JMJD6 is required for steady-state multilineage hematopoiesis, whereas Jmjd6-deficient HSCs have a normal apoptotic and cell cycle status. (A) Relative levels of Jmjd6 messenger RNA (normalized to Hprt) in cells isolated from BM of 8- to 12-week-old adult C57BL/6 mice: LSKCD48⁻CD150⁺ HSCs, LSKCD48⁻CD150⁻ MPPs, primitive HPCs (ie, LSKCD48⁺CD150⁻ HPC-1 and LSKCD48⁺CD150⁺ HPC-2 populations), LSKs, and LK MPPs (n = 3). Data are mean ± SEM. (B) The expression of Jmjd6 in HSCs (LSKCD34⁻CD135⁻), MPP1 (LSKCD34⁺CD135⁻CD150⁺CD48⁻), MPP2 (LSKCD34⁺CD135⁻CD150⁺CD48⁺), MPP3 (LSKCD34⁺CD135⁻CD150⁻CD48⁺), and lymphoid-primed multipotent progenitor (LMPP)/MPP4 (LSKCD34⁺CD135⁺) populations, as well as in CMP (LKCD34⁺FcγRII/III^low), GMP (LKCD34⁺FcγRII/III^high), and MEP (LKCD34⁻FcγRII/III^low) compartments, determined using single-cell SMART-seq2.²⁹  Data are mean ± SEM. (C) Jmjd6 expression in HSCs and indicated committed progenitor cell compartments determined by 10× genomics single-cell RNA-seq.³⁰  Bins represent clusters annotated via marker genes. Data are mean ± SEM. Ery, erythrocytes. (D) Genomic structure of the conditional Jmjd6 allele. Exon 3 is flanked by LoxP sites (red triangles). Following Cre-mediated recombination, exon 3 is excised, resulting in a frameshift mutation and a non-sense–mediated decay. (E) Levels of Jmjd6 messenger RNA in Jmjd6^fl/fl;Vav-iCre (Jmjd6^cKO) and control Jmjd6^fl/fl (Jmjd6^CTL) BM c-Kit⁺ cells (n = 4). Data are mean ± SEM. (F) Western blots for JMJD6 and β-actin from BM c-Kit⁺ cells from Jmjd6^cKO and Jmjd6^CTL mice (n = 3). (G) PB counts of white blood cells (WBCs), CD19⁺B220⁺ B cells, CD4⁺ and CD8⁺ T cells, and CD11b⁺Gr-1⁺ myeloid cells in 8- to 10-week-old Jmjd6^cKO and Jmjd6^CTL mice (n = 9-10). Data are mean ± SEM. (H) Total BM cellularity of Jmjd6^cKO and Jmjd6^CTL 8- to 10-week-old mice (2 femurs and 2 tibias) (n = 13-16). Data are mean ± SEM. (I) Total numbers of CMPs (LKCD34⁺FcγRII/III^low), GMPs (LKCD34⁺FcγRII/III^high), MEPs (LKCD34⁻FcγRII/III^low), and CLPs (Lin⁻Sca-1^lowc-Kit^lowCD127⁺CD135⁺) in BM from 8- to 10-week-old Jmjd6^cKO and Jmjd6^CTL mice (n = 13-16). Data are mean ± SEM. (J-K) Total numbers of HSCs, MPPs, HPC-1, and HPC-2 populations in BM of Jmjd6^cKO and Jmjd6^CTL mice. (J) Eight- to 10-week-old mice (n = 13-16). (K) Fifty-two–week-old mice (n = 6-9). Data are mean ± SEM. (L) Fold change in HSC, MPP, HPC-1, and HPC-2 populations in 52-week-old vs 8- to 10-week-old Jmjd6^cKO and Jmjd6^CTL mice (n = 6-13). (M) Percentage of DAPI⁻Annexin V⁺ and DAPI⁺Annexin V⁺ cells in HSCs, MPPs, HPC-1, and HPC-2 populations in BM from 8- to 10-week-old Jmjd6^cKO and Jmjd6^CTL mice (n = 6). Data are mean ± SEM. (N) Percentage of HSCs from 8- to 10-week-old Jmjd6^cKO and Jmjd6^CTL mice in the G₀ (DAPI⁻Ki67⁻), G₁ (DAPI⁻Ki67⁺), and G₂/S/M (DAPI⁺Ki67⁺) phases of the cell cycle (n = 6-7). Data are mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001, Mann-Whitney U test.