Nature Immunology: doi: /ni.3412

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2 Supplementary Figure 1 Gata1 expression in heamatopoietic stem and progenitor populations. (a) Unsupervised clustering according to 100 top variable genes across single pre-gm cells. The two main cell clusters are indicated. (b) List of genes differentially expressed between clusters A and B in (a). Gene IDs, P-values and false discovery rate (FDR) q-values are indicated. Genes were included if both P<0.001 and q<0.05. (c) Structure of the Gata1-EGFP transgene (not to scale). The Gata1 genomic locus was contained within the RP23-443E19 BAC. (d-e) Flow cytometry profiles and gating strategies for identification of pre-gm, GMP, pre-meg-e, MkP, pre-cfu-e and CFU- E progenitors (d) and LSKCD150 + Flt3 and LSKFlt3 hi cells (e).

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4 Supplementary Figure 2 Defining HSCs and LMPPs based on Gata1. (a-d) 22 weeks in vivo peripheral blood reconstitution after transplantation of 100 FACS purified GE - or GE + LSKCD150 + CD48 cells together with 250,000 congenic whole BM cells. Values show reconstitution of the compartment analyzed (mean ±SD); n=3 recipients/population in one experiment. WBC; white blood cells. (e) For evaluation of megakaryocyte (Mk) potential, LSKFlt3 hi GE - and GE + cells were manually plated at 1 cell per well into 60 well Terasaki plates and cultured for 8 or 12 days. Megakaryocytes were evaluated using an inverted microscope. Results are from 2 experiments, shown as percentage of colonies containing Mk cells of the total number of colonies formed. n=240 cells/population in 2 experiments. (f) Histogram plot showing expression of Flt3 and Gata1 in single sorted GE - (left) and GE + (right) pre-gms. Expression is shown as - (Ct(gene)-Ct(Kit)). (g) Combinations of one GE - pre-gm and three GE + pre-gms were cultured for 8 days. Single CD48 - HSCs were cultured for 25 days. Myeloid lineage potential readout of the cultures was based on morphology analysis after cytospins and MGG stain of the cultures. Number of cultures analyzed in indicated. Mo: monocytes, PMN: polymorphonuclear cells, Ma: mast cell. (h) Representative morphology of cells from cultures described in (g). Monocytes (Mo), polymorphonuclear granulocytes (PMN), and mast cells (Ma) are indicated. Scale bars: 25μm.

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6 Supplementary Figure 3 Gene expression of vitro differentiated cell populations. (a) Schematic overview of eosinophil favoring culture conditions used. (b) Morphology of peritoneal eosinophils (left panel) and peripheral blood neutrophils (right panel) purified by cell sorting after cytospin and May-Grünwald-Giemsa staining. Note the larger size and similar, but less condensed, nuclear morphology of eosinophils, compared to neutrophils. (c-f) Neutrophils (Ne), eosinophils (Eo), monocytes (Mo) and mast cells (Ma) from day 8 cultured pre-gm and GMPs as indicated were sorted based on markers as in Figure 4a. Their gene expression was analyzed by quantitative-pcr for genes associated with mast cells (c, red), neutrophils (d, yellow), eosinophils (e, purple) and Gata1/Gata2 (f, orange). Gene expression is presented relative to Hprt expression (mean ±SD) (n=2 biological replicates).

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8 Supplementary Figure 4 In vitro differentiation of hematopoietic progenitors. (a) Schematic overview of culture conditions used. Indicated progenitor populations were cultured for 3 days with mscf and mil-3, allowing the cells to reach a GMP stage, as defined by FcγRII/III expression. (b) Morphological analysis of EGFP-immunostained cytospins from cultures of the indicated in vitro generated EGFP - and EGFP + FcγRII/III + cells after manually plating at a density of 5 cells/well and cultured for 8 days. Cell types are shown as a percentage of total number of cultures analyzed (numbers above bars). (c) Flow cytometry analysis demonstrate expression of CD55, Ly6C, integrin beta 7 (Itgb7) and interleukin 1 receptor-like 1 (Il1rl1) with Gata1-EGFP in pre-gms and GMPs. Numbers indicate the percentage of cells within each quadrant. Representative plots of 2 (CD55, Ly6C and Il1rl1) or 1 experiment (Itgb7) are shown. (d) Quantification of identified cell population after 8-day cultures of CD55 + or Ly6C + pre-gm and GMP by FACS as in Figure 4a. n=6-8 biological replicates. Error bars show SD. Mo: monocyte, Ne: neutrophil, Eo: eosinophil, Ma: mast cell. (e) Flow cytometry analysis of T-cell cultures (OP9-DL1 stroma) of LMPP and pre- GM GE - cells cultured for 21 days (10 cells/culture). Plots show live cells. Stromal cells were gated out on FSC-SSC plot. Percentages are averages of cells in the gates or quadrants (+/-SD). (n=5 for LMPP, n=19 for pre-gm GE - ). Panels on the right show Thy1.2 vs CD25 expression profile on cells first gated as CD4 CD8.

9 Supplementary Figure 5 Effect of absence of Flt3L and GATA-1 on myeloid progenitors. (a) Flow cytometry analysis of wild type and Flt3L KO pre-gm cells from 17 week old male mice, showing Flt3 expression. Numbers indicate mean percentage of cells in each gate (+/- SD). n=5 from 2 experiments. (b) Quantification of number of myeloid progenitors by flow cytometry analysis shown as percentage of total bone marrow. Number of cells of indicated progenitors are shown as frequency of total number of bone marrow cells (+/- SD). n=5 from 2 experiments. (c) Schematic overview of Gata1 KO studies. Chimeric mice with wild type and conditional Gata1 KO blood cells are created by transplanting indicated cells into irradiated recipients. pic is injected to delete the floxed Gata1 gene mediated by MxCre. GE -

10 and GE + pre-gm and GMP cells were analyzed and sorted for in vitro culture. (d) Quantification of EGFP positive cells by flow cytometry of CD45.2 donor cells from experiment depicted in (c). Bars show percentage EGFP positive cells of the indicated CD45.2 progenitor population. Gata1 fl/y without MxCre are defined as wild type cells. n=2 for wild type cells and n=3 for Gata1 KO. SD is shown. (e) Indicated cells were manually plated at an average of 1 cell/culture and cultured for 8 days. Cell types of cultures are based on morphology analysis of cytospins. The number of cultures analyzed is indicated and are accumulated from 2 experiments. Mo: monocyte, PMN: polymorphonuclear granulocyte, Ma: mast cell (f) Representative picture of mast cells from cultured wild type and Gata1 KO GMP GE + single cells. Scale bar is 25µM.

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12 Supplementary Figure 6 In vivo myeloid reconstitution by GE and GE + myeloid progenitor cells. (a) Experimental design for direct comparison of in vivo potential from GE - and GE + GMPs and pre-gms. (b-c) Quantification of in vivo contribution of GE + and GE - GMP (b) and pre-gm (c) progenitors to mast cells, eosinophils, neutrophils and monocytes, 7 (red; n=3) and 10 (blue; n=5) days after intra-femur transplantation of cells. Mean (SD) donor derived cells per million analyzed MNCs per 1000 injected cells detected in injected femur (top) and spleen (bottom) is shown. Data is from three independent experiments. * below detection limit (see methods). (d) Total donor (CD CD45.1/2) contribution per million MNCs analyzed at 7 and days following IP or IF transplantation of indicated progenitor populations. Peritoneal fluid was analyzed from IP transplanted mice, and bone marrow cells from injected femur and spleen cells were analyzed from IF transplanted mice (n=3-5 mice) cells/mouse were injected IP and cells/mouse were injected IF. Data is adjusted to per 1000 injected donor cells per 1 million acquired MNCs. (e-h) Representative morphology of donor-derived monocytes (e), neutrophils (f), eosinophils (g) and mast cells (h) following IP transplantation of purified GMPs, pre-gms and LMPPs. (i) Gata1-EGFP expression in donor-derived (CD45.1/2 and CD45.2) mast cells, eosinophils, neutrophils and monocytes 7 and 11 days following I.P. transplantation of GMPs and pre-gms respectively. Data from transplanted mice included in Figure 8 is shown.

13 Supplementary Figure 7 Revised model of the hematopoietic hierarchy with an early bifurcation of distinct myeloid lineage pathways. (a) The classical model with initial segregation of myeloid/mk/e and lymphoid potential via a CMP and CLP, with a common CMP-derived GMP that gives rise to all monocyte-macrophage and granulocyte lineages. (b) Model incorporating the LMPP, where lymphoid and megakaryocyteerythroid potentials separate early, but both the LMPP and CMP produce the same GMP with combined potential for all monocyte-macrophage and granulocyte lineages. (c) Model supported by the work described herein based on an early branching point generating LMPPs and erythroidmegakaryocyte primed multi-potent progenitor (EMkMPP), where the monocyte-macrophage and granulocyte lineages separate along with the lymphoid and megakaryocyte/erythroid potentials, according to their Gata1 expression, generating progenitors restricted to eosinophil-mast cell (and likely basophil) fate (EoMP), or to neutrophil or monocyte-macrophage fate (prenm, NMP). The GATA-1 and Flt3 expression domains are

14 indicated. It should be noted that there is no direct evidence yet that the Flt3 fraction of prenms derive from multi-potent progenitors (MPPs), so this pathways remains hypothetical. (d) Composite model of the work described herein that includes a CMP placed upstream of both the EMkMPP and the prenm, assuming it contains all monocyte-macrophage and granulocyte potentials at the single cell level, something yet to be determined. For all models it should be noted that other commitment pathways are possible, given the lineage potentials of the progenitors involved. For example, direct commitment of MPPs or another multi-potent progenitor to an Mk-E fate, circumventing the CMP and/or EMkMPP cannot be ruled out. In addition, these models do not incorporate all established findings in the hematopoietic hierarchy, such as the sustained myeloid programming of CLPs and downstream lymphoid progenitors.

15 Supplementary Figure 8 Quantification of transcriptome coverage of pre-gm single-cell RNA-seq. (a) Quantification of the number of sequencing reads that were mapped to a unique genome position for each single pre-gm cell. (b) Percentage of total reads that were mapped to a unique genome position for each single pre-gm cell. (c) The total number of transcripts to which sequencing reads could be uniquely mapped for each single pre-gm cell, using a detection limit of 0.5 reads per million per kilobase of transcript. Cells where <2000 genes were detected were excluded from the analysis (sample 81).