Fig 2: Vav-Cre deletion of Brd4 blocks HSC development A schematic representation of targeted deletion of mouse Brd4. The exon–intron organization of the Brd4 locus (top). Targeting vector contained exon 3 flanked by LoxP sites (green) and a neomycin resistance gene flanked by FRT (white). Cre-mediated recombination should cause deletion of exon 3 resulting in loss of BRD4 expression (bottom).Genotyping of Brd4 fl/fl mice with ERT2-Cre (Cre) or without ERT2 -Cre (+/+). Tamoxifen treatment deleted 2.2 kb Brd4 fl/fl fragment from Cre/+ and Cre/Cre mice (top). Cre/+, Cre/Cre and +/+ mice were confirmed by the presence of ERT2 -Cre band (Cre) or its absence (WT) (bottom).WT and Brd4 KO cells were tested for mRNA (left) and the protein (right) by qRT–PCR and immunoblot assays. Values represent the average of three experiments ± SD.Vav-Cre-mediated Brd4 depletion was verified for cells from Brd4 fl/fl Vav-Cre mice (fetal liver or spleen) by qRT–PCR and immunoblot (left and right). Values represent the average of three experiments ± SD.Photographic images of E14.5 embryos from WT and KO mice.Corresponding flow cytometric profile of Lin- fetal liver cells stained with cKit and Sca-1 antibodies examining early hematopoietic progenitor population (LSK) (left). LSK populations were further divided by the Flt3 marker to detect HSCs and MPPs (middle). The number of total cells, e.g., LSKs, HSCs, MPPs, in a fetal liver was estimated on indicated days of embryonic development (E13.5, E14.5, and E17.5). Values are the average of 3–5 embryos ± SD (right).A schematic of hematopoietic stem cell and early progenitor cell pathways.Representative flow cytometric profile of CD71- cells stained for the myeloid and lymphoid markers. Values are the average of 3 E17.5 embryos ± SD.Data information: Statistical significance in all figures was determined by two-tailed unpaired t-test and is indicated by: *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significance.

Fig 3: Rad21 deficiency increases self-renewal and leads to impaired and myeloid-skewed differentiation of HSPCs. (A–D) Freshly isolated LSK cells were infected with an shRNA against Rad21 shRad21 or a control virus (shLuci) in culture and transplanted into lethally irradiated mice together with noninfected LSK cells from the same culture. (A and B) The number of HSCs (GFP+CD150+CD34-LSK cells) derived from infected LSK cells in the primary recipients (A) and the secondary recipients (B) that were transplanted with total BM cells of the primary recipients at 4 mo after the first transplantation. The dots represent individual recipient mice (n = 4–5 mice per group per experiment, n = 3 independent experiments, one of the three repeat experiments is shown, the other experiments showed similar results). Statistical values were calculated by Welch’s t test after log transformation. (C) The chimerism of GFP+ cells from the primary recipients was analyzed by FACS at 4 mo after transplantation in the indicated populations. HSC: CD150+CD34-LSK; MPP: CD34+LSK; CMP: common myeloid progenitor (Lin-c-Kit+Sca-1-CD34+Fc?R-); GMP: granulocyte macrophage progenitor (Lin-c-Kit+Sca-1-CD34+Fc?R+); MEP: megakaryocyte erythrocyte progenitor (Lin-c-Kit+Sca-1-CD34-Fc?R-), CLP: common lymphoid progenitor (Lin-c-KitlowSca-1lowIL-7Ra+Flt3+), Lin-: lineage- cells; BM: total BM cells. n = 4–5 mice per group per experiment, three independent experiments, one of the three experiments is shown, the other experiments showed similar results. (D) PB cells from the primary recipients were analyzed by FACS. The histogram shows the percentage of myeloid and lymphoid cells in GFP+CD45+ cells in the PB at the indicated time points after transplantation (n = 4–5 mice per group for each experiment, n = 3 independent experiments, one of the three experiments is shown; the other experiments showed similar results). Statistical significance for myeloid cells was assessed by Welch’s t test after logit transformation of the data. (E and F) LSK cells from young (2 mo old) wild-type mice were transduced with shLuci or shRad21 virus followed by transplantation into 2–3-mo-old recipients. 8 wk after transplantation, recipient mice were sacrificed and total BM was transplanted into secondary recipients. 4 wk after secondary transplantation, BM cells were analyzed to investigate the chimerism of shLuci- or shRad21-LSK cells in BM. Chimerism of shLuci- or shRad21-infected LSK cells in PB was monitored every 4 wk after each round of transplantation. (E) The data show the relative change of GFP+ cells in PB from the recipients at indicated time points after transplantation. The initial transduction efficiency was 71% for shLuci-LSK and 54% for shRad21-LSK (n = 5 recipient mice per group, per experiment in the primary transplantation, n = 10 recipient mice per group, per experiment in the secondary transplantation, n = 2 independent experiments, one of the two experiments is shown; the other experiment showed a similar result). (F) The histograms depict the chimerism of GFP+ cells in the indicated populations of the secondary recipients at 4 wk after transplantation. The dots represent individual mice (n = 4 mice per group for each experiment, n = 2 independent experiments, one of the two experiments is shown, the other experiment showed a similar result). Data in all panels of this figure depict mean ± SD; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Fig 4: Immuno-phenotypes of iHPCs after 10-day maturation (at day 21). Lin was defined as CD2-CD3-CD4-CD8-CD11b-Gr1-Ter119-CD19-NK1.1-TCR?d-. iHPCs were defined as Lin-c-kit+CD127+/CD135+/Sca1+.

Fig 5: cDC1 abundance in the medulla is locally sensed by preDCs and immature cDCs via Flt3L availability(A) Frequency of CD135+ cells among CD11c+MHCII- cells that contain preDCs in popliteal LNs of WT mice 16, 24, and 40 h after s.c. MVA infection into the foot hock.(B and C) Surface CD135 expression among preDCs (CD11c+MHCII-) (B) and immature (MHCIIloCD24+, left) or migratory (XCR1+CD8a-CD103+, right) cDC1s (C) in draining LNs of Zbtb46GFP mice 24 h after s.c. IFNa injection into the foot hock.(D and E) Surface CD135 expression among preDCs (CD11c+MHCII-) (D) and immature (MHCIIloCD24+, left) or migratory (XCR1+CD8a-CD103+, right) cDC1s (E) in draining LNs of Zbtb46GFP mice 16 h after i.p. Flt3L injection.(F) Surface CD135 expression among preDCs (CD11c+MHCII-) (left) and immature (MHCIIloCD24+, right) cDC1s in draining LNs of WT mice 24 h after s.c. IFNa injection into the foot hock and 8 h after Flt3 inhibitor administration.(G) Frequency of CD135+ cells among CD11c+MHCII- cells that contain preDCs in LNs of Xcr1DTR mice 2, 3, and 4 days after cDC1 depletion with DTx injection.(H and I) Mixed Xcr1DTR:Xcr1Venus (50:50) BM chimeras were treated with DTx 2 days before analysis to deplete only half of the cDC1 population. Frequency of CD135+ cells among CD11c+MHCII- cells that contain preDCs (H) and surface CD135 expression among Xcr1Venus immature (MHCIIloCD24+XCR1+, left) or migratory (XCR1+CD8a-CD103+, right) cDC1s in LNs (I).Data display pooled data from =2 independent experiments (A–I) (A, n = 6–8; B and C, n = 3–4; D and E, n = 3; F, n = 4; G, n = 4–6; H and I, n = 4–6). Error bars indicate the mean ± SD. Comparison between groups was calculated using one-way ANOVA or unpaired Student’s t tests. ***p value < 0.001, **p value < 0.01, *p value < 0.05.