Fig 1.
Conditional E2A-HLF transgenic mice.
(A) Schematic representation of wild type, targeted, and recombined E2A alleles. After Cre recombinase expression, 3’ E2A exons (13, E12, E47 and 16) and the PGKneo cassette are deleted and the human HLF cDNA linked with EGFP by an IRES element is fused in frame to E2A. The expression of Cre-recombinase was driven by the B-cell specific promoters CD19 or Mb1 (CD79a, Igα), or in hematopoietic stem cells by the Mx1 promoter. The phenotypes arising depending on the Cre-recombinase driven promoter are indicated on the right. MPD-like, myeloproliferative disease like. (B) Flow cytometry analysis shows GFP expression from fetal liver (FL) cells of an E2A-HLF/Mx1.Cre embryo and from total bone marrow (BM) of E2A-HLF/Mb1.Cre and E2A-HLF/CD19.Cre mice. (C) Representative western blots show E2A and E2A-HLF protein levels in FACS-sorted progenitor B cells from wild-type (WT, lin-CD19+CD43+) and 2-month-old transgenic (lin-CD19+CD43+GFP+) E2A-HLF/Mb1.Cre and E2A-HLF/CD19.Cre mice. For comparison, E2A and E2A-HLF expression is shown from the non-E2A-HLF cell line REH and from the E2A-HLF+ cell line HAL-01. GAPDH was used as loading control. The E2A/E2A-HLF ratio (shown below) was determined by densitometry.
Fig 2.
Conditional E2A-HLF transgenic mice develop B cell lymphopenia and hyposplenia.
(A) Total bone marrow cells from 2-month-old wild-type (WT, n = 12) and E2A-HLF/Mb1.Cre (n = 11) transgenic mice were enumerated by trypan blue exclusion assay. Horizontal bars denote the mean. Statistical analysis was done by Mann-Whitney U test. (B) Dot plots from flow cytometry analysis show mature B cell (B220+CD19+) subpopulations in peripheral blood (PB, upper panel) and progenitor B cell subpopulations (Lin-CD19-CD43+, Lin-CD19+CD43+, Lin-CD19+CD43-) in bone marrow (BM, lower panel) of representative wild-type and E2A-HLF/Mb1.Cre transgenic mice. Lin, lineage markers (CD3, CD4, CD8, NK1-1, Mac1, Gr1, Ter119). (C) Graph summarizes relative frequencies of mature B cells in peripheral blood (upper panel) and progenitor B cells in bone marrow (lower panel) of wild-type (WT, n = 9), E2A-HLF/CD19.Cre 2-month-old mice (n = 4), E2A-HLF/Mb1.Cre 2-month-old (n = 6) and 6-month-old (n = 3) mice. Columns denote mean and bars denote standard error of the mean. (D) Spleens are shown for representative WT (n = 3) and transgenic E2A-HLF/Mb1.Cre mice (n = 3). (E) Graph shows spleen weights from WT (n = 14), E2A-HLF/CD19.Cre (n = 4) and E2A-HLF/Mb1.Cre (n = 11) mice, horizontal bars denote the mean. Statistical analysis was done by Mann-Whitney U test; n.s., not significant.
Fig 3.
E2A-HLF conditional mice show increased B cell progenitor death.
(A) Dot plots show annexin V (AnnV) and propidium iodide (PI) staining in CD19+CD43+ gated cells of representative wild type, E2A-HLF/Mb1.Cre 2-month-old and 6-month-old transgenic mice. (B) Graph summarizes relative live (Ann-/PI-), apoptotic (AnnV+/PI-) and necrotic (AnnV+/PI+) fractions in CD19+CD43+ gated cells of bone marrow samples from wild type (WT, n = 5), E2A-HLF/Mb1.Cre 2-month-old (n = 3) and 6-month-old (n = 3) mice. Columns denote mean and bars denote standard error of the mean. Statistical analysis was performed using Mann-Whitney U test. (C) Bone marrow cells were sorted from wild type (WT, n = 3), E2A-HLF/Mb1.Cre transgenic 2-month-old (n = 3) and cultured in methylcellulose enriched with IL-7 (10 ng/ml). Colonies were counted after 7 days. Columns denote mean and bars denote standard error of the mean. (D) Total bone marrow cells from WT (n = 3) and 2 month-old E2A-HLF/Mb1.Cre mice (n = 3) were cultured in methylcellulose enriched with IL-7 (10 ng/ml). After 2 days, cells were analyzed for cleaved caspase3 (cCaspase 3) in B cell progenitor subpopulations (CD19-CD43+ and CD19+CD43+). (E) Flow cytometry analysis shows GFP expression in bone marrow cells from E2A-HLF/Mb1.Cre transgenic mouse before and after 2 days of culture. A representative from three experiments is shown.
Fig 4.
Expression of E2A-HLF in fetal liver hematopoietic progenitors results in embryonic lethality.
(A) Image shows embryo morphology (n = 8) at E12.5 E2A-HLF/Mx1.Cre (EH+Mx1+) embryos. An E2A-HLF/Mx1.Cre embryo in the lower part of the picture shows cranial hemorrhage and three other E2A-HLF/Mx1.Cre embryos were not viable. (B) Graph summarizing mortality of embryos depending on their genotype and developmental stage. Controls comprise wild type, E2A-HLF+/Mx1.Cre- and E2A-HLF-/Mx1.Cre+ embryos. A total of 118 embryos were analyzed. (C) Fetal liver cells of control (n = 3) and E2A-HLF/Mx1.Cre (n = 3) embryos at E11.5 were stained with annexin V (AnnV) and propidium iodide (PI) for apoptosis analysis. A representative experiment is shown. (D) Graph summarizes annexin V/propidium staining from fetal liver cells. Columns denote mean and bars denote standard error of the mean. Statistical analysis was done by Mann Whitney U test.
Fig 5.
E2A-HLF/Mb1.Cre transgenic mice develop myeloproliferative-like disease.
(A) Kaplan-Meier plots show disease-free survival of conditional E2A-HLF/Mb1.Cre mice (n = 54) and control E2A-HLF mice (n = 67). The incidence of myeloproliferative disease (MPD)-like at 12 months is shown on the right. Statistical difference between curves were calculated by log-rank (Mantel-Cox) test. (B) White blood cell counts (WBC, left panel) and hemoglobin concentration (HGB, right panel) at MPD-like disease presentation (n = 6) compared to wild type (n = 4) and healthy transgenic mice (n = 4). Each dot represents a mouse and horizontal bars denote the mean of analyzed mice. Statistical analysis was performed by Mann-Whitney U test. (C) Graph shows spleen (left panel) and liver weights (right panel) of wild type (WT, n = 9), healthy transgenic (n = 8) and MPD-like (n = 6) mice. Each dot represents a mouse and horizontal bars denote the mean of analyzed mice. Statistical analysis was performed by Mann-Whitney U test. (D) May-Grünwald Giemsa staining of cytospins from bone marrow (BM), spleen and peripheral blood smear (PB) show morphology of MPD-like cells. (E) Bone marrow cells were cultured in methylcellulose enriched with a myeloid cytokine-cocktail. Compact colonies were counted every seven days and then, cells were re-plated. Columns denote mean and bars standard error of the mean of triplicates. A representative of two bone marrow cells from WT and MPD-like mice is shown. (F) Secondary bone marrow transplantation (BMT) of 100,000 bone marrow cells from a mouse with MPD-like disease in five sublethally irradiated recipients. After 10 months of follow-up, no mice developed disease.