activina  (R&D Systems)

Journal: bioRxiv

Article Title: Eomes restricts Brachyury functions at the onset of mammalian gastrulation

doi: 10.1101/2023.01.27.525830

Figure Lengend Snippet: A) Schematic illustration of embryoid body (EB) differentiation. mESC – mouse embryonic stem cells; SF – serum free; ActA – ActivinA; d – day of differentiation; Dox – doxycycline. B) Schematic illustration of different cell lines used: Cells with single gene deletions of Eomes (EoKO) or Brachyury (BraKO), and double KO (dKO) cells were generated by the insertion of fluorescent reporters on one allele ( Eomes GFP and Brachyury Tomato ) and an out-of-frame deletion on the second allele for each gene. EOMES-GFP or BRACHYURY-GFP were introduced into the doxycycline-inducible locus (TRE) of EoKO, BraKO or dKO cells (EoKO+EoGFP, BraKO+BraGFP, dKO+EoGFP, dKO+BraGFP). C, D) Venn diagram of RNA-seq analyses showing the overlap of downregulated (C) or upregulated (D) genes in EoKO and in BraKO compared to WT EBs (adjusted p-value<0.05, log2(FC)>1.5 for upregulated genes, log2(FC)<-1.5 for downregulated genes). Key markers for different cell lineages are indicated on the right. DE – definitive endoderm, AM – anterior mesoderm, ExM – extraembryonic mesoderm, PS/NM – primitive streak/nascent mesoderm, Epi – epiblast. E) Heatmap showing normalized scaled counts of clustered, differentially expressed genes between WT and dKO cells (no dox), and rescue of expression levels by induction of EoGFP (clusters I and V) or BraGFP expression (clusters II and VI), or both (cluster III and IV). Adjusted p-value<0.05; log2(FC)>1.0 for upregulated genes, log2(FC)<-1.0 for downregulated genes. Scale represents centered scaled counts normalized by library size and gene-wise dispersion. Relevant marker genes in each group are indicated. F, G) High confidence target genes of Eomes or Brachyury defined by intersecting genes that are downregulated in EoKO (F) or BraKO (G) with EoGFP-rescued and BraGFP-rescued genes (E), respectively. Key markers for different cell lineages are indicated. DE – definitive endoderm, AM – anterior mesoderm.

Article Snippet: EBs were transferred into 60 mm non-adhesive dishes and differentiated in ESGRO Complete Basal medium with 30 ng/ml human recombinant ActivinA (ActA, R&D systems) for 3 days.

Techniques: Generated, RNA Sequencing, Expressing, Dispersion, Marker

Journal: bioRxiv

Article Title: Eomes restricts Brachyury functions at the onset of mammalian gastrulation

doi: 10.1101/2023.01.27.525830

Figure Lengend Snippet: A) Examples of ChIP-seq coverage tracks of EOMES- and/or BRACHYURY-bound regions close to target genes ( Nkx2-1, Dlk1, Gata5(os), Sox18, Bmp2, Meis2 ) in ActivinA-induced EBs (EoKO+EoGFP and BraKO+BraGFP). Counts normalized to RPKM are indicated. B) Binding affinity heatmaps of differentially bound regions comparing EOMES-GFP (EoGFP) with BRACHYURY-GFP (BraGFP) in differentiating EBs (EoKO+EoGFP and BraKO+BraGFP) analyzed by ChIP-seq of two independent experiments. C) Venn diagram showing the large overlap of associated genes (numbers indicated) to EOMES-GFP and BRACHYURY-GFP ChIP-seq binding peaks in differentiating EBs. D) Bar graphs show the frequency of genes associated to the different patterns of EOMES- and BRA-binding within all target genes, Eomes (Eo-HC), or Brachyury (Bra-HC) high-confidence target genes. The percentage is depicted as ratio of overlap of associated genes with all, Eo-HC and Bra-HC genes to the total number of all, Eo-HC and Bra-HC genes. Venn diagram is identical to using different labels to identify groups of genes associated to ChIP-peak distributions in the bar graph diagram. E, F) Representative transcription factor-binding motif enrichment within EO-unique over BRA-unique binding sites and vice versa . Prevalent Tbx-binding motifs were not included. P-values are indicated.

Article Snippet: EBs were transferred into 60 mm non-adhesive dishes and differentiated in ESGRO Complete Basal medium with 30 ng/ml human recombinant ActivinA (ActA, R&D systems) for 3 days.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Eomes restricts Brachyury functions at the onset of mammalian gastrulation

doi: 10.1101/2023.01.27.525830

Figure Lengend Snippet: A) Binding affinity heatmap of differentially bound regions of EOMES-GFP in dKO versus EoKO differentiating EBs identified by ChIP-seq shows that EOMES binding is almost unaffected by the presence or absence of BRACHYURY, since only 34 differentially bound regions were found. B) Binding affinity heatmap of differentially bound regions of BRACHYURY-GFP in dKO versus BraKO differentiating EBs shows that BRACHYURY binding is largely enhanced in the absence of EOMES in dKO cells, indicated by 1,673 regions with increased binding. C) Binding affinity heatmap of differentially accessible regions (DARs) between EoKO and BraKO differentiating EBs analyzed by ATAC-seq of two independent experiments. D) Schematic illustration of CHIR-induced embryoid body (EB) differentiation. mESC – mouse embryonic stem cells; SF – serum free; h – hour of differentiation; Dox – doxycycline. E) Immunofluorescence staining of EOMES and BRACHYURY protein in CHIR-induced EBs at indicated timepoints. Eomes-GFP expression is induced in WT+EoGFP EBs with no change in Brachyury expression. Scale bars 100 μm. F) Examples of ChIP-seq coverage tracks of EOMES- and/or BRACHYURY-bound region in ActvinA-induced EBs (EoKO+EoGFP and BraKO+BraGFP or dKO+EoGFP and dKO+BraGFP) close to Brachyury -dependent target genes ( Msgn1 and Tbx6 ). Brachyury -target genes show preferential BRACHYURY binding in absence of EOMES and loss of open chromatin in BraKO compared to EoKO and WT as shown by ATAC-seq coverage tracks of WT, EoKO and BraKO ActivinA-induced-EBs. Counts normalized to RPKM are indicated.

Article Snippet: EBs were transferred into 60 mm non-adhesive dishes and differentiated in ESGRO Complete Basal medium with 30 ng/ml human recombinant ActivinA (ActA, R&D systems) for 3 days.

Techniques: Binding Assay, ChIP-sequencing, Immunofluorescence, Staining, Expressing

Journal: bioRxiv

Article Title: Eomes restricts Brachyury functions at the onset of mammalian gastrulation

doi: 10.1101/2023.01.27.525830

Figure Lengend Snippet: A) Whole mount in situ hybridization of WT and WT+EoGFP CHIR-induced EBs at indicated timepoints for Brachyury -targets Aldh1a2, Rspo3, Msgn1, Tbx6 and the Eomes -target Cyp26a1 showing that Brachyury -target gene expression is reduced upon overexpression of Eomes . Scale bars 100 μm, n≥6. B) Examples of ChIP-seq coverage tracks of EOMES- and/or BRACHYURY-bound regions in ActvinA-induced EBs (EoKO+EoGFP and BraKO+BraGFP or dKO+EoGFP and dKO+BraGFP) close to Brachyury -target genes ( Rspo3 and Aldh1a2 ) or Eomes -target gene Cyp26a1. Brachyury -target genes show preferential BRACHYURY binding in absence of EOMES and loss of open chromatin in BraKO compared to EoKO and WT as shown by ATAC-seq coverage tracks of WT, EoKO and BraKO ActivinA-induced-EBs. Counts normalized to RPKM are indicated. C) Model for lineage-specific activities of EOMES and BRACHYURY. EOMES and BRACHYURY bind in overlapping fashion at promoter regions of common mesoderm and endoderm target genes, while enhancer binding is specific and conferred by different Tbx binding motifs for EOMES and BRACHYURY. Eomes activates gene programs that act early during gastrulation to specify AM and DE lineage programs (E6.5-7.5), concomitantly repressing Brachyury binding and activities, indicated by reduced enhancer accessibility. After E7.5 Eomes expression rapidly declines and the repression of Brachyury functions is released, allowing BRACHYURY to instruct lineage specific programs for PM and posterior PS derivatives.

Article Snippet: EBs were transferred into 60 mm non-adhesive dishes and differentiated in ESGRO Complete Basal medium with 30 ng/ml human recombinant ActivinA (ActA, R&D systems) for 3 days.

Techniques: In Situ Hybridization, Targeted Gene Expression, Over Expression, ChIP-sequencing, Binding Assay, Expressing