Beta-catenin is dispensable for hematopoiesis and lymphopoiesis

Abstract

Beta-catenin-mediated Wnt signaling has been suggested to be critically involved in hematopoietic stem cell maintenance and development of T and B cells in the immune system. Unexpectedly, here we report that inducible Cre-loxP-mediated inactivation of the beta-catenin gene in bone marrow progenitors does not impair their ability to self-renew and reconstitute all hematopoietic lineages (myeloid, erythroid, and lymphoid), even in competitive mixed chimeras. In addition, both thymocyte survival and antigen-induced proliferation of peripheral T cells is beta-catenin independent. In contrast to earlier reports, these data exclude an essential role for beta-catenin during hematopoiesis and lymphopoiesis.

Figure 1.

Inducible targeting of the β-catenin gene in the BM by the Mx-Cre recombinase. (a) Schematic representation of the β-catenin protein containing an NH₂-terminal region (N-term), 12 Armadillo repeats, and a carboxy terminus harboring the transactivation domain (C-term). The genomic organization of the β-catenin^lox/lox locus is shown with cylinders indicating coding exons and small black rectangles indicating noncoding exons. Exons 2–6 are flanked by two loxP sequences (triangles). β-catenin^lox/lox mice were crossed to mice carrying the IFN-α–inducible Mx-Cre transgene to inducibly inactivate β-catenin in the BM after pI-pC treatment. Arrows indicate approximate positions of the PCR primers used to verify the deletion of the floxed β-catenin locus. EcoRI restriction sites and the probe for Southern blot analysis are indicated. (b) Southern blot analysis of Eco-RI–digested genomic DNA from BM of WT, β-catenin^lox/lox, or β-catenin^−/− mice. The probe indicated in (a) reveals a 7-kb fragment for the WT allele, two fragments of 3.8 and 3.2 kb for the homozygously floxed β-catenin locus, and one 5.2-kb fragment for the β-catenin^−/− allele after successful deletion of the loxP flanked gene segment. (c) Survival curve of β-catenin^lox/lox and β-catenin^−/− mice after injection of pI-pC as indicated by arrows. Death of β-catenin^−/− mice starts 2 wk after the last injection. n = 8 for both β-catenin^lox/lox (controls) and β-catenin^−/−.

Figure 2.

β-catenin–deficient BM reconstitutes all major hematopoietic lineages. BM chimeras were analyzed 4–7 mo after reconstitution. (a) Absolute donor cell numbers for total BM and different subsets: early erythroblasts (Early Eryth., Ter119⁺), granulocytes (Gran., Gr1⁺), B cells (B220⁺), megakaryocytes (Mega., CD41⁺), and macrophages (Mac, Gr1⁻ CD11b⁺) of either β-catenin^lox/lox (open bars) or β-catenin^−/− (shaded bars) mice. The bars represent mean ± SD values (n = 5). (b) Absolute donor cell numbers for total thymocytes and thymocyte subsets: CD4⁻ CD8⁻ (DN), CD4⁺ CD8⁺ (DP), CD4⁺ CD8⁻ TCRβ⁺ (CD4), and CD8⁺ CD4⁻ TCRβ⁺ (CD8). The bars represent mean ± SD values for β-catenin^lox/lox (open bars) or β-catenin^−/− (filled bars) mice, where n = 5 for both β-catenin^lox/lox and β-catenin^−/− mice. (c) Absolute donor cell numbers for total splenocytes, B cells (B220⁺), and T cells (CD3⁺). n = 5 for control and β-catenin^−/− mice. (d) Southern blot analysis of Eco-RI–digested genomic DNA from donor thymocytes of control (β-catenin^lox/lox) or β-catenin^−/− BM chimeric mice (refer to Fig.1 a for details). (e) Immunoblot analysis of β-catenin protein expression in total donor thymocytes from control or β-catenin^−/− BM chimeras. The blots were probed with an antiserum specific to the COOH terminus of the β-catenin protein. To verify that equal amounts of protein were loaded, the membrane was reprobed with a monoclonal antibody against α-tubulin (bottom).

Figure 3.

Normal development of β-catenin^−/− hematopoietic lineages in a competitive situation. Mixed BM chimeric mice were analyzed 4–6 mo after reconstitution with a 1:2 mixture of WT (CD45.1⁺) and either β-catenin^lox/lox or β-catenin^−/− (CD45.2⁺) BM-derived populations. (a) A representative FACS^® analysis of BM stained with anti-CD117 and anti-Sca1 gated on lin⁻ CD45.2⁺ donor cells of either β-catenin^lox/lox or β-catenin^−/− mixed chimeras. Numbers indicate relative percentage of HSCs (CD117⁺ Sca1⁺) CLPs (CD117^low Sca1^low), and CMPs (CD117⁺ Sca1⁻) in each indicated region. (b) Percentage HSCs, CMPs, and CLPs gated on lin⁻ CD45.2⁺ cells from either β-catenin^lox/lox (open bars) or β-catenin^−/− mixed chimeras (shaded bars). The bars represent mean ± SD (n = 8). (c) Percentage early erythroblasts (Ter119⁺), granulocytes (Gr1⁺), and macrophages (Gr1⁻ CD11b⁺) after gating on CD45.2⁺ cells from either β-catenin^lox/lox or β-catenin^−/− mixed chimeras. The bars represent mean ± SD (n = 8).

Figure 4.

Normal development of β-catenin–deficient B and T cells in mixed BM chimeras. Representative FACS^® analyses (left and middle panels) gated on CD45.2⁺ donor cells. Bar diagrams (right panels) indicate relative number ± SD of the different subsets where n = 12 for both β-catenin^lox/lox (open bars) and β-catenin^−/− (shaded bars) mice. (a) BM stained with anti-B220 and anti-IgM antibodies. (b) BM cells stained with anti-CD43 and anti-IgM antibodies after gating on B220⁺ cells. (c) Total thymocytes stained with anti-CD4 and anti-CD8 antibodies. DN (CD4⁻ CD8⁻), DP (CD4⁺ CD8⁺), CD4 (CD4⁺ CD8⁻ TCRβ⁺), and CD8 (CD4⁻ CD8⁺ TCRβ⁺). (d) Thymocytes stained with anti-CD44 and anti-CD25 antibodies after gating on donor (CD45.2⁺)-derived lineage-negative cells. DN1 (CD44⁺ CD25⁻), DN2 (CD44⁺ CD25⁺), DN3 (CD44⁻ CD25⁺), and DN4 (CD44⁻ CD25⁻). (e) CD45.2⁺ (donor) and CD45.1⁺ (WT) cells derived from chimeric spleens were purified by FACS^® sorting to verify deletion of the floxed β-catenin alleles after reconstitution. FACS^® analysis before (left) and after the sort (right). Relative purity of the sorted populations are indicated. (f) Southern blot analysis of Eco-RI–digested genomic DNA from sorted CD45.1⁺ and CD45.2⁺ donor splenocytes. CD45.1⁺ cells were pooled from three different mice and CD45.2⁺ cells are derived from two individual mice.

Figure 5.

Absence of β-catenin does not affect spontaneous or glucocorticoid-induced death of thymocytes. (a) Percentage of surviving thymocytes gated on CD45.1⁺ (WT donor) or CD45.2⁺ (β-catenin^−/− donor) derived from mixed BM chimeras after 12 h of culture at 37°C. The percentage of surviving cells is normalized to control cells maintained at 4°C. Data represent mean ± SD of five experiments. (b) Glucocorticoid-induced cell death in WT (CD45.1⁺) and β-catenin^−/− (CD45.2⁺) CD4⁺ CD8⁺ DP thymocytes after 12 h of culture at 37°C. Data are normalized to untreated controls and represent mean ± SD from five individual experiments.

Figure 6.

Loss of β-catenin does not perturb antigen-induced T cell proliferation. CFSE-labeled splenocytes isolated from mixed BM chimeras were transferred i.v. into WT mice. The next day mice were injected i.p. with 20 μg SEB. 2 d later the draining popliteal LNs were removed and analyzed by FACS^®. After gating on CD45.1⁺ (WT donor) or CD45.2⁺ (β-catenin^−/− donor), the number of cell divisions in Vβ8⁻ (negative control), CD8⁺ Vβ8⁺, and CD4⁺ Vβ8⁺ populations are shown. One representative experiment out of four is shown.