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In situ mapping identifies distinct vascular niches for myelopoiesis

Abstract

In contrast to nearly all other tissues, the anatomy of cell differentiation in the bone marrow remains unknown. This is owing to a lack of strategies for examining myelopoiesis—the differentiation of myeloid progenitors into a large variety of innate immune cells—in situ in the bone marrow. Such strategies are required to understand differentiation and lineage-commitment decisions, and to define how spatial organizing cues inform tissue function. Here we develop approaches for imaging myelopoiesis in mice, and generate atlases showing the differentiation of granulocytes, monocytes and dendritic cells. The generation of granulocytes and dendritic cells–monocytes localizes to different blood-vessel structures known as sinusoids, and displays lineage-specific spatial and clonal architectures. Acute systemic infection with Listeria monocytogenes induces lineage-specific progenitor clusters to undergo increased self-renewal of progenitors, but the different lineages remain spatially separated. Monocyte–dendritic cell progenitors (MDPs) map with nonclassical monocytes and conventional dendritic cells; these localize to a subset of blood vessels expressing a major regulator of myelopoiesis, colony-stimulating factor 1 (CSF1, also known as M-CSF)1. Specific deletion of Csf1 in endothelium disrupts the architecture around MDPs and their localization to sinusoids. Subsequently, there are fewer MDPs and their ability to differentiate is reduced, leading to a loss of nonclassical monocytes and dendritic cells during both homeostasis and infection. These data indicate that local cues produced by distinct blood vessels are responsible for the spatial organization of definitive blood cell differentiation.

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Fig. 1: Stain development.
Fig. 2: Atlas of myelopoiesis.
Fig. 3: CSF1+ vessels organize myelopoiesis.
Fig. 4: Atlas of stress-induced myelopoiesis.

Data availability

Source data for quantifications described in the text or shown in graphs plotted in Figs. 14 and Extended Data Figs. 15, 79 are available with the manuscript. scRNAseq data shown in Fig. 3c and Extended Data Fig. 6 are reanalyses of published data sets: GSE128423 (ref. 27) and GSE108891 (ref. 26). These scRNAseq data underwent a preliminary annotation using the ICGS2 BioMarker database40, followed by a secondary analysis using the supervised classification tool cellHarmony40, comparing all cells to reference haematopoietic cells44 (GSE120409)44. Supplementary Table 2 shows the different ICGS2 marker genes and cellHarmony barcode assignments for the different cell clusters identified. Supplementary Table 1 lists all of the reagents and other resources used in the experiments described. Source data are provided with this paper.

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Acknowledgements

We thank J. Cancelas, M.-D. Filippi, D. Reynaud, D. Starczynowski and A. Hidalgo for feedback on the manuscript. We are grateful to L. G. Ng, I. Kwok and K. Leong for help in designing granulopoiesis experiments and reviewing the manuscript. We also thank the Confocal Imaging Core, the Research Flow Cytometry Core and the Veterinary Services at the University of Michigan and Cincinnati Children’s Medical Center for experimental and technical assistance. This work was partially supported by the National Heart Lung and Blood Institute (grants R01HL122661 to H.L.G. and R01HL136529 to D.L.). V.B.S.P. is supported by National Institutes of Health (NIH)/National Center for Advancing Translational Sciences (NCATS) grant U2CTR002818, NIH/National Heart, Lung and Blood Institute (NHLBI) grant U24HL148865, and NIH/National Institute of Allergy and Infectious Diseases (NIAID) grant U01AI150748. N.S. is supported by the Cincinnati Pediatric Cell Atlas Center. L.F.H. is supported by the Department of Defense (DoD) through a Peer Reviewed Cancer Research Program (PRCRP) award, W81XWH-20-1-0870(#CA191188). S.S.W. is supported by the NIH through grants R01AI120202, R01AI124657 and DP1AI131080 and by the Howard Hughes Medical Institute (HHMI) Faculty Scholar’s program, the March of Dimes Ohio Collaborative for Prematurity Research, and a Burroughs Wellcome Fund Investigator in Pathogenesis Award. J.X.J. is supported by NIH/National Institute of Aging (NIA) grant AG045040 and a Welch Foundation Grant, AQ-1507. A.S. is supported by grant T32 AI118697/AI/NIAID from the NIH Department of Health and Human Services (HHS) of the US. Data were generated using an SH800 cell sorter funded by NIH grant S10OD023410.

Author information

Affiliations

Authors

Contributions

D.L. conceptualized and managed the study. D.L., J.Z., H.L.G., N.S., J.D.E., J.M.K, A.D., S.S.W. and Q.W. designed experiments. J.Z. and Q.W. developed all of the stains required to analyse myelopoiesis in situ and performed most of the image analyses. C.B.J., A.S., M.M. and B.W. mapped haematopoietic cells for random simulations. G.P. and J.M.K. infected mice with L. monocytogenes. J.Z., Q.W. and A.O. performed FACS analyses. J.X.J generated the Csf1fl/fl mice. N.S., V.B.S.P. and L.F.H. performed bioinformatics analyses. D.L., J.Z. and Q.W. assembled the figures and wrote the manuscript, with editorial input from all authors.

Corresponding author

Correspondence to Daniel Lucas.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Jason Butler, Cristina Lo Celso and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Validation of stains to detect myeloid cells.

a, FACS plots showing the gating strategy used to identify MDPs and MOPs (as described in ref. 19). BM, bone marrow. b, Gating strategy used to identify GMPs, GPs and MOPs (as described in ref. 16). The lineage panel contains antibodies against Ly6G, CD11b, Ter119, B220 and CD3. c, Images showing that Ly6C labels arterioles, detected as either CD31+ CD144+ Sca1bright in wild-type mice or CD31+ CD144+ Nestinbright structures in Nestin–GFP mice; the histograms show quantifications demonstrating that all Sca1+ arterioles or Nestin–GFPbright arterioles are also Ly6C+. Scale bars, 50 μm. d, e, FACS plots (d) showing that only the CD11b+ gate contains CD117+ CD115+ or CD117+ Ly6C+ cells, and histogram (e) showing that GPs and MOPs are the only Ly6C+ cells in the Lin CD117+ gate. Together these data indicate that CD11b alone can be used to replace the lineage panel to exclude contamination of mature cells when detecting MDPs, MOPs and GPs. f, Cell numbers per femur detected by FACS of the indicated progenitors using previously described strategies16,19 (white) or that described in Fig. 1g (purple). g, Colony-forming activity (green, MDPs; orange, MOPs; red, GPs) of the indicated progenitors using previously described strategies16,19 (diamonds) or the one described in Fig. 1g (circles). For f, g, n = a total of three mice in two experiments. h, Frequency of total BM cells for each of the indicated populations in sternum when detected by FACS (white) or imaging (orange). n = 3. i, Representative image showing that CD11b CD117+ CD115+ Ly6C MDPs and CD11b CD117+ CD115+ Ly6C+ MOPs are GFP+ in Cx3cr1–gfp mice. Scale bar, 10 μm. j, qPCR results showing Gfi1 and Irf8 expression (relative to Gapdh expression) in FACS-purified GPs or MOPs; n = a total of six mice in three experiments. k, FACS analyses in Gfi1–tdTomato mice showing differential tdTomato expression in GPs and MOPs in Gfi1–tdTomato mice. l, Quantification of promyelocytes (PMs), myelocytes (MCs), metamyelocytes (MMs), banded cells (BCs) and polymorphonucleated neutrophils (PMNs) in cytospin preparations of FACS-purified PNs, INs and MNs. n = a total of two mice. Scale bar, 10 μm. m, The stains require discrimination of CD16/32, CD117 and Ly6G bright and dim cells. The panels show the gating strategy, experimental design, and quantification of frequencies of decanted CD16/32 and CD117 bright and dim cells or INs, PNs and MNs when compared with frequencies obtained by FACS before cytospinning. Each dot represents one image field from two experiments. n, o, Dendritic cells can be imaged as reticulated CX3CR1–GFP+ or CX3CR1–GFP+ MHC II+ cells in Cx3cr1–gfp reporter mice23,24. The images and histograms show that all reticulated GFP+ cells were also MHC II+ and CD11c+, indicating that MHC II expression and cell shape are sufficient to unambiguously identify dendritic cells and distinguish them from macrophages that are CXCR1–GFP CD11c cells24; n = a total of three mice. Scale bar, 10 μm. p, Image and histogram showing that CX3CR1–GFP+ MHCII+ dendritic cells are conventional dendritic cells, as they are CD11b+ but do not express B220 or CD8. Scale bar, 10 μm. n = a total of three mice. q, FACS gating strategy for isolation and imaging of the indicated cells. Dendritic cells are detected as MHCII+ reticulated cells in imaging analyses. In all bar graphs, one dot corresponds to one mouse. Statistical differences were calculated using two-tailed Student’s t-tests; P values are shown. Source data

Extended Data Fig. 2 Strategies to map myelopoiesis in whole mounted sternum.

a, Scheme showing the experimental pipeline to identify myeloid cells, obtain the X, Y and Z coordinates, and replace each myeloid cell with a colour-coded sphere centred on the cell to better visualize differentiation and to generate random distributions. Scale bar, 200 μm. b, Histograms showing the observed distribution of distances from each GMP (blue), MDP (green), MOP (orange) and GP (red) or random cell (white) to the closest indicated cell (n = 86 GMPs from 4 sternum sections of 4 mice; n = 243 MDPs from a total of 23 sternum sections of 15 mice; n = 458 MOPs from a total of 11 sternum sections of 11 mice; n = 338 GPs from a total of 15 sternum sections of 12 mice). c, Left, XY graphs showing the location of GPs, PNs and INs in mouse sternum sections; centre, the different colour-coded PN/IN clusters identified using the k-means algorithm; and right, PN/IN clusters containing (pink) or not containing (blue) GPs within the cluster. d, Number of PNs and INs in each type of cluster. n = 1,443 PNs from a total of 3 sternum sections from 3 mice in clusters with GPs; n = 1,050 PNs from a total 3 sternum sections from 3 mice in clusters without GPs; n = 880 INs from a total of 3 sternum sections from 3 mice in clusters with GPs; n = 866 PNs from a total of 3 sternum sections from 3 mice in clusters without GPs. e, Experimental design, representative image, and histogram showing the percentage of CFP-, GFP-, RFP- and YFP-positive cells in fate-mapping experiments using Ubc–creERT2:confetti mice. Scale bar, 10 μm. Each dot represents one sternum segment from a total of three confetti mice. TAM, tamoxifen. f, In the confetti model, GFP is detected in the nucleus whereas RFP is expressed in the cytoplasm21. The images show that by using antibodies conjugated to fluorochromes (Alexa Fluor488 and phycoerythrin (PE)) that spectrally overlap with GFP or RFP but that stain only the membrane, we could distinguish CD11b–Alexa488+ GFP, Ly6C–Alexa488+ GFP cells from GFP+ cells and CD115–PE+ RFP, and Ly6C–PE+ RFP from RFP+ cells. This allowed us to examine the relationships between YFP- or CFP-labelled cells. As we could not distinguish the membrane signal from the nuclear/cytoplasmic signal in GFP+ or RFP+ cells, these were discarded from the analyses. Scale bar, 10 μm. ‘α’ refers to an antibody against the indicated molecule. g, Percentage of PN clusters with at least one confetti-labelled PN that are oligoclonal (containing cells with at least two different origins: CFP+, YFP+, or no confetti label). Each dot represents one sternum segment from a total of three confetti mice. Statistical differences were calculated using two-tailed Student’s t-tests; P values are shown. Ob, observed; Rd, random. Source data

Extended Data Fig. 3 Confetti analyses of the differentiation of monocytes and dendritic cells.

a, Histograms showing the observed (colour) and random (white) distribution of distances from each MOP to the closest indicated cells (MOP–Ly6Chi monocyte, n = 137 MOPs from a total of 3 sternum sections from 3 mice; MOP–Ly6Clo monocyte, n = 171 MOPs from a total of 4 sternum sections from 4 mice; MOP–cDCs, n = 200 MOPs from a total of 5 sternum sections from 4 mice). b, Maps showing the location of Gfi1hi and Gfi1lo MOPs, MDPs, Ly6Chi monocytes and Ly6Clo monocytes in a sternum segment from Gfi1–tomato mice. Scale bar, 200 μm. c, Number of Gfi1hi and Gfi1lo MOPs per sternum segment. Each dot represents one sternum segment from three Gfi1–tomato mice in two experiments. d, Histograms showing the observed (colour) and random (white) distribution of distances from each Gfi1hi MOP (orange) and Gfi1loMOP (purple) to the closest indicated cells. n = 113 Gfi1hi MOPs and n = 72 Gfi1lo MOPs from a total of 3 sternum segments from 3 mice. e, Representative image showing the lack of contribution of a confetti-labelled MOP to surrounding monocytes. Tracked cells are YFP+, CFP+ or unlabelled CD117+ CD115+ CD11b Ly6C+ MOPs, CD117 CD115+ CD11b+ Ly6Chi monocytes and CD117 CD115+ CD11b+ Ly6Clo monocytes. Scale bar, 20 μm. f, Quantification of cell numbers for CFP (white) and CFP+ (blue) Ly6Chi monocytes (top) or Ly6Clo monocytes (bottom) found within the indicated distances to the closest CFP+ MOP cell. Each dot represents one CFP+ MOP (n = 9 MOPs from a total of 6 sternum segments from 4 confetti mice). g, h, Histograms showing the observed (colour) and random (white) distribution of distances from each Ly6Clo monocyte (yellow) and cDC (pink) to the six closest indicated neighbour cells. n = 1,603 Ly6Clo monocytes from a total of 3 sternum segments from 3 mice; n = 1,228 cDCs from a total of 6 sternum segments from 6 mice. i, Images showing the lack of contribution of a CFP+ MDP to surrounding Ly6Clo monocytes. Tracked cells are YFP+, CFP+ or unlabelled CD117+ CD115+ CD11b Ly6C MDPs, CD117+ CD115+ CD11b Ly6C+ MOPs, CD117 CD115+ CD11b+ Ly6Chi monocytes and CD117 CD115+ CD11b+ Ly6Clomonocytes. Scale bar, 20 μm. j, Images showing a lack of association between confetti-labelled Ly6Chi and Ly6Clo monocytes. Tracked cells are YFP+, CFP+ or unlabelled CD11b+ CD115+ CD117 Ly6Chi monocytes and CD11b+ CD115+ CD117 Ly6Clo monocytes. Scale bars for main and zoomed-in images, 40 μm and 10 μm respectively. The histograms show the observed (colour) and random (white) distribution of distances from each confetti-labelled Ly6Chi (blue) or Ly6Clo (yellow) monocytes to the closest Ly6Chi or Ly6Clo monocyte in the same colour. n = 48 confetti-labelled Ly6Chi monocytes, and n = 32 confetti-labelled Ly6Clo monocytes, from a total of 3 sternum sections from 3 mice. k, Images showing a lack of association between confetti labelled cDCs. Tracked cells are RFP+, GFP+, YFP+, CFP+ or unlabelled MCH II+ reticulated cDCs. Scale bars for main and zoomed-in images, 40 μm and 10 μm respectively. The histogram shows the observed (colour) and random (white) distribution of distances from each confetti-labelled cDC (pink) to the closest cDC in the same colour. n = 80 confetti-labelled cDCs from a total of 3 sternum sections from 3 mice. Unless otherwise indicated, for all graphs one dot corresponds to one cell. Horizontal blue bars indicate the median distance. Statistical differences were calculated using two-tailed Student’s t-tests; P values are shown. Source data

Extended Data Fig. 4 The architecture of myelopoiesis is similar in different sternum sections.

a, Observed distributions of distances for individual sternum sections (Ob), pooled values, or random (Rd) simulations for the data shown in Extended Data Fig. 2b. For distance to GMPs, n = 25, 22, 19, 20 GMPs per sternum section from a total of 4 sternum sections in 4 mice; n = 11, 12, 11, 10 MDPs per sternum section from a total of 4 sternum sections in 4 mice; n = 40, 37, 42, 46 MOPs per sternum section from a total of 4 sternum sections in 4 mice; n = 19, 21, 16, 26 GPs per sternum section from a total of 4 sternum sections in 4 mice. For distance to MDPs, n = 25, 22, 19, 20 GMPs per sternum section from a total of 4 sternum sections in 4 mice; n = 13, 14, 11, 9, 7, 14, 14, 14, 9, 11, 12, 10, 10, 11, 13, 11 MDPs per sternum section from a total of 16 sternum sections in 16 mice; n = 40, 37, 42, 46 MOPs per sternum section from a total of 4 sternum sections in 4 mice; n = 22, 24, 23, 28, 23, 16, 24, 18, 22, 21, 17, 26, 25, 20 GPs per sternum section from a total of 14 sternum sections in 14 mice. For distance to MOPs, n = 25, 22, 19, 20 GMPs per sternum section from a total of 4 sternum sections in 4 mice; n = 9, 11, 12, 10, 11, 13, 11, 10, 14, 11, 13, 10, 9, 11, 10, 14 MDPs per sternum section from a total of 16 sternum sections in 16 mice; n = 39, 49, 38, 33, 44, 36, 37, 44 MOPs per sternum section from a total of 8 sternum sections in 8 mice; n = 22, 24, 28, 23, 16, 24, 18, 22, 21, 17, 26, 25, 20 GPs per sternum section from a total of 13 sternum sections in 13 mice. For distance to GPs, n = 25, 22, 19, 20 GMPs per sternum section from a total of 4 sternum sections in 4 mice; n = 15, 12, 11, 10, 15, 9, 12, 11, 12, 11, 13, 10, 9, 11, 10, 15 MDPs per sternum section from a total of 16 sternum sections in 16 mice; n = 40, 37, 42, 46, 39, 39, 49, 53, 38, 34 MOPs per sternum section from a total of 10 sternum sections in 10 mice; n = 24, 23, 23, 28, 29, 22, 26, 17, 26 GPs per sternum section from a total of 9 sternum sections in 9 mice. b, c, As for a, but corresponding to the data shown in Fig. 2c, i. n = 24, 23, 28 GPs per sternum section from a total of 3 sternum sections in 3 mice. For MDPs to Ly6Chi and Ly6Clo monocytes, n = 11, 14, 8, 9, 9, 16 MDPs per sternum section from a total of 6 sternum sections in 4 mice. For MDPs to cDCs, n = 13, 14, 12, 9, 16, 15, 15, 10, 10, 10, 15 MDPs per sternum section from a total of 11 sternum sections in 6 mice. d, e, Throughout the paper, we have indistinctly used coronal and sagittal sternum sections. The maps (d) show granulopoiesis in coronal and sagittal sternum sections shown or analysed in Fig. 2a or Fig. 2c; the histograms show (e) observed and random distributions of distances for the indicated cells in each section or the pooled data for each type of section. n = 23 and 28 GPs in coronal sections and n = 24 and 23 GPs in sagittal sections. Scale bar, 200 μm. f, g, Representative maps showing the differentiation of monocytes and dendritic cells (f), comparing coronal and sagittal sternum sections with those shown or analysed in Fig. 2a, i; and observed and random distributions (g) of distances for the indicated cells in coronal or sagittal sections. n = 11 or 10 MDPs, n = 390 or 334 Ly6Clo monocytes, and n = 218 or 258 cDCs in coronal sections; n = 9 or 10 MDPs, n = 419 or 380 Ly6Clo monocytes, and n = 183 or 159 cDCs in sagittal sections. Scale bar, 200 μm. Statistical differences were calculated using two-tailed Student’s t-tests ; P values are shown. Source data

Extended Data Fig. 5 Interaction of myeloid progenitors with the microenvironment and with HSCs.

a, Representative images showing the simultaneous detection of PNs, INs, Ly6Clo monocytes and cDCs. Scale bar, 10 μm. b, Map showing the location of the indicated cells in the bone marrow. Each dot corresponds to one cell. Note that the radius of each dot is two times the average radius of the cell. Scale bar, 200 μm. c, Histograms showing the distance from each Ly6Clo monocyte (yellow dots) or cDC (pink dots) and their random simulation (white dots) to the closest indicated cell (Ly6Clo to PN/IN, n = 500 Ly6Clo monocytes; cDC to PN/IN, n = 727 cDCs; Ly6Clo to cDC, n = 1,322 Ly6Clo monocytes; from a total of 3 sternum sections from 3 mice). d, High-power images showing the relative positions of MDPs, MOPs, GPs and sinusoids. Scale bar, 10 μm. e, Histograms showing the distance from each MDP (green dots), MOP (orange dots), GP (red dots) or random distribution (white dots) to the closest indicated structure (for distances to arterioles, n = 62 MDPs from a total of 6 sternum sections of 6 mice; n = 218 MOPs and n = 114 GPs from a total of 5 sternum sections from 5 mice; for distances to endosteal surface, n = 98 MDPs, n = 410 MOPs, n = 217 GPs, from a total of 9 sterna from 6 mice). f, Representative images of multiple sternum segments, showing that MDPs are evenly distributed through the bone marrow, consistent with their sinusoidal location. Scale bars, 200 μm. g, Representative images showing the detection of HSCs and MDPs in a single stain. Scale bar, 10 μm. h, Quantification of MDP and Lin CD117+ CD48 CD41dim CD150+ HSCs in femurs by FACS (white) or imaging (orange) analyses. Each dot corresponds to one mouse femur or sternum image. n = 4. i, Representative images showing detection of HSCs and a population containing CD117+ Ly6C+ GPs and MOP in a single stain. Scale bar, 10 μm. j, Maps and histograms showing the relationships between HSCs and MDPs or GPs/MOPs in the bone marrow. In the map, the dot radius is three times the average cell radius. Scale bar, 200 μm. (n = 35 MDPs from a total of 4 sternum sections from 3 mice, and n = 191 GPs and MOPs from a total of 3 sternum sections from 3 mice.) Unless otherwise indicated, for all graphs one dot corresponds to one cell. Horizontal blue bars indicate the median distance. Statistical differences were calculated using two-tailed Student’s t-test; P values are shown. Source data

Extended Data Fig. 6 Broad comparison of stromal bone marrow compartments by scRNAseq.

Comparative analyses of two previously described scRNAseq data sets profiling stromal and haematopoietic cell populations in bone marrow (9,165 cells from ref. 26 and 89,007 cells from ref. 27). ac, Predictions of cell populations displayed on a UMAP plot from an unsupervised analysis of the two separate scRNAseq data sets (using ICGS version 2). Distinct captures are denoted by the gating strategy (col2.3, niche-col2.3, niche-LepR+ and niche-VEcad+). Populations are denoted as haematopoietic or stromal/mesenchymal on the basis of previously defined signatures of marker genes from scRNAseq cell populations (ICGS; see cluster labels in each panel). dl, Relative expression of marker genes from ICGS2-identified cell populations (identified in both data sets), projected onto the two UMAP plots to verify cell identity (gene expression is shown relative to capture strategy). Supplementary Table 2 shows the different ICGS2 marker genes and cellHarmony barcode assignments for the different cell clusters identified.

Extended Data Fig. 7 CSF1 from LepR+ cells is dispensable for myelopoiesis.

a, qPCR analyses showing Csf1 mRNA levels (relative to Gapdh) in Nestin–GFPdim perivascular cells (which largely overlap with LepR+ perivascular cells45). n = a total of four control mice and n = total of four Csf1ΔLepR mice in four experiments. b, c, Number of bone marrow cells or the indicated cell populations in the femur of control or Csf1ΔLepR mice. n = a total of six control and n = a total of eight Csf1ΔLepR mice in six experiments. d, Colony-forming activity (blue, GMPs; green, MDP; orange, MOPs; red, GPs) of the indicated progenitors, FACS-purified from control (diamonds, n = 5) or Csf1ΔLepR (circles, n = 5) mice in 4 experiments. e, Number of the indicated populations in the blood of control or Csf1ΔLepR mice. In all panels, one dot equals one mouse. n = a total of six control and n = a total of eight Csf1ΔLepR mice in six experiments. Statistical differences were calculated using two-tailed Student’s t-tests; P values are shown. Source data

Extended Data Fig. 8 CSF1 from a subset of endothelial cells is necessary for the generation of dendritic cells.

a, qPCR analyses showing Csf1 mRNA levels (normalized to endothelial control, Ctrl) in FACS-purified endothelial cells and the indicated haematopoietic cells in control or Csf1ΔEC mice; n = a total of four control and n = a total of four Csf1ΔEC mice in four experiments. Note that although Cdh5–cre also recombines in subsets of haematopoietic cells, it does not affect Csf1 expression in these cells. (n.d., none detected; n = a total of three control and n = a total of three Csf1ΔEC mice in three experiments.) EOS, eosinophil; Ery, erythrocyte. b, Colony-forming activity (blue, GMPs; green, MDPs; orange, MOP; red, GPs) of the indicated progenitors FACS-purified from control (diamonds, n = 5) or Csf1ΔEC (circles, n = 5) mice in four experiments. c, d, Number (c) and CFU-M activity (d) of GMPs or MOPs from control or Csf1ΔEC mice. n = a total of six control and n = a total of six Csf1ΔEC mice in five experiments. e, Number of Ly6Chi and Ly6Clo monocytes in the bone marrow and peripheral blood of control or Csf1ΔEC mice. n = a total of eight control and n = a total of eight Csf1ΔEC mice in six experiments. fh, Number of bone marrow cells from the indicated populations in the femur or blood of control or Csf1ΔEC mice. n = a total of eight control and n = a total of eight Csf1ΔEC mice in six experiments. i, Volume and number of vessels in sternum sections of control or Csf1ΔEC mice. Each dot represents one sternum segment, from three control and three Csf1ΔEC mice. j, Percentages of the indicated CD45.2+ cells in the blood of lethally irradiated CD45.1+ recipients after transplant of 106 bone marrow cells purified from control (white dots) or Csf1ΔEC (red dots) mice—both CD45.2+—together with 106 CD45.1+ competitor cells at the indicated time points after transplantation. Shown are means ± s.d. from 12 mice per group. k, Representative images showing anti-CSF1 or isotype control stains in the bone marrow of wild-type mice. Scale bars, 200 μm and 10 μm. l, Map of CSF1+ and CSF1 vessels and cDCs in Csf1ΔEC mice. Scale bar, 200 μm. m, High-power images showing a CSF1+ vessel and cDCs (pink dots) in control mice. The radius of the dot is two times the average cDC radius. Scale bar, 20 μm. n, Number of cDCs found within the indicated distances of CSF1+ and CSF1 vessels in wild-type (n = 76 CSF1+ vessels and n = 520 CSF1 vessels in a total of 4 sternum sections from 3 wild-type mice). o, Histograms showing the distance from each cDC to the closest sinusoid in control or Csf1ΔEC mice (n = 451 cDCs in a total of 2 sternum sections from 2 control mice; n = 343 cDCs in a total of 3 sternum sections from 3 Csf1ΔEC mice). p, Maps showing the relocation of MDPs away from sinusoids in Csf1ΔEC mice. Scale bars, 200 μm and 10 μm. The radius of the dots is three times (left) or one times (right) the average radius of the MDP. q, Maps showing the distribution of MDPs, Ly6Clo monocytes and cDCs in the sternum of control or Csf1ΔEC mice. Scale bar, 200 μm. The radius of the dot is three times (for MDPs) or two times (for all other cells) the average radius of the replaced cell. r, Histograms showing the distribution of distances from each MDP to the six closest Ly6Clo monocytes or cDCs in control or Csf1ΔEC mice. (For MDPs to Ly6Clo monocytes, n = 37 MDPs from a total of 4 sternum sections from 3 control mice; n = 18 MDPs from a total of 4 sternum sections from 3 Csf1ΔEC mice. For MDPs to cDCs, n = 47 MDPs from a total of 6 sternum sections from 3 control mice; n = 47 MDPs from a total of 9 sternum sections from 3 Csf1ΔEC mice.) Unless otherwise indicated, for ai each dot corresponds to one mouse; for lr each dot corresponds to one cell. Statistical differences were calculated using two-tailed Student’s t-tests; P values are shown. Source data

Extended Data Fig. 9 Changes in progenitor localization after infection.

a, Average percentage of the indicated cells per femur (normalized to numbers at day 0) at the indicated time points after infection of wild-type mice with L. monocytogenes. (n = 6 mice for days 0 and 2; n = 3 mice for day 4; n = 4 mice for day 6; n = 3 mice for day 8.) b, Histograms showing the distribution of distances from each MDP (green, n = 243 MDP from a total of 23 sternum sections from 15 uninfected wild-type mice and n = 37 MDPs from a total of 3 sternum sections from 3 wild-type mice 4 days after infection with L. monocytogenes), MOP (orange, n = 458 MOP from a total of 11 sternum sections from 11 uninfected wild-type mice and n = 377 MOPs from a total of 3 sternum sections from 3 wild-type mice 4 days after infection with L. monocytogenes) or GP (red, n = 338 GPs from a total of 15 sternum sections from 12 uninfected wild-type mice and n = 218 GPs from a total of 3 sternum sections from 3 wild-type mice 4 days after infection with L. monocytogenes) to the indicated progenitors 4 days after infection of wild-type mice with L. monocytogenes. c, Number of GPs or MOPs per cluster for the sternum sections analysed in b. Each dot represents a cluster; n = 17 GP clusters and n = 15 MOP clusters from 3 wild-type mice 4 days after infection with L. monocytogenes. d, Experimental design for in vivo fate mapping using Ubc–creERT2:confetti mice. e, Percentage of GP or MOP clusters with at least one confetti-labelled GP or MOP that are monoclonal (all cells in the cluster are labelled in the same confetti colour) or oligoclonal (containing cells with at least two different origins: CFP+, YFP+, or no confetti label). Each dot represents a GP or MOP cluster from a total of three sternum segments from two confetti mice in two experiments four days after infection with L. monocytogenes. f, Representative images showing a cluster composed of MDP-derived Gfi1lo MOPs and GMP-derived Gfi1hi MOPs in Gfi1–tomato mice four days after infection with L. monocytogenes. Scale bar, 10 μm. g, Percentage of GMP-derived Gfi1hi MOPs (orange dots) or MDP-derived Gfi1lo MOPs (purple dots) per cluster (each dot represents one cluster; n = 15 clusters in a total of 3 sternum sections from 2 Gfi1–tomato mice 4 days after infection with L. monocytogenes). h, Quantification of CD117 expression in PNs of wild-type mice at the indicated time points after infection. (n = 6 mice for day 0; n = 8 mice for days 2 and 4; n = 5 mice for day 6; n = 4 mice for day 8 in total 8 experiments). One dot indicates one mouse. i, Map showing the location of Gfi1hi and Gfi1lo MOPs, MDPs, and Ly6Chi and Ly6Clo monocytes in a sternum segment from Gfi1–tomato mice four days after infection. Scale bar, 200 μm. j, Histograms showing the distribution of distances from each Gfi1hi (orange) and Gfi1lo (purple) MOP and MDP (green) to the indicated cells in uninfected wild-type mice or four days after infection with L. monocytogenes. The values for day 0 (d0) are the same as shown in Fig. 2g, i. (n = 113 Gfi1hi MOP and n = 72 Gfi1lo MOPs from a total of 3 sternum sections from 3 uninfected Gfi1-tomato mice; n = 155 Gfi1hi MOP and n = 144 Gfi1lo MOP from total 3 sternum sections of 2 Gfi1–tomato mice 4 days after infection with L. monocytogenes; MDPto Ly6Chi monocyte, n = 67 MDPs from a total of 6 sternum sections from 4 mice; MDP to Ly6Clo monocyte, n = 67 MDPs from a total of 6 sternum sections from 4 wild-type uninfected mice; MDP to cDC, n = 139 MDPs from a total of 11 sternum sections from 6 wild-type uninfected mice; and n = 32 MDPs from a total of 3 sternum sections from 3 wild-type mice 4 days after infection with L. monocytogenes.) k, Representative image showing lack of contribution of a confetti-labelled MOP. Tracked cells are YFP+, CFP+ or unlabelled CD117+ CD115+ CD11b Ly6C+ MOPs, CD117 CD115+ CD11b+ Ly6Chi monocytes and CD117 CD115+ CD11b+ Ly6Clo monocytes. Scale bar, 10 μm. l, Quantification of cell numbers for CFP (white) and CFP+ (blue) Ly6Chi monocytes (left) or Ly6Clo monocytes (right) found within the indicated distances to the closest CFP+ MOP cell in confetti mice four days after infection with L. monocytogenes. Each dot represents one CFP+ MOP from a total of eight sternum segments from two confetti mice in two experiments four days after infection. m, Representative images showing the lack of contribution of a confetti-labelled MDP to surrounding monocytes. Tracked cells are YFP+, CFP+ or unlabelled CD117+ CD115+ CD11b Ly6C MDPs, CD117+ CD115+ CD11b Ly6C+ MOPs, CD117 CD115+ CD11b+ Ly6Chi monocytes and CD117 CD115+ CD11b+ Ly6Clo monocytes. Scale bar, 20 μm. n, qPCR analyses showing Csf1 mRNA levels (normalized to not infected) in bone marrow endothelial cells FACS-purified from wild-type mice in the steady-state or four days after infection. n = a total of three uninfected mice and n = a total of three infected mice in two experiments. o, Histogram showing the distance from each MDP to the closest sinusoid in control (pool of Cre:Csf1+/−, Csf1+/− and Csf1fl/-) or Csf1ΔEC mice four days after infection. n = 58 MDPs from a total of 4 sternum sections from 3 control mice and n = 36 MDPs from a total of 4 sternum sections from 3 Csf1ΔEC mice. p, q, Maps (p) showing the location of the indicated cells; and histogram (q) showing the distance from each MDP to the closest Ly6Clo monocyte and cDC in control or Csf1ΔEC mice four days after infection. n = 51 MDPs from a total of 3 sternum sections of 3 control mice and n = 29 MDP from total 3 sternum sections of 3 Csf1ΔEC mice. r, Number of the indicated cells per femur in control or Csf1ΔEC mice 4 days after infection. Each dot indicates one mouse. n = 3 control and n = 3 Csf1ΔEC mice. Unless otherwise indicated, one dot represents one cell. Statistical differences were calculated using two-tailed Student’s t-tests; P values are shown. Source data

Extended Data Fig. 10 Architecture of myelopoiesis at steady state and after infection.

The models show the spatial distributions of, and average distances between, the indicated cells at steady state and four days after infection with L. monocytogenes.

Supplementary information

Supplementary Figure 1

FACS gating strategies for quantification of: a, CDP in Fig. 3d, Fig. 4g, and ED Fig. 9a; b, Pre-DC in Fig. 3d, Fig. 4g, and ED Fig. 9a; c, cDC1 and cDC2 in Fig. 3d, Fig. 4a, and Fig. 4g; d, HSC, MPP, CMP in ED Fig. 7c and ED Fig. 8g; e, B and T cells in ED Fig. 7b, ED Fig. 7e, ED Fig. 8h and ED Fig. 8j; and f, macrophages in ED Fig. 7b and ED Fig. 8h.

Reporting Summary

Supplementary Table 1

List of antibodies, mouse models, instruments, software and other reagents used in the study.

Supplementary Table 2

Table showing the different ICGS2 marker genes and Cell barcode assignments for the difference cell clusters identified from the reanalyses of 19 independent 10x Genomics captures (GSE12842327). MarkerFinder cell-population specificity scores (Pearson correlation coefficient rho values) are given for each gene, indicating its specificity within the assigned population (AltAnalyze).

Video 1

Representative video showing detection of all fluorescent channels to a depth of 35μm.

Video 2

Representative video showing how we identified and annotated the different cell populations. Bone marrow cells were stained with CD11b (blue), CD115 (red), CD117(magenta), Ly6C (green), and Ly6G (white). MOP were identified as CD11b-CD117+CD115+Ly6C+ cells; Ly6Chi Mo were identified as CD11b+CD117-CD115+Ly6Chi cells; Ly6Clo Mo were identified as CD11b+CD117-CD115+Ly6Clo cells; MDP were identified as CD11b-CD117+CD115+Ly6C- cells; GP were identified as CD11b-CD117+CD115-Ly6C+ cells; Pre neutrophil (PN) were identified as CD11b+CD117dimCD115-Ly6Glo cells;  Immature neutrophil (IN) were identified as CD11b+CD117dimCD115-Ly6Ghi cells; and Mature neutrophil (MN) were identified as CD11b+CD117-CD115-Ly6Ghi cells.

Video 3

Representative video showing the spatial distribution of GMP, MDP, MOP, and GP in a mouse sternum. The position of each progenitor is indicated by a color-coded dot. Note that the radius of each dot is 3x the average radius of the replaced cell.

Video 4

Representative video showing the spatial distribution of GP, pre-neutrophils (PN), immature neutrophils (IN), and mature neutrophils (MN) in a mouse sternum. The position of each type of cell is indicated by a color-coded dot. The radius of each dot is 3x the radius of GP, 2x the radius of PN and In, and 1.5x the radius of MN.

Video 5

Representative video showing neutrophil differentiation around a GP. The position of each type of cell is indicated by a color-coded dot. The radius of each dot matches the radius of each cell.

Video 6

Representative video showing the spatial distribution of MDP, MOP, Ly6Chi monocyte, Ly6Clo monocyte, and cDC in a mouse sternum. The position of each type of cell is indicated by a color-coded dot. The radius of each dot is 3x the radius of MDP and MOP and 2x the radius of all other cells.

Video 7

Representative video showing Ly6Clo Mo and cDC localization near MDP. The position of each type of cell is indicated by a color-coded dot. The radius of each dot matches the radius of each cell.

Video 8

Representative video showing the interaction between MDP and sinusoids. The position of the MDP is denoted by a green dot, and the surface of the sinusoid was digitally reconstructed based on the signals for CD31 and CD144.

Video 9

Representative video showing the interaction between MOP and sinusoids. The position of the MOP is denoted by an orange dot, and the surface of the sinusoid was digitally reconstructed based on the signals for CD31 and CD144.

Video 10

Representative video showing the interaction between GP and sinusoids. The position of the GP is denoted by a red dot, and the surface of the sinusoid was digitally reconstructed based on the signals for CD31 and CD144.

Video 11

Representative video showing the interaction between cDC and CSF1+ sinusoids. Bone marrow cells were stained with antibodies against CD31 and CD144 (white), CSF1 (green), and MHC II (magenta). The position of each cDC is denoted by a magenta dot; the surface of the sinusoid was digitally reconstructed based on the signals for CD31 and CD144.  CSF1- vessels are shown in blue and CSF1+ vessels in yellow.

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Zhang, J., Wu, Q., Johnson, C.B. et al. In situ mapping identifies distinct vascular niches for myelopoiesis. Nature 590, 457–462 (2021). https://doi.org/10.1038/s41586-021-03201-2

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