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–cre^ERT2: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.

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