Abstract
In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta–gonad–mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.
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Acknowledgements
We thank the staff at the Advanced Imaging, animal facility, Molecular Biology Core facilities and Flow Cytometry of CRUK Manchester Institute for technical support and M. Lie-A-Ling for help with initiating the DamID-PIP bioinformatics project. We thank members of the Stem Cell Biology group, the Stem Cell Haematopoiesis groups and M. Gering for valuable advice and critical reading of the manuscript. Work in our laboratory is supported by the Leukaemia and Lymphoma Research Foundation (LLR), Cancer Research UK (CRUK) and the Biotechnology and Biological Sciences Research Council (BBSRC). S.C. is the recipient of an MRC senior fellowship (MR/J009202/1).
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R.T. designed and performed most of the experiments, analysed the data and wrote the manuscript, M.M. initiated the project, designed performed experiments and analysed the data. R.P., V.M., M.S. and C.L. designed and performed experiments. E.M. and Y.L. performed bioinformatics analysis on the sequencing data and microarray. T.C., T.M., C.R., C.M., S.C. and B.G. contributed valuable tools and protocols. V.K. and G.L. designed and supervised the research project, analysed the data, and wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Gfi1tomato mouse line.
(a) FACS analysis of Gfi1tomato and Gfi1GFP/+ bone marrow cells after staining for lymphoid (CD4/CD8) and myeloid (GR-1/MAC-1) markers. Bone marrow populations were gated into GFP− and GFP+ for Gfi1GFP/+ (top panel) and TOM− and TOM+ for Gfi1tomato (lower panel) fractions (representative FACS plot of one independent experiment). (b) IHC for CD31 (red), GFI1 (cyan) and GFI1B (green) on E10.5 AGM section counterstained with DAPI (representative images of 3 independent experiments). (c) Frequencies of GFI1+/GFI1− and GFI1+/GFI1+ cells in the CDH5 compartment of E10.5 and E11.5 AGMs (AGMs from 11 independent litters, n = 6 independent experiments for E10.5 and n = 4 independent experiments for E11.5 were analysed, p-values were determined by a two-tailed Student’s t-test. Error bars represent standard error of the mean (S.E.M.). The source data used to determine the statistical significance can be found in supplementary table 3. (d) FACS analysis of GFI1 and GFI1B cells in the CDH5 compartment of E11.5 AGMs (representative FACS plot from one independent experiment). Scale bar = 10 μm.
Supplementary Figure 2 GFI1+/GFI1B− cells gain GFI1B expression in vitro.
(a) CDH5+/GFI1+/GFI1B− cells were FACS sorted from Gfi1tomatoGfi1GFP/+ E10.5 AGM and cultured on OP-9 stromal cells. Images at day 2 and 7 of in vitro cultures (representative images of n = 4 independent experiments). (b) Day 7 cultures were analysed by FACS and indicated gates were used for sorting. (c) The initial sorted E10.5 CDH5+/GFI1+/GFI1B− AGM cells and the different cell populations isolated after 7 days of their cultures (GFI1+/GFI1B−, GFI1+/GFI1B+, GFI1−/GFI1B+) were analysed for Gfi1b expression level by q-PCR (representative data from one experiment).
Supplementary Figure 3 Heatmap of all cells and genes analysed by single cell PCR.
Heat map depicting the clustering all the single cells and genes analysed.
Supplementary Figure 4 E11.5 CDH5+/GFI1+ cells contribution to recipients.
(a) GFI1 and GFI1B expression mark all hematopoietic cells in the E10.5 AGM (i) schematic representation of the experimental design (ii and iii) E10.5 AGM cells were FACS sorted and re-plated into haematopoietic assay either before (one independent experiment with embryos from one litter) (ii) or after (one independent experiment with embryos from one litter) (iii) co-culture step with OP-9. Colonies were scored 9-11 days later. (b) FACS analysis of recipients transplanted with either CDH5+/GFI1+ or CDH5+/GFI1− E11.5 AGM cells (CD45.1/CD45.2 double positives) at 17 weeks after transplantation. Bone marrow LSK compartments of recipients (CD45.1) were analysed for CD45.1 and CD45.2 (representative FACS plots of one independent experiment). (c) Donor contribution to haematopoietic lineages determined by sub-gating on CD45.1/CD45.2 double positive population in the recipient. Donor contribution to T cells (CD4/CD8) in the spleen and thymus, B cells (IgM/B220), myeloid cells (GR-1/MAC-1) and erythroid cells (CD71/TER119) in different haematopoietic organs are shown (representative FACS plots of one independent experiment).
Supplementary Figure 5 GFI1 or GFI1B single knock out embryos can generate IAHC.
(a) IHC on E10.5 GFI1KOGFI1B+/+ and GFI1+/+GFI1BKO AGM section for CD31, GFP and c-KIT (counterstained with DAPI) (representative images of one independent experiment with embryos form different litters). (b) In situ hybridisation for Gfi1b (red dots) and IHC for CD31 (brown) on GFI1GFP/+GFI1BGFP/+ and GFI1KOGFI1BGFP/+ E10.5 embryo sections. (c) qPCRs on E10.5 AGM cells. (i) qPCR for Gfi1 and Gfi1b expression in AGM cell lysate of GFI1GFP/+GFI1BGFP/+ (het/het), GFI1GFP/GFPGFI1BGFP/+ (KO/het) and GFI1GFP/+GFI1BGFP/GFP (het/KO) embryos (representative data from one independent experiment with embryos from the same litter). (ii) PCR for Gfi1 and Gfi1b on sorted CDH5+/GFP+/c-KIT− HE cells of GFI1GFP/+GFI1BGFP/+ (het/het) and GFI1GFP/GFPGFI1BGFP/+ (KO/het) embryos (one independent experiment with embryos from the same litter). Scale bar = 10 μm.
Supplementary Figure 6 Conditional Lsd1 knock-out recapitulates LSD1 inhibition phenotype and leads to decrease in proliferation and apoptosis.
(a) FLK1+ cells from the conditional Lsd1Δ/lox line were isolated from day 3 EBs and cultured as monolayer. FACS analysis on 3 consecutive days with staining for TIE-2, CDH5 and CD41 are shown. Lsd1Δ/lox FLK1 + cells were either cultured with control ETOH or with 1 uM of 4OHT (in ETOH) to induce the activity of Cre-ERT2 and generate Lsd1Δ/Δ (representative FACS plots of 3 independent experiments). (b) EdU assay on Day 3 control (Lsd1Δ/lox) or Lsd1 deleted (Lsd1Δ/Δ). Blast cultures (Representative FACS plots of one independent experiment). (c) Annexin5/7-AAD staining on Day 3 control (Lsd1Δ/lox) or Lsd1 deleted (Lsd1Δ/Δ) Li-Blast cultures. Additionally, the cultures were stained with CD41 to differentiate between HE and non-HE cells.
Supplementary Figure 7 LSD1 is ubiquitously expressed and its inhibition leads to loss of EHT in E10.5 AGM.
(a) IHC for CD31, tomato and LSD1 on E10.5 AGM sections of Gfi1tomato embryos (counterstained with DAPI). (b) IHC for CDH5, GFI1 and CD45 on E9.5 P-Sp explants. (c) Single channel control images from live imagining of E10.5 Gfi1tomatoGfi1GFP/+ AGM slices wither treated with DMSO (ctrl) or 500nM of the LSD1 inhibitor. (di–iii) Screen shots of the Oit-3, Gata-2 and Hey-2 locus from the UCSC browser showing examples of GFI1:DAM (Cyan) and GFI1B:DAM (green) binding enrichment after sequencing. Region of interest is marked with a black box and specific peaks detected are highlighted with black bars below. Scale bar = 10 μm.
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41556_2016_BFncb3276_MOESM28_ESM.mov
Time-lapse imaging of FLK1+ cells blast monolayer cultures treated with 300 nM of the LSD1 inhibitor (LSD1 inhib). (MOV 4541 kb)
41556_2016_BFncb3276_MOESM29_ESM.mov
3D re-construction of z-stacks (4.2 um) of E9.5 P-Sp explants (2 days cultures) treated with DMSO and stained for CD31 (red), GFI1 (cyan) and GFI1B (green). (MOV 1359 kb)
41556_2016_BFncb3276_MOESM30_ESM.mov
3D re-construction of z-stacks (4.1 um) of E9.5 P-Sp explants (2 days cultures) treated with the LSD1 inhibitor (300 nM) and stained for CD31 (red), GFI1 (cyan) and GFI1B (green). (MOV 2698 kb)
41556_2016_BFncb3276_MOESM31_ESM.mov
Time-lapse imaging of FLK1+ cells from the cLsd1 ES line in Liquid Blast culture treated with EtOH (Lsd1Δ/lox). (MOV 9637 kb)
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Time-lapse imaging of FLK1+ cells from the cLsd1 ES line in Liquid Blast culture treated with 4-OHT (Lsd1Δ/Δ). (MOV 6479 kb)
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Thambyrajah, R., Mazan, M., Patel, R. et al. GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1. Nat Cell Biol 18, 21–32 (2016). https://doi.org/10.1038/ncb3276
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DOI: https://doi.org/10.1038/ncb3276
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