In vivo lineage potential of Ly6d⁻ and Ly6d⁺ bone marrow progenitors by intrathymic transplantation. (A) Intrathymic transplants (i.t.) of MPPs, ALPs, BLPs, and pre-pro-Bs (ppB) into nonirradiated recipients. Similar proportions of cells were transplanted as in Figure 2. Thymuses were harvested at day 9, and the distribution of donor cells within each lineage is shown. Displayed are the combined results from two independent experiments. (B) Absolute number of donor T-cell output (left) and B-cell output (right) recovered from the intrathymic transplants shown in A. (C) Distribution of donor T-lineage cells from MPPs, ALPs, and BLPs, listed as a fraction of total donor T cells. Representative stains for intrathymic analyses and definitions of all lineages can be found in Supplemental Figure S7.

B-cell specification occurs during the ALP-to-BLP transition. (A) Quantitative RT–PCR of B-lineage genes in hematopoietic progenitors and B-committed populations in wild-type bone marrow. Samples were normalized to β-actin transcription and shown relative to the population with the highest expression of each gene. (ND) Not detected (Ct > 32 cycles). The fold change between ALPs and BLPs is shown for select genes, with an asterisk indicating statistical significance (unpaired t-test, n = 2, P < 0.05). Error bars are shown for the MPP, ALP, and BLP samples. (B) Wild-type and age-matched E2A^−/− bone marrow were stained to examine changes in the proportions of MPP, ALP, BLP, and pre-pro-B cells. Only live lin⁻ (Mac1⁻Ter119⁻Gr1⁻CD3⁻) and CD27⁺ cells are shown. Percentages within each gate are shown. (C) Absolute numbers of progenitor populations in wild-type and E2A^−/− bone marrow. Absolute numbers are estimated for the two femurs, tibias, and hips of 6-wk-old mice. (D) Proposed model for the branching of the GM-cell, T-cell, and B-cell lineages from hematopoietic progenitors.

Prediction of surface markers up-regulated or down-regulated during B-cell development. (A) Prediction of genes encoding cell surface molecules up-regulated in B-cell development. The MiDReG algorithm uses Boolean implications from mouse data sets only. The first seed condition is Kit high AND Mpl high, and the second seed condition is CD19 high AND CD3ɛ low. The algorithm predicted gene X such that “(Kit high AND Mpl high) ⇒ X low” and “(Cd19 high AND Cd3ɛ low) ⇒ X high.” The gene list was filtered using membrane Gene Ontology classification and commercially available antibodies suitable for flow cytometry. (∧) AND, (¬) NOT, (→) “implies.” A diagram of the expression changes between Kit and Mpl, Gene X, and Cd19 and Cd3ɛ are depicted on the right. (B) Nineteen genes encoding cell surface molecules were predicted in this analysis. (*) Gene symbol truncated for brevity. Details of the MiDReG algorithm are described elsewhere (D Sahoo, J Seita, D Bhattacharya, M Inlay, I Weissman, S Plevritis, and D Dill, in prep.). (C) Prediction of down-regulated genes. Here, the algorithm predicted gene X such that “(Kit high AND Mpl high) ⇒ X high” and “(Cd19 high AND Cd3ɛ low) ⇒ X low.” Seven genes encoding cell surface molecules were identified after applying the membrane Gene Ontology and commercial antibodies filtrations. (D) Ly6d expression profile of MPPs (light gray), CLPs (white), and pre-pro-Bs (ppB, dark gray). Fluorescence minus one (FMO, black) control is shown for Lin⁻ cells. (E) Kit/Ly6d contour plot of MPPs, CLPs, and pre-pro-Bs. The stains and gating strategy leading to each of these populations are shown. Percentages of displayed cells within each gate are shown.

In vivo lineage potential of Ly6d⁻ and Ly6d⁺ bone marrow progenitors by intravenous transplantation. (A) MPPs, Ly6d⁻ CLPs (ALP), Ly6d⁺ CLPs (BLP), and pre-pro-Bs (ppB) were sorted and transplanted in physiologic proportions (∼10,000–20,000 cells per transplant) intravenously into sublethally irradiated recipients. At days 7 (top) and 14 (bottom), spleens and thymuses were harvested and analyzed for donor output in each of the four lymphoid lineages, as well as macrophages (M) and granulocytes (G). T-cell output was measured in the thymus (shown in the right column) and all other lineages in the spleen. Donor cells were congenic for two markers: (Donor) CD45.1, Thy1.2; (Host) CD45.2, Thy1.1. For spleen cells, the lineage output is shown as a fraction of total chimerism, with means indicated by horizontal bars. Thymuses were depleted of Thy1.1⁺ host cells prior to staining, and the absolute number of recovered T cells (DN1, DN2, DN3, DN4, DP, and SP) per thymus is shown in the right column. Data shown are a merge of four independent experiments. (na) Not analyzed. (B) Representative plots and gating strategy of donor lineages from the spleens and thymuses of intravenously transplanted MPPs, ALPs, and BLPs analyzed at day 7. The left column identifies the splenic chimerism for each sample, listed as the percentage of live cells. All other values are listed as a percentage of donor cells. Only the donor gate for the PBS-injected control (SHAM) is shown. Splenic and thymic lineages are defined as shown. In the thymus, cells shown are pregated as donor, and B220⁻CD19⁻CD11c⁻Mac1⁻Gr1⁻. Values of each thymus population are listed as a percentage of the total thymic donor cells (not shown).