Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors.
Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19(+) progenitors, these cells displayed an increased T cell potential in vivo and in vitro.This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling.This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.
Affiliation: Department of Clinical and Experimental Medicine, Experimental Hematopoiesis Unit, Faculty of Health Sciences, Linköping University, 58183 Linköping, Sweden.
- B-Cell-Specific Activator Protein/deficiency*/genetics
- Cell Differentiation/immunology*
- Cell Lineage/immunology*
- Gene Expression Regulation/genetics/immunology*
- Antigens, CD19/immunology
- Base Sequence
- Blotting, Western
- Chromatin Immunoprecipitation
- DNA Primers/genetics
- Flow Cytometry
- Loss of Heterozygosity
- Mice, Transgenic
- Molecular Sequence Data
- Real-Time Polymerase Chain Reaction
- Receptors, Notch/metabolism
- Sequence Analysis, RNA
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fig1: Combined transheterozygous loss of Pax5 and Ebf1 results in a partial block at the pro–B cell stage and lineage plasticity in CD19+ cells. (A) Graphs displaying the absolute numbers of 7AAD− pro–B cells (Lin−B220+CD19+CD43highIgM−), pre–B cells (Lin−B220+CD19+CD43low/negIgM−), and IgM+ B cells (Lin−B220+CD19+CD43low/negIgM+) in bone marrow from Wt (n = 15), Pax5+/− (n = 10), Ebf1+/− (n = 7), and Pax5+/−Ebf1+/− (TH) (n = 6) mice. (B) Q-PCR analysis of Ebf1 and Pax5 expression in pro–B cells from WT and Pax5+/−Ebf1+/− (TH) mice. Each square indicates a biological replicate analyzed by triplicate Q-PCR reactions. Mean of all mice is presented as a horizontal line. Statistical analysis was performed using unpaired Student’s t test. (C) Graphs illustrating the percent of donor (CD45.2+) of CD19+Thy1.2−, CD19−Thy1.2+, NK1.1+, and Gr1/Mac1+ cells in spleen of Rag1−/− CD45.1+ recipient mice 6–9 wk after transplantation with 2 × 106 sorted bone marrow CD19+IgM− cells from Wt (n = 8) or Pax5+/−Ebf1+/− (TH; n = 9) mice. Mice were from two independent transplantation experiments (4 Wt and 4 TH in experiment one and 4 Wt and 5 TH transplantations in experiment two). (D) Graphs and representative FACS plot illustrating the percent of IgM+, IgM+IgD+, and CD43highIgM− pro–B cells out of CD45.2+CD19+ from Rag1−/− mice as described in A. (E) Diagrams and representative FACS plot illustrating the percent of donor (CD45.2+) of CD4+CD8−, CD4−CD8+, CD4+CD8+, and Thy1.2+Tcrβ+ cells in the spleen of Rag1−/− mice. (F) Southern blot of generated PCR products analyzing immunoglobulin heavy chain VDJ recombination using genomic DNA from Wt pro–B, mouse ear, or sorted TCRβ+ cells from TH transplanted Rag1−/− mice. (G) Diagrams illustrating total CD45.2+ out of live cells in thymus, also the percent of donor (CD45.2+) of CD19+, NK1.1+, CD4+CD8−, CD4−CD8+, and CD4+CD8+ in the thymus of Rag1−/− mice as described in A. In A, C, E, and G, each open circle/square represents one mouse (Wt, n = 4; TH, n = 5) and the data are presented as median (horizontal line) ± interquartile range. Statistical analysis was performed using Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
To investigate the functional consequence of combined reductions of Ebf1 and Pax5 dose, we generated mice transheterozygous (TH) for mutations in the Ebf1 and Pax5 genes. These animals did not display any decrease in the total number of the pro–B cells as compared with that of the Wt or the single mutant mice (Fig. 1 A). In contrast, the Lin-B220+CD19+CD43low/negIgM− (pre–B cell) compartment was reduced in TH mice as compared with what was observed in any of the single mutant mice. The number of Lin−B220+CD19+CD43low/negIgM+IgD− cells was reduced to levels comparable to single Ebf1+/− mice in the Pax5+/−Ebf1+/− animals (Fig. 1 A). The formation of early progenitor cells in the Pax5+/−Ebf1+/− mice suggested that the combined dose reduction in Ebf1 and Pax5 would not result in a complete disruption of the autoregulatory loop between Ebf1 and Pax5 and collapse of the genetic program. Consistent with this notion, Q-PCR analysis of primary pro–B cells from Wt and Pax5+/−Ebf1+/− mice (Fig. 1 B) suggested a twofold down-regulation of Ebf1 and Pax5 transcripts, respectively in pro–B cells from the TH mice, as would be expected from loss of one functional allele.