c-Myb is required for pro-B cell differentiation - PubMed (original) (raw)

c-Myb is required for pro-B cell differentiation

Shawn P Fahl et al. J Immunol. 2009.

Abstract

The c-Myb transcription factor is required for normal adult hematopoiesis. However, the embryonic lethality of Myb-null mutations has been an impediment to identifying roles for c-Myb during lymphocyte development. We have used tissue-specific inactivation of the Myb locus in early progenitor cells to demonstrate that c-Myb is absolutely required for the differentiation of CD19(+) B-lineage cells and B cell differentiation is profoundly blocked beyond the pre-pro-B cell stage in Myb(f/f) Mb1-cre mice. We demonstrate that c-Myb is required for the intrinsic survival of CD19(+) pro-B cells as well as the proper expression of the alpha-chain of the IL-7 receptor (CD127) and Ebf1. However, survival of c-Myb-deficient CD19(+) pro-B cells cannot be rescued by transduction with CD127-producing retrovirus, suggesting that c-Myb controls a survival pathway independent of CD127. Furthermore, c-Myb-deficient progenitor cells inefficiently generate CD19(+) B-lineage cells during stromal cell culture but this process can be partially rescued with exogenous Ebf1. Thus, c-Myb does not appear to be required for commitment to B cell differentiation but is crucial for B cell differentiation to the CD19(+) pro-B cell stage as well as survival of CD19(+) pro-B cells. Surprisingly, forced c-Myb expression in lymphoid-primed multipotent progenitors favors differentiation toward the myeloid lineage, suggesting that proper c-Myb expression is crucial for B-lineage development.

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Figures

FIGURE 1

FIGURE 1

Expression of Myb mRNA in bone marrow B-lineage progenitors. Total cellular RNA was isolated from HSC (Lin− CD117+ Sca1+ CD135−), LMPP (Lin− CD117+ Sca1+ CD135hi), CLP (Lin− CD19− B220− CD127+ CD135+), pre-pro-B cell (Ly6C− NK1.1− B220+ CD43+ CD19−), pro-B cell (Ly6C− NK1.1− B220+ CD43+ CD19+), pre-B cell (B220+ CD43−), immature B cell (B220+ IgM+), and mature B cell (B220hi IgM+) subsets isolated from C57BL/6J bone marrow. Myb expression was analyzed by quantitative RT-PCR and normalized to the expression of HPRT. * p<0.01

FIGURE 2

FIGURE 2

Deletion efficiency of the floxed Myb allele in Mybf/+ Mb1-cre mice. Deletion at the Myb locus was analyzed in B-lineage subsets from the bone marrow as described in Figure 1 as well as CD19+ splenocytes (top panel) and thymic DN (CD4− CD8−), DP (CD4+ CD8+), CD4 SP (CD4+ CD8−), and CD8 SP (CD4− CD8+) subsets as well as CD4+ and CD8+ splenocytes (bottom panel). Control PCR from Mybf/+ (f/+), Mybf/− (f/−) and Myb+/− (+/−) splenocytes was included as markers.

FIGURE 3

FIGURE 3

Lack of peripheral B cells in Mybf/f Mb1-cre mice. A, Bone marrow, spleen, and inguinal lymph node cells were analyzed for surface expression of B220 and CD19 by flow cytometry. Viable cells were identified as PI−. Number next to gates is percent of total PI− cells. Data is representative of at least 10 mutant and control mice. B, Peritoneal fluid cells were analyzed for surface expression of B220 and CD19 (top panel) or CD5 and IgM (bottom panel) to detect B1a and B1b or B1a B-cells defined as B220lo CD19+ and CD5+ IgM+, respectively. Viable cells were identified as PI−. Data is representative of 3 mutant and control mice of each strain. C, Absolute number of total nucleated cells, B220+ CD19+ B-cells, CD4+ T-cells, and CD8+ T-cells from Mybf/f and Mybf/f Mb1-cre spleen (left panel). Total splenic cellularity, CD4+ T-cells and CD8+ T-cells were significantly reduced in Mybf/f Mb1-cre mice compared to controls (p<0.05). No statistically significant difference in total thymic cellularity was detected between Mybf/f and Mybf/f Mb1-cre mice (right panel). D, Spleen, inguinal lymph node and thymus cells were analyzed for surface expression of CD4 and CD8 by flow cytometry. Viable cells were identified as PI−.

FIGURE 4

FIGURE 4

B-cell development in Mybf/f Mb1-cre mice. A, Bone marrow from Mybf/f Mb1-cre and Mybf/f control mice was stained for surface expression of Ly6C, NK1.1, B220, IgM, CD43, CD19, and CD24. Viable cells were identified as DAPI−. Top tier represents B220 versus IgM after gating out Ly6C+ and NK1.1+ cells. Mature B-cells were defined as Ly6C− NK1.1− B220hi IgM+ and immature B-cells were defined as Ly6C− NK1.1− B220+ IgM+. Middle tier represents B220 versus CD43 through a B220+ IgM− gate. Pre-B cells were defined as Ly6C− NK1.1− B220+ IgM− CD43−. Bottom tier represents CD19 versus CD24 through B220+ CD43+ gate. Pro-B cells were defined as Ly6C− NK1.1− B220+ IgM− CD43+ CD19+ CD24+ and pre-pro-B cells were defined as Ly6C− NK1.1− B220+ IgM− CD43+ CD19− CD24−. Data is representative of at least 10 mutant and control mice. B, Bone marrow was stained for surface expression of lineage markers [CD3e, CD11b, Ly6C, Ter119], CD19, B220, CD135, and CD127. Viable cells were identified as DAPI−. CLPs were defined as Lin− CD19− B220− CD127+ CD135+. Data is representative of three mutant and three control mice. C, Bone marrow was stained for surface expression of lineage markers [B220, CD3e, CD11b, Ly6C, Ter119], CD127, CD117, and Sca1. Viable cells were identified as DAPI−. HSCs were defined as Lin− CD127− CD117+ Sca1+. Data is representative of three mutant and three control mice. D, Absolute number of HSCs, CLPs, pre-pro-B cells, pro-B cells, pre-B cells, immature B cells, and mature B cells detected in Mybf/f Mb1-cre and Mybf/f mice. Data is compiled from three mutant and control mice. No statistically significant difference in HSCs, CLPs, and pre-pro-B cells between Mybf/f Mb1-cre mice and LMC. Asterisks (*) mark populations that are severely reduced in Mybf/f Mb1-cre mice.

FIGURE 5

FIGURE 5

B-cell development in Myb+/+ Rag2−/−, Myb+/− Rag2−/−, and Mybf/f CD19-cre Rag2−/− mice. A, Absolute number of pre-pro-B cells and pro-B cells in Myb+/+ Rag2−/− (black bar), Myb+/− Rag2−/− (grey bar), and Mybf/f CD19-cre Rag2−/− (white bar) mice. None are statistically significant. B, Bone marrow was stained for surface expression of Ly6C, NK1.1, B220, IgM, CD24, and CD19. Top tier, B220 versus IgM after gating out Ly6C+ and NK1.1+ cells. Bottom tier, CD19 versus CD24 through the B220+ IgM− gate. Pro-B cells were defined as Ly6C− NK1.1− B220+ IgM− CD19+ CD24+ and pre-pro-B cells were defined as Ly6C− NK1.1− B220+ IgM− CD19− CD24−. Histogram represents surface expression of CD24 on CD19+ pro-B cells, defined as Ly6C− NK1.1− B220+ IgM− CD19+, from Myb+/+ Rag2−/−, Myb+/− Rag2−/−, and Mybf/f CD19-cre Rag2−/− mice. C, Relative recovery of CD19+ pro-B cells isolated from Myb+/+ Rag2−/−, Myb+/− Rag2−/−, and Mybf/f CD19-cre Rag2−/− mice after 24, 48, and 72 hours in liquid culture without exogenous IL-7. Viable cell counts were determined by trypan blue exclusion. Relative recovery was determined by normalization to the total number of cells plated.

FIGURE 6

FIGURE 6

c-Myb-deficient CD19+ pro-B cells have an intrinsic survival defect. A, CD19+ pro-B cells isolated from Mybf/f Rag2−/− mice were transduced with MIG-R1 or MIG-Cre and cultured without exogenous IL-7. Cells were analyzed 24, 48, and 72 hours post-transduction for expression of GFP and total cells per well. B, CD19+ pro-B cells isolated from Myb+/+ Rag2−/− mice were transduced with MIG-Cre and cultured without exogenous IL-7. Cells were analyzed 24 and 48 hours post-transduction for the expression of GFP and total cells per well.

FIGURE 7

FIGURE 7

Reduced expression of Il7r and Ebf1 mRNA in the absence of c-Myb. A, Surface expression of CD117 (c-kit) and CD127 (IL-7Rα) on Mybf/f Rag2−/− CD19+ pro-B cells transduced with MIG-R1 or MIG-Cre and analyzed 24, 36, and 48 hours post-transduction. Viable cells were identified as 7AAD−. B, Relative expression of Il7r and Ebf1 mRNA in Mybf/f Rag2−/− CD19+ pro-B cells transduced with tNGFR or tNGFR-Cre was measured by semi-quantitative RT-PCR 24, 36, and 48 hours post-transduction and normalized to the expression of ActB. Transduced cells were isolated after treatment with biotinylated anti-tNGFR antibody and streptavidin coated magnetic beads prior to making RNA. C, Intracellular expression of Bcl-xL and Mcl1 in Mybff Rag2−/− CD19+ pro-B cells transduced with tNGFR or tNGFR-Cre was measured by flow cytometry 24 and 48 hours post-transduction. D, Semi-quantitative RT-PCR analysis of Bcl2, Bcl-xL, Mcl1, and Bim performed on 3-fold serial dilutions of cDNA prepared from Mybf/f Rag2−/− CD19+ pro-B cells transduced with tNGFR or tNGFR-Cre 24 and 36 hours post-transduction. ActB serves as a loading control.

FIGURE 8

FIGURE 8

Overexpression of IL-7Rα or EBF is unable to rescue the survival defect in _Myb_-deficient CD19+ pro-B cells. A, CD19+ pro-B cells from Mybf/f Rag2−/− mice were cotransduced with tNGFR-Cre and MIG-R1, MIG-cMyb, MIG-IL7Rα, or MIG-Ebf1, cultured in the absence of exogenous IL-7, and analyzed 24, 36, and 48 hours post-transduction for expression of tNGFR and GFP and total cells per well. Relative recovery was determined by normalization to the number of cotransduced cells 24 hours post-transduction. B, CD19+ pro-B cells from Mybf/f Rag2−/− mice were cotransduced with tNGFR or tNGFR-Cre and MIG-R1 or MIG-IL7Rα, cultured in the presence of exogenous IL-7, and analyzed 24 and 48 hours post-transduction for expression of tNGFR and GFP and total cells per well. C, Sub2n DNA content of Mybf/f Rag2−/− CD19+ pro-B cells cotransduced with tNGFR or tNGFR-Cre and MIG-R1 or MIG-IL7Rα was determined by flow cytometry 48 hours post-transduction.

FIGURE 9

FIGURE 9

Ebf1 can partially rescue B-lineage development from Mybf/f Mb1-cre LMPPs. A. Surface expression of CD127 on pre-pro-B cells, defined as Ly6C− NK1.1− B220+ CD43+ CD19− CD24−, from Mybf/f Mb1-cre and Mybf/f mice compared to FMO control. B, Five thousand LMPPs from Mybf/f and Mybf/f Mb1-cre mice were seeded on OP-9 stromal cells in the presence of SCF, Flt3L, and IL-7 and analyzed 14 days later by flow cytometry for surface expression of CD19 and Gr1. Bars represent percentage of positive cells from each culture. Data are representative of 4 independent experiments. C, Total number of CD19+, Gr1+, and CD19− Gr1− cells that grew out of OP-9 stromal cell coculture with Mybf/f and Mybf/f Mb1-cre LMPPs. Data are representative of 4 independent experiments. D, LMPPs were isolated from Mybf/f Mb1-cre bone marrow, seeded at 5,000 cells per well on OP-9 stromal cells and transduced with MIG-R1, MIG-cMyb, MIG-IL7Rα, or MIG-Ebf1. LMPPs were cultured in the presence of SCF, Flt3L, and IL-7 and analyzed 14 days later by flow cytometry for expression of B220 and CD19 and the number of GFP+ B220+ CD19+ cells were determined. Data are representative of 4 independent experiments. E, Mybf/f and Mybf/f Mb1-cre LMPPs were transduced with MIG-R1 or MIG-cMyb and cultured on OP9 stromal cells in the presence of SCF, Flt3L, and IL-7. Cultures were analyzed 14 days later by flow cytometry for the surface expression of CD19 and Gr1. Number in quadrants indicates percent cells in each well. Data are representative of four independent experiments. F, Day 14.5 fetal liver progenitors from Myb+/+ embryos were transduced with MIG-R1 or MIG-cMyb and cultured on OP-9 stromal cells in the presence of SCF, Flt3L, and IL-7. Cultures were analyzed 14 days later by flow cytometry for the surface expression of CD19 and Gr1. Number in quadrants indicates percent cells in each.

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