HEB is required to specify the B-cell fate. (A) Representative FACS plots of LMPPs from WT and HEB^f/fTie2Cre bone marrow. Lineage (LIN) includes CD11B, GR1, TER119, CD3ε, LY6C, NK1.1, CD19, and CD11C. (B) Representative FACS plots of LY6D⁻ CLPs and LY6D⁺ CLPs from WT and HEB^f/fTie2Cre bone marrow. (C) Total cellularity of LMPP, LY6D⁻ CLPs, and LY6D⁺ CLPs in WT and HEB^f/fTie2Cre bone marrow. Each dot represents the number from a single mouse, and the horizontal lines indicate the mean in each group. Data shown are pooled from two independent experiments. **P < 0.01; ***P < 0.001. (D) Representative FACS plots of CD19⁺B220⁺ cells from WT and HEB^f/fTie2Cre bone marrow. LIN includes CD11B, GR1, and TER119. (E) Total cell numbers of B-cell progenitor populations within the CD19⁺B220⁺ bone marrow B-cell populations. All populations were first gated as CD19⁺B220⁺. Pro-B, KIT⁺IgM⁻IgD⁻; Pre-B, KIT⁻IgM⁻IgD⁻; Im. B, KIT⁻IgM⁺IgD⁻; Mat. B, KIT⁻IgM^+/low IgD⁺. Each dot represents the number from a single mouse, and the horizontal lines are the mean in each group. Data shown are pooled from two independent experiments. *P < 0.05; ***P < 0.001. F Left displays the cell cycle distribution (quantified by Ki67 and 7AAD FACS staining) of WT and HEB-deficient CLPs (n = 3 in each group). F Right shows a representative plot of Annexin V staining comparing WT and HEB-deficient CLPs (total n = 3 in each group). For F Left and F Right, CLPs are defined as depicted in B.

HEB and E2A act in concert to specify the B-cell fate. (A) Representative FACS plots comparing bone marrow stained for LY6D⁺ CLPs in WT, HEB^f/fVaviCre, E2A^f/+VaviCre, HEB^f/fE2A^f/+VaviCre, and E2A^f/fVaviCre mice. (B) Microarrays analysis displaying genes that are changed by a factor of at least twofold between WT and HEB^f/fTie2Cre LY6D⁻ CLPs. E2A^−/− displays how this subset of genes is affected in E2A^−/− LY6D⁻ CLPs. Displayed data are derived from the means from two or more microarray replicas using material from independent sorts. (C) Graph shows the number of CCR9⁺ LY6D⁻ CLPs in WT and HEB^f/fTie2Cre mice. Each dot represents the number from a single mouse, and the horizontal lines are the mean in each group. Representative data from one of a total of two experiments are shown. **P < 0.01. D Left, right column shows the result of microarray analysis, displaying genes that are changed by a factor of at least twofold between WT and E2A^−/− LY6D⁻ CLPs. In addition, the center column displays how these genes are affected in HEB^f/fTie2Cre LY6D⁻ CLPs. Highlighted in the left column is the location of genes of interest for lymphocyte development (a full gene list is in Table S1). D Right displays box plots of cluster analysis showing how genes patterns in D Left are changed in WT and HEB- and E2A-deficient LY6D⁻ CLPs. ***P < 0.001. Displayed data are the means derived from two or more microarray replicas using material from independent sorts.

HEB is expressed in bone marrow progenitors. (A) Indicated FACS-sorted progenitor populations were analyzed by real-time PCR for the abundance of E2A, HEB, Id2, and Id3 mRNA. Values were normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) expression and are shown as mean ± SEM using purified mRNA from two independent sorts. (B) Absolute numbers of bone marrow cells (from femurs, tibias, and cresta iliac) of sex- and age-matched WT and HEB^f/fTie2Cre mice. Each dot represents the number from a single mouse. The horizontal lines indicate the mean in each group. The asterisk indicates a P value < 0.05. C Left represents FACS plots of the gating strategies used to identify LK, MPP, and HSC populations. Lineage (LIN) includes CD11B, GR1, CD3ε, and NK1.1. C Right displays the absolute cell numbers of each population in WT and HEB^f/fTie2Cre mice. Each dot represents the number from a single mouse, and the horizontal lines are the mean in each group. Data shown are pooled from two independent experiments. D Upper shows representative FACS plots of the gating strategy to identify erythromyeloid progenitor populations within the LK population. D Lower shows the absolute cell number of each population in WT and HEB^f/fTie2Cre bone marrow. Each dot represents the number from a single mouse, and the horizontal lines are the mean in each group. Data shown are pooled from two independent experiments. MkP, megakaryocyte progenitor; GMP, granulocyte macrophage progenitor; preGM, pregranulocyte macrophage; preMegE, premegakarycocyte erythrocyte; preCFUE, pre-CFU erythrocyte; CFUE, CFU erythrocyte; proEry, proerythrocyte.

HEB promotes the transition from the LY6D⁻ to LY6D⁺ CLP compartment. (A) Schematic overview of B-cell development in the bone marrow. (B) Plot of cell number ratios from the consecutive developmental stages depicted in A. The total cellularity of an early population for a mouse is divided by the total cell number of the proceeding population. Each dot represents the ratio from a single mouse, and the horizontal lines are the mean in each group. Data shown are pooled from two independent experiments. *P < 0.05; ***P < 0.001. n.s., not significant. C Left shows an example of D_H–J_H rearrangement PCR readouts from LY6D⁺ CLPs. C Right shows the distribution of cells within the LY6D⁺ CLP compartment with respect to D_H–J_H rearrangement status. A total of 192 cells/population from two independent sorts was assayed. Displayed are the mean ± SEM of cells producing at least one PCR product. (D) Result of limiting dilution assay for in vitro B-cell potential. CLPs from WT and HEB-deficient mice were sorted onto preplated OP9 stroma cells and cultured in conditions promoting B-lymphoid development. The number of cells plated per well is plotted vs. the percentages of wells negative for B-cell growth (CD19⁺ cells). Full lines represent the best fit regression. The frequency of cells with B-cell potential was determined as the cell concentration where 37% of the cells were not generating B cells. The frequency for WT and HEB^f/fTie2Cre cells is indicated. Data shown are from a representative experiment from a total of three experiments.

Direct regulation of Foxo1 expression in CLPs by the E proteins. (A) LY6D⁻ CLPs from WT, HEB^f/fTie2Cre, and E2A^−/− mice were analyzed by real-time PCR for the abundance of Foxo1 mRNA. Values were normalized to HPRT expression and are shown as mean ± SEM using purified mRNA from two independent sorts. (B) E2A (red; top row) occupancy, H3K4me1 (blue; middle row), and H3K4me3 (blue; bottom row) epigenetics mark across the Foxo1 locus in EBF1-deficient cells identified by ChIP followed by genome-wide deep sequencing. Numbers on the left indicate the number of tags observed. The box on the left indicates a Foxo1 locus promoter region with E-box sites; the box on the right indicates a region where E2A occupancy is associated with H3K4me1. (C) Transcriptional activity of H3K4me1 islands (corresponding to the boxes in B) with associated E2A occupancy and presence of E boxes. Δ, deletion within the E-box sequences. Data shown are the mean ± SEM derived from two independent experiments. *P < 0.05. (D) Schematic diagram depicting B-cell development and the activities of E2A and HEB in B-cell specification.