Fig 1.
Decreased B cells and increased myeloid cells in Nfix expressing cells in the periphery.
C57Bl/6 mice were reconstituted with BM cells transduced with control MigR1 or Nfix vectors. (A) Graph of percentage number of GFP+ B cells (B220+, CD19+), myeloid cells (Gr-1+CD11b+), and (C) T cells (CD4+, CD8+) in the PB of MigR1 and Nfix chimeric animals 6 weeks post-transplant. (B) Representative flow cytometric analysis of PB cells in MigR1 and Nfix chimeric animals, 10 wk post-transplant, showing engraftment of GFP (left panel), B220+CD19+ B cells in the GFP- and GFP+ fractions (middle panel, percentages given) and the Gr-1+CD11b+ myeloid cells in the GFP- and GFP+ fractions (right panels, percentages given). Results are representative of 2 independent BMT experiments.
Fig 2.
Decreased B cells and increased myeloid cells in Nfix expressing cells in the bone marrow and spleen.
C57Bl/6 mice were reconstituted with BM cells transduced with control MigR1 or Nfix vectors. Graph of percentage of B cells (left, B220+CD19+) and myeloid cells (right, Gr-1+CD11b+) in the (A) BM and (B) Spleen of MigR1 and Nfix chimeric animals (GFP+) 10 wks post-transplant. Representative flow cytometric analysis in MigR1 and NFIX chimeric animals, 10 wk post transplant, showing GFP engraftment (left panel), B220+CD19+ B cells (middle panel, percentages given) and Gr-1+CD11b+ myeloid cells (right panel) in the GFP- and GFP+ fractions of the bone marrow (C) and spleen (D). Results are representative of 2 independent BMT experiments.
Fig 3.
Nfix expression in stem and progenitor populations.
(A) Analysis of Nfix expression during murine B cell differentiation using the B-cell lineage microarray dataset (GSE11110)[41]. Error bars represent max and min Nfix expression values. (B) Quantitative RT-PCR analysis of Nfix expression in murine B cell populations fractionated by flow cytometry. Data are representative of three independent experiments, performed in duplicate. Error bars denote ±SD. (C) Analysis of NFIX expression in human B cell development from the human hematopoiesis microarray dataset (GSE24759) (21). Plots represent raw values, error bars represent max and min values. (D) Analysis of Nfix expression by qRT-PCR in murine stem and progenitor cells isolated from bone marrow. (E) Graph of percentage expression of CD43+, B220+, and B220loCD43+ populations in BM cells from chimeras established with MigR1 or Nfix progenitors 8–10 wks previously. Error bars denote +/- SD of 2 independent experiments (n = 3). (F) Representative flow cytometric analysis, with gates and percentages showing B220loCD43+ populations (early B cells) in GFP- and GFP+ populations.
Fig 4.
Nfix blocks B cell differentiation in vitro in favour of myelopoiesis.
(A) Total FL was transduced with control MigR1 or Nfix, plated on OP9 cells and analyzed by flow cytometry on day 16. FACS plots of GFP+ B cells (B220+CD19+, top panels) and GFP+ myeloid cells (CD11b+Gr-1+, lower panels) representative of 3 replicates from 2 independent experiments. (B) Graph of average percentage of B220+CD19+ (left) and CD11b+Gr-1+ (right) from (A). Error bars denote +/- SD of 2 independent experiments (n = 3). Graph shows a statistically significant decrease in Nfix derived B220+CD19+ cells (p = 0.02) and a significant increase in Nfix derived CD11b+Gr-1+ cells (p = 0.01) when compared with MigR1 controls. (C) E14.5 FL HSPCs (Lin-Sca1+cKit+Flt3-) transduced with control MigR1 or Nfix retrovirus were plated on OP9 cells (Day 0) and analysed by flow cytometry on day 12 with FACs plots of GFP expression (left histograms) and GFP- and GFP+ B220+CD19+ cells (right dot plots) representative of 2 independent experiments. (D) Graph of average percentage of B220+CD19+ cells from (C), error bars denote +/- SD of 2 independent experiments. Experiment shows significant decrease in Nfix expressing B cells compared with control MigR1 (p = 0.01) (E) Total BM cells were sorted for MigR1 and Nfix expression 24 hr post transduction and qRT-PCR was performed. Graphical presentation of relative mRNA expression of Nfix, E2A, ID2 and ID3 genes and presented relative to control MigR1 cells. Error bars denote +/- SD of 3 technical replicates. Data representative of 2 biological replicates.
Fig 5.
Efficient differentiation of myeloid cells in Nfix expressing cells.
(A) MigR1 and Nfix transduced total BM cells were plated in methylcellulose media (M3434) and colonies scored after 9–11 days according to morphological criteria. The mean percentage of erythroid (E), granulocyte/erythrocyte/monocyte/megakaryocyte (GEMM), granulocyte/monocyte (GM) macrophage (M) and granulocyte (G) colonies are shown from 3 independent experiments +/- SEM. A statistically significant decrease in CFU-EE colonies indicated. (B) Representative flow cytometric analysis of total cells from colony assay in (A). (C) Graph of methylcellulose colony numbers from HSPCs, CMPs and GMPs sorted and transduced with control MigR1 or Nfix. Colonies were counted on day 12 and graph represents mean of triplicate plates normalized to GFP expression +/- SD. (D) Representative flow cytometric analysis of cells from colony assay in (C). 32D cells transduced with MigR1 or Nfix maintained in either IL3 (E) or G-CSF (F) for 5 days, and analyzed for F4/80 expression by flow cytometry. The mean percentages of 3 independent experiments are shown +/- SD. (G) 32D cells and (H) Ba/F3 cells transduced with MigR1 and Nfix and qRT-PCR performed. Graphical presentation of relative mRNA expression of Nfix, MMP9, G-CSFr and C/EBPalpha genes and presented relative to control MigR1 cells. Error bars denote +/- SD of 3 technical replicates. Data representative of 2 biological replicates.
Fig 6.
Loss of Nfix perturbs myelopoiesis while driving B cell differentiation.
Total BM from WT or Nfix deficient mice was plated onto OP9 cells and analysed by flow cytometry on day 4 (A) and day 6 (B). Bar chart shows the mean percentages of B220+ cells from 3 independent experiments +/- SD. (C) Representative flow cytometric analysis of cells expressing B220 at day 6. (D) Total BM from WT or Nfix deficient mice was plated in methylcellulose (M3434). Colonies were counted after 11 days. Bar chart represents the mean number of colonies from 2 independent experiments +/- SD. (E) Bar chart shows the mean percentages of Gr1 + and F4/80+ cells from 2 independent experiments +/- SD. (F) Representative flow cytometric analysis of cells from colony assay which were stained with Gr-1 and F4/80. (G) Graphical presentation of relative mRNA expression of indicated genes assessed using high-throughput qPCR on the 48.48 Dynamic Array IFC system (Fluidigm). Bars represent the average of 3 biological Nfix-/- replicates normalized to WT control, and error bars denote +/- SD.