XB-ART-57790EMBO J May 3, 2021; 40 (9): e104913.
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Combinatorial transcription factor activities on open chromatin induce embryonic heterogeneity in vertebrates.
During vertebrate gastrulation, mesoderm is induced in pluripotent cells, concomitant with dorsal-ventral patterning and establishing of the dorsal axis. We applied single-cell chromatin accessibility and transcriptome analyses to explore the emergence of cellular heterogeneity during gastrulation in Xenopus tropicalis. Transcriptionally inactive lineage-restricted genes exhibit relatively open chromatin in animal caps, whereas chromatin accessibility in dorsal marginal zone cells more closely reflects transcriptional activity. We characterized single-cell trajectories and identified head and trunk organizer cell clusters in early gastrulae. By integrating chromatin accessibility and transcriptome data, we inferred the activity of transcription factors in single-cell clusters and tested the activity of organizer-expressed transcription factors in animal caps, alone or in combination. The expression profile induced by a combination of Foxb1 and Eomes most closely resembles that observed in the head organizer. Genes induced by Eomes, Otx2, or the Irx3-Otx2 combination are enriched for maternally regulated H3K4me3 modifications, whereas Lhx8-induced genes are marked more frequently by zygotically controlled H3K4me3. Taken together, our results show that transcription factors cooperate in a combinatorial fashion in generally open chromatin to orchestrate zygotic gene expression.
PubMed ID: 33555045
PMC ID: PMC8090851
Article link: EMBO J
Species referenced: Xenopus tropicalis
Genes referenced: cer1 chrd.1 crebbp crx ctcf dkk1 dmbx1 eomes fgf8 foxa1 foxb1 foxd4l1.1 foxi4.2 foxj1 frzb fst gata3 gata4 grhl1 grhl3 gsc irx1 irx3 klf2 klf5 lhx1 lhx8 mespb mix1 mixer nog otx2 pcdh8 pou5f3.1 pou5f3.3 snai1 sox11 sox17a sox17b.1 sox2 sp5 tbxt tfap2a upk3b vegt ventx1.1 ventx1.2 ventx2.1 wnt11b zic1 zic3
GO keywords: axis specification
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|SYNOPSIS Mesoderm induction, dorsal‐ventral patterning, and establishment of the dorsal axis involve transcriptional heterogeneity at the gastrula stage of vertebrate embryogenesis. Single‐cell chromatin‐accessibility and transcriptomic analyses identify the contribution and cooperation of key transcription factors in zygotic gene expression. The extent of chromatin opening closely reflects lineage potential. Integration of single cell chromatin‐accessibility and transcriptomic data allows identification of developmental key regulators. Bipotent neuromesodermal, head‐organizer and trunk‐organizer cells are found as early as the early gastrula. Transcription factors Foxb1, Eomes, Irx3 and Otx2 cooperate in a combinatorial fashion in zygotic gene regulation.|
|Figure 1.Chromatin accessibility during early development Genome browser view showing chromatin accessibility (ATAC‐seq) and ChIP‐seq (p300 and H3K4me3) profiles at stage 9, 10½, 12, and 16 at sox2 gene locus. The number at the left (black line) indicates the Y‐axis scale of the profile. ATAC‐seq peaks were found at promoter (H3K4me3) and enhancer (p300‐bound) regulatory regions. Chromatin accessibility and p300 binding at differential ATAC‐seq peaks visualized using K‐means clustering. Boxplots showing pair‐wise sequential‐stage comparisons of fold change in accessibility (ATAC‐seq) and corresponding changes in gene expression (RNA‐seq data set; Owens et al, 2016). The data represents two biological replicates. The central band within the boxplot represents the median (50th percentile), the box represents the range between the first and third quartile (25th–75th percentile), and the whiskers show 1.5× the interquartile range (IQR).|
|Figure EV1.Chromatin accessibility at promoters and enhancers Clustering of ATAC‐seq signal at ATAC‐seq peaks (center of heatmap, ±5 kb) along with p300 and H3K4me3 ChIP‐seq signals at stage 9 (blastula), 10½ (early gastrula), 12 (late gastrula), and 16 (neurula). Clustering (k 8) on all peaks. Clustering (k 6) on differential ATAC‐seq peaks. H3K4me3 marked promoter regions have mostly stable accessibility across the stages, whereas differential accessibility seems restricted to enhancers (p300‐bound). Expression profiles of genes associated to cluster Fig 1B. In the graph, the dots represent median expression values (transcripts per million) of genes. The solid lines connect these expression values across each stage.|
|Figure 2.Chromatin accessibility in animal cap (AC) and dorsal marginal zone (DMZ) Genome browser view of AC and DMZ accessibility profiles for ectoderm‐expressed (tfap2a and grhl3) and organizer‐expressed (gsc and chrd) marker genes. Boxplots showing differential gene expression (AC versus DMZ) and associated ATAC‐seq signals (Two biological replicates). The central band within the boxplot represents the median and the whiskers show 1.5 times the range between the first and third quartile (IQR). Hierarchical clustering of AC and DMZ ATAC‐seq data on H3K4me3‐positive (top) and p300‐positive (bottom) ATAC‐seq peaks. Heatmap showing accessibility signal (log1p of fold over background) at p300‐positive ATAC‐seq peaks surrounding pluripotency genes (ventx1/ventx2, pou5f3 and sox2). The row labeled “random” shows accessibility signals at random genomic loci. Single‐cell ATAC‐seq UMAP projection of cells derived from gastrula stage embryos (stage 10½), colored by cluster. Genomic tracks showing aggregated accessibility of single‐cell ATAC‐seq clusters at the t (tbxt), tfap2a, gsc, ctcf, lhx1 and sox11 loci.|
|Figure EV2.Region‐specific chromatin accessibility A. Genome browser view of AC and DMZ ATAC‐seq at regulatory regions of key pluripotency genes (pou5f3.3, ventx1.2, sox2, and ventx1.1). Most of the genes show relatively higher accessibility for animal cap cells and lower signals in DMZ. B, C. Sample statistics for single‐cell ATAC‐seq clusters A1‐A3. The ridge plots display the distribution of transcription start site (TSS) enrichment (B) and unique nuclear fragments (log10 of nFrags; C) over the cells in clusters A1‐A3. The TSS enrichment is determined by the peak signal (centered at the TSS of annotated genes) relative to the flanking regions (± 1,900–2,000 bp). Unique nuclear fragments represent those fragments that do not map to mitochondrial genome.|
|Figure 3.Cellular heterogeneity and developmental trajectories in blastula and gastrula stages A, B. UMAP visualization of whole embryo scRNA‐seq for stages 8, 10, and 12 colored by stage (A) and cell type annotations (B).|
|Figure EV3.Single‐cell transcriptome analysis of early development Analysis of single‐cell RNA‐seq data (Briggs et al, 2018). Panels depict 7,705 filtered cells of stage 8, 10 and 12 embryos, shown in Uniform Manifold Approximation and Projection (dimensionality reduction). Cell type annotation based on marker gene expression (Briggs et al, 2018). Cell clusters (Louvain clusters L0–L20) based on hypervariable genes, labeled with predominant cell type annotation. Cluster labels contain cluster number, followed by predominant cell annotations and the stage it was derived from in brackets. Abbreviations: NoNEct, non‐neural ectoderm; Neur, neural plate; O, organizer; End, endoderm; Not, notochord; VMes, ventral mesoderm; Ect, ectoderm; Blas, blastula; NEct, neural ectoderm; InvDMes, involuted dorsal mesoderm; MZ, marginal zone; Tlbd, tailbud; Cil, ciliated epidermal progenitor; Misc, miscellaneous.|
|Figure 4.scRNA‐seq of hand‐picked cells from stage 10½ dissected AC and DMZ explants Dimensionality reduction using UMAP. Each dot represents a cell. Colors indicate AC and DMZ. As panel (A), colors indicate clusters (C0‐C6). Feature maps, showing the expression of selected genes in single cells (cf. Appendix Fig S2). Color scale represents the gene expression value (log1p‐transformed counts per 10,000 unique reads) for each cell for a given gene, from low (gray) to high (orange). Heatmap depicting top 100 hypervariable genes in each cluster (cf. Dataset EV5). Color scale represents scaled gene expression (z‐score values). The z‐score values are ranging from −2 to 2. Spatially restricted AC and DMZ cell clusters embedded in UMAP of whole embryo scRNA‐seq data (cf. Fig 3).|
|Figure EV4.Correlations of region‐specific and whole embryo single cell clusters Correlation of AC and DMZ single cell clusters (C0‐C6) with whole embryo single cell clusters of whole embryos of stage 8, 10 and 12.|
|Figure 5.Integration of single‐cell transcriptomics and chromatin accessibility, for identifying regulators driving cluster‐specific gene expression Heatmap showing motif activity inferred from differential chromatin accessibility of stage 10½‐AC‐DMZ. Heatmap showing motifs identified based on regulatory regions closest to cluster‐specific genes, and regression to cluster gene expression. Heatmap of transcription factor (TF)‐motif combinations showing cluster‐specific motif activity (z‐score, color) and gene expression (size of dot). Motifs and the Motif‐TF combinations were hierarchically clustered.|
|Figure EV5.Transcription factor motifs and expression of single cell clusters A. Heatmap showing motif activity inferred from differential chromatin accessibility of single‐cell ATAC‐seq. B. Schematic overview, outlining the steps involved in the integrative analysis: (1) Identifying transcription factor motifs associated with regulatory regions closest to cluster‐specific genes, and regression to cluster gene expression; (2) Prioritizing transcription factors based on their gene expression and the corresponding motif activity in specific clusters. Combining the information (lower right panel) on motif activity (color of dots) and corresponding transcription factor expression (size of dots) allows prediction of factors that may play a role in cell cluster‐specific gene expression. C, D. Heatmaps of transcription factor‐motif combinations showing cluster‐specific motif activity (z‐score, color) and transcription factor expression (size of dot) for animal cap (panel C) and dorsal marginal zone (panel D) peak sets.|
|Figure 6.Induction of organizer gene expression in AC cells Heatmap showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1, Foxb1‐Eomes, Eomes, Irx3, Irx3‐Otx2, Otx2, and Lhx8. Correlation heatmap of overexpression RNA‐seq samples and single‐cell clusters. Fold enrichment of genes with zygotically defined (ZyD) H3K4me3 at their promoter in AC with transcription factor overexpression. Asterisk indicates hypergeometric P‐value ≤ 0.01.|
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