Figure 5
Figure 5. Canonical Wnt signaling activity in hematopoietic cells in vivo as measured with the conductinlacZ Wnt reporter. (A) Histograms show β-galactosidase (LacZ) activity (conductin expression) in total thymocytes and gated spleenic CD4+ T cells and CD4−8− cells (B cells) in conductin+/LacZ compared with conductin+/+ controls. (B) β-Catenin was deleted in primary recipients and 10 days later LacZ activity (conductin expression) was determined in gated CD45.2+ LSK cells of the indicated genotypes. Numbers indicate the geometric mean of the green fluorescence intensity of the total population (top left) and the percentage of cells in the indicated gate. The data shown are representative of 2 experiments. (C) Primary recipients were treated and analyzed as described above. In addition, we also used secondary recipients, which were repopulated with β-catenin–deleted BM for more than 8 weeks. LacZ activity was determined in gated CD45.2+ DN4 (lin− CD44− CD25−) and DP (CD4+ CD8+) thymocytes. In panels A-C, numbers on graphs are percentage of total cells in the delineated region. (D) Genomic DNA isolated from chimeras reconstituted with deletion-induced Mx-cre:β-cateninΔ/Δ:γ-catenin−/− BM cells was amplified by PCR specific for the floxed, un-recombined β-catenin allele (loxP) for the indicated number of cycles. The osf2 locus was amplified as a control. (E) Total cellular lysates from BM cells of the indicated types of chimeric mice were subjected to immunoblot analysis with mAbs to the COOH terminus of β-catenin (clone 14) and to tubulin (to ensure equal protein loading). Note that full-size β-catenin protein was not detected after Cre-mediated recombination. (F) LacZ activity was determined in gated DN4 (lin− CD44− CD25−) and DP (CD4+ CD8+) thymocytes of TCF-1-deficient conductin+ LacZ mice. Numbers on graphs are percentage of total cells in the delineated region. (C,E) Numbers indicate the geometric mean of the green fluorescence in the total population. In mutant mice, the mean percentage (± SD) of the geometric mean was calculated relative to the wild-type control (100%) after subtracting the background staining (from conductin+/+ mice). Data are from 3 to 5 independent experiments; a single determination was performed for DN4 cells in β-/γ-catenin–deficient cells.

Canonical Wnt signaling activity in hematopoietic cells in vivo as measured with the conductinlacZ Wnt reporter. (A) Histograms show β-galactosidase (LacZ) activity (conductin expression) in total thymocytes and gated spleenic CD4+ T cells and CD4−8− cells (B cells) in conductin+/LacZ compared with conductin+/+ controls. (B) β-Catenin was deleted in primary recipients and 10 days later LacZ activity (conductin expression) was determined in gated CD45.2+ LSK cells of the indicated genotypes. Numbers indicate the geometric mean of the green fluorescence intensity of the total population (top left) and the percentage of cells in the indicated gate. The data shown are representative of 2 experiments. (C) Primary recipients were treated and analyzed as described above. In addition, we also used secondary recipients, which were repopulated with β-catenin–deleted BM for more than 8 weeks. LacZ activity was determined in gated CD45.2+ DN4 (lin− CD44− CD25−) and DP (CD4+ CD8+) thymocytes. In panels A-C, numbers on graphs are percentage of total cells in the delineated region. (D) Genomic DNA isolated from chimeras reconstituted with deletion-induced Mx-cre:β-cateninΔ/Δ:γ-catenin−/− BM cells was amplified by PCR specific for the floxed, un-recombined β-catenin allele (loxP) for the indicated number of cycles. The osf2 locus was amplified as a control. (E) Total cellular lysates from BM cells of the indicated types of chimeric mice were subjected to immunoblot analysis with mAbs to the COOH terminus of β-catenin (clone 14) and to tubulin (to ensure equal protein loading). Note that full-size β-catenin protein was not detected after Cre-mediated recombination. (F) LacZ activity was determined in gated DN4 (lin− CD44− CD25−) and DP (CD4+ CD8+) thymocytes of TCF-1-deficient conductin+ LacZ mice. Numbers on graphs are percentage of total cells in the delineated region. (C,E) Numbers indicate the geometric mean of the green fluorescence in the total population. In mutant mice, the mean percentage (± SD) of the geometric mean was calculated relative to the wild-type control (100%) after subtracting the background staining (from conductin+/+ mice). Data are from 3 to 5 independent experiments; a single determination was performed for DN4 cells in β-/γ-catenin–deficient cells.

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