1. Simultaneous loss of β- and γ-catenin does not perturb hematopoiesis or lymphopoiesis
by Ute Koch, Anne Wilson, Monica Cobas, Rolf Kemler, H. Robson MacDonald, and Freddy Radtke
Blood
Volume 111(1):
January 1, 2008
©2008 by American Society of Hematology

2. Normal hematopoiesis in the combined absence of β- and γ-catenin.
Normal hematopoiesis in the combined absence of β- and γ-catenin. (A) Experimental strategy: CD45.2+ FL cells (E12.5-E14.5) from γ-catenin−/− β-cateninlox/lox (control) and γ-catenin−/− β-cateninlox/lox,Mx-Cre (β/γ dko) embryos were isolated and transplanted into lethally irradiated congenic wt mice (CD45.1+). At 8 weeks after reconstitution, all mice were injected 5 times at 2-day intervals with pI-pC to inactivate the floxed β-catenin alleles via induction of the Mx-Cre transgene. Mice were analyzed 5 to 7 weeks after the last pI-pC injection, and a fraction of T cell–depleted BM cells derived from these FL chimeras were used to set up mixed BM chimeras and serial transplantations, which were both analyzed between 6 weeks and 6 months after transplantation. B. (Left) Western blot analysis performed on embryos from either wt (control) or γ-catenin−/− mice showing the absence of γ-catenin protein in the conventional γ-catenin knock-out embryos. Tubulin was used as a loading control. (Right) Southern blot analysis of EcoRI-digested genomic DNA derived from sorted CD45.2+ BM cells from 2 control and 2 β/γ dko chimeras 7 weeks after inactivation of the floxed β-catenin alleles are shown. Floxed indicates the floxed β-catenin alleles, and deleted indicates the inactivated alleles (98%-100%). (C) Absolute numbers of either total or CD45.2+ BM cells derived from control (n = 7; □) and β/γ dko (n = 8; ■) chimeras. (D) Representative FACS analysis of KLS HSCs defined by CD117 and Sca1 after gating on Lin− BM (top row) and by CD135 and CD34 after gating on KLS (bottom) BM cells derived from control and β/γ dko chimeras. (E) Absolute numbers of KLS HSCs (CD117+Lin−Sca1+), LT-HSCs (CD117+Sca1+CD34−CD135−), ST-HSCs (CD117+Sca1+CD34+CD135−), MPPs (CD117+Sca1+CD34+CD135+), and CMPs (CD117+Sca−) gated on Lin− CD45.2+ BM cells derived from control (n = 7; □) and β/γ dko (n = 8; ■) chimeras. The error bars represent mean (± SD) in panels C and E.
Ute Koch et al. Blood 2008;111:
©2008 by American Society of Hematology

3. Simultaneous lack of β- and γ-catenin in HSCs of mixed BM chimeras and serial transplants does not affect their repopulation capacity, nor does it influence B- or T-cell development.
Simultaneous lack of β- and γ-catenin in HSCs of mixed BM chimeras and serial transplants does not affect their repopulation capacity, nor does it influence B- or T-cell development. (A) Mixed BM chimeras were analyzed between 6 and 26 weeks after reconstitution with a 1:2 mixture of CD45.1+ and either control or β/γ dko CD45.2+ BM cells (left panel). BM cells from primary chimeric mice of either control or β/γ dko were serially transplanted into CD45.1+ recipients and analyzed at 12 and 24 weeks after reconstitution (right panel). Percentages of CD45.2+ KLS (CD117+lin−Sca1+), LT-HSCs (CD117+lin−Sca1+CD34−CD135−), ST-HSCs (CD117+lin−Sca1+CD34+CD135−), and MPPs (CD117+lin−Sca1+CD34+CD135+) from either control (□) or β/γ dko (■) mixed BM chimeras (n = 6 for control and n = 7 for β/γ dko) and the serial transplants (n = 5 for control and n = 5 for β/γ dko) are shown. (B) Percentages of myeloid lineages, including granulocytes (Gr; Gr1+Mac1+), macrophages (MØ; Gr1−Mac1+), megakaryocytes (Mega; CD41+), early erythroblasts (EB; Ter119+CD71+), and red blood cells (RBC; Ter119+CD71−) gated on CD45.2+ donor-derived cells derived from either control (□) or β/γ dko (■) mixed BM chimeras (n = 6 for control and n = 7 for β/γ dko). (C) Percentages of BM-derived B-cell subsets, including total BM B cells (B220+), pro-/pre-B cells (B220+CD43+BP1−), large pre-BII cells (preBII; B220+CD43+BP1+), small pre-BII cells (spreBII; B220+CD43−BP1+IgM−), and immature B cells (Imm; B220+CD43−BP1−IgM+), gated on CD45.2+ donor-derived cells derived from either control (□) or β/γ dko (■) mixed BM chimeras (n = 6 for control and n = 7 for β/γ dko). (D) Representative FACS analysis of CD45.2+ total thymocytes stained with anti-CD4 and anti-CD8 antibodies (top row), or with anti-CD44 and anti-CD25 antibodies (bottom row) after gating on Lin− cells (and excluding CD44+CD117− cells) derived from control and β/γ dko mixed chimeras. (E) Bar graphs represent relative percentages of CD45.2+ thymocyte subsets; DN (double negative; CD4−CD8−TCRβ−), DN1 (CD117+CD44+CD25−), DN2 (CD117+CD44+CD25+), DN3 (CD44−CD25+), DN4 (CD44−CD25−), DP (double positive; CD4+CD8+), CD4 (CD4+CD8−), and CD8 (CD4−CD8+) derived from control (□) and β/γ dko (■) mixed chimeras (n = 6 for control and n = 7 for β/γ dko). The error bars represent mean (± SD) in panels A, B, C, and E. No statistically significant differences (P values ranged between .06 and .89) were observed between control and β/γ dko chimeras in all populations analyzed.
Ute Koch et al. Blood 2008;111:
©2008 by American Society of Hematology