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An ancestral Wnt–Brachyury feedback loop in axial patterning and recruitment of mesoderm-determining target genes

Nature Ecology & Evolution volume 6, pages 1921–1939 (2022)Cite this article

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Abstract

Transcription factors are crucial drivers of cellular differentiation during animal development and often share ancient evolutionary origins. The T-box transcription factor Brachyury plays a pivotal role as an early mesoderm determinant and neural repressor in vertebrates; yet, the ancestral function and key evolutionary transitions of the role of this transcription factor remain obscure. Here, we present a genome-wide target-gene screen using chromatin immunoprecipitation sequencing in the sea anemone Nematostella vectensis, an early branching non-bilaterian, and the sea urchin Strongylocentrotus purpuratus, a representative of the sister lineage of chordates. Our analysis reveals an ancestral gene regulatory feedback loop connecting Brachyury, FoxA and canonical Wnt signalling involved in axial patterning that predates the cnidarian–bilaterian split about 700 million years ago. Surprisingly, we also found that part of the gene regulatory network controlling the fate of neuromesodermal progenitors in vertebrates was already present in the common ancestor of cnidarians and bilaterians. However, while several endodermal and neuronal Brachyury target genes are ancestrally shared, hardly any of the key mesodermal downstream targets in vertebrates are found in the sea anemone or the sea urchin. Our study suggests that a limited number of target genes involved in mesoderm formation were newly acquired in the vertebrate lineage, leading to a dramatic shift in the function of this ancestral developmental regulator.

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Fig. 1: Lineages sampled in this study and Brachyury protein and mRNA expression.
Fig. 2: Distribution of Brachyury binding sites and motif analyses.
Fig. 3: Brachyury binding sites at selected target genes in Nematostella and Strongylocentrotus.
Fig. 4: Validation of Brachyury target genes.
Fig. 5: Apomorphic and synapomorphic target genes of Brachyury.
Fig. 6: Comparison of regulatory functions of Brachyury in the sea anemone, sea urchin and vertebrates.

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Data availability

The raw files from the ChIP–seq and RNA-seq experiments have been deposited to the NCBI GEO database under the accession numbers GSE182573 and GSE198320 for N. vectensis and S. purpuratus, respectively.

Code availability

All scripts developed for this study are available at GitHub at https://github.com/technau/brachyury_grn, https://github.com/xxxmichixxx/MouseEmbryoSingleCell and https://github.com/fmi-basel/gbuehler-MiniChip.

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Acknowledgements

We are grateful to I. Blitz for critically reading the manuscript. We would like to thank the members of the Technau and the Arnone laboratory for discussions. This work was supported by the EU-ITN EVONET to U.T. and M.I.A. and by grants of the Austrian Science Fund FWF to U.T. (P34404, P31018) and by EU-ITN EVOCELL (grant no. 766053) to M.I.A.

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Author notes

  1. Michaela Schwaiger

    Present address: Friedrich Miescher Institute for Biomedical Research, Swiss Institute of Bioinformatics, Basel, Switzerland

  2. Carmen Andrikou

    Present address: Department of Biological Sciences, University of Bergen, Bergen, Norway

  3. These authors contributed equally: Michaela Schwaiger, Carmen Andrikou, Rohit Dnyansagar.

Authors and Affiliations

  1. Department of Neurosciences and Developmental Biology, Faculty of Life Sciences,University of Vienna, Vienna, Austria

    Michaela Schwaiger, Rohit Dnyansagar, Patricio Ferrer Murguia, Bob Zimmermann, Tatiana Lebedeva, Grigory Genikhovich & Ulrich Technau

  2. Stazione Zoologica Anton Dohrn, Villa Comunale, Naples, Italy

    Carmen Andrikou, Periklis Paganos, Danila Voronov, Giovanna Benvenuto & Maria Ina Arnone

  3. Center for Integrative Bioinformatics Vienna, Max Perutz Labs, University of Vienna, Vienna, Austria

    Heiko A. Schmidt

  4. Max Perutz Labs, University of Vienna, Vienna, Austria

    Ulrich Technau

  5. Research Platform ‘Single Cell Regulation of Stem Cells’, University of Vienna, Vienna, Austria

    Ulrich Technau

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Contributions

U.T. and M.I.A. conceived the study. M.S., C.A., P.F.M., P.P. and T.L. performed experiments. M.S., R.D., D.V., B.Z., H.A.S., G.G., G.B., M.I.A. and U.T. analysed the data. M.S., C.A., R.D., M.I.A. and U.T. wrote the paper.

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Correspondence to Maria Ina Arnone or Ulrich Technau.

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Extended data

Extended Data Fig. 1 Specificity of the Brachyury antibodies and target gene detection strategy.

Specificity of the Brachyury antibodies and target gene detection strategy. (A) Ectopic expression of Brachyury at aboral pole of Nematostella confirms the specificity of the antibody. Zygotes were injected with a plasmid of EF1a::mCherry-p2A-Brachyury. Embryos with mosaic expression were stained for mCherry and Brachyury antibodies. (B) Western Blot of Brachyury antibody in control and Morpholino-mediated knockdown of brachyury in Nematostella (C) Immunocytochemistry of gastrula stage embryos of Nematostella (oral views) in controls and after morpholino-mediated knockdown of Brachyury. (D) Quantitative summary of Brachyury knockdown with morpholino oligonucleotides. (E) Western blot of anti-Brachyury in Strongylocentrotus. The estimated protein size is approx. 50kD. Developmental stages tested: 6h, 12h, 48h, 72h. Recombinant protein (RP) size: 9 kD. (F, G) Heatmap of Brachyury binding sites in Bra_AB1 and Bra_AB2 of Nematostella and Strongylocentrotus related to the TSS of all target genes. (H) Summary of the sources of the datasets used in this study.

Extended Data Fig. 2 Brachyury binding sites and relationship to chromatin modifications.

Brachyury binding sites and relationship to chromatin modifications. (A) Heatmap of Nematostella Brachyury binding sites from this study with chromatin modification sites earlier identified in Schwaiger et al. 2014. (B) Venn diagram showing the overlap of Brachyury binding sites in Nematostella identified in this study overlaps with the enhancer/promoter sites previously identified in51. (C) Venn diagram of Brachyury binding sites identified in this study in Strongylocentrotus showing the overlap with the previously identified open chromatin sites identified with ATAC-seq data. (D) Brachyury target selection strategy. Two closest genes on either side of the binding site were considered and their respective orthologs in the species under study were identified. A gene was prioritized as a target, if it was also a target gene in one or more species.

Extended Data Fig. 3 PCA analyses and summary of DEG analyses of RNA-seq experiments.

PCA analyses and summary of DEG analyses of RNA-seq experiments. (A–D) Principal Component Analysis (PCA) of RNA-seq datasets in Nematostella, Strongylocentrotus, Xenopus and Mouse respectively. The blue dots represent the control/WT samples while as red dots indicate KD/KO samples/ In case of Nematostella both pre-mRNA splicing (spl) and translation-blocking (tra) morpholinos were used. (E) Summary of differentially expressed genes after Bra KD/KO. Differential expression analysis was done using DEseq2 R package with 0.05 alpha value. Data for Nematostella, Strongylocentrotus and Xenopus is from morpholino induced knockdown of Bra transcripts while mouse data is a result of Bra knockout. (F) Overlap of direct (ChIP–seq detected) and indirect targets (DEGs) across different species. Each species was used as a query species (query dataset) and the genes determined that are differentially expressed, and also ChIP targets (‘column ‘direct targets in query’) or not ChIP targets (column ‘indirect targets in query’). The numbers refer to the number of orthologs in each one of the other species. The numbers are low, since the overlap between ChIP–seq targets and DEGs from RNA-seq experiments is only 1–10% within a given species.

Extended Data Fig. 4 Motif analyses of Brachyury ChIP-peaks and Gene Ontology analyses of target genes.

Motif analyses of Brachyury ChIP-peaks and Gene Ontology analyses of target genes. (A) Brachyury ChIP metaplots around Brachyury peaks containing palindrome or half-palindrome (single) or no Brachyury binding motifs. The average read count (normalized to a million reads) was calculated for Brachyury ChIP–seq reads for regions around peak summits spanning 2kb in 20bp windows. The shaded area around the lines represents the 95% confidence interval across peaks in a category. Note that in all four species, peaks with palindromes show significantly higher ChIP–seq read counts, which may serve as a proxy for the strength of Brachyury binding. (B) Intersection of motifs with the peak region. The Brachyury peak region (as identified using the Peakzilla algorithm) was scanned for presence of other transcription factor binding sites using Fimo (MEME-ChIP suite) with default settings. The resulting binding motifs were grouped by the transcription factor families Paired box (Pax), basic helix–loop–helix (bHLH), Forkhead box (Fox), homeobox (homeo), High mobility group (hmg), T-box (tbox). Presence of these motifs together with the Brachyury motif is highlighted in orange in the upset plots (iii, vi, ix, xii). Distance of these motifs from the peak centre was also tracked and is shown in adjoining plots (I, ii, iv, v, vii, viii, x, xi). (C) ChIP/DE gene set overlap and GO analysis. Overlap (dark grey) between ChIP targets (black) and differentially expressed (light grey) genes in M. musculus (i), X. tropicalis (iv), S. purpuratus (vi), N. vectensis (x). In C. owczarzaki (xiii) only the number of ATAC-seq peaks with Bra motifs is shown. Gene ontology analysis of Brachyury ChIP targets for M. musculus (ii), X. tropicalis (v), S. purpuratus (vii), N. vectensis (xi) and C. owczarzaki (xiv) and of differentially expressed genes after Bra KO/KD (iii, vi, ix, xii). Only gene ontology terms for biological process and molecular function were reported. The colour of the dot represents the score (−log(p-value)) assigned by topGO while the size of the dot represents the number of genes associated with the term. (D) GO analyses of target genes of peaks with or without a Bra consensus motif. Note that no significant difference can be detected.

Extended Data Fig. 5 Expression analysis of Brachyury ChIP targets by WMISH after knockdown in Nematostella.

Expression analysis of Brachyury ChIP targets by WMISH after knockdown in Nematostella. (A) Morpholino-mediated knockdown of target genes with complex expression pattern show partial down or upregulation in the ectodermal layer. (B) Neuronal target genes that are not affected by Brachyury knockdown. Note that all unaffected genes are expressed in the inner layer of the embryo. (C) Knockdown of SoxB1 (a homologue of vertebrate Sox2) shows no effect on neuronal target genes regulated by Brachyury.

Extended Data Fig. 6 Expression analyses of Brachyury target genes in Strongylocentrotus upon knockdown.

Expression analyses of Brachyury target genes in Strongylocentrotus upon knockdown. (A) ChIP target genes that are not affected by Brachyury knockdown. (B) Expression analysis of Brachyury RNA-seq targets by WMISH after morpholino induced knockdown (C) Expression analysis of Brachyury RNA-seq targets by immunohistochemistry after morpholino induced knockdown. Arrows show the embryonic domain in which we see an effect (red arrow: mesodermally derived; blue ectodermally derived). (D) Differentially expressed genes after Morpholino induced knockdown that are also ChIP targets at 24h. Key genes playing a crucial role in endoderm development are highlighted in yellow, key genes playing a role in mesoderm development are highlighted in red while key genes playing a role in ectoderm development are highlighted in blue. Asterisks indicate genes that are also ChIP targets. l/v: lateral view; v/v vegetal view; o/v oral view; a/v aboral view. up: upregulated gene; down; downregulated gene. The scale bar is 20 μm.

Extended Data Fig. 7 Shared Brachyury targets between lineages of Metazoa, Bilateria, Chordata and Vertebrata.

Shared Brachyury targets between lineages of Metazoa, Bilateria, Chordata and Vertebrata. Upset plot of shared orthologous genes as detected by OMA between different lineage combinations. Note that brachyury is the only target gene found in all investigated organisms. The large number of shared target genes between mouse and Xenopus indicates that this screen is robust against slight differences in developmental staging, source of cells, experimental design and sensitivity.

Extended Data Fig. 8 Protein phylogenies of selected target genes.

Protein phylogenies of selected target genes. (A) T-box family phylogenetic tree T-Box family tree constructed with T-box genes from Apis mellifera (Ame), Branchiostoma floridae (Bfl), Capsaspora owczarzaki (CAOG), Ciona intestinalis (Ciona), Lottia gigantean (Lgi), Mus musculus (mmus), Nematostella vectensis (NVE), Strongylocentrotus purpuratus (Spu), Xenopus tropicalis (xtro), Rattus norvegicus (Rat), Saccoglossus kowalevskii (Sko). Tree was constructed using maximum likelihood method with 1000 UFboot samples, the values at the nodes represent the support values. UFboot values below 50% are not shown and the nodes are marked with a red circle. The values at nodes with 100% support are also not shown. The tree was rooted on a T-box gene from the fungus Paramicrosporidium saccamoebae. Brachyury target genes in Nematostella and Strongylocentrotus are indicated by arrows. (B) Sox family phylogenetic tree Sox family tree constructed with Sox genes from Apis mellifera (ame), Acropora millepora (Ami), Amphimedon queenslandica (Aq), Ciona intestinalis (ci), Mus musculus (mmu), Nematostella vectensis (NVE), Strongylocentrotus purpuratus (Spu), Xenopus tropicalis (xtro). Tree was constructed with a maximum likelihood method with 1000 UFboots samples, the values at the nodes represent the support values. UFboots values below 50% are not shown and the nodes are marked with a red circle. The values at nodes with 100% support are also not shown. The tree was rooted with a sponge A. queenslandica Sox protein. Brachyury target genes in Nematostella and Strongylocentrotus are indicated by arrows. (C) Zic family phylogeny ZIC family tree constructed from ZIC genes from Amphimedon queenslandica (Aque), Branchiostoma floridae (Bflo), Capitella teleta (Ctel), Capsaspora owczarzaki (Cowc), Ciona intestinalis (Cint), Drosophila melanogaster (Dmel), Homo sapiens (Hsap), Mus musculus (Mmus), Nematostella vectensis (Nvec), Strongylocentrotus purpuratus (Spur), Tribolium castaneum (Tcas), Xenopus tropicalis (Xtro). This protein maximum likelihood tree was constructed using IQ-Tree with 10000 UFboot samples. The values at the nodes represent the UFboot support, where values below 50 % are not shown. The tree is rooted between the ZIC and GLI/GLIS subfamilies. Sequences from Nematostella are marked bold in green, those from Strongylocentrotus in bold and blue. Brachyury target genes in Nematostella and Strongylocentrotus are indicated by arrows. (D) TFAP2 family phylogeny TFAP2 family tree constructed from TFAP2 genes from Amphimedon queenslandica (Aque), Branchiostoma floridae (Bflo), Capitella teleta (Ctel), Ciona intestinalis (Cint), Drosophila melanogaster (Dmel), Homo sapiens (Hsap), Mus musculus (Mmus), Nematostella vectensis (Nvec), Strongylocentrotus purpuratus (Spur), Tribolium castaneum (Tcas), Xenopus tropicalis (Xtro). This protein maximum likelihood tree was constructed using IQ-Tree with 10000 UFboot samples. The values at the nodes represent the UFboot support, where values below 50 % are not shown. The tree is shown midpoint-rooted with Figtree. Sequences from Nematostella are marked bold in green, those from Strongylocentrotus in bold and blue. Brachyury target genes in Nematostella and Strongylocentrotus are indicated by arrows. (E) RNF family phylogeny RNF family tree constructed from RNF genes from Amphimedon queenslandica (Aque), Branchiostoma floridae (Bflo), Capitella teleta (Ctel), Capsaspora owczarzaki (Cowc), Ciona intestinalis (Cint), Drosophila melanogaster (Dmel), Homo sapiens (Hsap), Mus musculus (Mmus), Nematostella vectensis (Nvec), Strongylocentrotus purpuratus (Spur), Tribolium castaneum (Tcas), Xenopus tropicalis (Xtro). This protein maximum likelihood tree was constructed using IQ-Tree with 10000 UFboot samples. The values at the nodes represent the UFboot support, where values below 50 % are not shown. The tree is shown midpoint-rooted with Figtree. Sequences from Nematostella are marked bold in green, those from Strongylocentrotus in bold and blue. Brachyury target genes in Nematostella and Strongylocentrotus are indicated by arrows. (F) RFX family phylogeny RFX family tree constructed from RFX genes from Amphimedon queenslandica (Aque), Branchiostoma floridae (Bflo), Capitella teleta (Ctel), Capsaspora owczarzaki (Cowc), Ciona intestinalis (Cint), Drosophila melanogaster (Dmel), Homo sapiens (Hsap), Mus musculus (Mmus), Nematostella vectensis (Nvec), Strongylocentrotus purpuratus (Spur), Tribolium castaneum (Tcas), Xenopus tropicalis (Xtro). This protein maximum likelihood tree was constructed using IQ-Tree with 10000 UFboot samples. The values at the nodes represent the UFboot support, where values below 50 % are not shown. The tree is shown midpoint-rooted with Figtree. Sequences from Nematostella are marked bold in green, those from Strongylocentrotus in bold and blue. Brachyury target genes in Nematostella and Strongylocentrotus are indicated by arrows. (G) NR2 family phylogeny NR2f family tree constructed from NR2 genes from Amphimedon queenslandica (Aque), Branchiostoma floridae (Bflo), Capitella teleta (Ctel), Ciona intestinalis (Cint), Drosophila melanogaster (Dmel), Homo sapiens (Hsap), Mus musculus (Mmus), Nematostella vectensis (Nvec), Strongylocentrotus purpuratus (Spur), Tribolium castaneum (Tcas), Xenopus tropicalis (Xtro). This protein maximum likelihood tree was constructed using IQ-Tree with 10000 UFboot samples. The values at the nodes represent the UFboot support, where values below 50 % are not shown. The tree is shown midpoint-rooted with Figtree. Sequences from Nematostella are marked bold in green, those from Strongylocentrotus in bold and blue. Brachyury target genes in Nematostella and Strongylocentrotus are indicated by arrows.

Extended Data Fig. 9 Expression of apomorphic and synapomorphic target genes of Brachyury in mouse E8.5 neuronal, endodermal, and mesodermal cell types.

Expression of apomorphic and synapomorphic target genes of Brachyury in mouse E8.5 neuronal, endodermal, and mesodermal cell types. This is the same analysis as shown in Figure 6, except that definitive endoderm cells were removed from the analysis (A–C) or the single-cell gene expression dataset from Grosswendt et al.121 (E, F) was used. A–C: The expression of the target genes in neuronal versus mesodermal cell types was annotated using single-cell RNA-seq data from E8.0 and E8.5 mouse embryos, as in Figure 6. However, since definitive endoderm cells in this dataset express notochord marker genes, we decided to remove these cells from the analysis before continuing as in Figure 6. (A) Heatmap of the log2 fold change (logFC) of neuronal (turquoise) or endodermal (yellow) vs mesodermal (purple) gene expression for each gene using the corresponding mouse gene symbols as they appeared in the single-cell dataset in each node (see Methods for details). B) Boxplot of the log2 fold change (y-axis) of endodermal vs mesodermal gene expression for all genes per node (x-axis). Note that node II is enriched in endodermal expression while node V is enriched in mesodermal expression (p-value = 0.003, Wilcoxon Rank Sum Test). (C) Boxplot of the log2 fold change (y-axis) of neuronal vs mesodermal gene expression for all genes per node (x-axis). Note that node II is enriched in neuronal expression while node V is enriched in mesodermal expression (p-value = 0.008, Wilcoxon Rank Sum Test). D–F: The expression of the target genes in neuronal versus mesodermal cell types was annotated using single-cell RNA-seq data from E8.5 mouse embryos (read counts per gene/cell from GEO accession GSE122187). To annotate cells as neuronal, endodermal or mesodermal, we used the information from Supplementary Table 2 of Grosswendt et al. For endodermal, we used Lineage = Eendo, for mesodermal we used Lineage = Emeso, and for neuronal we used the following cell states: 1,11,24, and 39, which, according to Supplementary Fig. 1i of Grosswendt et al corresponds to: neural ectoderm anterior, neural ectoderm posterior, fore/midbrain, and future spinal cord. (D) Heatmap of the log2 fold change (logFC) of neuronal (turquoise) or endodermal (yellow) vs mesodermal (purple) gene expression for each gene using the corresponding mouse gene symbols as they appeared in the single-cell dataset in each node (see Methods for details). (E) Boxplot of the log2 fold change (y-axis) of neuronal vs mesodermal gene expression for all genes per node (x-axis). Note that node II is enriched in neuronal expression while node V is enriched in mesodermal expression (p-value = 0.037, Wilcoxon Rank Sum Test). (F) Boxplot of the log2 fold change (y-axis) of endodermal vs mesodermal gene expression for all genes per node (x-axis). Note that when, as is the case in this dataset, ‘gut’ is the only annotated endodermal cell type, node II is not more enriched in endodermally expressed genes compared to node V (p-value = 0.23, Wilcoxon Rank Sum Test). The boxes range from the 25th to the 75th percentile and the horizontal lines represent the median. Outliers are shown as dots.

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Schwaiger, M., Andrikou, C., Dnyansagar, R. et al. An ancestral Wnt–Brachyury feedback loop in axial patterning and recruitment of mesoderm-determining target genes. Nat Ecol Evol 6, 1921–1939 (2022). https://doi.org/10.1038/s41559-022-01905-w

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