The T-box transcription factor Eomesodermin governs haemogenic competence of yolk sac mesodermal progenitors

Abstract

Extra-embryonic mesoderm (ExM)—composed of the earliest cells that traverse the primitive streak—gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.

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Fig. 1: Eomes is expressed in extra-embryonic mesodermal progenitors that give rise to yolk sac haematopoietic and vascular cells.
Fig. 2: Eomes, SCL and Runx1 expression during haematopoietic development in vitro and in vivo.
Fig. 3: Eomes functional loss disrupts primitive and definitive haematopoiesis but not endothelial development.
Fig. 4: Eomes-null cultures lack Runx1+ haemogenic endothelial cells.
Fig. 5: Eomes regulates chromatin accessibility at SCL-bound cis-regulatory regions.
Fig. 6: Genomic regions transiently marked by H3K27Ac are bound by Eomes, Tead4 and Smad2/3 during early stages of haematopoietic mesoderm development.
Fig. 7: Comparison of scRNA-seq profiles of WT and Eomes-null Flk-1hi/PdgfRa cells generated in vitro to WT cells from E6.5–E8.5 mouse embryos.
Fig. 8: Runx1 re-expression in Eomes-null/Runx1-null EHT cultures rescues definitive haematopoiesis.

Data availability

The RNA-seq, scRNA-seq, ChIP-seq and ATAC-seq data have been deposited in the Gene Expression Omnibus (GSE140005). Previously published sequencing data that were re-analysed here are available under accession codes GSE110164, GSE128466 and GSE47085. Source data are provided with this paper. All other data supporting the findings of this study and biological materials presented in this study are available upon reasonable request.

Code availability

All of the computational code is available from E.J.R. upon reasonable request.

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Acknowledgements

We acknowledge M. Maj and L. Ericsen (flow cytometry facility at the Dunn School) and K. Clark (flow cytometry facility at the Weatherall Institute of Molecular Medicine (WIMM)) for providing cell sorting services. The WIMM is supported by the MRC HIU, MRC MHU (MC_UU_12009), NIHR Oxford BRC and John Fell Fund (131/030 and 101/517), EPA fund (CF182 and CF170) and WIMM Strategic Alliance awards G0902418 and MC_UU_12025. We thank N. Ashley for help with 10× sample preparation and sequencing. The WIMM Single Cell Core Facility was supported by the MRC MHU (MC_UU_12009), the Oxford Single Cell Biology Consortium (MR/M00919X/1) and WT-ISSF (097813/Z/11/B#) funding. The WIMM facility was supported by WIMM Strategic Alliance awards G0902418 and MC_UU_12025. We also thank the High-Throughput Genomics Group (Wellcome Trust Centre for Human Genetics, funded by the Wellcome Trust (090532/Z/09/Z)) for generating sequencing data. We thank V. Kouskoff for providing the iRunx1 embryonic stem cell line, S. Thongjuea and G. Wang for advice on the scRNA-seq analysis, J. Riepsaame for advice on the CRISPR experiments, and D. Higgs, H. Chagraoui, D. Owens, A. Nelson and A. Mould for helpful discussions. M.F.T.R.d.B. and C.P. are supported by programmes in the MRC Molecular Hematology Unit Core award (grant number MC_UU_12009/2 to M.F.T.R.d.B. and MC_UU_12009/9 to C.P.). L.G. was supported by a Clarendon PhD studentship and the MRC Molecular Haematology Unit. The work was supported by grants from the Wellcome Trust (214175/Z/18/Z to E.J.R. and 10281/Z/13/Z to L.T.G.H.). L.T.G.H. was supported by a Clarendon Fund Scholarship and Trinity College Titley Scholarship. E.J.R. is a Wellcome Trust Principal Fellow.

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Contributions

L.T.G.H., E.K.B., C.P., M.F.T.R.d.B. and E.J.R. designed the study. L.T.G.H., C.S.S., I.C. and A.D.S. performed the experiments. L.T.G.H., I.I.-R., J.C.M. and B.G. performed the scRNA-seq analyses. L.G. generated the Runx1-Venus reporter line. L.T.G.H., E.K.B., M.F.T.R.d.B. and E.J.R. wrote the manuscript with input from all of the authors.

Corresponding authors

Correspondence to Marella F. T. R. de Bruijn or Elizabeth J. Robertson.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks Valerie Kouskoff and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Eomes and Flk-1 expression at the outset of mouse gastrulation.

a,b, Immunofluorescence staining of E6.5 (a) and E7.5 (b) wildtype embryos for Flk-1 (red) and Eomes (green); Images representative of 5 embryos (E6.5) and 8 embryos (E7.5). Nuclei are stained with DAPI (blue). Dotted white lines indicate the extraembryonic/embryonic boundary. White boxes denote the zoomed in areas displayed in the far-right panel. White arrows indicate Flk-1+ extra-embryonic mesodermal cells. Orange arrows indicate Flk-1+ embryonic mesodermal cells. Scale bars, 100 μM.

Extended Data Fig. 2 Generation of the EomesiCre allele.

a, Targeting strategy used to generate the EomesiCre allele. The targeting vector introduces a mammalian codon-improved Cre (iCre), β-globin polyA cassette into the endogenous Eomes initiator methionine and a LoxP flanked PGK-Neo positive drug selection cassette and TK negative selection cassette. Red and blue lines indicate the locations of 5’ external (red) and 3’ external probes (blue, exon 6) used for Southern blotting. Orange arrows indicate the location of primers used for PCR genotyping. E=EcoRV. b, Southern blot showing wildtype (15 kb) and targeted (10 kb) alleles. (Red probe) c, Southern blot after Cre-mediated excision of the Neo drug selection cassette showing excised targeted (6 kb) and wild type (15 kb) alleles and loss of the targeted allele (8 kb). (Blue probe) d, PCR genotyping of EomesiCre mice; iCre band size is 440 bp. (b-d) Southern blots and PCR genotyping blots are representative of at least 3 independent experiments. e, Wholemount in-situ hybridization (WISH) comparing Eomes and iCre expression in EomesiCre/+ embryos. ExE, extra-embryonic ectoderm; ch, chorion; PS, primitive streak; APS, anterior primitive streak. Scale bars, 100 μM f, Evaluating the expression of extra-embryonic mesodermal markers upon Eomes conditional inactivation from the epiblast (EomesΔepi). (i) Wholemount X-gal staining of E7.5 control (top) and mutant EomesΔepi (bottom) embryos carrying a Flk-1-LacZ reporter allele. (ii) WISH analysis of ER71 expression in control (top) and mutant EomesΔepi (bottom) embryos. Scale bars, 100 μm. LS, late streak; EB, early allantoic bud stage. (e-f) Images are representative of at least 3 embryos from each gestational stage and genotype. Source data

Extended Data Fig. 3 Generation of the Runx1-Venus ESC reporter line.

a, Targeting strategy used to generate the Runx1-Venus allele. A 3xFlag-P2A-Venus was inserted in the last exon of Runx1 (exon 6), before the stop codon. Pink arrow indicates the locations of Cas9-gRNA induced DNA double stand break immediately preceding the Runx1 stop codon. Orange arrows indicate locations of primers used for PCR genotyping b, Integration of the targeting vector was confirmed using long-range PCR; Runx-1Venus band size is 5.8 kb, WT band size is 4.4 kb. A clone with the Venus reporter integrated in both alleles (RV11 – red box) was used for all experiments. This experiment was performed once. c, Assessing Eomes’ requirement for cell autonomous Runx1 expression during hematopoiesis (left panel). At day 0 WT (grey) cells were mixed in equal proportions with either Eo+/+ Runx1-Venus ESCs (orange) or Eo-/- Runx1-Venus ESCs (purple). Flow cytometric analysis was performed on bulk EHT RV-Eo+/+:WT (top) and RV-Eo-/-:WT co-cultures at day 6 to assess Runx1-venus, cKit and CD41 expression. Runx1-venus was expressed at varying levels (left flow cytometry panel); Runx1-negative, grey dots; Runx1-lo, blue dots; Runx1 + , yellow dots. Percentages of Runx1-Venuslo (blue) and Runx1-Venus+ (yellow) cells within the CD41hi cell population are indicated by coloured numbers in the middle and right flow cytometry panels; Representative of 1 Runx-1 Venus Eomes+/+ and 2 Runx1-Venus Eomes-/- clones.

Extended Data Fig. 4 Re-generation of the Eomes loss-of-function allele in the SCL-mCherry and Runx1-Venus ESC reporter lines and the iRunx1 Runx1-/- ESC using CRISPR-Cas9.

a, The targeting strategy used to re-generate the Eomes-loss of function allele23 in SCL-mCherry and Runx1-Venus ESC reporter lines. An ssODN was used to patch an EcoRV site in between introns 1 and 5 to delete a 3.5 kb region, including exons 2-5 of the Eomes locus. Pink arrows indicate the locations of Cas9-gRNA induced DNA double strand breaks in intron 1 and intron 5. Blue lines (exon 6) indicate the location of a 3’ external Southern blotting probe. Orange arrows indicate the location of primers used for PCR genotyping. E = EcoRV. b, Southern blots showing wild type (15 kb) and targeted (2.5 kb) alleles in Runx1-Venus (top) and SCL-mCherry (bottom) ESC lines. Representative of 2 independent experiments. c, PCR genotyping of Eomes-null ESCs; WT band size is 401 bp, Eomes-null band size is 535 bp. d, The targeting strategy used to re-generate the Eomes-loss of function allele in the iRunx1 Runx1-/- ESC line. ssODNs were used to patch an Sph1/Spe1 restriction sites in between introns 1 and 5 to delete a 3.5 kb region, including exons 2-5 of the Eomes locus. Pink arrows indicate the locations of Cas9-gRNA induced DNA double strand breaks in intron 1 and intron 5. Orange arrows indicate the location of primers used for PCR genotyping. e, PCR genotyping of Eomes-null ESCs; WT band size is 499 bp, Eomes-null band size is 633 bp and digestion products of the Eomes-null PCR product are 221 bp and 412 bp. iRunx1 Runx1-/- Eomes-/- clones had insertion of both Sph1 and Spe1 restriction sites, indicating a homozygous deletion. Representative of 2 independent experiments. Source data

Extended Data Fig. 5 Generation of the EomesV5/V5 ESC line for ChIP-Seq.

a, The targeting strategy used to insert a 3XGly-V5 tag directly upstream of the UAG translational stop codon (exon 6) at the C-terminus of the Eomes locus using an ssODN. The pink arrow indicates the location of the Cas9-gRNA induced DNA double strand break. Orange arrows indicate the location of primers used for PCR genotyping. b, Western blotting of whole cell protein lysates from EoV5/V5 and WT day 4 EBs shows that the three isoforms of Eomes protein (upper panel) are V5 tagged (lower panel) in both EoV5/V5 clones. This experiment was performed once. c, PCR genotyping of Eomes-V5 targeted clones; WT band size is 250 bp, Eo-V5 band size is 301 bp. Representative of at least 3 independent experiments. d, e, V5 protein expression was evaluated in day 3-5 EomesV5/V5 EBs using intracellular flow cytometry. d Equivalent stage WT EBs were used as a gating control (upper panel). e, Additionally, EBs were stained for Flk-1 and PdgfRa expression; purple dots indicate cells that are V5 + and numbers indicate the proportion of cells in the Flk-1hi/PdgfRa- that are V5+; Representative of 1 wildtype and 2 EoV5/V5 clones. f, Representative immunofluorescence staining of EoV5/V5 day 4 EBs for Eomes (red) and V5 (green); 2 biologically independent samples. Nuclei are counter stained with DAPI (blue). Scale bars, 100 μM. g, Representative flow cytometric analysis of Flk-1/PdgfRa (top) and CD41/c-Kit (bottom) expression in WT (left) and EoV5/V5 (right) day 4 and day 7 EBs, respectively; Representative of1 WT and 2 EoV5/V5 clones. Source data

Extended Data Fig. 6 Eomes binds multiple Runx1 cis-regulatory regions.

IGV snapshots of EomesV5/V5, Eomes-V5/ Eomes-GFP51, Tead449, Smad2/350 and Scl39 ChIP-Seq peaks. H3K27Ac ChIP-Seq peaks and DNase1 hypersensitivity (DN1 HS) at different stages of hematopoietic development in vitro; HB, hemangioblast (T+ /Flk-1+); HE, hemogenic endothelium (Tie2+/Kit+); HP, hematopoietic progenitor (CD41+)49. Yellow bars highlight Eomes bound sites identified in the Eomes-V5 dataset51. The numbers above coloured columns indicate the relative location of these sites in kilobases to the TSS at P1 of the Runx1 locus. Red numbers indicate sites which have reduced chromatin accessibility in Eomes-/- Flk-1hi/PdgfRa- cells. P1, promoter 1; P2, promoter 2.

Extended Data Fig. 7 Comparing Eomes bound ChIP-Seq peaks with the subset of ATAC-Seq peaks that are Eomes-dependent and normally bound by SCL at various stages of hematopoietic differentiation.

a, Venn diagram depicting the overlap of Eo-V5 ChIP-Seq peaks in D4 EBs, SCL ChIP-Seq peaks in D4 Flk-1+ EBs39 and sites of reduced chromatin accessibility in day 4 Eo-/- Flk-1+/PdgfRa- cells. Heatmaps showing ATAC signals (blue) from WT or Eomes-/- day 4 Flk-1hi/PdgfRa- cells and SCL ChIP-Seq signal (green) from day 4 WT Flk-1+ cells39. All heatmaps show a 4 kb region flanking peak centres of the 338 Eomes bound sites. b, Venn diagram depicting the overlap of Eo-V5 ChIP-Seq peaks in D4 EBs with sites of reduced chromatin accessibility in day 4 Eo-/- Flk-1hi/PdgfRa- cells that are normally bound by SCL in WT day 4 Flk-1+ cells39. Heatmaps show DNAseI hypersensitivity signal (red), ChIP-Seq signal for H3K27Ac (green) histone modifications and SCL occupancy (purple) in different cell populations that develop during hematopoietic differentiation. Mes, mesoderm (T+/Flk-1-); HB, hemangioblast (T+/Flk-1+); HE, hemogenic endothelium (Tie2+/Kit+); HP, hematopoietic progenitor (CD41+)49. All heatmaps show 4 kb regions flanking peak the peak regions indicated in the Venn diagram.

Extended Data Fig. 8 scRNA-Seq shows that hematoendothelial progenitor and endothelial subsets are still specified in the absence of Eomes function.

a, Gating strategy used to sort Flk1hi/PdgfRa- cells from day 4 and day 5 wildtype and Eomes-/- EBs for 10X scRNA-Seq. b, c, Uniform manifold approximation and projection (UMAP) plots. WT (blue) and Eomes-/- (red) Flk-1hi/PdgfRa- cells from day 4 (left) and day 5 (right) EBs; n = 3805 cells/genotype/day. c, WT (left) and Eomes-/- (right) cells coloured by their Seurat cluster/cell type annotation denoted in the legend. The black box highlights clusters 7 and 8 that are diminished in the Eomes-/- cultures. d, Log2 fold change in the abundance of Eomes-/- cells with respect to WT cells in each Seurat cluster. e, Violin plots showing mesodermal, hematovascular and allantoic gene expression levels in WT (blue) and Eomes-/- (red) cells within the various Seurat clusters; n = 7610 WT cells and n = 7610 Eomes-/- cells. Violin plots show normalized expression values, genes are indicated on the x-axis and clusters on the y-axis. f, Normalized expression of Scl (top) and Runx1 (bottom) overlaid on UMAPs for all single cells collected from day 4/5 Flkhi/PdgfRa- wildtype (left) or Eomes-/- (right) EBs. HEP, hematoendothelial progenitors; Endo, endothelium; Meso, mesoderm; PS, primitive streak.

Extended Data Fig. 9 Eomes-null endothelial cells immunophenocopy Runx1-null endothelial cells.

a, b, Flow cytometric analysis of day 6 EB (a) and day 7 EHT (b) for expression of Cdh5/cKit and CD41 within the Cdh5+/cKit+ compartment. Pink gate highlights a CD41lo subset absent in SCL-null cultures. This experiment was performed once. c, Proportion of Cdh5+ cells in day 8 iRunx1 Eomes+/+ and iRunx1 Eomes -/- EHT cultures in which Runx1 has been uninduced (0 ng/mL dox) or induced (90 and 300 ng/mL dox) from day 6-8. Graphical representations depict mean± SEM of n = 3 independent differentiations for iRunx1 Eo+/+ 90 ng/mL and iRunx1 Eo+/+ 300 ng/mL and n = 4 independent differentiations for iRunx1 Eo+/+ 0 ng/mL, iRunx1 Eo-/- 0 ng/mL, iRunx1 Eo-/- 90 ng/mL and iRunx1 Eo-/- 300 ng/mL. Statistical source data are provided in Source Data Fig. 8. d, Representative examples of definitive erythro-myeloid colonies generated from iRunx1 Eomes-/- day 8 EHT cultures in which Runx1 expression was induced from day 6-8 via dox addition; 3 independent differentiations. Ery-D, definitive erythrocyte; GM, granulocyte-macrophage; GEMM, granulocyte-erythrocyte-myeloid. Scale bars, 100 μM. Source data

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. List of conserved marker genes across genotypes for each Seurat cluster. Only positive markers for each cluster are listed. Supplementary Table 2. Primer sequences. Primer sequences used for real-time quantitative reverse transcription PCR and genotyping are listed. Product lengths indicate the size of the PCR product in base pairs. Supplementary Table 3. Antibody information. Antibodies used for immunofluorescence, flow cytometry, ChIP, western blotting and MACS experiments are listed. Supplementary Table 4. iCre probe sequence. The sequence for the iCre probe used for whole-mount in situ hybridization is shown. Supplementary Table 5. CRISPR–Cas9 reagents. Sequences for gRNAs and repair templates are listed that were used to generate the Runx1-Venus, Eomes-V5 and Eomes-null ESC lines. Asterisks indicate phosphorothioated DNA bases. Supplementary Table 6. Eomes-V5-bound genomic regions. List of the Eomes-V5-bound genomic regions and nearby associated genes, as identified using GREAT. Additionally, lists on the right indicate whether the corresponding Eomes-V5-bound peak is co-occupied by Tead4 (ref. 49) and Smad2/3 (ref. 50) in the indicated datasets.

Source data

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 2

Unprocessed southern blots.

Source Data Extended Data Fig. 4

Unprocessed Southern blots/gels.

Source Data Extended Data Fig. 5

Unprocessed western blots/gels.

Source Data Extended Data Fig. 9

Statistical source data.

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Harland, L.T.G., Simon, C.S., Senft, A.D. et al. The T-box transcription factor Eomesodermin governs haemogenic competence of yolk sac mesodermal progenitors. Nat Cell Biol 23, 61–74 (2021). https://doi.org/10.1038/s41556-020-00611-8

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