZE) distributions of overlapped proteins at the 256-cell, Dome, and 50% epiboly stages. The reporter ion intensity of proteins from CZE or RPLC were averaged across biological duplicates at each stage, and the reporter ion intensity from the 256-cell, Dome, and 50% epiboly stages were divided by that at the 64-cell stage for normalization. The obtained intensity ratios from RPLC were divided by that from CZE at the same stage and the produced ratios were used in the figure. (E) Boxplots of log₂(reporter ion intensity ratios) at the 256-cell, Dome, and 50% epiboly stages from the combined CZE and RPLC data. The reporter ion intensity of those three stages were normalized to the 64-cell stage for CZE and RPLC separately. For the overlapped proteins, the ratios from CZE and RPLC were averaged for each stage. Otherwise, the CZE or RPLC data were used. In total, 4846 proteins were quantified and used in the figure.

Figure 2

Cluster analysis of differentially expressed proteins quantified with RPLC-MS/MS and CZE-MS/MS methods (A) Heatmap in the middle showing distinct expression profiles of different clusters at each developmental stage. Fuzzy clustering of the expression data along zebrafish embryo early development stages are shown at left. The functional enrichment data of the proteins in each cluster are shown at right (p-value<0.1). The smaller the p-value is, the higher enrichment of genes in the annotation categories is. (B) Cellular component distribution of the differentially expressed proteins in each cluster. DAVID Bioinformatics Resources (https://david.ncifcrf.gov/) was used for the functional enrichment and cellular component analyses.

Figure 3

Expression level changes of the quantified transcription factors (TFs) (A) The quantified TFs and those with a significant change in expression level across four development stages were classified into families according to their DNA-binding domain composition. Families with fewer than three members were excluded. (B) DNA-binding domain distribution of the TFs upregulated during the MZT. (C and D) Expression profiles of TFs that show significant upregulation before the ZGA. (E) Expression profiles of TFs that have significant increase in abundance after the initiation of ZGA.

Figure 4

Expression patterns of potential regulators beyond TFs show significant abundance changes during the MZT Expression patterns of (A) histones, (B) histone-modifying enzymes, (C) RNA helicases, (D) P-body proteins, and (E) proteasomes. (F) Protein-protein interaction analysis of the differentially expressed TFs, histone-modifying enzymes, RNA helicases, and P-body proteins. The STRING (https://string-db.org/) was used for the analysis. Network type is full STRING network, and the edges indicate both physical and functional associations of proteins. The network edge suggests the confidence of protein-protein connection and the line thickness indicates the strength of data support with thicker lines as more confident connections. The minimum required interaction score is medium confidence (0.4). Others are default settings.

Figure 5

Discordance of temporal patterns in zebrafish transcript and protein expression during the MZT (A) Mutual information between the temporal pattern of mRNA and protein expression presented as co-clustering into three main trends (mRNA) and four main trends (protein). The grayscale background reflects the number of genes in each group. Only proteins or mRNAs that show significant fold changes with p-values ≤0.05 were considered as differentially expressed ones for the co-clustering analysis. The four time points covered by the proteome data are 64-cell, 256-cell, dome, and 50% epiboly stages. The time points covered by the transcriptome data are 128-cell, 1k-cell, dome, and 50% epiboly stages. Top 10 enriched biological processes of genes corresponding to the degraded mRNAs (B), the accumulated mRNAs (C), and the no-change mRNAs (D). The size of circle represents gene count involved in each biological process. The enriched biological processes are colored by adjusted p-value. The smaller the p-value is, the more significant enrichment of the particular biological process associated with the group of genes is.