Fig 2: Myb function is required for both Hoxb8 and non-Hoxb8 hematopoietic progenitors during fetal liver hematopoiesis. (A-C) Representative flow cytometry plots of Ter119- Kit+ cells gated for tdTomato from the fetal liver (FL) of (A) E11.5, (B) E12.5 and (C) E14.5 Myb+/+ and Myb-/- embryos. (D,E) Representative flow cytometry plots of CD16/32+ EMPs (CD41+ Kit+) gated for tdTomato from fetal livers of E12.5 (D) Myb+/+ and (E) Myb-/- embryos. (F-H) Number of Hoxb8 lineage (B8L) and non-Hoxb8 lineage (nB8L) cells in the Kit+ population in fetal livers of (F) E11.5, (G) E12.5 and (H) E14.5 Myb+/+ and Myb-/- embryos. E11.5: n=2 biological replicates per group; two-way ANOVA with post-hoc analysis comparing Myb+/+ with Myb-/- in B8L (P=0.0351) and nB8L (P<0.0001). E12.5: n=2 biological replicates per group; two-way ANOVA with post-hoc analysis comparing Myb+/+with Myb-/- in B8L (P=0.0047) and nB8L (P<0.0001). E14.5: n=5 biological replicates (Myb+/+) and n=3 biological replicates (Myb-/-); two-way ANOVA with post-hoc analysis comparing Myb+/+ with Myb-/- in B8L (P<0.0001) and nB8L (P=0.9406). (I) Number of B8L and nB8L cells in the CD16/32+ EMP population of the E12.5 fetal liver. n=2 biological replicates per group; two-way ANOVA with post-hoc analysis comparing Myb+/+ with Myb-/- in B8L (P=0.0040) and nB8L (P=0.0150). Gray circles are individual data points. Data are mean±s.e.m. n.s., non-significant, *P<0.05, **P<0.01, ****P<0.0001. Data are from two or three independent experiments per time point.

Fig 3: Impact of hypoxia on bone marrow cell egress and composition.(a) BrdU+ blood lymphocytes (CD3 and CD19+) and (b) BrdU+ blood neutrophil proportion in mice treated with LPS and housed in normoxia (N) and hypoxia (H) for 24 hours (Naïve N and H n = 3, LPS N and H n = 4). Data representative of 2 experiments (c) Schematic showing hematopoietic hierarchy with Lin-Sca-C-Kit+ (LSK) compartment and progenitors (HSC-hematopoietic stem cell, MPP- multipotent progenitor, HPC- hematopoietic progenitors, CMP- common myeloid progenitor, LMPP- lymphoid-primed multipotent progenitor, CLP- common lymphoid progenitor, GMP- granulocyte/monocyte progenitor, MEP-Megakaryocyte/erythrocyte progenitor). (d) Schematic showing erythrocytosis and monopoiesis (Pre-GM-pre-granulocyte/monocyte precursor, Pre-MegE - megakaryocyte-erythrocyte precursor, Pre-CFU-E - pre-colony forming unit erythroid, CFU-E - Colony forming unit erythroid, GMP – granulocyte-monocyte precursor, MkP- megakaryocyte precursor) gating strategy for bone marrow common myeloid progenitor progeny on CD41-CD16/32- cells. (e) Blood hematocrit at 24 hours (n = 3/ group, data representative of 2 experiments) or (f) 5 days in mice treated with LPS and housed in normoxia (N) and hypoxia (H) (n = 3/group, data representative of 2 experiments) was measured. a, b Mean±SD. Statistical testing performed using one-way ANOVA with Tukey’s multiple comparisons test, e, f Mean±SD. f, statistical testing performed using unpaired two-sided t-test.

Fig 4: Systemic hypoxia alters BM hematopoiesis toward increased erythropoiesis.a, Representative dot plots gated on live CD45+Lin-(CD3/CD19/Ly6G)CD115+Ly6Chi cells and proportion of BrdU+ monocytes in naive or LPS-challenged mice housed in normoxia or hypoxia for 24 h and pulsed with BrdU for the last 12 h (naive, n = 5–6; LPS treated, n = 8 in). b, Absolute numbers of BM LSK cells, CD48+CD150- HPC-1, CD48+CD150+ HPC-2 (n = 6 per group) and Lin-cKit+Sca1-CD127-CD16/32-CD34+ CMPs (n = 5–6 group) in mice as in a. c, Proportion of BM pre-GMs, pre-Meg-E, pre-CFU-E and CFU-E CD41-CD32/16- cells in mice as in a (n = 6 per group). d,e, Representative UMAP analysis of BM cells gated on live CD45+Lineage-Sca1-C-Kit+CD41-CD32/16- cells (d) and summary data of proportions of pre-GM and CFU-E cells (e) measured in the BM of mice treated with LPS and housed in normoxia (N) or hypoxia (H) for 5 d (n = 6 per group). Data are shown as the mean ± s.e.m. Each datapoint represents an individual mouse. Data in a–e are pooled from two independent experiments. Statistical testing: for a–c is by one-way ANOVA with Tukey’s multiple comparison test and for e by unpaired, two-tailed Student’s t-test.

Fig 5: CITE-seq characterization of CD31+ cells from E10.5 AGM.A UMAP representation of single-cell unsupervised clustering based on transcriptome data. B Genes associated with each cluster in (A) that identify cell types. C, D Expression of antibody-derived tag (ADT) for (C) each cluster previously identified represented by ridge plots and D scatter plots of the relevant subpopulations. E Heatmap of surface expression, based on ADT, of the selected markers on cells forming the EHT cluster. F Identification of phenotypic HE within the “EHT’ subcluster on a UMAP projection. Cells labeled in blue are negative for CD117, CD41, CD43, and CD45 based on ADT expression and red cells expressed at least one of the EHT markers.