Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Related to . ( A ) Cdh5-tracking mouse lines. Tamoxifen switches on green fluorescence in cells that express the Cre-recombinase and their cell progeny. Confocal microscopy images of representative BM sections from Cdh5-Cre ERT2 (PAC)/ZsGreen ( B ) and Cdh5-Cre ERT2 (BAC)/ZsGreen ( C ) adult mice showing tamoxifen-induced ZsGreen fluorescence co-staining of most Endomucin + cells. Control BM sections from representative Cre + mouse treated with peanut oil (no tamoxifen) display occasional ZsGreen + Endomucin + cells but no tamoxifen-independent ZsGreen fluorescence is detected in representative Cre − mice. ( D ) Flow cytometry analysis of adult BM cells from Cdh5-Cre ERT2 (PAC)/ZsGreen ( n = 6–8) and Cdh5-Cre ERT2 (BAC)/ZsGreen mice ( n = 10) shows that ZsGreen fluorescence identifies most ECs 4 weeks after tamoxifen administration but also tracks a small proportion of EC expressing tamoxifen-independent fluorescence in Cre + but not Cre - mice. ( E ) Percent CD45 + ZsGreen + cells of viable BM cells from Cre − control ( n = 6), Cre + control (peanut oil treated, no tamoxifen; n = 8) and Cre + tamoxifen-treated Cdh5-Cre ERT2 (PAC) /ZsGreen ( n = 6) or Cdh5-Cre ERT2 (BAC)/ZsGreen mice ( n = 9). Mice were 8–12 weeks old at the time of tamoxifen administration. ( F ) Percent ZsGreen + cells of peripheral blood mononuclear cell (PBMC) from Cre − control, Cre + control (peanut oil treated), and Cre + tamoxifen-treated Cdh5-Cre ERT2 (PAC)/ZsGreen and Cdh5-Cre ERT2 (BAC)/ZsGreen mice ( n = 9/group). Mice were 8–12 weeks old at the time of tamoxifen administration. Representative flow cytometry gating of CD45 + EGFP + cells from BM and blood of Cdh5-CreERT2/mTmG mice ( G , relates to ), and CD45 + ZsGreen + cells from BM and blood of Cdh5-Cre ERT2 /ZsGreen mice ( H , relates to Figure S1E, F). ( I ) Representative confocal images showing a nucleated (DAPI + ) BM ZsGreen + CD45 + cell in the BM from a Cdh5-Cre ERT2 (PAC)/ZsGreen mouse treated with tamoxifen. ( J ) Percent ZsGreen + cells of PBMC in individual Cdh5-Cre ERT2 (BAC)/ZsGreen mice before or four weeks after tamoxifen administration. Each dot represents the results from 50 to 250 µl blood/mouse. The lines link results from individual mice ( n = 15, 8–12 weeks old). ( K ) Percent B and T-lymphocytes, granulocytes, and monocytes among EGFP + PBMC of Cre + tamoxifen-treated Cdh5-Cre ERT2 (PAC)/mTmG mice ( n = 15, 8–12 weeks old). ( L ) Percent EGFP + cells in peripheral blood cell populations of Cre + peanut oil-treated ( n = 9) and tamoxifen-treated ( n = 16) Cdh5-Cre ERT2 (PAC)/mTmG mice (8–12 weeks old). Dots represent individual mice. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t -test.

Article Snippet: Sequence-based reagent , mTmG mouse strain genotyping primers (5′–3′) , CTT TAA GCC TGC CCA GAA GA TAG AGC TTG CGG AAC CCT TC AGG GAG CTG CAG TGG AGT AG , JAX: 007676 , .

Techniques: Fluorescence, Confocal Microscopy, Staining, Control, Flow Cytometry, Expressing

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: ( A ) Experimental design: tamoxifen was administered to 8- to 12-week-old Cdh5-Cre mice to induce fluorescent labeling of VE-Cadherin + cells and their cell progeny. Four weeks later, BM and blood were analyzed. ( B ) CD31 + EGFP + BM ECs in Cre − mice ( n = 10) and Cre + mice treated with oil ( n = 13) or tamoxifen ( n = 10); flow cytometry results. ( C, D ) CD45 + EGFP + cells in BM and blood from Cre − mice ( n = 8) and Cre + mice treated with oil ( n = 6–10) or tamoxifen ( n = 15–18). Representative flow cytometry gating in . ( E ) Representative blood smear from a tamoxifen-treated Cdh5-Cre ERT2 (PAC)/ZsGreen mouse showing ZsGreen + CD45 + DAPI + cells (arrows). ( F ) Kinetics of ZsGreen + cell detection in BM ECs (CD45⁻VE-Cadherin + ) and blood white blood cells (WBCs) post-tamoxifen; mouse n = 8–10/group. ( G ) EGFP + B and T-lymphocytes, granulocytes, and monocytes in BM of tamoxifen-treated mice ( n = 14) as percent of total EGFP + cells; three experiments. ( H ) EGFP + BM LSK, lymphocytes, granulocytes, and monocytes as percent of total EGFP +/− cell type; Cdh5-Cre ERT2 (PAC)/mTmG mice (oil n = 10; tamoxifen n = 15), three experiments. ( I ) Uniform Manifold Approximation and Projection (UMAP) plots of Lin − BM hematopoietic stem and progenitor cell (HSPC) from tamoxifen-treated Cdh5-Cre ERT2 (PAC)/ZsGreen mice ( n = 26; 1 femur/mouse) showing FlowSOM clustering of all (ZsGreen +/− ) and ZsGreen + populations. ( J ) Violin plots showing ZsGreen + cell distribution across HSPC subsets from ( I ). Dots represent individual mice; data shown as mean ± SD except shown as median in ( G ). *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t -test.

Article Snippet: Sequence-based reagent , mTmG mouse strain genotyping primers (5′–3′) , CTT TAA GCC TGC CCA GAA GA TAG AGC TTG CGG AAC CCT TC AGG GAG CTG CAG TGG AGT AG , JAX: 007676 , .

Techniques: Labeling, Flow Cytometry

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: ( A ) Transplantation experiment: donor LSK sorting, recipient irradiation, transplantation, tamoxifen treatment, and analysis (top). Tabular representation of possible outcomes of the experiment designed to address the question ‘Do HSPCs/other hematopoietic cells in Cdh5Cre ERT2 /ZsGreen mice express Cdh5-Cre ERT2 ?’ (bottom). Blood WBC counts ( B ), percent ZsGreen + peripheral blood mononuclear cell (PBMC) ( C ), and time course of ZsGreen + PBMC detection ( D ) in transplant recipients of ZsGreen − LSK (5 × 10 4 or 2.5 × 10 4 cells/mouse; n = 3/group) and ZsGreen-enriched LSKs (2.8 × 10 3 cells/mouse; n = 2). Results in B and C are from week 24 post-tamoxifen. ( E ) Experiment: WT BM transplantation (BMTP) into lethally irradiated Cdh5-Cre/mTmG mice ( n = 9). Four weeks later, tamoxifen was administered; blood was monitored for 16 weeks. EGFP + PBMC detection before and after tamoxifen or peanut oil administration ( F ) and cell type distribution of EGFP + and EGFP − PBMCs at week 12 post-tamoxifen or peanut oil ( G ) in Cdh5-Cre/mTmG recipients ( n = 9) of WT BM (5 × 10 6 cells). Statistical significance reflects comparisons between EGFP + and EGFP − cells in the tamoxifen versus peanut oil groups. Dots represent individual mice. Data are shown as mean ± SD. ***p < 0.001, ns, not significant by Student’s t -test.

Article Snippet: Sequence-based reagent , mTmG mouse strain genotyping primers (5′–3′) , CTT TAA GCC TGC CCA GAA GA TAG AGC TTG CGG AAC CCT TC AGG GAG CTG CAG TGG AGT AG , JAX: 007676 , .

Techniques: Transplantation Assay, Irradiation

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Related to . ( A ) Schematic representation of the Col1a2 tracking lines. ( B ) Representative confocal image of a BM section from a tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen mouse, showing widespread distribution of ZsGreen + cells. Flow cytometric identification of RUNX1 + VE-Cadherin + CD45 − endothelial cells (ECs) in the BM of peanut oil-treated ( n = 6) and tamoxifen-treated ( n = 6) Col1a2-Cre ERT2 /ZsGreen adult mice; Cre − mice ( n = 5) ( C ); WT C57Bl/6 mice ( n = 6) and Fluorescence Minus One (FMO) control ( n = 5) ( D ); and Cdh5-Cre ERT2 (PAC)/ZsGreen mice treated with peanut oil ( n = 6) or tamoxifen ( n = 6); Cre − mice ( n = 5) ( E ). Left panels: quantification of cells identified by the indicated gates as a percentage of total BM ECs; each dot represents one mouse (1 femur + 1 tibia). Middle and right panels: representative gating strategies. ( F ) Representative confocal microscopy image of a BM section from a tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen adult mouse showing a ZsGreen + Endomucin + cell lining a vascular structure (white arrows). ( G ) Representative confocal image of a blood smear from a Col1a2-Cre ERT2 /ZsGreen mouse treated with tamoxifen showing the presence of a nucleate CD45 + cell tracked by ZsGreen/Col1a2 fluorescence (pointed by the arrow). ( H ) Representative confocal image of a blood smear from a Col1a2-Cre ERT2 /mTmG mouse treated with tamoxifen showing the presence of a nucleated CD45 + cell tracked by EGFP/Col1a2 fluorescence (pointed by the arrow). Dots represent individual mice. Data are shown as mean ± SD. ***p < 0.001 by Student’s t -test.

Article Snippet: Sequence-based reagent , mTmG mouse strain genotyping primers (5′–3′) , CTT TAA GCC TGC CCA GAA GA TAG AGC TTG CGG AAC CCT TC AGG GAG CTG CAG TGG AGT AG , JAX: 007676 , .

Techniques: Fluorescence, Control, Confocal Microscopy

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Percent EGFP + CD45 + cells in bone marrow (BM) and blood of tamoxifen-treated ( n = 6) or oil-treated ( n = 5) Col1a2-Cre ERT2 /mTmG mice ( A ) and tamoxifen-treated ( n = 4) or oil-treated ( n = 3) Col1a2-Cre ERT2 /ZsGreen mice ( B ). Cre-control mice ( n = 5 in A, and n = 2 in B). ( C ) Transplant experiment: sorted VE-Cadherin + CD45 − ZsGreen + /Col1a2 + cells from tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen mice are transplanted into 5-FU-conditioned WT recipients. Detection ( D ) and characterization ( E ) of ZsGreen + CD45 + cells in BM and blood of WT 5-FU-conditioned mice ( n = 5), 4 weeks post-transplant of VE-Cadherin + CD45⁻ZsGreen + /Col1a2 + cells. Control FU-conditioned WT mice ( n = 4) received no cell transplant ( D ). ( F ) Time course of ZsGreen + peripheral blood mononuclear cell (PBMC) detection in control (Cdh5-Cre + /ZsGreen + ) and Runx1 EC-KI (Cdh5-Cre + /ZsGreen + /Runx1-KI) mice ( n = 10 per group). Representative images ( G ) and quantification ( H ) of ZsGreen + cells from OP9 cell-supported cultures of BM cells from tamoxifen-treated Cdh5-Cre + /ZsGreen + ( n = 11) and Runx1 EC-KI mice ( n = 5). Representative flow cytometry plots ( I ) and quantification ( J ) of CD45 + ZsGreen + cells from OP9 cell-supported BM cell cultures ( n = 5/group). Dots represent individual mice. Data are shown as mean ± SD. **p < 0.01, ***p < 0.001 by Student’s t -test.

Article Snippet: Sequence-based reagent , mTmG mouse strain genotyping primers (5′–3′) , CTT TAA GCC TGC CCA GAA GA TAG AGC TTG CGG AAC CCT TC AGG GAG CTG CAG TGG AGT AG , JAX: 007676 , .

Techniques: Control, Flow Cytometry