American Society of Hematology

Figure 4.

$Figure 4. Decreased bone marrow B cells in Fzd9 KO mice. (A) Bone marrow from 4-month-old KO (bottom row), WT (top row), and Fzd9 heterozygous littermate (middle row) mice was stained for CD43 and B220 (i), and B220+CD43+ (ii, Hardy fractions A-C) were examined for 6C3 (BP1) and CD24 expression. B220intCD43lo/- cells (iii, predominantly Hardy fraction D-E) and B220hiCD43-/lo (iv, Hardy fraction E-F) were examined for surface IgM and IgD by flow cytometry. Percentages of total BM cells (i) or of gated cells (ii-iv) are shown. Total absolute numbers of bone marrow cells in Fzd9-/- mice varied from equal to 50% of WT levels but were not statistically significantly reduced (data not shown). In this representative experiment, bilateral tibia and femur yielded 2.15 × 107 cells in KO, 1.88 × 107 cells in heterozygotes, and 2.54 × 107 in WT mice. The CD43+B220+ gate contains essentially no sIgM+ or sIgD+ cells (data not shown). Data shown are representative of 14 pairs of KO and WT control mice studied (for CD43, B220, and sIg staining; CD24/6C3 staining was technically more variable with analyzable data in 7 pairs). (B) Individual Fzd9-/- animals are plotted as a function of the relative numbers of each B-cell subset in comparison with WT mice analyzed at the same time, with a value of 1.0 indicating equal numbers and values less than 1 indicating decreased cells in KO animals. Subsets are, from left to right, all B cells (B220+), all Hardy A to C cells (B220+CD43+), Hardy D to E (B220intCD43-), and Hardy F (B220hiCD43-); n = 14; bars denote the mean value for each subset. Individual Hardy subsets are not presented separately due to variability in the antibody combinations used in individual experiments such that data on each subset are unavailable for all data points. B220 and CD43 staining, however, was always performed, and the B220+CD43+, B220intCD43-, and B220hiCD43- subsets consistently contained a vast majority of Hardy A to C, D to E, and F cells, respectively, as determined by staining for CD25, CD24, 6C3, IgM, and IgD (panel A and data not shown).$

Decreased bone marrow B cells in Fzd9 KO mice. (A) Bone marrow from 4-month-old KO (bottom row), WT (top row), and Fzd9 heterozygous littermate (middle row) mice was stained for CD43 and B220 (i), and B220⁺CD43⁺ (ii, Hardy fractions A-C) were examined for 6C3 (BP1) and CD24 expression. B220^intCD43^lo/- cells (iii, predominantly Hardy fraction D-E) and B220^hiCD43^-/lo (iv, Hardy fraction E-F) were examined for surface IgM and IgD by flow cytometry. Percentages of total BM cells (i) or of gated cells (ii-iv) are shown. Total absolute numbers of bone marrow cells in Fzd9^-/- mice varied from equal to 50% of WT levels but were not statistically significantly reduced (data not shown). In this representative experiment, bilateral tibia and femur yielded 2.15 × 10⁷ cells in KO, 1.88 × 10⁷ cells in heterozygotes, and 2.54 × 10⁷ in WT mice. The CD43⁺B220⁺ gate contains essentially no sIgM⁺ or sIgD⁺ cells (data not shown). Data shown are representative of 14 pairs of KO and WT control mice studied (for CD43, B220, and sIg staining; CD24/6C3 staining was technically more variable with analyzable data in 7 pairs). (B) Individual Fzd9^-/- animals are plotted as a function of the relative numbers of each B-cell subset in comparison with WT mice analyzed at the same time, with a value of 1.0 indicating equal numbers and values less than 1 indicating decreased cells in KO animals. Subsets are, from left to right, all B cells (B220⁺), all Hardy A to C cells (B220⁺CD43⁺), Hardy D to E (B220^intCD43^-), and Hardy F (B220^hiCD43^-); n = 14; bars denote the mean value for each subset. Individual Hardy subsets are not presented separately due to variability in the antibody combinations used in individual experiments such that data on each subset are unavailable for all data points. B220 and CD43 staining, however, was always performed, and the B220⁺CD43⁺, B220^intCD43^-, and B220^hiCD43^- subsets consistently contained a vast majority of Hardy A to C, D to E, and F cells, respectively, as determined by staining for CD25, CD24, 6C3, IgM, and IgD (panel A and data not shown).

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