Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow
- Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health, United States
- School of Life Sciences, Northeast Normal University, China
- Experimental Immunology Branch, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health, United States
- Laboratory of Genome Integrity, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health, United States
- Sequencing Facility Bioinformatics Group, Bioinformatics and Computational Science Directorate, Frederick National Laboratory for Cancer Research, United States
- Pediatric Oncology Branch, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health, United States
Figures
Figure 1 with 2 supplements
Lineage tracking discloses a contribution of endothelial cells (ECs) to hematopoiesis in adult bone marrow (BM).
(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 Figure 1—figure supplement 1G. (E) Representative blood smear from a tamoxifen-treated Cdh5-CreERT2(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-CreERT2(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-CreERT2(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.
Figure 1—figure supplement 1
Contribution of endothelial cells (ECs) to hematopoiesis in adult bone marrow (BM) is revealed by Cdh5-CreERT2 mouse tracking lines.
Related to Figure 1. (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-CreERT2(PAC)/ZsGreen (B) and Cdh5-CreERT2(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-CreERT2(PAC)/ZsGreen (n = 6–8) and Cdh5-CreERT2(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-CreERT2(PAC) /ZsGreen (n = 6) or Cdh5-CreERT2(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-CreERT2(PAC)/ZsGreen and Cdh5-CreERT2(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 Figure 1C, D), and CD45+ZsGreen+ cells from BM and blood of Cdh5-CreERT2/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-CreERT2(PAC)/ZsGreen mouse treated with tamoxifen. (J) Percent ZsGreen+ cells of PBMC in individual Cdh5-CreERT2(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-CreERT2(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-CreERT2(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.
Figure 1—figure supplement 2
Characterization of tracked hematopoietic progenitors and mature cells in adult bone marrow (BM) and peripheral blood of Cdh5-Cre reporter mice.
Related to Figures 1—3. Percent B lymphocytes, T lymphocytes, granulocytes, and monocytes of all ZsGreen+ cells (A) and percent ZsGreen+ cells of total BM LSK, B and T-lymphocytes, granulocytes, and monocytes (B). Each dot reflects results from individual mice (1 femur plus 1 tibia combined, n = 11); group means ± SD (error bars) are shown by the horizontal lines. Percent B lymphocytes, T lymphocytes, granulocytes, and monocytes of all ZsGreen+ peripheral blood mononuclear cell (PBMC) in Cdh5-CreERT2(PAC)/ZsGreen mice treated with tamoxifen (C, n = 10) and percent ZsGreen+ cells of peripheral blood B lymphocytes, T lymphocytes, granulocytes, and monocytes in Cdh5-CreERT2(PAC)/ZsGreen mice (D) treated with peanut oil (n = 15) or tamoxifen (n = 20). (E) Gating strategy for identification of hematopoietic stem and progenitor cell (HSPC) subsets in BM. Representative flow cytometry plots show sequential gating of lineage negative (Lin−) Sca1+ cKit+ (LSK) cells into long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), multipotent progenitors (MPP), common lymphoid progenitors (CLP), common myeloid progenitors (CMP), megakaryocyte–erythroid progenitors (MEP), and granulocyte–macrophage progenitors (GMP). Dots represent individual mice. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test.
Figure 2 with 1 supplement
Bone marrow (BM) endothelial cells (ECs) generate engraftable hematopoietic cells ex vivo.
(A) BM cells from tamoxifen-treated mice were cultured on high-attachment Primaria flasks or OP9 cell monolayers. Representative images show ZsGreen+ cells at weeks 1, 3, and 8. (B) Workflow for culturing unsorted and sorted BM cell populations. Post-sort purity of ZsGreen+ ECs is shown in the bottom left panel. All cells s were cultured (8 weeks) on OP9 cell monolayers supplemented with WT BM cells. Culture medium and floating cells were removed twice/week for 7 weeks. At the start of week 8, one final WT BM and medium supplementation was implemented prior to harvest at the end of week 8. Representative flow cytometry plots (C) and quantification (D) of CD45+ZsGreen+ cells from each of the 8 week cultures (n = 5). (E) Floating/loosely adherent ZsGreen+ cells from unsorted BM 8-week cell cultures were sorted and transplanted (5 × 104, 2.5 × 104, 1.25 × 104, or 6.25 × 103 cells) into lethally irradiated (11 Gy) WT mice (n = 2/group). Representative flow cytometry image (F) and quantification (G) of low-adherent cells harvested after 8 weeks of culture, showing that >95% (group average) of ZsGreen+ low-adherent cells are CD45+. These ZsGreen+CD45+ cells were sorted for transplantation. White blood cell (WBC) counts from five control mice (no irradiation or transplant) (H) and percent ZsGreen+, ZsGreen dim, and ZsGreen− cells (I, J) in blood of transplant recipients 10 months post-transplant (n = 6). Dots represent individual mice. Data are shown as mean ± SD. ns, not significant by Student’s t-test.
Figure 2—figure supplement 1
Bone marrow (BM) endothelial cells (ECs) generate engraftable hematopoietic cells ex vivo.
Related to Figure 2. (A) Representative image (relates to Figure 2B) showing the appearance of sorted ZsGreen+ ECs after 4-week culture on OP9 monolayer. (B) Representative cytospin image of floating and low-adherent cells from ex vivo culture of BM cells from a tamoxifen-induced Cdh5- CreERT2(PAC)/ZsGreen mouse.
Figure 3 with 1 supplement
Adult bone marrow (BM) endothelial cells (ECs) give rise to hematopoietic cells following transfer into conditioned recipients.
(A) Transplant experiment: donor ECs from BM of tamoxifen-treated mice were FACS-sorted and transplanted into WT C57Bl/6 recipients conditioned with 5-FU or PBS. (B) ZsGreen+ ECs detected in BM of 5-FU-conditioned (n = 5) or PBS-conditioned (n = 15) recipients of ECs 4 weeks post-transplant. ZsGreen+CD45+ cells (C) and cell type distribution (D) in the BM and blood of 5-FU-conditioned transplant recipients of BM ECs or no cell controls (n = 5/group). Age-dependent decline of ZsGreen+CD45+ cells (E) but not ZsGreen+VE-Cadherin+ cells (F) in the BM of Cdh5-CreERT2(BAC)/ZsGreen mice (n = 35) treated with tamoxifen 4 weeks prior to harvest. Cell number (G; mouse n = 8–12) and cell type distribution (H; mouse n = 6) in the peritoneal cavity (PerC) of PBS- or thioglycolate (TGL)-pretreated (4 hr) mice. (I) ZsGreen+ and ZsGreen− PerC cell types in TGL-pretreated mice (n = 12). (J) Representative histograms depicting pHrodo Red fluorescence detection of E. coli phagocytosis. E. coli+ phagocytosis by ZsGreen+ and ZsGreen− PerC neutrophils (K) and macrophages (L) in TGL-pretreated mice (n = 4). (M) Representative histograms depicting CellRox Orange fluorescence for cell-associated ROS detection. CellRox mean fluorescence intensity (MFI) in ZsGreen+ and ZsGreen− PerC neutrophils (N) and macrophages (O) in TGL-pretreated mice (n = 4). Dots represent individual mice. Data are shown as mean ± SD. *p < 0.05, ***p < 0.001, ns, not significant by Student’s t-test.
Figure 3—figure supplement 1
Adult bone marrow (BM) endothelial cells (ECs) give rise to hematopoietic cells following transfer into conditioned recipients.
Related to Figure 3. (A) Gating strategy for sorting VE-Cadherin+ ZsGreen+ ECs from the BM of Cdh5-CreERT2(PAC)/ZsGreen mice. (B) Purity analysis of sorted VE-Cadherin+ ZsGreen+ ECs. (C) Gating strategy used for detecting ZsGreen+CD45+ hematopoietic cells in WT C57Bl/6 recipients of ECs sorted from the BM of Cdh5-CreERT2(PAC)/ZsGreen donors. (D) Percent CD45+ZsGreen+ cells recovered from the peritoneal cavity (PerC) of Cre+Cdh5-CreERT2/ZsGreen mice treated with peanut oil (n = 8) or tamoxifen (n = 15). Cre− mice (n = 5). (E) Percent ZsGreen+ cells within PerC cell populations recovered from mice (n = 15) under steady-state conditions. Cell type identification; B1 cells: CD19+, CD3−, CD45R(B220)−, CD5+, CD43+; B2 cells: CD19+, CD3−, CD45R(B220)+, CD5−, CD43−; T cells: CD3+, CD11b−; monocytes: CD11b+, CD19−, Ly6G−, Ly6Chigh; neutrophils: CD11b+, CD19−, Ly6G+, Ly6Clow; and macrophages: CD11b+, CD19−, F4/80+. Dots represent individual mice. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test.
Figure 4 with 1 supplement
Independence of adult endothelial-to-hematopoietic transition (EHT) from preexisting hematopoietic stem and progenitor cell (HSPC).
(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 Cdh5CreERT2/ZsGreen mice express Cdh5-CreERT2?’ (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 × 104 or 2.5 × 104 cells/mouse; n = 3/group) and ZsGreen-enriched LSKs (2.8 × 103 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 × 106 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.
Figure 4—figure supplement 1
Bone marrow (BM) plasmacytoid dendritic cells (pDCs), but not other BM hematopoietic cells, express Cdh5 and Ptprc, encoding CD45.
Related to Figure 4. (A) Uniform Manifold Approximation and Projection (UMAP) plot showing unsupervised clustering of sc transcriptomic data from a public dataset of mouse BM hematopoietic cells. UMAP plots displaying expression of Cdh5 (B) and Ptprc (encoding CD45, C) in the dataset shown in (A). (D) Dot plot illustrating the expression of selected marker genes across clusters shown in (A). The red rectangle highlights gene expression by cluster 13 cells, identifying pDCs. Dot size represents the percentage of cells expressing the gene within each cluster, and color intensity reflects the mean expression level. (E) Gating strategy for selecting ZsGreen- and ZsGreen+ LSK progenitors. Analysis of purity of sorted LSK populations enriched for ZsGreen+ cells (F) and depleted of ZsGreen+ cells (G) from the BM of Cdh5-CreERT2(PAC)/ZsGreen mice (not treated with tamoxifen).
Figure 5 with 2 supplements
Polylox sc lineage tracing links adult bone marrow (BM) endothelial cells (ECs) to hematopoietic progenitors and mature blood cell progeny.
(A) Schematic of Polylox barcode and transcriptome profiling. FACS-enriched ECs (ZsGreen+VE-Cadherin+Endomucin+) and EC-depleted (ZsGreen−VE-Cadherin−Endomucin−) BM cells from tamoxifen-treated Cdh5-CreERT2/ZsGreen/PolyloxExpress mice (n = 3, 10-week-old at the time of tamoxifen treatment) were mixed (1:1), and encapsulated (147,446 cells loaded; 93,553 processed). Indexed cDNA was used for scRNA-seq and barcode detection by PacBio sequencing after nested PCR enrichment; barcode-transcriptome integration was accomplished via shared cell indices. (B) Uniform Manifold Approximation and Projection (UMAP) clustering and cell type annotation. Clusters 0, 1, 13, and 22 comprise ECs; cluster 14 comprises Mesenchymal-type cells. Heatmaps showing ‘true’ Polylox barcodes (pGen <1 × 10−6) linking hematopoietic stem and progenitor cells (HSPCs) to hematopoietic cells (C), ECs to hematopoietic and other cells (D), and Mesenchymal-type cells to other cells (E). The numbers within the colored boxes identify cell number; the labels at the bottom of each column denote the barcode shared by all cells in that column; the number on the right side of the heatmaps reflects the total number of cells in each row. (F) UpSet plot showing cells (identified by colored dots) sharing the same ‘true’ barcode (identified by lines connecting the colored dots); bar graph at the top of the plot reflects (height and number on each bar) the number of ‘true’ barcodes. Colors of dots: EC (red), Mesenchymal-type (orange), ECs connecting with Mesenchymal-type cells (blue), cells other than ECs and Mesenchymal-type cells (black). (G) Violin plots showing selected gene expression profile in Mesenchymal-type cells (cluster 14) and ECs (clusters 0, 1, 13, 22 combined).
Figure 5—figure supplement 1
Single-cell RNA-seq analysis of bone marrow (BM) ZsGreen+ cells from tamoxifen-treated Cdh5-Cre/ZsGreen/Polylox mice.
Related to Figure 5. (A) Uniform Manifold Approximation and Projection (UMAP) plot showing unsupervised clustering of sc RNA-seq data, identifying 34 distinct cell clusters within BM ZsGreen+ cells. (B) Histogram of doublet score distribution. A threshold of 0.2 was applied to match the expected doublet rate from 10x Genomics Chromium GEM-X chips. (C) Doublet score distribution (gray violin plots, left y-axis) and corresponding doublet percentages (blue bars, right y-axis) across Leiden clusters. (D) UMAP plots of clusters after doublet removal, and expression of ZsGreen1, Cdh5, Pecam1, Eng, CreERT2, and Ptprc (CD45). (E) Dot plot showing expression of selected marker genes across Leiden clusters identified in panel A. Dot size indicates the proportion of cells expressing the gene; color intensity reflects the average expression level of each cluster.
Figure 5—figure supplement 2
Identification and distribution of ‘True’ Polylox barcodes across cell types.
Related to Figure 5. (A) Bubble plot showing individual Polylox barcodes plotted against their corresponding pGen values (log10 scale). The y-axis indicates representative barcodes (one label is shown for every five barcodes). Bubble size reflects the number of cells harboring each barcode. The red dashed line denotes the log10(pGen) = −6 cutoff, which was used to define true barcodes. (B) Uniform Manifold Approximation and Projection (UMAP) plot showing the distribution of ‘True’ Polylox barcodes across the 34 Leiden-defined clusters identified in (A). Cells containing ‘True’ barcodes (n = 721) are shown in orange; cells with ‘Not True’ barcodes (n = 3348) are shown in blue. A total of 388 barcodes were detected, including 274 ‘True’ and 125 ‘Not True’. (C) Heatmap displaying the distribution and abundance of ‘True’ Polylox barcodes across annotated cell types. Each row corresponds to a unique barcode (1 out of every 5 barcodes shown); color intensity represents the number of cells carrying that barcode within each listed cell type. (D) Heatmap showing cell cycle phase distribution (G1, G2/M, and S) across Leiden clusters identified in (A). Color intensity and numerical values represent the percentage of cells in each phase within the indicated cell type.
Figure 6
Sc transcriptomic analysis of prospective hemogenic endothelial cells (ECs).
(A) Uniform Manifold Approximation and Projection (UMAP) clustering of 434,810 cells from eight public scRNA-seq datasets. (B) Dot plot showing relative Cdh5 and Runx1 co-expression across clusters; clusters 8 and 50 co-express both genes. (C) UMAP highlighting clusters 8 and 50; all other clusters shown in grey. (D) Violin plots of doublet scores across Leiden clusters. Clusters 50 and 8 show no evidence of doublet enrichment. (E) Datasets proportional contribution to clusters 50 and 8; each dataset is color-coded. (F) Dot plot showing expression of selected marker genes in clusters 50 and 8 (from the public sc RNA-seq datasets listed in Figure 7D) and from clusters 0, 1, 13, 22, and 14 (from Polylox scRNA-seq; Figure 5B). Results reflect mean expression and fraction of cells in group. (G) Cdh5, Runx1, and Col1a2 co-expression in the indicated clusters as a fraction of cells in the cluster. (H, I) t-SNE plot of ECs from 11 murine tissues (G) and Venn diagram (H) showing rare co-expression of Cdh5, Runx1, and Col1a2 in these tissues.
Figure 7 with 2 supplements
Contribution of Col1a2 and Runx1 expression to endothelial cells (ECs) hemogenic activity.
Percent EGFP+CD45+ cells in bone marrow (BM) and blood of tamoxifen-treated (n = 6) or oil-treated (n = 5) Col1a2-CreERT2/mTmG mice (A) and tamoxifen-treated (n = 4) or oil-treated (n = 3) Col1a2-CreERT2/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-CreERT2/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 Runx1EC-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 Runx1EC-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.
Figure 7—figure supplement 1
Characterization of Col1a2-tracked cell populations in bone marrow (BM) and blood.
Related to Figure 7. (A) Schematic representation of the Col1a2 tracking lines. (B) Representative confocal image of a BM section from a tamoxifen-treated Col1a2-CreERT2/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-CreERT2/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-CreERT2(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-CreERT2/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-CreERT2/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-CreERT2/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.
Figure 7—figure supplement 2
Analysis and hemogenic potential of Col1a2-tracked adult bone marrow (BM) endothelial cells (ECs).
Related to Figure 7. (A) Representative FACS gating strategy used to isolate BM hematopoietic cells, ECs, stromal cells, and Col1a2-tracked ECs from tamoxifen-treated Col1a2-CreERT2/ZsGreen mice. (B) Gene expression profiling of unsorted BM and sorted BM populations defined in (A). Results from qRT-PCR are normalized by Gapdh and unsorted BM. Dots reflect experimental triplicates. (C) Representative confocal image of a BM section from a transplant recipient showing a CD45+CD11b+ZsGreen+ cell (arrow), indicating hematopoietic derivation from transplanted ZsGreen+VE-Cadherin+CD45− (Col1a2+) cells. (D) Representative image of a blood smear from a WT recipient mouse transplanted with ZsGreen (Col1a2)+ BM ECs from Col1a2-CreERT2/ZsGreen mice. ZsGreen tracked cells are pointed by the arrows. (E) Schematic diagram of the Cdh5-CreERT2/ZsGreen/Runx1-Knock-in (Runx1EC-KI) mouse line used to trace ECs with Runx1 expression induced upon tamoxifen treatment.
Author response image 1
Expression of CreERT2, Cdh5, Ptprc and ZsGreen in BM cell populations enriched with ECs and hematopoietic cells.
The single-cell RNAseq results are derived from ZsGreen-enriched BM ECs and ZsGreen-enriched BM hematopoietic cells were derived from Polylox lineage-tracing experiments (data shown in Fig. 5; 37,667 ECs and 48,065 BM hematopoietic cells) and from LSKs (23,017 cells) independently isolated from tamoxifen-treated Cdh5-CreERT2/ZsGreen mice without ZsGreen enrichment (unpublished data).
Author response image 2
Expression of CreERT2, Cdh5, Ptprc, Pecam1, Emcn, ZsGreen1, Col1a2, Cd19, Cd3e, Itgam (CD11b), Ly6a (Sca-1), Kit(cKit), Cd34, Cd48, Slamf1 (CD150), and Siglech in enriched BM ECs and LSKs from Cdh5-CreERT2/ZsGreen mice treated with tamoxifen 4 weeks prior to harvest (same cell source as indicated in Author response image 1).
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | BD Horizon BV786 Rat Anti-Mouse CD117 (rat monoclonal, clone 2B8) | BD Biosciences | 564012; RRID:AB_2732005 | Flow cytometry 2 mg/107 cells |
| Antibody | Brilliant Violet 421 anti-mouse CD117 (c-Kit) Antibody (rat monoclonal, clone 2B8) | BioLegend | 105828; RRID:AB_11204256 | Flow cytometry 2 mg/107 cells |
| Antibody | Brilliant Violet 785 anti-mouse CD117 (c-Kit) Antibody (rat monoclonal, clone 2B8) | BioLegend | 105841; RRID:AB_2629799 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Pharmingen APC Rat Anti-CD11b (rat monoclonal, clone M1/70) | BD Biosciences | 553312; RRID:AB_398535 | Flow cytometry 2 mg/107 cells; immunostaining: 2 ng/ml |
| Antibody | PerCP/Cyanine5.5 anti-mouse CD150 (SLAM) Antibody (rat monoclonal, clone TC15-12F12.2) | BioLegend | 115922; RRID:AB_2303663 | Flow cytometry 2 mg/107 cells |
| Antibody | APC/Fire 750 anti-mouse CD150 (SLAM) Antibody (rat monoclonal, clone TC15-12F12.2) | BioLegend | 115940; RRID:AB_2629587 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Pharmingen APC-Cy7 Rat Anti-Mouse CD19 (rat monoclonal, clone 1D3) | BD Biosciences | 557655; RRID:AB_396770 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Horizon BUV737 Rat Anti-Mouse CD19 (rat monoclonal, clone 1D3) | BD Biosciences | 612781; RRID:AB_2870110 | Flow cytometry 2 mg/107 cells |
| Antibody | Brilliant Violet 510 anti-mouse CD3 Antibody (rat monoclonal, clone 17A2) | BioLegend | 100234; RRID:AB_2562555 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Pharmingen Purified Rat Anti-Mouse CD31 (rat monoclonal, clone 390) | BD Biosciences | 553370; RRID:AB_394816 | Flow cytometry 2 mg/107 cells |
| Antibody | BV421 anti-mouse Cd31 (rat monoclonal, clone 390) | BD Biosciences | 563356; RRID:AB_2738154 | Flow cytometry 2 mg/107 cells |
| Antibody | Brilliant Violet 421 anti-mouse CD31 Antibody (rat monoclonal, clone 390) | BioLegend | 102424; RRID:AB_2650892 | Flow cytometry 2 mg/107 cells |
| Antibody | Brilliant Violet 605 anti-mouse CD31 Antibody (rat monoclonal, clone 390) | BioLegend | 102427; RRID:AB_2563982 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Pharmingen Alexa Fluor 700 Rat Anti-Mouse CD45 (rat monoclonal, clone 30-F11) | BD Biosciences | 560510; RRID:AB_1645208 | Flow cytometry 2 mg/107 cells; immunostaining: 2 ng/ml |
| Antibody | BD OptiBuild BUV615 Rat Anti-Mouse CD45 (rat monoclonal, clone 30-F11) | BD Biosciences | 751170; RRID:AB_2875194 | Flow cytometry 2 mg/107 cells |
| Antibody | PE anti-mouse CD45 Antibody (rat monoclonal, clone 30-F11) | BioLegend | 103106; RRID:AB_312971 | Flow cytometry 2 mg/107 cells; immunostaining: 2 ng/ml |
| Antibody | PerCP/Cyanine5.5 anti-mouse CD45 Antibody (rat monoclonal, clone 30-F11) | BioLegend | 103131; RRID:AB_893344 103132; RRID:AB_893340 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Horizon BV510 Hamster Anti-Mouse CD48 (Armenian hamster monoclonal, clone HM48-1) | BD Biosciences | 563536; RRID:AB_2738266 | Flow cytometry 2 mg/107 cells |
| Antibody | APC/Cyanine7 anti-mouse CD48 Antibody (Armenian hamster monoclonal, clone HM48-1) | BioLegend | 103432; RRID:AB_2561463 | Flow cytometry 2 mg/107 cells |
| Antibody | Brilliant Violet 510 anti-mouse CD48 Antibody (Armenian hamster monoclonal, clone HM48-1) | BioLegend | 103443; RRID:AB_2650826 | Flow cytometry 2 mg/107 cells |
| Antibody | ‘Collagen I Polyclonal Antibody, Biotin’ (rabbit polyclonal) | Invitrogen | PA1-28530; RRID:AB_1956957 | Flow cytometry 2 mg/107 cells |
| Antibody | ‘Endomucin Monoclonal Antibody (eBioV.7C7 (V.7C7)), eFluor 660’ (rat monoclonal, clone eBioV.7C7) | Invitrogen | 50-5851-82; RRID:AB_11220465 | Flow cytometry 2 mg/107 cells; immunostaining: 2 ng/ml |
| Antibody | Anti-Endomucin Antibody (V.7C7) AF546 (rat monoclonal, clone V.7C7) | Santa Cruz Biotechnology | sc-65495 AF546; RRID:AB_2100037 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Pharmingen PE-Cy7 Rat Anti-Mouse Ly-6G (rat monoclonal, clone 1A8) | BD Biosciences | 560601; RRID:AB_1727562 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Horizon BUV395 Rat Anti-Mouse Ly-6G (rat monoclonal, clone 1A8) | BD Biosciences | 563978; RRID:AB_2716852 | Flow cytometry 2 mg/107 cells |
| Antibody | ‘BD Pharmingen APC Mouse Lineage Antibody Cocktail, with Isotype Control’ (rat/hamster monoclonal cocktail) | BD Biosciences | 558074; RRID:AB_1645213 | Flow cytometry 2 mg/107 cells |
| Antibody | ‘Goat anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594’ (goat polyclonal) | Invitrogen | A48264; RRID:AB_2896333 | Flow cytometry 2 mg/107 cells |
| Antibody | ‘RUNX1 Monoclonal Antibody (RXDMC), PE, eBioscience’ (rat monoclonal, clone RXDMC) | Invitrogen | 12-9816-80; RRID:AB_11151519 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Pharmingen PE-Cy7 Rat Anti-Mouse Ly-6A/E (rat monoclonal, clone D7) | BD Biosciences | 561021; RRID:AB_2034021 | Flow cytometry 2 mg/107 cells |
| Antibody | PE/Cy7 anti-mouse Ly-6A/E (Sca-1) (rat monoclonal, clone D7) | BioLegend | 108114; RRID:AB_493596 | Flow cytometry 2 mg/107 cells |
| Antibody | PerCP/Cyanine5.5 anti-mouse TER-119/Erythroid Cells Antibody (rat monoclonal, clone TER-119) | BioLegend | 116228; RRID:AB_893636 | Flow cytometry 2 mg/107 cells |
| Antibody | Brilliant Violet 605 anti-mouse TER-119/Erythroid Cells Antibody (rat monoclonal, clone TER-119) | BioLegend | 116239; RRID:AB_2562447 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Pharmingen Alexa Fluor 647 Rat Anti-Mouse CD144 (rat monoclonal, clone 11D4.1) | BD Biosciences | 562242; RRID:AB_2737608 | Flow cytometry 2 mg/107 cells |
| Antibody | BD Pharmingen PE Rat Anti-Mouse CD144 (rat monoclonal, clone 11D4.1) | BD Biosciences | 562243; RRID:AB_2737609 | Flow cytometry 2 mg/107 cells |
| Antibody | BUV737 Rat Anti-Mouse CD144 (rat monoclonal, clone 11D4.1) | BD Biosciences | 741792; RRID:AB_2871138 | Flow cytometry 2 mg/107 cells |
| Antibody | Alexa Fluor 647 anti-mouse CD144 (VE-cadherin) Antibody (rat monoclonal, clone BV13) | BioLegend | 138006; RRID:AB_10569114 | Flow cytometry 2 mg/107 cells |
| Antibody | BD OptiBuild RB780 Rat Anti-Mouse CD144 (rat monoclonal, clone 11D4.1) | BD Biosciences | 755945; RRID:AB_3683567 | Flow cytometry 2 mg/107 cells |
| Antibody | PE anti-mouse CD144 (VE-cadherin) Antibody (rat monoclonal, clone BV13) | BioLegend | 138010; RRID:AB_10641139 | Flow cytometry 2 mg/107 cells |
| Antibody | PE/Cyanine7 anti-mouse CD144 (VE-cadherin) Antibody (rat monoclonal, clone BV13) | BioLegend | 138015; RRID:AB_2562885 138016; RRID:AB_2562886 | Flow cytometry 2 mg/107 cells |
| Antibody | PE anti-mouse CD144 (VE-cadherin) Antibody (rat monoclonal, clone VECD1) | BioLegend | 138105; RRID:AB_2077941 | Flow cytometry 2 mg/107 cells |
| Chemical compound, drug | 5-FLUOROURACIL | Sigma-Aldrich | F6627 | |
| Chemical compound, drug | 7-AAD Viability Stain SOLN | Life Technologies Corp | 00-6993-50 | |
| Chemical compound, drug | 7-Aminoactinomycin D | Sigma-Aldrich | A9400 | |
| Chemical compound, drug | ACETONE | MALLINCKRODT | 2440 | |
| Chemical compound, drug | ACK lysing Buffer | Lonza | 10-548E | |
| Chemical compound, drug | Acrylamide/Bis 19:1, 40% (wt/vol) solution | Invitrogen | AM9024 | |
| Chemical compound, drug | Acrylamide/Bis-acrylamide 19:1 | Sigma-Aldrich | A2917 | |
| Chemical compound, drug | Acridine Orange/Propidium Iodide Stain | Logos Biosystems | F23001 | |
| Chemical compound, drug | AMPure beads | Beckman Coulter | A63881 | |
| Chemical compound, drug | Antibiotic-Antimycotic | Gibco | 15240062 | |
| Chemical compound, drug | Anti-Rat Ig, κ/Negative Control (BSA) Compensation Plus (7.5 µm) Particles Set | BD Biosciences | 560499 | |
| Chemical compound, drug | Anti-Rat Ig, κ/Negative Control Compensation Particles Set | BD Biosciences | 552844 | |
| Chemical compound, drug | APC/Fire 750 Streptavidin | BioLegend | 405250 | |
| Chemical compound, drug | Azide-Free Fc Receptor Blocker | INNOVEX | NB335-60 | |
| Chemical compound, drug | Bovine serum albumin solution | MP Biomedicals | 160069 | |
| Chemical compound, drug | CellROX Orange Flow Cytometry Assay Kit | Invitrogen | C10493 | |
| Chemical compound, drug | Collagenase Type 2 | Worthington Biochemical Corporation | LS004176 | |
| Chemical compound, drug | Dispase | Worthington Biomedical | LS02109 | |
| Chemical compound, drug | Deoxyribonuclease | Worthington Biomedical | LS006344 | |
| Chemical compound, drug | DAPI (4′,6-Diamidino-2-Phenylindole, Dilactate) | BioLegend | 422801 | |
| Chemical compound, drug | Dihydroethidium (Hydroethidine) | Invitrogen | D1168 | |
| Chemical compound, drug | Donkey serum | Sigma-Aldrich | D9663 | |
| Chemical compound, drug | DRAQ5 | Biostatus | DR50200 | |
| Chemical compound, drug | EDTA (0.5 M, pH 8.0) | KD Medical | RGF3130 | |
| Chemical compound, drug | ETHYL ALCOHOL (200 PROOF ANHYDROUS) | Warner-Graham Co | 201096 | |
| Chemical compound, drug | Ethylenediamine-N,N,N′,N′-tetra-2-propanol | Sigma-Aldrich | 8219401000 | |
| Chemical compound, drug | Fetal bovine serum | Millipore Sigma | F2442-500ML (Lot 24G002) | |
| Chemical compound, drug | Fc Receptor Blocker | Innovex | NB309-30 | |
| Chemical compound, drug | Formalin solution, neutral buffered, 10% | Sigma-Aldrich | HT501128-4L | |
| Chemical compound, drug | Gelatin | Sigma-Aldrich | G9391 | |
| Chemical compound, drug | Goldenrod Animal Lancet | Braintree Scientific Inc | GR-3MM | |
| Chemical compound, drug | HBSS | Gibco | 14025075 | |
| Chemical compound, drug | HEPES | Gibco | 15630080 | |
| Chemical compound, drug | Immu-Mount mountant | Epredia | 9990402 | |
| Chemical compound, drug | Isoflurane | Baxter | 10019036040 | |
| Chemical compound, drug | Mag-Bind TotalPure NGS | Omega Bio-tek | M1378-01 | |
| Chemical compound, drug | Methanol, HPLC Grade | Avantor | JT-9093-03 | |
| Chemical compound, drug | MojoSort Mouse CD45 Nanobeads | BioLegend | 480028 | |
| Chemical compound, drug | Kwik Stop Stypic Power | Miracle Care | SKU 423615 | |
| Chemical compound, drug | Lineage Cell Depletion Kit, mouse | MiltenyiBiotec | 130-090-858 | |
| Chemical compound, drug | Lipopolysaccharides | Sigma-Aldrich | L2880-25MG | |
| Chemical compound, drug | Microscope Slides | MATSUNAMI GLASS IND | SUMGP12 | |
| Chemical compound, drug | Neutral Protease, Partially Purified, Animal Free/AF | Worthington Biochemical Corporation | LS02109 | |
| Chemical compound, drug | Paraformaldehyde (formaldehyde) aqueous solution (20%) | Electron Microscopy Sciences | 15713-S | |
| Chemical compound, drug | Penicillin–streptomycin | Gibco | 15140-122 | |
| Chemical compound, drug | Peanut oil | Sigma-Aldrich | P2144 | |
| Chemical compound, drug | Phusion Green Hot Start II High-Fidelity PCR Master Mix | Thermo Scientific | F566L | |
| Chemical compound, drug | pHrodo Red E. coli BioParticles | Invitrogen | P35361 | |
| Chemical compound, drug | Polyvinylpyrrolidone | Sigma-Aldrich | P5288 | |
| Chemical compound, drug | Propidium iodide | Sigma-Aldrich | P4170 | |
| Chemical compound, drug | Q5 Hot Start High-Fidelity 2X Master Mix | NEB | M0494S | |
| Chemical compound, drug | Richard-Allan Scientific Cover glass | Epredia | 102424 | |
| Chemical compound, drug | RNeasy Micro Kit | QIAGEN | 74004 | |
| Chemical compound, drug | Saponin | Sigma-Aldrich | 47036 | |
| Chemical compound, drug | SPRIselect Beads | Beckman Coulter | B23318 | |
| Chemical compound, drug | Sucrose | Sigma-Aldrich | S8501 | |
| Chemical compound, drug | SuperScript IV First-Strand Synthesis System | Invitrogen | 18091050 | |
| Chemical compound, drug | Tamoxifen | Sigma-Aldrich | T5648 | |
| Chemical compound, drug | TaqMan Fast Advanced Master Mix | Thermo Fisher Scientific | 4444557 | |
| Chemical compound, drug | Thioglycollate Broth | Sigma-Aldrich | 70157 | |
| Chemical compound, drug | Tissue-Tek O.C.T. Compound | SAKURA | 4583 | |
| Chemical compound, drug | Triton X-100 | Sigma-Aldrich | T9284 | |
| Chemical compound, drug | Trypsin-EDTA (0.25%), phenol red | Gibco | 25200056 | |
| Chemical compound, drug | TruBond 380 Adhesion Slide | Electron Microscopy Sciences | 63700-Y10 | |
| Chemical compound, drug | UNI-TRIEVE | INNOVEX | NB325 | |
| Chemical compound, drug | VA-044 (Water soluble Azo initiators) | FUJIFILM Labchem Wako | VA-044 | |
| Chemical compound, drug | VECTASHIELD Vibrance Antifade Mounting Medium | Vector Laboratories | H-1700-10 | |
| Chemical compound, drug | Zombie Aqua Fixable Viability Kit | BioLegend | 423101 | |
| Chemical compound, drug | Zombie NIR Fixable Viability Kit | BioLegend | 423105 | |
| Chemical compound, drug | Zombie UV Fixable Viability Kit | BioLegend | 423107 | |
| Chemical compound, drug | Zombie Yellow Fixable Viability Kit | BioLegend | 423103 | |
| Chemical compound, drug | Zymosan A from Saccharomyces cerevisiae | Sigma-Aldrich | Z4250 | |
| Commercial assay or kit | Chromium GEM-X Single Cell 3′ Kit | 10× Genomics | 1000686 | |
| Commercial assay or kit | Chromium GEM-X Single Cell 3′ Chip Kit v4 | 10× Genomics | 1000690 | |
| Commercial assay or kit | Chromium Next GEM Chip G Single Cell Kit | 10× Genomics | 1000127 | |
| Commercial assay or kit | Chromium Next GEM Single Cell 3′ Kit v3.1 | 10× Genomics | 1000269 | |
| Commercial assay or kit | Dual Index Kit TT Set | 10× Genomics | 1000215 | |
| Commercial assay or kit | NextSeq 2000 P4 Reagents (100 Cycles) | Illumina | 20100994 | |
| Commercial assay or kit | NextSeq 2000 P3 Reagents (100 Cycles) | Illumina | 20040559 | |
| Other | Polylox PacBio long-read sequencing data | This study | PRJNA1079369 | Dataset accession associated with this study; eLife usually requests datasets in the submission metadata rather than in the Key Resources Table. See Materials and methods for details. |
| Other | Single-cell RNA-seq data | This study | PRJNA1079369 | Dataset accession associated with this study; eLife usually requests datasets in the submission metadata rather than in the Key Resources Table. See Materials and methods for details. |
| Cell line (Mus musculus) | OP9 cells | Dr. Nakano; same line deposited in ATCC | ATCC # CRL-2749; RRID:CVCL_4398 | Mouse bone marrow stromal cell line. |
| Genetic reagent (Mus musculus) | Cdh5-Cre(PAC)ERT2 | Drs. R. Adams and M. Boehm; also available from Taconic Biosciences | MGI:3848982 Taconic # 13073 | Mouse line used in this study. |
| Genetic reagent (Mus musculus) | Cdh5-Cre(BAC)ERT2 | Drs. Y. Kubota and Y. Mukoyama | MGI:5705396 | Mouse line used in this study. |
| Genetic reagent (Mus musculus) | Col1a2-CreERT | JAX, #029567 | MGI:3785760 | Mouse line used in this study. |
| Genetic reagent (Mus musculus) | ZsGreen (Ai6) | JAX, #007906 | MGI:3809522 | Mouse line used in this study. |
| Genetic reagent (Mus musculus) | mTmG | JAX, #007676 | MGI:3716464 | Mouse line used in this study. |
| Genetic reagent (Mus musculus) | PolyloxExpress | Drs. H. Rodewald and A. Bhandoola | MGI:6470648 | Mouse line used in this study. |
| Genetic reagent (Mus musculus) | Runx1 Knock-In | Drs. Q. Ma and N. Speck | MGI:7490340 | Mouse line used in this study. |
| 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 | |
| Sequence-based reagent | ZsGreen mouse strain genotyping primers (5′–3′) | AAG GGA GCT GCA GTG GAG TA CCG AAA ATC TGT GGG AAG TC GGC ATT AAA GCA GCG TAT CC AAC CAG AAG TGG CAC CTG AC | JAX: 007906 | |
| Sequence-based reagent | Cdh5-CreERT2(PAC) mouse strain genotyping primers (5′–3′) | TCC TGA TGG TGC CTA TCC TC CCT GTT TTG CAC GTT CAC CG CAC CCT GTT CTT TGC CTC CT | This study | |
| Sequence-based reagent | Cdh5-CreERT2(BAC) mouse strain genotyping primers (5′–3′) | ATA CCG GAG ATC ATG CAA GC ATG TGA ACC AGC TCC CTG TC CTA GGC CAC AGA ATT GAA AGA TCT GTA GGT GGA AAT TCT AGC ATC ATC C | JAX: Protocol 029211 | |
| Sequence-based reagent | Col1a2-CreERT mouse strain genotyping primers (5′–3′) | CAT GTC CAT CAG GTT CTT GC TGA AAA AGT CCA CTA ATT AAA ACC A CTA ACA ACC CTT TCT CTC AAG GT CAG GAG GTT TCG ACT AAG TTG G | JAX: Protocol 19893 | |
| Sequence-based reagent | Runx1 Knock-In mouse strain genotyping primers (5′–3′) | GAG TTC TCT GCT GCC TCC TGG CGA GGG CAG CCA TAG CAA CTC CGA GGC GGA TCA CAA GCA ATA | Qi et al., 2017 | |
| Sequence-based reagent | PolyloxExpress mouse strain genotyping primers (5′–3′) | AAG GGA GCT GCA GTG GAG TA TAA GCC TGC CCA GAA GAC TCC AAG ACC GCG AAG AGT TTG TCC | Pei et al., 2020 | |
| Software, algorithm | FlowJo v10 | BD | https://www.flowjo.com RRID:SCR_008520 | |
| Software, algorithm | GraphPad Prism 10 | Dotmatics | www.graphpad.com RRID:SCR_002798 | |
| Software, algorithm | Snakemake | Köster et al. | https://snakemake.github.io/ RRID:SCR_003475; v9.6.0 | |
| Software, algorithm | Lima | PacBio | https://lima.how/ RRID:SCR_025520 | |
| Software, algorithm | Iso-Seq | PacBio | https://isoseq.how/ RRID:SCR_025481 | |
| Software, algorithm | Minimap2 | Li, 2021 | https://github.com/lh3/minimap2 RRID:SCR_018550; Minimap2-2.28 (r1209) | |
| Software, algorithm | Cell Ranger | 10x Genomics | V8.0.1, RRID:SCR_017344 | |
| Software, algorithm | Polylox barcode recovery | This study; Zhao, 2026 | https://github.com/CCRSF-IFX/SF_Polylox-BC | Custom or study-specific computational resource used in this study. |
| Software, algorithm | pGen calculation | Pei et al., 2020; Rößler, 2017 | https://github.com/hoefer-lab/polylox | |
| Software, algorithm | Scanpy | Scanpy Community | https://scanpy.readthedocs.io RRID:SCR_018139 | |
| Software, algorithm | rapids-singlecell | Rapids-SingleCell | https://rapids-singlecell.readthedocs.io | |
| Software, algorithm | decoupler | Badia-I-Mompel et al., 2022 | https://decoupler-py.readthedocs.io | |
| Software, algorithm | scANVI | Gayoso et al., 2022 | https://github.com/scverse/scvi-tools; scvi-tools 1.4.0 | |
| Software, algorithm | CellTypist | Xu et al., 2023 | https://celltypist.readthedocs.io/ | |
| Other | Primaria dishes/flasks | Corning | 353808; 353810; 353846 | Tissue culture surface used for primary endothelial cell culture without OP9 cells. |
| Other | Regular Cell Culture Flask | Corning | 430641U |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/109553/elife-109553-mdarchecklist1-v1.docx
-
Supplementary file 1
Distribution of Polylox barcodes among bone marrow cell populations.
- https://cdn.elifesciences.org/articles/109553/elife-109553-supp1-v1.docx
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Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow
eLife 15:RP109553.
https://doi.org/10.7554/eLife.109553.3
