Id1 regulates the maintenance of steady-state bone marrow cellularity, LKS cell frequency and myeloerythroid lineage determination. (A) Id1 protein is readily detectable in the nuclei of a subset of Lin⁻ but not Lin⁺ bone marrow cells. Arrows point to representative overlay images of Id1 (red fluorescence) and nuclear DNA DAPI staining (blue fluorescence) (B) Id1 mRNA level is highest in the most primitive hematopoietic cells and decreases after myeloid commitment. FACS-purified subsets of immature bone marrow hematopoietic cells were purified by multiparameter flow-cytometric sorting and analyzed for Id1 RNA expression by qPCR. Id1 expression was normalized to HPRT to obtain relative mRNA levels. The results represent an average (±SEM) of four measurements from two independent sorting experiments. (C) Decreased frequency of primitive LKS hematopoietic cells in Id1^−/− animals. Shown profiles are representative of flow-cytometric staining for Lin⁻ c-kit⁺ Sca-1⁺ (HSC and MPP) and Lin⁻ c-kit⁺ Sca-1⁻ (HPC) in Id1^+/+ and Id1^−/− bone marrow. The numbers indicate average (±SEM) percentage of nucleated bone marrow cells. (n = 15; ∗∗, P < 0.01). (D and E) Representative flow-cytometric profiles of LT-HSC, ST-HSC, MPP, and myeloid progenitor subsets (CMP, GMP, MEP) in Id1^+/+ and Id1^−/− bone marrow by using the published multiparameter surface staining criteria. The numbers indicate the average (±SEM) percentage of LKS⁺ and LKS⁻ populations, respectively (n = 4; ∗, P < 0.05).

Increased cycling status of Id1^−/− LKS cells. (A) Enhanced in vitro proliferation of sorted Id1 LKS⁺ cells in response to cytokine stimulation. LKS cells were sorted into a 96-well plate (1 cell per well), and clonal expansion was followed for 4 days by manual cell counting under the microscope. The results show the average (±SEM) numbers of cells per well (50 wells were scored for each genotype) and are depicted on a log scale. These data are representative of two independent sorting experiments. (B) Higher rate of S-phase entry in Id1^−/− LKS compartment in vivo. After 3 days of BrdU administration in drinking water, bone marrow cells were simultaneously stained for surface markers and BrdU incorporation into newly synthesized DNA. Gray histograms indicate isotype Ab control; red histograms indicate anti-BrdU Ab. The profiles shown are representative of those seen in individual mice. Numbers indicate the percentage (±SEM) of BrdU⁺ LKS⁺ cells (n = 4; ∗, P < 0.05).

Myeloid lineage priming in Id1^−/− LKS cells. (A and B) Sorted subsets of Id1^+/+ and Id1^−/− cells were analyzed by Affymetrix oligonucleotide array transcript profiling. Myeloid and HSC-associated gene lists were compiled from the experimentally obtained CMP and HSC-associated clusters (SI Data Sets 1 and 2) by using published microarray studies (30–32) and a detailed literature search. (A) In the CMP cluster, 19 of 26 probe sets show similar expression in Id1^−/− LKS and CMPs, that is, higher than in the wild-type LKS cells. (B) Most of the probe sets in the LKS cluster show similar expression in Id1^+/+ and Id1^−/− LKS cells, that is, higher than in the CMPs. (C) Quantitative mRNA expression (qPCR) in Id1^−/− LKS cells of genes relevant for myeloid commitment, self-renewal, and cell cycle regulation in HSCs. The results were normalized to HPRT expression and expressed as average fold change (±SEM) for Id1^−/− over Id1^+/+ LKS cells. Three independent RT-PCR measurements were performed for each gene (∗, P < 0.05; ∗∗, P < 0.01).

Decreased self-renewal capacity of multilineage repopulating HSCs in the absence of Id1. (A) Robust competitive long-term lymphomyeloid repopulating ability of Id1^−/− bone marrow cells. Flow-cytometric analysis of donor myeloid (Gr1⁺) and B-lymphoid (B220⁺) cell contribution in the peripheral blood 5 months after transplant (based on percentage of CD45.2⁺ cells). Bar graphs represent average (±SEM) values for three mice per group. The results are representative of two independent experiments. (B and C) Defective maintenance of the HSC self-renewal capacity in the absence of Id1. Bar graph represents average (±SEM) values of donor contribution to hematopoiesis in the peripheral blood and Lin⁻ bone marrow in groups of secondary recipients of Id1^+/+ (gray bars) vs. Id1^−/− (red bars) bone marrow cells (based on percentage of CD45.2⁺ cells) (n = 6).

Premature myeloid commitment within the Id1^−/− LKS compartment. (A) Spleen colony-forming assay shows increased CFU-S_d8 activity by using sorted Id1^−/− LKS⁺ cells. Five hundred sorted LKS⁺ cells from Id1^+/+ or Id1^−/− bone marrow were transplanted into lethally irradiated recipients, and spleen colonies were scored on days 8 and 12 after transplant. Numbers indicate average colony numbers (±SEM) (n = 12) from three independent experiments. (B and C) In vitro stem/progenitor cell differentiation depends on the cell intrinsic expression of Id1. The results shown are representative of three independent experiments. The numbers indicate the percentage of cells remaining in the lineage-negative gate after 5 days in liquid culture. The accelerated expression of lineage markers in the absence of Id1 expression (red arrow) is reversed by retroviral expression of Id1 in primitive Id1^−/− LKS⁺ cells (blue arrow) (B) but not Id1^−/− LKS⁻ cells (C).